Bioanalysis of aminoglycosides using high-performance liquid chromatography


doi: http://dx.doi.org/10.5599/admet.1183   27 

ADMET & DMPK 10(1) (2022) 27-62; doi: https://doi.org/10.5599/admet.1183  

 
Open Access : ISSN : 1848-7718  

http://www.pub.iapchem.org/ojs/index.php/admet/index   

 

Review 

Bioanalysis of aminoglycosides using high-performance liquid 
chromatography 

Seth K. Amponsah
1
, Joseph A. Boadu

2
, Daniel K. Dwamena

1
, Kwabena F. M. Opuni

2,*
 

1
Department of Medical Pharmacology, University of Ghana Medical School, University of Ghana, Ghana 

2
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Ghana, Ghana 

*Corresponding Author:  E-mail: kfopuni@ug.edu.gh; Tel.: +233208260595 

Received: November 19, 2021; Revised: December 30, 2021; Published: January 11, 2022  

 

Abstract 

Aminoglycosides are broad-spectrum antibiotics used in the treatment of gram-negative bacterial 
infections. Due to their nephrotoxic and ototoxic potential (narrow therapeutic index), the use of 
aminoglycoside for clinical indications requires monitoring. The objective of this review was to identify 
relevant literature reporting liquid chromatographic methods for the bioanalysis of aminoglycosides in 
both preclinical and clinical settings/experiments. Data on liquid chromatographic methods were collected 
from articles in an online academic database (PubMed, Science Direct, Scopus, and Google Scholar). All 71 
articles published from 1977 to 2020 were included in the review. Reversed-phase liquid chromatography 
was the most used method for the bioanalysis of aminoglycosides. Fluorescence or ultraviolet detection 
methods were mostly used from 1977 to 2002 (51 articles), while mass spectrometry was predominantly 
used as a detector from 2003 to 2020 (15 articles). Sixty-seven articles reported calibration ranges, which 
varied significantly for the various drugs assayed: some in the range of 0.1-0.5 ng/mL and others 1250-
200000 ng/mL. Also, 61 articles reported R

2
 values (0.964-1.0) for almost all analytes under consideration. 

Sixty-three articles reported percent recoveries mostly between 61.0 % to 114.0 %, with only two articles 
reporting recoveries of 4.9 % and 36 %. Out of the 71 reviewed articles, 56 reported intermediate precision 
values ranging between 0.331 % to 19.76 %, which is within the acceptable limit of 20 %. This review will 
serve as a guide for research and/or routine clinical monitoring of aminoglycosides in biological matrices. 

©2022 by the authors. This article is an open-access article distributed under the terms and conditions of the Creative Commons 
Attribution license (http://creativecommons.org/licenses/by/4.0/). 

Keywords 

PK studies; Therapeutic drug monitoring; Biological matrices. 

 

Introduction 

Aminoglycosides are broad-spectrum antibiotics that are used in the treatment of gram-negative 

bacterial infections [1,2]. Aminoglycosides elicit their pharmacological effect by binding to the 16S rRNA 

ribosomal subunit of bacteria and blocking mRNA translation, altering protein synthesis. Structurally, this 

class of antibiotics has amino sugars in their core connected via glycosidic linkages to a dibasic aminocyclitol 

[3]. Aminoglycosides are relatively hydrophilic, hence, rarely undergo biotransformation in vivo. 

Aminoglycosides, to some extent, bind to plasma proteins and are excreted entirely unchanged in urine [4]. 

Streptomycin, netilmicin, tobramycin, kanamycin, spectinomycin, gentamicin, neomycin, amikacin, and 

http://dx.doi.org/10.5599/admet.1183
https://doi.org/10.5599/admet.1183
http://www.pub.iapchem.org/ojs/index.php/admet/index
mailto:kfopuni@ug.edu.gh
http://creativecommons.org/licenses/by/4.0/


Seth K. Amponsah et al.   ADMET & DMPK 10(1) (2022) 27-62 

28  

paromomycin are examples of aminoglycosides [5,6]. 

Aminoglycosides are known to possess nephrotoxic and ototoxic potentials, limiting their clinical use [2]. 

In addition, due to their narrow therapeutic index, therapeutic monitoring is required for aminoglycosides. 

Especially among patients with underlying renal problems, monitoring aminoglycosides in biological 

matrices ensures optimal therapy and reduces toxicity [7].  

There are several validated bioanalytical methods used to quantitatively determine levels of 

aminoglycosides in biological matrices. Microbiological assays, radioimmunoassay (RIA), radioenzymatic 

assays, fluorescence polarization immunoassay (FPIA), high-performance liquid chromatography (HPLC), gas 

chromatography (GC) and mass spectrometric techniques are some common examples [8-12]. Of these 

methods, HPLC is the most preferred or routinely used [13]. Indeed, several studies have used HPLC for the 

bioanalysis of aminoglycosides [14-16]. Although there are recently published reviews on aminoglycosides, 

the focus of these reviews have been a) pre-treatment and analysis methods of aminoglycosides in food 

[17], b) challenges in the development of analytical test procedures for aminoglycosides [18], and c) 

determination of kanamycin by high-performance liquid chromatography [19]. 

The current review focuses on liquid chromatographic methods employed for the bioanalysis of 

aminoglycosides. A total of 20 aminoglycosides were reported by 71 articles (total number reviewed). The 

aminoglycosides were gentamicin [20], netilmicin [21], amikacin [22], tobramycin [23], dibekacin [24], 

sisomicin [25], astromicin [25], micronomicin [25], kanamycin [26], streptomycin [27], neomycin [14], 

isepamicin [28], geneticin [29], dihydrostreptomycin [30], paromomycin [31], apramycin [5], hygromycin 

[5], etimicin [32], arbekacin [33], and spectinomycin [34]. The aforementioned aminoglycosides obtained 

from either natural products or semi-synthetic derivatives of soil actinomycetes notably Streptomyces have 

suffix -mycin (examples are streptomycin, dihydrostreptomycin, kanamycin, apramycin, paromomycin, 

neomycin, tobramycin, spectinomycin, and hygromycin); and those obtained from other actinomycetes 

notably Micromonospora have the suffix -micin (examples are gentamicin, netilmicin, isepamicin, sisomicin, 

etimicin, geneticin, astromicin, and micronomicin). There are other exceptions, such as amikacin, arbekacin, 

and dibekacin. The constitute structures of these 20 aminoglycosides are presented in Figure 1. In the 

review, gentamicin was the most reported aminoglycoside (24 articles). This is not surprising since 

gentamicin is often used clinically because of its low cost and high efficacy against gram-negative aerobes. 

Fifty (50) articles reported bioanalysis of at least one of the 20 aminoglycosides, whilst 21 articles reported 

analysis of more than one aminoglycoside. The highest number of aminoglycosides simultaneously assayed 

was 13 [5].  

Although the list of articles used in this review may not be exhaustive, suitable liquid chromatographic 

conditions used in assaying aminoglycosides in biological matrices have been identified. Performance 

metrics of the various liquid chromatographic assays have also been appraised. Also, highlights of current 

procedures, scope, characteristics, and limitations of chromatographic methods used in assaying 

aminoglycosides in biological matrices have been provided. Although this is not a systematic review, it will 

serve as a comprehensive reference for subsequent related research that may involve the assay of 

aminoglycosides.  

Methods 

This study reviewed relevant and accessible articles on liquid chromatographic assays of 

aminoglycosides in biological matrices from 1977 to 2020. Articles were retrieved from journals in online 

academic databases (PubMed, Science Direct, Scopus, and Google Scholar) and limited to only the English 



ADMET & DMPK 10(1) (2022) 27-62 Bioanalysis of aminoglycozides by HPLC 

doi: http://dx.doi.org/10.5599/admet.1183 29 

language. Keywords used during the search were aminoglycosides, assay, HPLC, plasma, serum, milk, 

cerebrospinal fluid, and urine. The searched terms used were ‘‘chromatographic assay’’, ‘‘aminoglycosides’’ 

and ‘‘biological matrix’’. Articles were excluded if they were not pertinent. 

 

 

Figure 1. Structures of the 20 aminoglycosides reported by the various articles reviewed  

Aminoglycosides 

Sixty-seven (67) out of the 71 articles reported the use of HPLC in the bioanalysis of aminoglycosides, 

while four articles used ultra-performance liquid chromatography (UPLC) [35]. The relevant aspect of liquid 

chromatographic conditions used in the various articles, such as matrix, sample preparation, flow rate, 

column selection, mobile phase, and detection, have been summarized (Table 1). 

  

Streptomycin Dihydrostreptomycin Kanamycin Apramycin

Paromomycin Neomycin

Tobramycin Spectinomycin Hygromycin Gentamicin 

Amikacin

Netilmicin

Isepamicin

Sisomicin

Geneticin Arbekacin EtimicinDibekacin

Astromicin Micronomicin

http://dx.doi.org/10.5599/admet.1183


Seth K. Amponsah et al.   ADMET & DMPK 10(1) (2022) 27-62 

30  

Table 1. HPLC conditions for bioanalysis of aminoglycosides. 
D

e
te

ct
io

n
 

M
o

d
e

 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
4

0
 

n
m

; 
e

m
is

si
o

n
, 

K
V

4
1

8
 f

il
te

r)
 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
6

0
 

n
m

; 
e

m
is

si
o

n
, 

4
3

0
 n

m
) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

2
2

0
 

n
m

; 
e

m
is

si
o

n
, 

K
V

4
7

0
 f

il
te

r)
 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
4

0
 

n
m

; 
e

m
is

si
o

n
, 

4
1

8
 n

m
) 

  

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
 

fi
lt

e
rs

, 
7

-5
4

/7
-

6
0

; 
e

m
is

si
o

n
 

fi
lt

e
rs

, 
4

-7
6

/3
-

7
2

) 

D
e

ri
v

a
ti

za
ti

o
n

 a
g

e
n

t 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

d
a

n
sy

l 
ch

lo
ri

d
e

 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

  

o
-p

h
th

a
la

ld
e

h
y

d
e

 

M
o

b
il

e
 P

h
a

se
; 

E
lu

ti
o

n
; 

F
lo

w
 r

a
te

 (
m

L/
m

in
) 

 

0
.2

 M
 N

a
2
S

O
4
, 

0
.0

2
 M

 s
o

d
iu

m
 p

e
n

ta
n

e
 s

u
lf

o
n

a
te

, 

a
n

d
 

0
.1

%
 

(v
/v

) 
a

ce
ti

c 
a

ci
d

 
in

 
a

 
w

a
te

r/
m

e
th

a
n

o
l 

(9
7

:3
, 

v
/v

);
 I

S
O

; 
2

.0
 

m
e

th
a

n
o

l/
w

a
te

r 
(7

9
:2

1
, 

v
/v

) 
co

n
ta

in
in

g
 

2
 

g
/L

 
o

f 

tr
ip

o
ta

ss
iu

m
 E

D
T

A
; 

IS
O

; 
2

.0
 

a
ce

to
n

it
ri

le
/w

a
te

r 
(9

5
:5

, 
v

/v
);

 I
S

O
; 

1
.0

 

0
.2

 m
o

l 
o

f 
N

a
2
S

O
4
, 

0
.0

2
 m

o
l 

o
f 

so
d

iu
m

 p
e

n
ta

n
e

 

su
lf

o
n

a
te

, 
a

n
d

 1
7

.4
 m

m
o

l 
o

f 
a

ce
ti

c 
a

ci
d

/m
e

th
a

n
o

l 

(9
7

:3
, 

v
/v

);
 I

S
O

; 
2

.0
 

0
.1

 m
o

l 
o

f 
N

a
2
S

O
4
, 

0
.0

2
 m

o
l 

o
f 

so
d

iu
m

 p
e

n
ta

n
e

 

su
lf

o
n

a
te

, 
a

n
d

 1
7

.4
 m

m
o

l 
o

f 
a

ce
ti

c 
a

ci
d

; 
IS

O
; 

2
.0

 

0
.1

 m
o

l 
o

f 
N

a
2
S

O
4
, 

0
.0

2
 m

o
l 

o
f 

so
d

iu
m

 p
e

n
ta

n
e

 

su
lf

o
n

a
te

, 
a

n
d

 1
7

.4
 m

m
o

l 
o

f 
a

ce
ti

c 
a

ci
d

; 
IS

O
; 

2
.0

 

0
.5

 
m

o
l/

L 
T

ri
s 

b
u

ff
e

r 
(p

H
 

7
.9

)/
tr

im
e

th
y

la
m

in
e

, 

re
a

d
ju

st
e

d
 

p
H

 
7

.9
 

w
it

h
 

co
n

ce
n

tr
a

te
d

 
su

lp
h

u
ri

c 
a

ci
d

/m
e

th
a

n
o

l 
(2

5
0

:1
0

:7
4

0
, 

v
/v

/v
);

 I
S

O
; 

2
.0

 

0
.5

 
m

o
l/

L 
T

ri
s 

b
u

ff
e

r 
(p

H
 

7
.9

)/
tr

im
e

th
y

la
m

in
e

, 

re
a

d
ju

st
e

d
 

p
H

 
7

.9
 

w
it

h
 

co
n

ce
n

tr
a

te
d

 
su

lp
h

u
ri

c 

a
ci

d
/m

e
th

a
n

o
l 

(2
5

0
:1

0
:7

4
0

, 
v

/v
/v

);
 I

S
O

; 
2

.0
 

M
e

th
a

n
o

l/
tr

ip
o

ta
ss

iu
m

 
e

th
y

le
n

e
 

d
in

it
ri

lo
te

tr
a

a
ce

ta
te

 (
2

 g
/L

) 
(7

9
:2

1
, 

v
/v

);
 I

S
O

; 
2

.0
 

S
ta

ti
o

n
a

ry
 

P
h

a
se

 

C
1

8
  

C
1

8
  

C
1

8
 

(A
m

b
ie

n
t)

 

C
1

8
  

  

C
1

8
 

S
a

m
p

le
 

P
re

p
a

ra
ti

o
n

 

Io
n

-e
xc

h
a

n
g

e
 g

e
l 

ch
ro

m
a

to
g

ra
p

h
y

 

(S
P

E
) 

S
P

E
 

P
P

T
 (

A
C

N
) 

S
P

E
 

  

P
P

T
 (

A
C

N
) 

M
a

tr
ix

 

h
-s

e
ru

m
 

h
-s

e
ru

m
 

d
-s

e
ru

m
 

 h
-p

la
sm

a
 

h
-s

e
ru

m
 

  

h
-s

e
ru

m
  

In
te

rn
a

l 

S
ta

n
d

a
rd

 

n
.i

. 

n
.i

. 

n
.i

. 

N
-a

ce
ty

l 

g
e

n
ta

m
ic

in
 

C
1

 

T
o

b
ra

m
y

ci
n

 

A
m

ik
a

ci
n

 

n
.i

. 

A
n

a
ly

te
 

G
e

n
ta

m
ic

in
 

G
e

n
ta

m
ic

in
 C

1
 

G
e

n
ta

m
ic

in
 C

1
a

 

G
e

n
ta

m
ic

in
 C

2
 

N
e

ti
lm

ic
in

 

G
e

n
ta

m
ic

in
 

A
m

ik
a

ci
n

 

T
o

b
ra

m
y

ci
n

 

N
e

ti
lm

ic
in

 

T
o

b
ra

m
y

ci
n

 

G
e

n
ta

m
ic

in
 

R
e

f 

[2
0

] 

[3
6

] 

[2
1

] 

[2
2

] 

[2
3

] 

 



ADMET & DMPK 10(1) (2022) 27-62 Bioanalysis of aminoglycozides by HPLC 

doi: http://dx.doi.org/10.5599/admet.1183 31 

Table 1. Contined... 
D

e
te

ct
io

n
 

M
o

d
e

 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
4

0
 

n
m

; 
e

m
is

si
o

n
, 

4
1

8
 

n
m

) 

U
V

 (
3

6
5

 n
m

) 

U
V

 (
2

3
0

 n
m

) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

2
7

5
 

n
m

; 
e

m
is

si
o

n
, 

4
1

8
 

n
m

) 

U
V

 (
3

6
0

 n
m

) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
4

0
 

n
m

; 
e

m
is

si
o

n
, 

4
1

8
 n

m
) 

F
lu

o
re

sce
n

ce
 

(e
xcita

tio
n

, 3
6

5
 

n
m

; e
m

issio
n

, 

4
4

0
 n

m
) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
4

0
 

n
m

; 
e

m
is

si
o

n
, 

4
5

5
 n

m
) 

D
e

ri
v

a
ti

za
ti

o
n

 a
g

e
n

t 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

1
-f

lu
o

ro
-2

,4
-

d
in

it
ro

b
e

n
ze

n
e

 

B
e

n
ze

n
e

 s
u

lp
h

o
n

y
l 

ch
lo

ri
d

e
 

F
lu

o
re

sc
a

m
in

e
 

1
-f

lu
o

ro
-2

,4
-

d
in

it
ro

b
e

n
ze

n
e

 

o
-

P
h

th
a

li
cd

ic
a

rb
o

xa
ld

e

h
y

d
e

 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

M
o

b
il

e
 P

h
a

se
; 

E
lu

ti
o

n
; 

F
lo

w
 r

a
te

 (
m

L/
m

in
) 

m
e

th
a

n
o

l/
w

a
te

r/
a

ce
to

n
it

ri
le

 (
6

2
:3

5
.1

:2
.9

, 
v
/v

/v
) 

co
n

ta
in

in
g

 2
.5

 g
 t

ri
p

o
ta

ss
iu

m
 

e
th

y
le

n
e

d
ia

m
in

e
te

tr
a

a
ce

ti
c 

a
ci

d
; 

IS
O

; 
1

.6
 

1
 g

/L
 t

ri
s(

h
y

d
ro

xy
m

e
th

y
l)

a
m

in
o

m
e

th
a

n
e

 a
d

ju
st

e
d

 w
it

h
 

h
y

d
ro

ch
lo

ri
c 

a
ci

d
 t

o
 p

H
 7

/a
ce

to
n

it
ri

le
 (

3
0

:7
0

, 
v
/v

);
 I

S
O

; 

1
.5

 

a
ce

to
n

it
ri

le
/m

e
th

y
le

n
e

 c
h

lo
ri

d
e

/w
a

te
r/

m
e

th
a

n
o

l 

(8
0

:1
0

:8
:4

, 
v

/v
/v

/v
);

 I
S

O
; 

4
.0

 

a
ce

to
n

it
ri

le
/p

h
o

sp
h

o
ri

c 
a

ci
d

 (
5

 g
/L

) 
(7

0
:3

0
);

 I
S

O
; 

2
.0

 

a
ce

to
n

it
ri

le
/w

a
te

r 
(6

8
:3

2
, 

v
/v

);
 I

S
O

; 
1

.0
 

a
ce

to
n

it
ri

le
/t

ri
s(

h
y

d
ro

xy
m

e
th

y
l)

a
m

in
o

m
e

th
a

n
e

 (
1

 

g
/L

) 
a

d
ju

st
e

d
 t

o
 p

H
 3

.0
 w

it
h

 1
M

 h
y

d
ro

ch
lo

ri
c 

a
ci

d
 

(7
0

:3
0

, 
v
/v

);
 I

S
O

; 
1

.5
 

0
.1

 M
 d

is
o

d
iu

m
 1

,2
 e

th
a

n
e

d
is

u
lf

o
n

a
te

 a
n

d
 0

.0
0

5
 M

 

so
d

iu
m

 o
ct

a
n

e
su

lf
o

n
a

te
 a

d
ju

st
e

d
 t

o
 a

 p
H

 3
.5

 w
it

h
 

a
ce

ti
c 

a
ci

d
/a

ce
to

n
it

ri
le

 (
8

5
:1

5
, 

v
/v

);
 I

S
O

; 
0

.8
 

m
e

th
a

n
o

l/
w

a
te

r/
e

th
y

le
n

e
d

ia
m

in
e

te
tr

a
-a

ce
ti

c 
a

ci
d

 

p
H

 7
.2

 (
8

0
:1

5
:5

, 
v
/v

/v
);

 I
S

O
; 

1
.0

 

S
ta

ti
o

n
a

ry
 

P
h

a
se

 

C
N

 (
A

m
b

ie
n

t)
 

C
1

8
 (

R
T

) 

C
1

8
 

C
a

ti
o

n
-

e
xc

h
a

n
g

e
 

co
lu

m
n

 

S
il

ic
a

; 
C

1
8
 

(6
0

-1
0

0
 °

C
) 

C
8
 (

2
5

 °
C

) 

C
1

8
 (

5
0

 °
C

) 

C
1

8
 (

2
2

 °
C

) 

S
a

m
p

le
 

P
re

p
a

ra
ti

o
n

 

P
P

T
 (

A
C

N
) 

P
P

T
 (

A
C

N
) 

P
P

T
 (

A
C

N
) 

P
P

T
 (

A
C

N
) 

P
P

T
 (

M
e

O
H

) 

P
P

T
 (

A
C

N
) 

S
P

E
 

P
P

T
 

S
P

E
 

M
a

tr
ix

 

h
-s

e
ru

m
 

h
-u

ri
n

e
 

h
-s

e
ru

m
 

h
-s

e
ru

m
 

r-
p

la
sm

a
 

rb
-u

ri
n

e
 

g
-p

la
sm

a
 

h
-p

la
sm

a
 

h
-s

e
ru

m
 

 h
-u

ri
n

e
 

h
-s

e
ru

m
 

h
-s

e
ru

m
 

In
te

rn
a

l 

S
ta

n
d

a
rd

 

G
e

n
ta

m
ic

in
 C

2
 

n
.i

. 

N
e

ti
lm

ic
in

 

n
.i

. 

n
.i

. 

N
e

ti
lm

ic
in

 

n
.i

. 

T
o

b
ra

m
y

ci
n

 

T
o

b
ra

m
y

ci
n

 

A
n

a
ly

te
 

T
o

b
ra

m
y

ci
n

 

G
e

n
ta

m
ic

in
 C

1
a

 

G
e

n
ta

m
ic

in
 C

1
+

C
2

 

G
e

n
ta

m
ic

in
 

G
e

n
ta

m
ic

in
 

A
m

ik
a

ci
n

 

G
e

n
ta

m
ic

in
 (

C
1

, 

C
1

a
, 

a
n

d
 C

2
) 

G
e

n
ta

m
ic

in
 

G
e

n
ta

m
ic

in
 

N
e

ti
lm

ic
in

 

R
e

f 

[3
7

] 

[3
8

] 

[3
9

] 

[4
0

] 

[4
1

] 

[4
2

] 

[4
3

] 

[4
4

] 

http://dx.doi.org/10.5599/admet.1183


Seth K. Amponsah et al.   ADMET & DMPK 10(1) (2022) 27-62 

32  

Table 1. Contined... 
D

e
te

ct
io

n
 

M
o

d
e

 

U
V

 (
3

6
5

 n
m

) 

U
V

 (
3

4
0

 n
m

) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

U
G

 

1
 f

il
te

r;
 

e
m

is
si

o
n

, 
K

V
 

4
1

8
 f

il
te

r)
. 

   

U
V

 (
3

4
0

 n
m

) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
6

0
 

n
m

; 
e

m
is

si
o

n
, 

4
5

0
 n

m
) 

D
e

ri
v

a
ti

za
ti

o
n

 a
g

e
n

t 

1
-F

D
N

B
 

2
,4

,6
-

tr
in

it
ro

b
e

n
ze

n
e

 

su
lf

o
n

ic
 a

ci
d

 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

   

T
N

B
S

 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

M
o

b
il

e
 P

h
a

se
; 

E
lu

ti
o

n
; 

F
lo

w
 r

a
te

 (
m

L/
m

in
) 

a
ce

to
n

it
ri

le
/w

a
te

r/
a

ce
ti

c 
a

ci
d

 (
4

7
0

:5
3

0
:1

);
 I

S
O

; 
2

.5
 

a
ce

to
n

it
ri

le
/5

0
 m

m
o

l/
L 

p
h

o
sp

h
a

te
 b

u
ff

e
r 

a
d

ju
st

e
d

 

to
 p

H
 3

.5
 w

it
h

 p
h

o
sp

h
o

ri
c 

a
ci

d
 (

7
0

:3
0

, 
v

/v
);

 I
S

O
; 

3
.0

 

7
4

%
 m

e
th

a
n

o
l-

w
a

te
r 

8
0

%
 m

e
th

a
n

o
l-

w
a

te
r 

0
.1

 M
 s

o
d

iu
m

 a
ce

ta
te

, 
p

H
 7

.4
; 

G
R

A
; 

1
.0

 

9
5

%
 m

e
th

a
n

o
l-

w
a

te
r 

0
.2

 M
 s

o
d

iu
m

 a
ce

ta
te

, 
p

H
 5

.0
; 

G
R

A
; 

1
.0

 

8
0

%
 m

e
th

a
n

o
l-

w
a

te
r;

 G
R

A
; 

1
.0

 

0
.1

 M
 s

o
d

iu
m

 a
ce

ta
te

, 
p

H
 7

.4
; 

G
R

A
; 

1
.0

 

a
ce

to
n

it
ri

le
/p

h
o

sp
h

a
te

 b
u

ff
e

r 
(5

2
:4

8
, 

v
/v

);
 I

S
O

; 
2

.0
 

2
5

 m
M

 s
o

d
iu

m
 p

-t
o

lu
e

n
e

su
lp

h
o

n
a

te
; 

so
d

iu
m

 

p
e

rc
h

lo
ra

te
a

n
h

g
jy

d
ro

u
s;

 G
R

A
; 

0
.8

 (
C

8
);

 1
.5

 (
C

1
8
) 

S
ta

ti
o

n
a

ry
 

P
h

a
se

 

C
1

8
 

C
1

8
 (

5
0

 °
C

) 

C
1

8
 (

2
5

 °
C

) 

   

C
8

 (
5

0
 °

C
) 

C
8

; 

C
1

8
 (

5
0

 °
C

) 

S
a

m
p

le
 

P
re

p
a

ra
ti

o
n

 

S
P

E
 

S
P

E
, 

P
P

T
 

S
P

E
 

   

P
P

T
/S

P
E

 

P
P

T
, 

S
P

E
 

M
a

tr
ix

 

h
-s

e
ru

m
 

h
-s

e
ru

m
 

h
-s

e
ru

m
 

   

h
-s

e
ru

m
 

h
-s

e
ru

m
 

In
te

rn
a

l 

S
ta

n
d

a
rd

 

K
a

n
a

m
y

ci
n

 

S
is

o
m

ic
in

 

N
e

a
m

in
e

 

G
e

n
ta

m
ic

in
 

C
1

a
 

n
.i

. 

T
o

b
ra

m
y

ci
n

 

T
o

b
ra

m
y

ci
n

 

T
o

b
ra

m
y

ci
n

 
T

o
b

ra
m

y
ci

n
 

T
o

b
ra

m
y

ci
n

 

K
a

n
a

m
y

ci
n

 

T
o

b
ra

m
y

ci
n

 

A
st

ro
m

ic
in

 

N
e

ti
lm

ic
in

 

S
is

o
m

ic
in

 

A
n

a
ly

te
 

A
m

ik
a

ci
n

 

T
o

b
ra

m
y

ci
n

 

A
m

ik
a

ci
n

 

T
o

b
ra

m
y

ci
n

 

N
e

ti
lm

ic
in

 

G
e

n
ta

m
ic

in
 C

1
 

G
e

n
ta

m
ic

in
 C

1
a

 

G
e

n
ta

m
ic

in
 C

2
 

D
ib

e
k

a
ci

n
 

S
is

o
m

ic
in

 

 

A
m

ik
a

ci
n

 

S
is

o
m

ic
in

 

N
e

ti
lm

ic
in

 

A
st

ro
m

ic
in

 

M
ic

ro
n

o
m

ic
in

 

R
e

f 

[4
5

] 

[4
6

] 

[2
4

] 

   

[4
7

] 

[2
5

] 



ADMET & DMPK 10(1) (2022) 27-62 Bioanalysis of aminoglycozides by HPLC 

doi: http://dx.doi.org/10.5599/admet.1183 33 

Table 1. Contined... 
D

e
te

ct
io

n
 

M
o

d
e

 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
6

5
 

n
m

; 
e

m
is

si
o

n
, 

4
4

0
 n

m
) 

F
lu

o
re

sc
e

n
ce

 
(e

xc
it

a
ti

o
n

, 
3

4
1

 

n
m

; 
e

m
is

si
o

n
, 

4
4

0
 n

m
) 

 

U
V

 (
1

9
5

 n
m

) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
5

1
 

n
m

; 
e

m
is

si
o

n
, 

4
2

0
 n

m
) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
4

5
 

n
m

; 
e

m
is

si
o

n
, 

4
3

3
 n

m
 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
0

2
 

n
m

; 
e

m
is

si
o

n
, 

4
2

0
 n

m
) 

D
e

ri
v

a
ti

za
ti

o
n

 a
g

e
n

t 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

  





























 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

N
in

h
y

d
ri

n
 

M
o

b
il

e
 P

h
a

se
; 

E
lu

ti
o

n
; 

F
lo

w
 r

a
te

 (
m

L/
m

in
) 

0
.1

 M
 d

is
o

d
iu

m
 1

,2
-e

th
a

n
e

d
is

u
lf

o
n

a
te

 a
n

d
 0

.0
0

5
 M

 

so
d

iu
m

 o
ct

a
n

e
su

lf
o

n
a

te
 i

n
 w

a
te

r/
m

e
th

a
n

o
l 

m
ix

tu
re

 

(6
4

:3
6

, 
v
/v

),
 a

d
ju

st
e

d
 t

o
 p

H
 3

.5
 w

it
h

 a
ce

ti
c 

a
ci

d
; 

G
R

A
; 

2
.0

 

2
2

 m
M

 d
is

o
d

iu
m

 1
, 

2
-e

th
a

n
e

d
is

u
lf

o
n

a
te

 a
n

d
 5

 m
M

 
so

d
iu

m
 o

ct
a

n
e

 s
u

lf
o

n
a

te
 i

n
 a

 w
a

te
r/

a
ce

to
n

it
ri

le
 

m
ix

tu
re

 (
8

0
:2

0
, 

v
 /

v
) 

a
d

ju
st

e
d

 w
it

h
 a

ce
ti

c 
a

ci
d

 t
o

 

a
b

o
u

t 
p

H
 3

.5
; 

IS
O

; 
1

.5
 

3
7

 m
M

 d
is

o
d

iu
m

 1
,2

-e
th

a
n

e
su

lf
o

n
a

te
 a

n
d

 5
 m

M
 

so
d

iu
m

 o
ct

a
n

e
 s

u
lf

o
n

a
te

 i
n

 a
 w

a
te

r/
a

ce
to

n
it

ri
le

 

m
ix

tu
re

 (
8

0
:2

0
, 

v
/v

) 
a

d
ju

st
e

d
 w

it
h

 a
ce

ti
c 

a
ci

d
 t

o
 

a
b

o
u

t 
p

H
 3

.5
; 

IS
O

; 
1

.5
 

3
.7

6
 g

 o
f 

so
d

iu
m

 l
-h

e
xa

n
e

su
lp

h
o

n
a

te
 a

n
d

 9
.5

0
 g

 o
f 

tr
ib

a
si

c 
so

d
iu

m
 p

h
o

sp
h

a
te

 d
o

d
e

ca
h

y
d

ra
te

 d
is

so
lv

e
d

 

in
 w

a
te

r 
(1

 L
),

 p
H

 3
.0

 a
d

ju
st

e
d

 w
it

h
 p

h
o

sp
h

o
ri

c 

a
ci

d
/a

ce
to

n
it

ri
le

 (
9

2
:8

, 
v

/v
);

 I
S

O
; 

1
.0

 

2
0

 m
M

 d
is

o
d

iu
m

 1
,2

-e
th

a
n

e
d

is
u

lf
o

n
a

te
, 

5
 m

M
 

so
d

iu
m

 o
ct

a
n

e
su

lf
o

n
a

te
, 

a
n

d
 0

.4
 m

M
 N

Q
S

 i
n

 a
 

w
a

te
r/

a
ce

to
n

it
ri

le
 (

8
0

:2
0

, 
v
/v

),
 a

d
ju

st
e

d
 t

o
 a

b
o

u
t 

p
H

 3
.3

 w
it

h
 a

ce
ti

c 
a

ci
d

; 
IS

O
; 

1
.5

 

so
lv

e
n

t 
A

: 
1

0
 m

M
 s

o
d

iu
m

 s
u

lf
a

te
, 

8
 m

M
 s

o
d

iu
m

 

p
a

n
ta

n
e

su
lf

o
n

a
te

 a
n

d
 2

0
 m

M
 a

ce
ti

c 
a

ci
d

; 
G

R
A

; 
0

.8
 

 

so
lv

e
n

t 
B

: 
6

0
 m

M
 s

o
d

iu
m

 s
u

lf
a

te
, 

8
 m

M
 s

o
d

iu
m

 

p
a

n
ta

n
e

su
lf

o
n

a
te

 a
n

d
 2

0
 m

M
 a

ce
ti

c 
a

ci
d

; 
G

R
A

; 
0

.8
 

 

2
0

 m
M

 s
o

d
iu

m
 o

ct
a

n
e

 s
u

lf
o

n
a

te
, 

a
n

d
 5

 m
M

 

n
in

h
y

d
ri

n
 i

n
 a

 w
a

te
r/

a
ce

to
n

it
ri

le
 (

8
0

:2
0

, 
v
/v

),
 

a
d

ju
st

e
d

 t
o

 a
b

o
u

t 
p

H
 3

.3
 w

it
h

 a
ce

ti
c 

a
ci

d
; 

IS
O

; 
1

.5
 

S
ta

ti
o

n
a

ry
 

P
h

a
se

 

C
1

8
 

C
1

8
 

 

C
1

8
 (

5
0

 °
C

) 

C
1

8
 (

6
5

 °
C

) 

C
1

8
 (

5
0

 °
C

) 

C
1

8
 (

5
0

-9
5

 

°C
) 

S
a

m
p

le
 

P
re

p
a

ra
ti

o
n

 

P
P

T
 (

M
e

O
H

) 

P
P

T
 (

3
.5

 %
 

p
e

rc
h

lo
ri

c 
a

ci
d

) 

 

S
P

E
 

P
P

T
 (

3
.5

 %
 

p
e

rc
h

lo
ri

c 
a

ci
d

) 

S
P

E
 

P
P

T
 (

3
.5

 %
 

p
e

rc
h

lo
ri

c 
a

ci
d

) 

M
a

tr
ix

 

h
-s

e
ru

m
 

h
-s

e
ru

m
 

 

h
-s

e
ru

m
 

h
-s

e
ru

m
 

rb
-s

e
ru

m
 

h
-s

e
ru

m
 

In
te

rn
a

l 

S
ta

n
d

a
rd

 

S
is

o
m

ic
in

 

N
e

ti
lm

ic
in

 

S
is

o
m

ic
in

 

n
.i

.  

D
ih

y
d

ro
st

p
t

o
m

y
ci

n
 

n
.i

. 

n
.i

. 

n
.i

. 

A
n

a
ly

te
 

T
o

b
ra

m
y

ci
n

 

S
is

o
m

ic
in

 

N
e

ti
lm

ic
in

 

K
a

n
a

m
y

ci
n

 
 

D
ib

e
k

a
ci

n
 

S
tr

e
p

to
m

y
ci

n
 

S
tr

e
p

to
m

y
ci

n
 

G
e

n
ta

m
ic

in
 C

1
 

G
e

n
ta

m
ic

in
 C

1
a

 

G
e

n
ta

m
ic

in
 C

2
 

S
tr

e
p

to
m

y
ci

n
 

R
e

f 

[4
8

] 

[2
6

] 

 

[2
7

] 

[4
9

] 

[5
0

] 

[5
1

] 

http://dx.doi.org/10.5599/admet.1183


Seth K. Amponsah et al.   ADMET & DMPK 10(1) (2022) 27-62 

34  

Table 1. Contined... 
D

e
te

ct
io

n
 

M
o

d
e

 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

2
6

0
 

n
m

; 
e

m
is

si
o

n
, 

4
1

8
 n

m
) 

U
V

 (
3

6
5

 n
m

) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
4

0
 

n
m

; 
e

m
is

si
o

n
, 

4
5

0
 n

m
) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
4

0
 

n
m

; 
e

m
is

si
o

n
, 

4
5

5
 n

m
) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
4

0
 

n
m

; 
e

m
is

si
o

n
, 

K
V

 4
1

8
) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
3

8
 

n
m

; 
e

m
is

si
o

n
, 

4
1

8
 n

m
) 

F
lu

o
re

sce
n

ce
 

(e
xcita

tio
n

, 3
4

0
 

n
m

; e
m

issio
n

, 

4
1

8
 n

m
) 

U
V

 (
3

4
0

 n
m

) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
4

0
 

n
m

; 
e

m
is

si
o

n
, 

4
1

5
 n

m
) 

D
e

ri
v

a
ti

za
ti

o
n

 a
g

e
n

t 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

F
D

N
B

 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

M
o

b
il

e
 P

h
a

se
; 

E
lu

ti
o

n
; 

F
lo

w
 r

a
te

 (
m

L/
m

in
) 

1
 %

 T
E

A
 s

o
lu

ti
o

n
 (

a
d

ju
st

e
d

 t
o

 p
H

 6
.2

 ±
 0

.1
 w

it
h

 

p
h

o
sp

h
o

ri
c 

a
ci

d
)/

m
e

th
a

n
o

l 
(7

9
:2

1
, 

v
/v

);
 I

S
O

; 
2

.0
 

w
a

te
r/

a
ce

to
n

it
ri

le
/a

ce
ti

c 
a

ci
d

 (
3

0
0

:7
0

0
:1

, 
v

/v
/v

);
 

IS
O

; 
2

.2
 

m
e

th
a

n
o

l/
so

d
iu

m
 1

-h
e

p
ta

n
e

 s
u

lf
o

n
a

te
 (

2
.5

g
)/

a
ce

ti
c 

a
ci

d
/w

a
te

r 
(8

0
0

:2
0

0
:4

2
:2

0
8

, 
v

/v
/v

/v
);

 I
S

O
; 

0
.9

 

3
0

 %
 e

th
y

le
n

e
 g

ly
co

l 
in

 0
.0

5
 M

 p
h

o
sp

h
a

te
 b

u
ff

e
r 

(p
H

 7
);

 I
S

O
; 

0
.5

 

so
lv

e
n

t 
A

: 
E

th
y

le
n

e
d

ia
m

in
e

te
tr

a
a

ce
ti

c 
a

ci
d

, 

tr
ip

o
ta

ss
iu

m
 s

a
lt

 (
2

.0
 g

m
) 

d
is

so
lv

e
d

 i
n

 1
 L

 
w

a
te

r/
m

e
th

a
n

o
l,

 (
3

0
0

:7
0

0
, 

v
/v

) 

so
lv

e
n

t 
B

: 
M

e
th

a
n

o
l;

 G
R

A
; 

1
.7

 

m
e

th
a

n
o

l/
b

u
ff

e
r 

so
lu

ti
o

n
 c

o
n

ta
in

in
g

 0
.0

1
 M

 

so
d

iu
m

 h
e

xa
n

e
su

lp
h

o
n

a
te

, 
0

.1
 M

 s
o

d
iu

m
 s

u
lp

h
a

te
 

a
n

d
 1

7
 m

M
 a

ce
ti

c 
a

ci
d

 (
1

0
:9

0
, 

v
/v

);
 I

S
O

; 
1

.1
 

0
.2

 M
 s

o
d

iu
m

 s
u

lp
h

a
te

, 
0

.0
2

 M
 s

o
d

iu
m

 p
e

n
ta

n
e

 

su
lp

h
a

te
 a

n
d

 1
 m

l 
a

ce
ti

c 
a

ci
d

 i
n

 1
L 

d
is

ti
ll

e
d

 w
a

te
r;

 

IS
O

; 
1

.2
 

a
ce

to
n

it
ri

le
/2

-m
e

th
o

xy
e

th
a

n
o

l/
te

tr
a

h
y

d
ro

fu
ra

n
-

g
la

ci
a

l 
a

ce
ti

c 
a

ci
d

/t
ri

s(
h

y
d

ro
xy

m
e

th
y

l)
-a

m
in

o
 

e
th

a
n

e
 (

1
%

 a
q

u
e

o
u

s 
so

lu
ti

o
n

) 

(4
1

:4
.5

2
:4

.2
4

:0
.2

1
:5

0
, 

v
/v

);
 G

R
A

; 
P

ro
g

ra
m

m
e

d
 f

lo
w

 

ra
te

 

0
.0

5
 M

 N
a

2
S

O
4
 a

n
d

 0
.0

0
5

 M
 s

o
d

iu
m

 o
ct

y
ls

u
lf

a
te

, 

p
H

 3
.5

 a
d

ju
st

e
d

 w
it

h
 g

la
ci

a
l 

a
ce

ti
c 

a
ci

d
/m

e
th

a
n

o
l 

(7
0

:3
0

, 
v
/v

);
 I

S
O

; 
1

.5
 

S
ta

ti
o

n
a

ry
 

P
h

a
se

 

C
1

8
 

C
1

8
 (

2
5

 º
C

) 

C
1

8
 

C
1

8
 

S
H

P
 

C
1

8
 (

2
5

 º
C

) 

C
1

8
 

C
1

8
 (

5
8

 º
C

) 

C
1

8
 (

4
5

 º
C

) 

S
a

m
p

le
 

P
re

p
a

ra
ti

o
n

 

S
P

E
 

P
P

T
 (

A
C

N
) 

S
P

E
 

S
P

E
 

S
P

E
 

S
P

E
 

S
P

E
 

U
lt

ra
fi

lt
ra

ti
o

n
 

P
P

T
 (

1
0

 %
w

/v
 

T
C

A
) 

M
a

tr
ix

 

h
-s

e
ru

m
 

h
-s

e
ru

m
 

g
-s

e
ru

m
 

h
-D

B
S

 

rb
-s

e
ru

m
 

c-
m

il
k

 

h
-s

e
ru

m
 h

-

u
ri

n
e

 

h
-s

e
ru

m
 

h
-s

e
ru

m
 h

-

u
ri

n
e

 

d
-p

la
sm

a
 

In
te

rn
a

l 

S
ta

n
d

a
rd

 

n
.i

. 

G
e

n
ta

m
ic

in
 

C
1

a
 

n
.i

. 

n
.i

. 

n
.i

. 

D
ib

e
k

a
ci

n
 

T
o

b
ra

m
y

ci
n

 

n
.i

. 

n
.i

. 

A
n

a
ly

te
 

G
e

n
ta

m
ic

in
 C

1
 

G
e

n
ta

m
ic

in
 C

1
a

 

G
e

n
ta

m
ic

in
 C

2
 

 

N
e

ti
lm

ic
in

 

S
is

o
m

ic
in

 

S
is

o
m

ic
in

 

N
e

o
m

y
ci

n
 

Is
e

p
a

m
ic

in
 

A
m

ik
a

ci
n

 

A
m

ik
a

ci
n

 

A
m

ik
a

ci
n

 

R
e

f 

[5
2

] 

[6
] 

[5
3

] 

[5
4

] 

[1
4

] 

[2
8

] 

[5
5

] 

[5
6

] 

[5
7

] 



ADMET & DMPK 10(1) (2022) 27-62 Bioanalysis of aminoglycozides by HPLC 

doi: http://dx.doi.org/10.5599/admet.1183 35 

Table 1. Contined... 
D

e
te

ct
io

n
 

M
o

d
e

 

U
V

 (
3

3
0

 n
m

) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
3

7
 

n
m

; 
e

m
is

si
o

n
, 

4
3

7
 n

m
) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

2
6

0
 

n
m

; 
e

m
is

si
o

n
, 

3
1

5
 n

m
) 

U
V

 (
3

4
0

 n
m

) 

F
lu

o
re

sc
e

n
ce

 
(e

xc
it

a
ti

o
n

, 
3

4
0

 

n
m

; 
e

m
is

si
o

n
, 

4
3

0
 n

m
) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
3

8
 

n
m

; 
e

m
is

si
o

n
, 

4
1

7
 n

m
) 

F
lu

o
re

sc
e

n
ce

 
(e

xc
it

a
ti

o
n

, 
2

6
0

 

n
m

; 
e

m
is

si
o

n
, 

3
1

5
 n

m
) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

2
6

3
 

n
m

; 
e

m
is

si
o

n
, 

4
3

5
 n

m
) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

2
6

0
 

n
m

; 
e

m
is

si
o

n
, 

3
1

5
 n

m
) 

D
e

ri
v

a
ti

za
ti

o
n

 a
g

e
n

t 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

F
M

O
C

-C
1

 

D
N

F
B

 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

F
M

O
C

-C
1

 

n
.i

. 

F
M

O
C

-C
1

 

M
o

b
il

e
 P

h
a

se
; 

E
lu

ti
o

n
; 

F
lo

w
 r

a
te

 (
m

L/
m

in
) 

1
-h

e
p

ta
n

e
su

lf
o

n
ic

 a
ci

d
 (

5
 g

) 
d

is
so

lv
e

d
 i

n
 a

ce
ti

c 

a
ci

d
/w

a
te

r/
M

e
0

H
 (

5
0

:2
5

0
:7

0
0

);
 I

S
O

; 
0

.5
 

so
lv

e
n

t 
A

: 
w

a
te

r/
a

ce
ti

c 
a

ci
d

/h
e

p
ta

n
e

su
lp

h
o

n
ic

 a
ci

d
 

(0
.1

 M
) 

(8
0

:1
0

:1
0

, 
v
/v

/v
) 

so
lv

e
n

t 
B

: 
a

ce
to

n
it

ri
le

; 
G

R
A

; 
2

.0
 

a
ce

to
n

it
ri

le
/w

a
te

r 
(9

0
:1

0
, 

v
/v

);
 I

S
O

; 
1

.0
 

so
lv

e
n

t 
A

: 
a

ce
to

n
it

ri
le

 a
n

d
 w

a
te

r 
(5

0
:5

0
) 

so
lv

e
n

t 
B

: 
A

ce
to

n
it

ri
le

; 
G

R
A

; 
1

.0
 

0
.0

1
1

 M
 p

e
n

ta
n

e
 s

u
lf

o
n

ic
 a

ci
d

 s
o

d
iu

m
 s

a
lt

, 
0

.0
0

5
6

 
M

 s
o

d
iu

m
 s

u
lp

h
a

te
 a

n
d

 0
.1

 %
 a

ce
ti

c 
a

ci
d

 i
n

 

w
a

te
r/

m
e

th
a

n
o

l 
(8

2
:1

8
);

 I
S

O
; 

1
.5

 

so
lv

e
n

t 
A

: 
0

.0
1

 M
 h

e
xa

n
e

su
lp

h
o

n
a

te
/0

.0
1

7
 M

 (
0

.1
 

%
) 

a
ce

ti
c 

a
ci

d
 i

n
 w

a
te

r 

so
lv

e
n

t 
B

: 
0

.0
1

 M
 h

e
xa

n
e

su
lp

h
o

n
a

te
/0

.0
 1

7
 

M
./

a
ce

ti
c 

a
ci

d
/0

.1
0

 M
 s

o
d

iu
m

 s
u

lp
h

a
te

/3
.5

3
 M

 

(1
5

%
) 

m
e

th
a

n
o

l;
 G

R
A

; 
1

.1
 

a
ce

to
n

it
ri

le
–

w
a

te
r 

(9
0

:1
0

, 
v
/v

);
 I

S
O

; 
1

.0
 

0
.8

 g
 o

f 
1

,2
-n

a
p

h
th

o
q

u
in

o
n

e
-4

-s
u

lf
o

n
ic

 a
ci

d
 (

N
Q

S
) 

in
 0

.0
1

M
 s

o
d

iu
m

 h
e

xa
n

e
-1

-s
u

lf
o

n
ic

 

a
ci

d
/A

ce
to

n
it

ri
le

 (
8

8
0

:1
2

0
, 

v
/v

);
 I

S
O

; 
0

.5
 

a
ce

to
n

it
ri

le
/w

a
te

r 
(8

4
.5

:1
5

.5
, 

v
/v

);
 I

S
O

; 
2

.5
 

S
ta

ti
o

n
a

ry
 

P
h

a
se

 

C
1

8
 

C
1

8
 

C
1

8
 

C
1

8
 (

2
5

 °
C

) 

C
1

8
 (

2
5

 °
C

) 

C
1

8
 

C
1

8
 (

2
0

-2
5

 
°C

) 

C
8
 (

2
5

 °
C

) 

C
1

8
 (

3
0

 °
C

) 

S
a

m
p

le
 

P
re

p
a

ra
ti

o
n

 

S
P

E
 

LL
E

 

S
P

E
 

P
P

T
 

(M
e

O
H

/T
C

A
) 

S
P

E
, 

P
P

T
 (

3
0

 %
 

T
C

A
) 

P
P

T
 (

m
e

th
y

le
n

e
 

ch
lo

ri
d

e
),

 S
P

E
 

S
P

E
 

S
P

E
 

LL
E

 

M
a

tr
ix

 

c-
m

il
k

 

h
-s

e
ru

m
 

h
-s

e
ru

m
 

m
-p

la
sm

a
 

c-
m

il
k

 

h
-s

e
ru

m
 

h
-u

ri
n

e
 

h
-s

e
ru

m
 

c-
m

il
k

 

h
-s

e
ru

m
 

In
te

rn
a

l 

S
ta

n
d

a
rd

 

N
e

ti
lm

ic
in

 

G
e

n
ta

m
ic

in
 

n
.i

. 

n
.i

. 

n
.i

. 

D
ib

e
k

a
ci

n
 

n
.i

. 

n
.i

. 

N
e

o
m

y
ci

n
 

A
n

a
ly

te
 

G
e

n
ta

m
ic

in
 

N
e

ti
lm

ic
in

 

G
e

n
ta

m
ic

in
 C

1
 

G
e

n
ta

m
ic

in
 C

1
a

 
G

e
n

ta
m

ic
in

 C
2

 

G
e

n
ta

m
ic

in
 C

2
a

 

G
e

n
e

ti
ci

n
 

G
e

n
ta

m
ic

in
 

Is
e

p
a

m
ic

in
 

N
e

o
m

y
ci

n
 

N
e

ti
lm

ic
in

 

S
is

o
m

ic
in

 

S
tr

e
p

to
m

y
ci

n
 

D
ih

y
d

ro
st

re
p

to

m
y

ci
n

 

G
e

n
ta

m
ic

in
 

R
e

f 

[5
8

] 

[5
9

] 

[6
0

] 

[2
9

] 

[6
1

] 

[6
2

] 

[6
3

] 

[3
0

] 

[1
6

] 

http://dx.doi.org/10.5599/admet.1183


Seth K. Amponsah et al.   ADMET & DMPK 10(1) (2022) 27-62 

36  

Table 1. Contined... 
D

e
te

ct
io

n
 

M
o

d
e

 

U
V

 (
3

6
5

 n
m

) 

U
V

 (
2

3
0

 n
m

) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
4

0
 

n
m

; 
e

m
is

si
o

n
, 

4
1

8
 n

m
) 

U
V

 (
2

3
0

 n
m

) 

M
S
/E

S
I 

(+
) 

M
S
/E

S
I 

(+
) 

M
S
/E

S
I 

(+
) 

E
LS

D
 

C
L 

D
e

ri
v

a
ti

za
ti

o
n

 a
g

e
n

t 

F
N

D
B

 

N
IT

C
 

o
-p

h
th

a
la

ld
e

h
y

d
e

 

N
IT

C
 

     

M
o

b
il

e
 P

h
a

se
; 

E
lu

ti
o

n
; 

F
lo

w
 r

a
te

 (
m

L/
m

in
) 

a
ce

to
n

it
ri

le
/T

ri
s 

b
u

ff
e

r 
(8

.3
 m

m
o

l/
L,

 t
it

ra
te

d
 t

o
 p

H
 

7
.0

 w
it

h
 H

C
l)

 (
6

8
0

:3
2

0
, 

v
/v

);
 I

S
O

; 
1

.2
 

w
a

te
r/

a
ce

to
n

it
ri

le
 (

5
7

:4
3

, 
v
/v

);
 I

S
O

; 
0

.8
 

m
e

th
a

n
o

l/
g

la
ci

a
l 

a
ce

ti
c 

a
ci

d
/w

a
te

r 
(8

0
0

:2
0

:1
8

0
, 

v
/v

/v
) 

co
n

ta
in

in
g

 0
.0

2
 M

 s
o

d
iu

m
 h

e
p

ta
n

e
 s

u
lf

o
n

ic
 

a
ci

d
, 

p
H

 3
.4

; 
IS

O
; 

1
.0

 

w
a

te
r/

a
ce

to
n

it
ri

le
 (

5
0

:5
0

, 
v
/v

);
 I

S
O

; 
1

.3
 

so
lv

e
n

t 
A

: 
w

a
te

r 
co

n
ta

in
in

g
 2

 m
M

 a
m

m
o

n
iu

m
 

a
ce

ta
te

, 
0

.1
 %

 (
v
/v

) 
fo

rm
ic

 a
ci

d
 

so
lv

e
n

t 
B

: 
M

e
th

a
n

o
l 

co
n

ta
in

in
g

 2
 m

M
 a

m
m

o
n

iu
m

 

a
ce

ta
te

, 
0

.1
%

 (
v

/v
) 

fo
rm

ic
 a

ci
d

) 

so
lv

e
n

t 
C

: 
S

o
lv

e
n

t 
A

 c
o

n
ta

in
in

g
 H

e
p

ta
fl

u
o

ro
b

u
ty

ri
c 

a
ci

d
 (

H
B

F
A

) 
(1

0
 m

M
)/

2
0

%
 S

o
lv

e
n

t 
B

; 
G

R
A

; 
2

.5
 

so
lv

e
n

t 
A

: 
a

ce
to

n
it

ri
le

, 
2

 m
M

 a
m

m
o

n
iu

m
 a

ce
ta

te
 

a
n

d
 f

o
rm

ic
 a

ci
d

 (
5

/9
5

/0
.2

, 
v
/v

/v
) 

so
lv

e
n

t 
B

: 
a

ce
to

n
it

ri
le

, 
2

 m
M

 a
m

m
o

n
iu

m
 a

ce
ta

te
 

a
n

d
 f

o
rm

ic
 (

9
5

/5
/0

.2
, 

v
/v

/v
);

 G
R

A
; 

0
.6

 

so
lv

e
n

t 
A

: 
a

ce
to

n
it

ri
le

/1
0

 m
M

 a
m

m
o

n
iu

m
 

a
ce

ta
te

/f
o

rm
ic

 a
ci

d
 (

5
:9

5
:0

.2
, 

v
/v

/v
) 

so
lv

e
n

t 
B

: 
a

ce
to

n
it

ri
le

/1
0

 m
M

 a
m

m
o

n
iu

m
 

a
ce

ta
te

/f
o

rm
ic

 a
ci

d
 (

9
5

:5
:0

.2
, 

v
/v

/v
);

 G
R

A
; 

0
.6

 

w
a

te
r/

a
ce

to
n

it
ri

le
 5

5
:4

5
 c

o
n

ta
in

in
g

 1
.5

0
 m

l/
L 

H
F

B
A

 

(1
1

.6
 m

M
);

 I
S

O
; 

1
.0

 

1
0

−
2

 m
o

l/
l 

p
o

ta
ss

iu
m

 h
y

d
ro

g
e

n
 p

h
th

a
la

te
 a

t 
p

H
 

3
.3

5
, 

a
d

ju
st

e
d

 w
it

h
 d

il
u

te
d

 s
o

d
iu

m
 

h
y

d
ro

xi
d

e
/a

ce
to

n
it

ri
le

 (
9

0
:1

0
, 

v
/v

);
 I

S
O

; 
1

.0
 

S
ta

ti
o

n
a

ry
 

P
h

a
se

 

C
1

8
 (

2
5

 º
C

) 

C
8
 (

7
0

 º
C

) 

C
1

8
 

C
1

8
 

C
1

8
 (

2
5

 º
C

) 

H
IL

IC
 

H
IL

IC
 

C
1

8
 (

4
5

 º
C

) 

C
1

8
 

S
a

m
p

le
 

P
re

p
a

ra
ti

o
n

 

S
P

E
 

LL
E

 

S
P

E
 

P
P

T
 

P
P

T
 (

A
C

N
) 

S
P

E
 

S
P

E
 

S
P

E
 

P
P

T
 

M
a

tr
ix

 

h
-s

e
ru

m
 

d
-s

e
ru

m
 

h
-u

ri
n

e
 

d
-u

ri
n

e
 

h
-s

e
ru

m
 

h
-u

ri
n

e
 

h
-s

e
ru

m
 

h
-s

e
ru

m
 

h
-s

e
ru

m
 

h
-s

e
ru

m
 

h
-s

e
ru

m
 

h
-u

ri
n

e
 

h
-s

e
ru

m
 

h
-u

ri
n

e
 

In
te

rn
a

l 

S
ta

n
d

a
rd

 

n
.i

. 

N
a

p
h

th
a

le
n

 

N
e

ti
lm

ic
in

 

A
n

th
ra

ce
n

e
 

S
is

o
m

ic
in

 

n
.i

. 

n
.i

. 

n
.i

. 

n
.i

. 

A
n

a
ly

te
 

G
e

n
ta

m
ic

in
 C

1
 

G
e

n
ta

m
ic

in
 C

1
a

 

G
e

n
ta

m
ic

in
 C

 2
 

A
m

ik
a

ci
n

 

G
e

n
ta

m
ic

in
 

T
o

b
ra

m
y

ci
n

 

T
o

b
ra

m
y

ci
n

 

A
m

ik
a

ci
n

 

G
e

n
ta

m
ic

in
 

K
a

n
a

m
y

ci
n

 

N
e

o
m

y
ci

n
 

P
a

ro
m

o
m

yc
in

 
T

o
b

ra
m

y
ci

n
 

N
e

o
m

y
ci

n
 

T
o

b
ra

m
y

ci
n

 

A
m

ik
a

ci
n

 

R
e

f 

[6
4

] 

[6
5

] 

[6
6

] 

[6
7

] 

[6
8

] 

[3
1

] 

[6
9

] 

[7
0

] 

[7
1

] 



ADMET & DMPK 10(1) (2022) 27-62 Bioanalysis of aminoglycozides by HPLC 

doi: http://dx.doi.org/10.5599/admet.1183 37 

Table 1. Contined... 
D

e
te

ct
io

n
 

M
o

d
e

 

M
S
/E

S
I 

(+
) 

M
S
/E

S
I 

(+
) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

4
6

5
 

n
m

; 
e

m
is

si
o

n
, 

5
3

1
 n

m
) 

M
S
/E

S
I 

(+
) 

P
E

D
 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

2
5

0
 

n
m

; 
e

m
is

si
o

n
, 

3
9

5
 n

m
) 

F
lu

o
re

sc
e

n
t 

(e
xc

it
a

ti
o

n
, 

4
9

0
 

n
m

; 
e

m
is

si
o

n
, 

5
1

8
 n

m
) 

M
S
/E

S
I 

(+
) 

D
e

ri
v

a
ti

za
ti

o
n

 a
g

e
n

t 

  

7
-f

lu
o

ro
-4

-

n
it

ro
b

e
n

z-
2

-o
xa

-1
,3

-

d
ia

zo
le

 

  

6
-a

m
in

o
q

u
in

o
ly

l-
N

-

h
y

d
ro

xy
 s

u
cc

in
im

id
y

l 

ca
rb

a
m

a
te

 

F
IT

C
 

 

M
o

b
il

e
 P

h
a

se
; 

E
lu

ti
o

n
; 

F
lo

w
 r

a
te

 (
m

L/
m

in
) 

so
lv

e
n

t 
A

: 
2

0
 m

  
so

lv
e

n
t:

 B
 2

0
 m

M
 P

F
P

A
 i

n
 w

a
te

r/
a

ce
to

n
it

ri
le

 

(5
0

/5
0

, 
v
/v

);
 I

S
O

; 
0

.3
M

 P
F

P
A

 i
n

 w
a

te
r 

 

so
lv

e
n

t 
A

: 
2

0
 m

M
 f

o
rm

ic
 a

ci
d

 a
n

d
 1

0
 m

M
 N

F
P

A
 i

n
 

w
a

te
r 

so
lv

e
n

t 
B

: 
2

0
 m

M
 f

o
rm

ic
 a

ci
d

 a
n

d
 1

0
 m

M
 N

F
P

A
 i

n
 

m
e

th
a

n
o

l;
 G

R
A

; 
0

.5
 

0
.0

1
M

 s
o

d
iu

m
 a

ce
ta

te
 p

H
 3

/a
ce

to
n

it
ri

le
 (

8
5

:1
5

, 

v
/v

);
 I

S
O

; 
1

.2
 

2
 m

M
 a

m
m

o
n

iu
m

 a
ce

ta
te

 (
p

H
 3

.2
)/

 5
%

 a
ce

to
n

it
ri

le
 

(9
5

:5
, 

v
/v

);
 I

S
O

; 
0

.5
 

1
.5

 g
 s

o
d

iu
m

-1
-o

ct
a

n
e

su
lp

h
o

n
a

te
, 

2
0

 g
 a

n
h

y
d

ro
u

s 

so
d

iu
m

 s
u

lp
h

a
te

, 
1

5
 m

l 
te

tr
a

h
y

d
ro

fu
ra

n
, 

2
5

0
 m

l 
0

.2
 

M
 p

h
o

sp
h

a
te

 b
u

ff
e

r 
p

H
 3

, 
w

a
te

r 
u

p
 t

o
 1

0
0

0
 m

L;
 

IS
O

; 
1

.0
 

2
0

 m
M

 K
H

2
P

O
4
 c

o
n

ta
in

in
g

 8
 m

M
 T

E
A

 (
p

H
 

7
.0

)/
a

ce
to

n
it

ri
le

 (
7

8
:2

2
, 

v
/v

);
 I

S
O

; 
1

.0
 

a
ce

to
n

it
ri

le
/m

e
th

a
n

o
l/

g
la

ci
a

l 
a

ce
ti

c 
a

ci
d

/w
a

te
r 

(4
2

0
:6

0
:5

:5
1

5
, 

v
/v

/v
/v

);
 I

S
O

; 
1

.0
 

p
h

a
se

 A
: 

a
ce

to
n

it
ri

le
/w

a
te

r 
(5

0
:9

5
0

, 
v
/v

) 
co

n
ta

in
in

g
 

2
0

 m
M

 H
F

B
A

 

p
h

a
se

 B
: 

a
ce

to
n

it
ri

le
/w

a
te

r 
(5

0
0

:5
0

0
, 

v
/v

) 

co
n

ta
in

in
g

 2
0

 m
M

 H
F

B
A

; 
G

R
A

; 
0

.3
 

S
ta

ti
o

n
a

ry
 

P
h

a
se

 

C
1

8
 (

3
0

 °
C

) 

 

C
1

8
 

C
1

8
 (

6
0

 °
C

) 

C
1

8
 (

4
0

 °
C

) 

C
1

8
 (

4
0

 °
C

) 

C
1

8
 

(A
m

b
ie

n
t)

 

C
1

8
 (

3
0

 °
C

) 

S
a

m
p

le
 

P
re

p
a

ra
ti

o
n

 

LL
E

, 
P

P
T

 (
T

C
A

),
 

S
P

E
 

  

P
P

T
 (

A
C

N
) 

P
P

T
 (

A
C

N
) 

P
P

T
 

(A
C

N
/p

h
o

sp
h

a
te

 

b
u

ff
e

r)
 

P
P

T
 (

A
C

N
) 

S
P

E
 

S
P

E
 

M
a

tr
ix

 

c-
m

il
k

 

h
-s

e
ru

m
 r

b
-

se
ru

m
 

h
-s

e
ru

m
 

h
-s

e
ru

m
 

h
-C

S
F

 

h
-s

e
ru

m
 

h
-u

ri
n

e
 

c-
m

il
k

 

In
te

rn
a

l 

S
ta

n
d

a
rd

 

S
tr

e
p

to
m

y
ci

n
 

K
a

n
a

m
y

ci
n

 

n
.i

. 

S
is

o
m

ic
in

 

n
.i

. 

A
m

ik
a

ci
n

 

N
e

o
m

y
ci

n
 

n
.i

. 

A
n

a
ly

te
 

D
ih

y
d

ro
st

re
p

to
-

m
y

ci
n

 

(N
e

o
m

y
ci

n
) 

N
e

o
 1

8
0

4
 A

7
 

N
e

o
 1

8
0

4
 A

8
 

N
e

o
 1

8
0

4
 A

9
 

N
e

o
 1

8
0

4
 B

4
 

A
m

ik
a

ci
n

 

T
o

b
ra

m
y

ci
n

 

A
m

ik
a

ci
n

 

Is
e

p
a

m
ic

in
 

T
o

b
ra

m
y

ci
n

 

N
e

o
m

yc
in

 
S

tr
e

p
to

m
yc

in
 

D
ih

yd
ro

st
re

p
to

m
yc

in
 

A
m

ik
a

ci
n

 

K
a

n
a

m
yc

in
 

P
a

ro
m

o
m

yc
in

 

T
o

b
ra

m
yc

in
 

S
p

e
ct

in
o

m
yc

in
 

A
p

ra
m

yc
in

 
H

yg
ro

m
yc

in
 

G
e

n
ta

m
ic

in
(C

1
) 

G
e

n
ta

m
ic

in
(C

1
a

) 
G

e
n

ta
m

ic
in

 (
C

2
0

 

R
e

f 

[7
2

] 

[7
3

] 

[7
4

] 

[7
5

] 

[1
5

]*
 

[7
6

] 

[7
7

] 

[5
]*

 

http://dx.doi.org/10.5599/admet.1183


Seth K. Amponsah et al.   ADMET & DMPK 10(1) (2022) 27-62 

38  

Table 1. Contined... 
D

e
te

ct
io

n
 

M
o

d
e

 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

2
6

5
 

n
m

; 
e

m
is

si
o

n
, 

3
1

5
 n

m
) 

M
S
/E

S
I 

(+
) 

R
R

S
D

 

(e
xc

it
a

ti
o

n
/e

m
i

ss
io

n
, 

3
7

0
 n

m
) 

M
S
/E

S
I 

(+
) 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

3
9

0
 

n
m

; 
e

m
is

si
o

n
, 

4
8

0
 n

m
) 

M
S
/E

S
I 

(+
) 

M
S
/E

S
I 

(+
) 

D
e

ri
v

a
ti

za
ti

o
n

 a
g

e
n

t 

F
M

O
C

-C
1

 

   

F
lu

o
re

sc
a

m
in

e
 

  

M
o

b
il

e
 P

h
a

se
; 

E
lu

ti
o

n
; 

F
lo

w
 r

a
te

 (
m

L/
m

in
) 

w
a

te
r/

a
ce

to
n

it
ri

le
 (

3
2

:6
8

, 
v
/v

);
 I

S
O

; 
1

.0
 

b
u

ff
e

r 
A

 (
w

a
te

r/
fo

rm
ic

 a
ci

d
 0

.0
5

 %
) 

0
.1

 %
 t

ri
fl

u
o

ro
a

ce
ti

c 
a

ci
d

 (
p

H
 2

.2
);

 I
S

O
; 

0
.4

 

w
a

te
r/

a
ce

to
n

it
ri

le
 e

a
ch

 c
o

n
ta

in
in

g
 0

.0
0

5
 %

(v
/v

) 

tr
if

lu
o

ro
a

ce
ti

c 
a

ci
d

 a
n

d
 0

.1
 %

 (
v
/v

) 
fo

rm
ic

 a
ci

d
; 

G
R

A
; 

0
.7

 

m
e

th
a

n
o

l/
w

a
te

r 
(6

0
:4

0
, 

v
/v

);
 I

S
O

; 
1

.0
 

so
lv

e
n

t 
A

: 
p

u
ri

fi
e

d
 w

a
te

r 

so
lv

e
n

t 
B

: 
a

ce
to

n
it

ri
le

 1
0

0
 %

 

so
lv

e
n

t 
C

: 
p

e
rf

lu
o

ro
p

e
n

ta
n

o
ic

 a
ci

d
 (

2
0

0
 

m
M

)/
a

m
m

o
n

iu
m

 a
ce

ta
te

 (
1

3
0

 m
M

) 
in

 p
u

ri
fi

e
d

 

w
a

te
r;

 G
R

A
; 

0
.4

 

so
lv

e
n

t 
A

: 
1

0
 m

M
 n

o
n

fl
u

o
ro

p
e

n
ta

n
o

ic
 a

ci
d

 (
N

F
P

A
) 

so
lv

e
n

t 
B

: 
a

ce
to

n
it

ri
le

 i
n

 1
0

 m
M

 N
F

P
A

; 
G

R
A

; 
0

.3
 

S
ta

ti
o

n
a

ry
 

P
h

a
se

 

C
8
 (

3
0

 °
C

) 

C
1

8
 (

3
5

 °
C

) 

C
1

8
 (

3
0

 °
C

) 

C
1

8
 

C
1

8
 

C
1

8
 (

3
0

 °
C

) 

C
1

8
 

S
a

m
p

le
 

P
re

p
a

ra
ti

o
n

 

P
P

T
 (

A
C

N
) 

P
P

T
 (

A
C

N
) 

P
P

T
 (

A
C

N
) 

P
P

T
 (

0
.3

 M
 

p
e

rc
h

lo
ri

c 
a

ci
d

) 

P
P

T
 (

A
C

N
) 

P
P

T
 (

T
C

A
) 

P
P

T
 (

T
C

A
) 

M
a

tr
ix

 

r-
p

la
sm

a
 

h
-s

e
ru

m
 

h
-s

e
ru

m
 

h
-u

ri
n

e
 

h
-s

e
ru

m
 

h
-p

la
sm

a
 

h
-p

la
sm

a
 

b
-m

il
k

 

In
te

rn
a

l 

S
ta

n
d

a
rd

 

A
m

ik
a

ci
n

 

Q
u

in
o

xa
li

n
e

 

n
.i

. 

D
ib

e
k

a
ci

n
 

n
.i

. 

K
a

n
a

m
y

ci
n

 B
 

n
.i

. 

A
n

a
ly

te
 

Is
e

p
a

m
ic

in
 

A
m

ik
a

ci
n

 

G
e

n
ta

m
ic

in
 

A
m

ik
a

ci
n

 

N
e

ti
lm

ic
in

 
E

ti
m

ic
in

 

A
rb

e
k

a
ci

n
 

T
o

b
ra

m
y

ci
n

 

A
m

ik
a

ci
n

 

G
e

n
ta

m
ic

in
 C

1
 

G
e

n
ta

m
ic

in
 C

1
a

 
G

e
n

ta
m

ic
in

 C
1

2
 

S
p

e
ct

in
o

m
y

ci
n

 

T
o

b
ra

m
y

ci
n

 

G
e

n
ta

m
ic

in
 

K
a

n
a

m
y

ci
n

 

H
y

g
ro

m
y

ci
n

 

A
p

ra
m

y
ci

n
 

S
tr

e
p

to
m

y
ci

n
 

D
ih

y
d

ro
st

re
p

to
-

m
y

ci
n

 
A

m
ik

a
ci

n
 

N
e

o
m

y
ci

n
 

R
e

f 

[7
8

] 

[7
9

] 

[3
2

] 

[3
3

] 

[8
0

] 

[8
1

] 

[3
4

] 



ADMET & DMPK 10(1) (2022) 27-62 Bioanalysis of aminoglycozides by HPLC 

doi: http://dx.doi.org/10.5599/admet.1183 39 

Table 1. Contined... 
D

e
te

ct
io

n
 

M
o

d
e

 

F
lu

o
re

sc
e

n
ce

 

(e
xc

it
a

ti
o

n
, 

2
6

5
 

n
m

; 
e

m
is

si
o

n
, 

3
1

5
 n

m
) 

M
S
/E

S
I 

(+
) 

M
S
/E

S
I 

(+
) 

M
S
/E

S
I 

(+
) 

M
S

-E
S

I 
(+

) 

h
-s

e
ru

m
, 

h
u

m
a

n
 s

e
ru

m
; 

d
-s

e
ru

m
, 

d
o

g
 s

e
ru

m
; 

h
-p

la
sm

a
, 

h
u

m
a

n
 p

la
sm

a
; 

h
-u

ri
n

e
, 

h
u

m
a

n
 u

ri
n

e
; 

r-
p

la
sm

a
, 

ra
t 

p
la

sm
a

; 
rb

-u
ri

n
e

, 
ra

b
b

it
 u

ri
n

e
; 

g
-p

la
sm

a
, 

g
u

in
e

a
 p

ig
 p

la
sm

a
; 

rb
-s

e
ru

m
, 

ra
b

b
it

 s
e

ru
m

; 
h

-D
B

S
, 

h
u

m
a

n
 d

ri
e

d
 

b
lo

o
d

 s
p

o
t;

 c
-m

ilk
, 

co
w

 m
ilk

; 
m

-p
la

sm
a

, 
m

o
u

se
 p

la
sm

a
; 

h
-C

S
F,

 h
u

m
a

n
 c

e
re

b
ra

l 
sp

in
a

l 
fl

u
id

; 
M

e
O

H
, 

m
e

th
a

n
o

l;
 C

N
, 

cy
a

n
o

; 
A

C
N

, 
F

A
, 

fo
rm

ic
 a

ci
d

; 
a

ce
to

n
it

ri
le

; 
T

C
A

, 
tr

ic
h

lo
ro

a
ce

ti
c 

a
ci

d
; 

R
T

, 
ro

o
m

 t
e

m
p

e
ra

tu
re

; 
IS

O
, 

is
o

cr
a

ti
c;

 

G
R

A
, 

g
ra

d
ie

n
t;

 S
P

E
, 

so
lid

 p
h

a
se

 e
xt

ra
ct

io
n

; 
P

P
T

, 
p

ro
te

in
 p

re
ci

p
it

a
ti

o
n

; 
LL

E
, 

liq
u

id
-l

iq
u

id
 e

xt
ra

ct
io

n
; 

C
L,

 c
h

e
m

ilu
m

in
e

sc
e

n
ce

 d
e

te
ct

io
n

; 
E

D
T

A
, 

e
th

yl
e

n
e

 d
ia

m
in

e
 t

e
tr

a
 a

ce
ti

c 
a

ci
d

; 
E

LS
D

, 
e

va
p

o
ra

ti
ve

 l
ig

h
t 

sc
a

tt
e

ri
n

g
 d

e
te

ct
o

r;
 

E
S

I:
 e

le
ct

ro
sp

ra
y 

io
n

iz
a

ti
o

n
; 

H
F

B
A

: 
h

e
p

ta
fl

u
o

ro
b

u
ty

ri
c 

a
ci

d
; 

i.
d

.,
 i

n
te

rn
a

l 
d

ia
m

e
te

r;
 M

S
, 

m
a

ss
 s

p
e

ct
ro

m
e

tr
y;

 E
S

I,
 e

le
ct

ro
sp

ra
y 

io
n

iz
a

ti
o

n
; 

N
a

2
S

O
4

, 
so

d
iu

m
 s

u
lp

h
a

te
; 

N
F

P
A

, 
n

o
n

a
fl

u
o

ro
p

e
n

ta
n

o
ic

 a
ci

d
; 

n
.i

.:
 n

o
t 

in
d

ic
a

te
d

; 

P
E

D
: 

p
u

ls
e

 e
le

ct
ro

ch
e

m
ic

a
l 

d
e

te
ct

io
n

; 
U

H
P

LC
, 

u
lt

ra
-h

ig
h

 p
e

rf
o

rm
a

n
ce

 l
iq

u
id

 c
h

ro
m

a
to

g
ra

p
h

y;
 U

V
-V

IS
: 

u
lt

ra
vi

o
le

t-
vi

si
b

le
 l

ig
h

t;
 F

D
N

B
, 

1
-f

lu
o

ro
-2

,4
-d

in
it

ro
b

e
n

ze
n

e
; 

F
M

O
C

-C
1

, 
9

-f
lu

o
re

n
yl

m
e

th
yl

ch
lo

ro
fo

rm
a

te
; 

N
IT

C
, 

1
-

n
a

p
h

th
yl

 is
o

th
io

cy
a

n
a

te
; 

H
IL

IC
, 

h
yd

ro
p

h
ili

c 
in

te
ra

ct
io

n
 l

iq
u

id
 c

h
ro

m
a

to
g

ra
p

h
y;

 R
R

S
D

, 
re

so
n

a
n

ce
 R

a
y

le
ig

h
 s

ca
tt

e
ri

n
g

 d
e

te
ct

io
n

; 
*

, 
m

e
th

o
d

s 
th

a
t 

ca
n

 b
e

 o
p

ti
m

is
e

d
 f

o
r 

th
e

 a
n

a
ly

si
s 

o
f 

a
ll 

th
e

 a
m

in
o

g
ly

co
si

d
e

s 
u

n
d

e
r 

re
vi

e
w

. 

 

D
e

ri
v

a
ti

za
ti

o
n

 a
g

e
n

t 

F
M

O
C

-C
1

 

    

M
o

b
il

e
 P

h
a

se
; 

E
lu

ti
o

n
; 

F
lo

w
 r

a
te

 (
m

L/
m

in
) 

a
ce

to
n

it
ri

le
/w

a
te

r 
(7

0
:3

0
, 

v
/v

);
 I

S
O

; 
0

.4
 

so
lv

e
n

t 
A

: 
0

.2
 %

 (
v

/v
) 

H
F

B
A

 i
n

 w
a

te
r 

so
lv

e
n

t 
B

: 
0

.2
%

 (
v
/v

) 
H

F
B

A
 i

n
 A

C
N

; 
G

R
A

; 
0

.3
 

so
lv

e
n

t 
A

: 
w

a
te

r 
co

n
ta

in
in

g
 0

.1
 %

 f
o

rm
ic

 a
ci

d
, 

0
.0

1
 

%
 H

F
B

A
 

so
lv

e
n

t 
B

: 
a

ce
to

n
it

ri
le

 c
o

n
ta

in
in

g
 0

.1
%

 f
o

rm
ic

 a
ci

d
 

a
n

d
 0

.0
1

%
 H

F
B

A
; 

G
R

A
; 

0
.4

 

so
lv

e
n

t:
 p

u
ri

fi
e

d
 w

a
te

r 
w

it
h

 0
.1

 %
 f

o
rm

ic
 

a
ci

d
 a

n
d

 0
.0

1
 %

 o
f 

H
F

B
A

 

so
lv

e
n

t 
B

: 
d

 a
ce

to
n

it
ri

le
 w

it
h

 0
.1

 0
.1

%
 f

o
rm

ic
 a

ci
d

 

a
n

d
 0

.0
1

 %
 o

f 
H

F
B

A
; 

G
R

A
; 

0
.4

 

5
 m

M
 H

F
B

A
 i

n
 w

a
te

r/
a

ce
to

n
it

ri
le

 (
7

:3
, 

v
/v

) 
m

ix
tu

re
 

(3
.5

 m
M

 H
F

B
A

 i
n

 t
h

e
 m

ix
tu

re
);

 I
S

O
; 

0
.4

 

S
ta

ti
o

n
a

ry
 

P
h

a
se

 

C
1

8
 (

3
0

 º
C

) 

C
1

8
 (

4
0

 º
C

) 

C
1

8
 (

4
0

 º
C

) 

C
1

8
 (

4
0

 º
C

) 

C
1

8
 (

4
0

 º
C

) 

S
a

m
p

le
 

P
re

p
a

ra
ti

o
n

 

P
P

T
 (

A
C

N
) 

S
P

E
 

P
P

T
 (

0
.1

 %
 F

A
) 

P
P

T
 (

0
.1

 %
 F

A
) 

P
P

T
 (

2
0

 %
w

/v
 

T
C

A
) 

M
a

tr
ix

 

h
-s

e
ru

m
 

g
-

p
e

ri
ly

m
p

h
/

C
S

F
 

h
-s

e
ru

m
 

h
-p

la
sm

a
 

h
-p

la
sm

a
 

In
te

rn
a

l 

S
ta

n
d

a
rd

 

n
.i

. 

T
o

b
ra

m
y

ci
n

 

K
a

n
a

m
y

ci
n

 

K
a

n
a

m
y

ci
n

 

D
e

u
te

ra
te

d
 

P
a

ro
m

o
m

yc
in

 

a
ce

ti
c 

a
ci

d
 

A
n

a
ly

te
 

A
m

ik
a

ci
n

 

N
e

o
m

y
ci

n
 

 

A
m

ik
a

ci
n

 

A
m

ik
a

ci
n

 

P
a

ro
m

o
m

yc
in

 

R
e

f 

[8
2

] 

[8
3

] 

[8
4

] 

[8
5

] 

[3
5

] 

http://dx.doi.org/10.5599/admet.1183


Seth K. Amponsah et al.   ADMET & DMPK 10(1) (2022) 27-62 

40  

Sample cleanup 

The matrix in which a drug is found can affect bioanalysis. Also, matrices can compromise the sensitivity 

and selectivity of bioanalysis methods [86-89], which is deemed a “matrix effect”. The matrix effect could 

be due to endogenous or exogenous agents. Some endogenous substances include salts, carbohydrates, 

amines, urea, lipids, peptides, and metabolites [90,91]. Exogenous substances may include anticoagulants, 

blood preservatives, and mobile-phase additives such as buffer salts [92,93]. Sample cleanup is required to 

isolate analyte(s) of interest from a matrix. 

Sample cleanup tends to reduce or remove matrix components and concentrate the analyte(s). This 

process improves assay sensitivity and selectivity. An optimal sample cleanup system should be capable of 

minimizing matrix effect while ensuring reliable extraction recovery [94]. Basically, there are three sample 

preparation methods that can be applied during HPLC bioanalysis of aminoglycosides; protein precipitation 

(PPT), solid-phase extraction (SPE), and liquid-liquid extraction (LLE) [4]. All the articles reviewed provided 

sample preparation methods; 41 used PPT, 33 SPE, and 4 LLE. One article reported the use of ultrafiltration 

as a sample preparation method [56], 6 articles reported a combination of PPT and SPE [25,42,46,47,61,62], 

and 1 article used a combination of PPT, SPE, and LLE [72]. 

Solvents that were used for PPT included acetonitrile for the extraction of netilmicin, amikacin, 

tobramycin, gentamicin, etimicin, neomycin, and isepamicin [6,15,21,23,32,37-40,42,68,73-76,78,79,82,86]; 

trichloroacetic acid for the extraction of geneticin, spectinomycin, tobramycin, gentamicin, kanamycin, 

hygromycin, apramycin, streptomycin, dihydrostreptomycin, amikacin, neomycin, and paromomycin 

[29,34,35,57,61,72,81]; methanol for the extraction of geneticin, amikacin, tobramycin, sisomicin, and 

netilmicin [29,41,48]; perchloric acid for the extraction of kanamycin, dibekacin, arbekacin, and 

streptomycin [26,33,49,51]; formic acid for the extraction of amikacin [84,85]; and methylene chloride for 

the extraction of isepamicin [62]. PPT aids in reducing interference during derivatization [7], and this may 

have accounted for its use by 41 articles. 

SPE is useful in isolating polar analytes such as aminoglycosides. SPE has also been proven to be useful 

when the volume of a matrix is high [4]. Preparation of matrices containing aminoglycosides using SPE 

tends to give well reproducible recovery [95]. Despite these merits, SPE is relatively expensive compared to 

PPT. In this review, 33 of the articles reported the use of SPE as a sample preparation technique for the 

extraction of gentamicin, amikacin, tobramycin, sisomicin, netilmicin, astromicin, micronomicin, 

streptomycin, neomycin, isepamicin, dihydrostreptomycin, kanamycin, paromomycin, spectinomycin, 

apramycin, and hygromycin in biological matrices. 

Also, four articles adopted the LLE as a sample preparation technique for the extraction of netilmicin, 

gentamicin, amikacin, and dihydrostreptomycin. Aminoglycosides are hydrophilic in nature, and LLE may 

not be ideal for extraction. 

Chromatographic conditions 

All articles reviewed reported chromatographic conditions, and these are highlighted in this review. 

Mode of chromatography 

The most-reported mode of chromatography used for bioanalysis of aminoglycosides was reversed-

phase HPLC (66 articles). This may be due to the polarity of the analytes of interest [96]. Other modes of 

chromatography for the separation of aminoglycosides used were hydrophilic interaction liquid 

chromatography (2 articles) for the analysis of amikacin, gentamicin, kanamycin, neomycin, paromomycin, 



ADMET & DMPK 10(1) (2022) 27-62 Bioanalysis of aminoglycozides by HPLC 

doi: http://dx.doi.org/10.5599/admet.1183 41 

and tobramycin [31,69]; hydrophobic interaction chromatography (1 article) for the analysis of neomycin 

[14]; and ion-exchange chromatography (1 article) for the analysis of gentamicin [40]. One article reported 

the use of mixed-mode chromatography using both reversed-phase and normal chromatography for the 

analysis of amikacin [41]. 

Stationary phase 

The selection of a chromatographic mode of separation is dependent on the choice of the column. In 

this review, 63 articles reported the use of C18 columns for the separation of any of the twenty 

aminoglycosides; while 3 other articles used C8 columns for the separation of streptomycin, 

dihydrostreptomycin, and amikacin [30,47,65]. Also, ion exchange for the separation of gentamicin [40]; 

cyano for the separation of tobramycin [37]; shielded hydrophobic phase for the separation of neomycin 

[14]; silica for the separation of amikacin [41]; and hydrophilic interaction columns for the separation of 

amikacin, gentamicin, kanamycin, neomycin, paromomycin, and tobramycin [31,69] have been reported. 

This implies that C18 columns may be the most appropriate for liquid chromatographic bioanalysis of 

aminoglycosides. In this review, columns were kept at temperatures ranging from 22 °C to as high as 100 °C 

[41]. Overall, the most widely used column temperature was 25 °C for the separation of netilmicin, 

amikacin, tobramycin, gentamicin, dibekacin, sisomicin, isepamicin, geneticin, streptomycin, 

dihydrostreptomycin, and neomycin [6,24,28-30,42,61,64,68,73]; followed by 30 °C for the separation of 

neomycin, streptomycin, dihydrostreptomycin, amikacin, kanamycin, paromomycin, tobramycin, 

spectinomycin, apramycin, hygromycin, gentamicin, netilmicin, etimicin, and isepamicin 

[5,16,32,72,78,81,82]; and 50 °C for the separation of sisomicin, netilmicin, astromicin, micronomicin, 

streptomycin, gentamicin, tobramycin, and amikacin [25,27,43,46,47,50].  

Internal standard 

Internal standards are normally employed to offset injection volume errors and/or losses during sample 

extraction [97]. Forty-four (44) articles used internal standards, which include N-acetyl gentamicin C1 [22], 

tobramycin [22,24,25,44,55,83], amikacin [22,76,78], gentamicin C2 [37], netilmicin [25,39,42,48,58,66], 

kanamycin [45,47,73,84,85], sisomicin [25,46,48,68,75], neamine [24], gentamicin C1a [6,24], astromicin 

[25], dihydrostreptomycin [27], dibekacin [28,33,62], gentamicin [59], neomycin [16,77], naphthalene [65], 

anthracene [67], streptomycin [72], quinoxaline [79], kanamycin B [81], and deuterated paromomycin 

acetic acid [6]. Of the internal standards used, 13 were aminoglycosides, 3 were modified aminoglycosides 

[6,22,81], and 4 were not aminoglycosides or congeners of aminoglycosides [24,65,67,79]. 

Mode of elution 

Although the mobile phase is not directly responsible for chromatographic separation, it can affect 

chromatographic resolution, selectivity, and efficiency [96]. The selection of a suitable mobile phase for the 

HPLC system is dependent on the physicochemical properties of the analyte [98]. Since reversed-phase 

chromatography was reported by most articles, mobile phase solvents used consisted of an aqueous buffer 

and a non-ultraviolet active water-miscible organic solvent [96]. The solvents included acetonitrile for the 

separation of netilmicin, gentamicin, and amikacin [21,39,40,47,60]; methanol and water mixture for the 

separation of gentamicin, amikacin, tobramycin, netilmicin, dibekacin, and sisomicin [20,22,24,36,44]; and 

heptanesulphonic acid for separation of gentamicin, and netilmicin [58,59]. Other solvents such as 

tetrahydrofuran for the separation of amikacin [15]; octanesulphonate for the separation of tobramycin, 

sisomicin, and netilmicin [48]; and ethylene glycol for the separation of sisomicin [54] were also reported. 

In the case where ionisable analytes were present, the pH of the mobile phase had a significant effect on 

the ionization state(s) of the analyte(s), which eventually affected resolution. Thus, the buffer reported by 

http://dx.doi.org/10.5599/admet.1183


Seth K. Amponsah et al.   ADMET & DMPK 10(1) (2022) 27-62 

42  

most articles were aqueous, which included tris buffer [23], sodium acetate [24], and potassium phosphate 

buffer [15]. Basic analytes such as aminoglycosides are protonated at low pH when ionized.  

Poor elution of analytes could contribute to peak broadening [96,99]. Out of 71 articles reviewed, 50 

employed isocratic elution for the separation of streptomycin, gentamicin, amikacin, dihydrostreptomycin, 

kanamycin, netilmicin, isepamicin, sisomicin, paromomycin, neomycin, tobramycin, etimicin, and dibekacin; 

whilst the remaining 21 used gradient elution for the separation of streptomycin, gentamicin, amikacin, 

dihydrostreptomycin, kanamycin, netilmicin, apramycin, isepamicin, sisomicin, paromomycin, geneticin, 

neomycin, tobramycin, arbekacin, spectinomycin, hygromycin, dibekacin, astromicin, and micronomicin. 

For complex multicomponent samples, gradient elution is used since all components cannot be eluted 

between the retention factor of 1 and 10. An isocratic mode is sufficient if the measured ratio is less than 

0.25, however, if the ratio is greater than 0.25, then an elution gradient is deemed suitable [96,99].  

Derivatization and mode of detection 

Of the 71 articles reviewed, fluorescence (36 articles), UV (15 articles), mass spectrometry (15 articles), 

resonance Rayleigh scattering (1 article) [32], pulsed electrochemical (1 article) [15], chemiluminescence (1 

article) [71], and evaporative light scattering (1 article) [70] were used in the detection of aminoglycosides. 

Fluorescence (~50 %) was the most used method for the detection of streptomycin, gentamicin, 

amikacin, dihydrostreptomycin, kanamycin, netilmicin, isepamicin, sisomicin, neomycin, tobramycin, 

dibekacin, astromicin, and micronomicin. However, non-fluorescence drugs such as aminoglycosides are 

mostly difficult to detect by this mode due to the absence of a fluorophore. This shortcoming can be 

mitigated by derivatization [100]. Additionally, the mobile phase used must be selected with care, as highly 

polar solvents or halide ions can quench fluorescence. It is noteworthy that fluorescence is mostly 

preferred to UV detection due to its high sensitivity and selectivity [101]. Aminoglycosides assayed in 

various studies with fluorescence detectors were achieved at an excitation wavelength range of 220 nm 

[21] to 490 nm [77] and emission wavelength range of 415 nm [57] to 531 nm [74].  

UV-visible detectors are not easily influenced by the mobile phase and surrounding temperature [102]. 

UV-visible detectors interact with compounds containing chromophores. Since aminoglycosides do not 

have chromophores, derivatization of these compounds is necessary for their detection. UV-visible was 

used for the detection of gentamicin, amikacin, tobramycin, streptomycin, netilmicin, and geneticin 

[6,27,29,38,39,41,45,47,58,64,65,67]. In this review, the UV wavelength range used for the detection of 

aminoglycosides was 195 nm [27] to 365 nm [6,38,45,64]. 

In all the 51 articles that used fluorescence or UV detection, only 3 articles did not use derivatization for 

the detection of streptomycin, dihydrostreptomycin, and sisomicin [27,30,54]. The most common 

derivatizing agent used in the bioanalysis of aminoglycosides was o-phthalaldehyde; reported by 24 articles 

for the derivatization of gentamicin, amikacin, kanamycin, netilmicin, isepamicin, sisomicin, neomycin, 

tobramycin, dibekacin, astromicin, and micronomicin [6,14,20,22-26,28,36,37,43,44,48,50,52,53,55,57-

59,61,62,66]. Dansyl chloride for the derivatization of netilmicin [21]; 1-fluoro-2,4-dinitrobenzene for the 

derivatization of geneticin, gentamicin, and amikacin [29,38,41,45,56,64]; 7-fluoro-4-nitrobenz-2-oxa-1,3-

diazole for the derivatization of amikacin [74]; benzene sulphonyl chloride for the derivatization of 

gentamicin [39]; 6-aminoquinolyl-N-hydroxysucciminidylcarbamate for the derivatization of isepamicin 

[76]; fluorescamine for the derivatization of gentamicin, and tobramycin [40,80]; 1-naphthyl isothiocyanate 

for the derivatization of amikacin, and tobramycin [65,67]; fluorescein isothiocyanate for the derivatization 

of tobramycin [77]; o-phthalicdicarboxaldehyde for the derivatization of gentamicin [42]; 2,4,6-

trinitrobenzene sulfonic acid for the derivatization of tobramycin, and amikacin [46,47]; -naphthoquinone-



ADMET & DMPK 10(1) (2022) 27-62 Bioanalysis of aminoglycozides by HPLC 

doi: http://dx.doi.org/10.5599/admet.1183 43 

4-sulfonate for the derivatization of streptomycin [49]; ninhydrin for the derivatization of streptomycin 

[51]; and 9-fluorenylmethylchloroformate for the derivatization of gentamicin, neomycin, netilmicin, 

sisomicin, isepamicin, and amikacin [16,60,63,78,82] were other derivatizing agents used in the bioanalysis 

of aminoglycosides. 

Challenges associated with UV and fluorescence detections (need for chromophore or fluorophore 

necessitating derivatization) can be circumvented using mass spectrometry. Also, mass spectrometry can 

analyze small sample volumes with high precision, sensitivity, and selectivity [68,81]. In this review, 15 

articles reported the use of mass spectrometry as a detector for the bioanalysis of neomycin, streptomycin, 

dihydrostreptomycin, amikacin, kanamycin, paromomycin, tobramycin, spectinomycin, apramycin, 

hygromycin, gentamicin, and arbekacin [5,31,33-35,68,69,72,73,75,79,81,83-85]. Interestingly, articles 

published between 1977 [20] to 2002 [67] were dominated by fluorescence or UV detection methods. 

More importantly, mass spectrometry was the mostly used detection mode from 2014 to 2020 

[34,35,81,83-85], except in one case [82]. This is not surprising as mass spectrometry appears to be an 

effective detection method [103]. 

In this review, HPLC coupled with resonance Rayleigh scattering detection was used in analyzing three 

aminoglycosides; amikacin, netilmicin, and etimicin [32]. An advantage of the resonance Rayleigh scattering 

detector over other spectroscopic techniques is that the detection limit is lower by several orders of 

magnitude [104].  

The pulsed electrochemical detector is mostly used to analyze carbohydrates and polyalcohol. They are 

also used in analyzing amines, amino acids, and sulphur-containing compounds [105, 106]. One (1) article 

reported the use of HPLC coupled with the pulsed electrochemical detector in the bioanalysis of amikacin 

[15]. This approach was used to address the shortfall of using derivatization for the detection of 

aminoglycosides [15]. 

Chemiluminescence allows the detection of analytes at ultra-high sensitivity. In this review, 1 article 

reported the use of chemiluminescence for the detection of amikacin, and the chemiluminescence reagent 

used was luminol in combination with hydrogen peroxide and Cu
2+

 [71]. 

HPLC coupled with evaporative light scattering detector is rapidly becoming a quasi-universal detector, 

mitigating the need for derivatization of non-absorbing analytes. In this review, one article reported the use 

of an evaporative light scattering detector for the direct determination of tobramycin [70]. 

Performance metrics 

Performance metrics are quantifiable terms that indicate the quality of an analytical process. Some 

performance metrics include specificity, sensitivity, linearity, the lower limit of quantification (LLOQ), limit 

of detection (LOD), precision, accuracy, and calibration range. In this review, all the 71 articles reported 

some aspects of method performance characteristics. Method performance characteristics reported by 

most articles included calibration range, linearity, recovery, repeatability, and intermediate precision (Table 

2). Out of the 71 articles reviewed, only four reported on matrix effect [63,83-85]. Resolution was reported 

by one article [75].  

The calibration range is often obtained from a calibration curve [107]. Sixty-seven (67) articles reported 

calibration ranges, whilst 4 did not [5,23,59,79] (Table 2). These calibration ranges of HPLC varied 

significantly for the various drugs assayed, with some in the range of 0.1-0.5 ng/mL [14] and others 1250-

200000 ng/mL [83]. Of the 20 aminoglycosides reported, only eight had established a therapeutic reference 

range. The reported calibration range of some of the analytes reported was outside the established 

http://dx.doi.org/10.5599/admet.1183


Seth K. Amponsah et al.   ADMET & DMPK 10(1) (2022) 27-62 

44  

therapeutic reference range [14,15,24,25,30,31,34,37,48,55,57,61,65,67,71,74,75,80-82], which invalidates 

the measurements obtained from the bioanalysis. 

The quality of a bioanalytical method is highly dependent on the linearity of the calibration curve [108]. 

The linearity of the calibration curve is usually expressed as a coefficient of correlation (R
2
). The coefficient 

of correlation close to 1 (R
2
 ≈1) is mostly considered ideal. In the current review, 61 articles reported R

2
 

values for almost all analytes under consideration, whilst 10 articles did not 

[23,28,33,39,45,47,50,55,56,84]. The reported coefficient of correlation was between 0.964 [75] and 1.0 

[48,60,76,82]. Additionally, y-intercept, the slope of the regression of line, and residual sum of squares can 

also be used in evaluating linearity [109]. Out of 71 articles reviewed, 39 articles reported values for both 

the slope of regression and the y-intercept. 

Accuracy of a bioanalytical method is normally expressed as the percent recovery by the assay of the 

known added amount of analyte. In this study, 63 articles reported percent recoveries that ranged mostly 

between 61.0 % [72] to 114.0 % [5]. Two (2) articles reported recoveries of 4.9 % [28] and 36 % [76], 

respectively. 

LLOQ is defined as the amount of analyte in a biological matrix that can be quantitatively determined 

with suitable precision and accuracy [110]. Thirty-six (36) articles reported LLOQ for aminoglycosides 

assayed between 1 ng/mL [72] to 2340 ng/mL [79]. Forty-one (41) articles also reported LOD values in the 

range 0.3 ng/mL [61] to 75000 ng/ml [56]. There were instances where the same article reported both the 

LLOQ and LOD. 

Repeatability expresses the closeness of results obtained with the same sample using the same 

procedure, operators, measuring system, operating conditions, and location over a short period of time. 

Out of 71 articles, 58 reported repeatability values ranging from 0.28 % [69] to 36 % [30]. Intermediate 

precision also refers to laboratory variations such as different days, instruments, and analyses. Out of the 

71 reviewed articles, 56 reported intermediate precision values ranging between 0.331 % [80] to 19.76 % 

[52], which is within the acceptable limit of 20 % [111]. 

 

 

 
 
 
 
  



ADMET & DMPK 10(1) (2022) 27-62 Bioanalysis of aminoglycozides by HPLC 

doi: http://dx.doi.org/10.5599/admet.1183 45 

Table 2. Performance metrics of HPLC method for bioanalysis of aminoglycosides. 
In

te
rm

e
d

ia
te

 

p
re

ci
si

o
n

 (
%

) 

n
.i

. 

6
 

6
 

6
 

n
.i

. 

3
.6

 

2
.8

 

3
.4

 

<
8

 

<
8

 

<
8

 

4
.8

3
-7

.9
1

 

(S
e

ru
m

) 

3
.0

0
-6

.0
0

 

(U
ri

n
e

) 

n
.i

. 

n
.i

. 

<
2

.0
 

n
.i

. 

R
e

p
e

a
ta

b
il

it
y

 

(%
) 

n
.i

. 

4
.2

-5
.6

 

4
.4

-7
.5

 

3
.9

-5
.1

 

n
.i

. 

n
.i

. 

3
.2

 

2
.3

 

<
8

 

<
8

 

<
8

 

3
.7

9
-5

.6
0

 

(S
e

ru
m

) 

n
.i

. 
(U

ri
n

e
) 

n
.i

. 

n
.i

. 

3
.5

 

n
.i

. 

LL
O

Q
 

(n
g

/m
L)

 

n
.i

. 

n
.i

.   

n
.i

. 

n
.i

.   

5
0

0
 

1
0

0
0

 

5
0

0
 

2
0

0
 

(S
e

ru
m

) 

2
0

0
 

(U
ri

n
e

) 

n
.i

. 

2
0

0
 

n
.i

. 

n
.i

. 

LO
D

 

(n
g

/m
L)

 

n
.i

. 

n
.i

.   

5
0

0
 

n
.i

.   

n
.i

.   

n
.i

.  

n
.i

. 

n
.i

. 

1
0

0
0

 

1
0

0
0

 

R
e

co
v

e
ry

 (
%

) 

>
9

5
 

8
0

-1
0

5
 

  

n
.i

. 

>
9

5
 

9
3

 

9
3

 

9
6

.7
-1

1
0

 

9
4

.1
-9

8
.3

 

9
1

.5
-9

1
.8

 

n
.i

. 

(S
e

ru
m

) 

8
3

.1
-9

4
.3

 

(U
ri

n
e

) 

8
3

.0
-8

4
.0

 

n
.i

. 

9
3

 

n
.i

. 

y
-I

n
te

rc
e

p
t 

1
.3

6
 

0
.6

2
 

  

-0
.0

0
4

 

1
.3

6
 

0
.0

4
4

 

-0
.0

2
4

 

n
.i

.   

-0
.0

0
9

2
6

 

(S
e

ru
m

) 

-0
.0

4
0

6
0

 

(U
ri

n
e

) 

n
.i

. 

n
.i

. 

-0
.2

1
 –

 

0
.7

3
 

-0
.1

0
 –

 

1
.6

0
 

S
lo

p
e

 

4
.7

2
 

0
.8

7
 

  

0
.5

6
3

 

4
.7

2
 

0
.1

3
6

 

0
.2

7
8

 

n
.i

.   

0
.1

1
4

0
 

(S
e

ru
m

) 

0
.3

4
4

0
 

(U
ri

n
e

) 

n
.i

. 

n
.i

. 

2
.4

9
 -

 

4
.2

6
 

1
.9

0
 -

 

2
.3

3
 

R
2
 

0
.9

9
7

 

0
.9

9
 

  

0
.9

9
9

 

0
.9

9
7

 

0
.9

9
9

2
 

0
.9

9
9

7
 

n
.i

.   

0
.9

9
9

0
 

(S
e

ru
m

) 

0
.9

9
9

0
 

(U
ri

n
e

) 

n
.i

. 

n
.i

. 

0
.9

9
7

7
-

0
.9

9
9

9
 

0
.9

9
6

3
-

0
.9

9
9

7
 

R
e

fe
re

n
ce

 r
a

n
g

e
 

(n
g

/m
L)

 

5
0

0
0

-1
0

0
0

0
b

)  

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

1
0

0
0

0
-1

6
0

0
0

c)
 

5
0

0
0

-1
0

0
0

0
b

)  

1
5

0
0

0
-2

5
0

0
0

b
)  

5
0

0
0

-1
0

0
0

0
b
 

1
0

0
0

0
-1

6
0

0
0

c)
 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

 

R
a

n
g

e
 (

n
g

/m
L)

 

1
0

0
0

-1
0

0
0

0
 

0
-2

0
0

0
0

 

  

5
0

0
-1

0
0

0
0

 

1
0

0
0

-1
0

0
0

0
 

2
0

0
0

-3
2

0
0

0
 

2
0

0
0

-1
5

0
0

0
 

n
.i

.   

1
0

0
0

-7
5

0
0

 

(S
e

ru
m

)a
)  

1
0

0
0

-7
5

0
0

 

(U
ri

n
e

)a
)  

1
0

0
0

-1
6

0
0

0
 

0
-1

0
0

0
0

 

0
-4

0
0

0
0

 

(P
la

sm
a

) 

0
-7

1
0

0
0

 

(U
ri

n
e

) 

R
e

so
lu

ti
o

n
 

n
.i

. 

n
.i

.   

n
.i

. 

n
.i

.   

n
.i

.   

n
.i

.  

n
.i

. 

n
.i

. 

n
.i

.  

M
a

tr
ix

 

e
ff

e
ct

 

n
.i

. 

n
.i

.   

n
.i

. 

n
.i

.   

n
.i

.   

n
.i

.  

n
.i

. 

n
.i

. 

n
.i

.  

A
n

a
ly

te
 

G
e

n
ta

m
ic

in
 

G
e

n
ta

m
ic

in
 C

1
 

G
e

n
ta

m
ic

in
 C

1
a

 

G
e

n
ta

m
ic

in
 C

2
 

N
e

ti
lm

ic
in

 

G
e

n
ta

m
ic

in
 

A
m

ik
a

ci
n

 

T
o

b
ra

m
y

ci
n

 

N
e

ti
lm

ic
in

 

T
o

b
ra

m
y

ci
n

 

G
e

n
ta

m
ic

in
 

T
o

b
ra

m
y

ci
n

 

 

G
e

n
ta

m
ic

in
 

C
1

a
/G

e
n

ta
m

ic
in

 

C
1

+
C

2
 

G
e

n
ta

m
ic

in
 

G
e

n
ta

m
ic

in
 

 

R
e

f.
 

[2
0

] 

[3
6

] 

  

[2
1

] 

[2
2

] 

  

[2
3

] 

  

[3
7

] 

 

[3
8

] 

[3
9

] 

[4
0

] 

 

  

http://dx.doi.org/10.5599/admet.1183


Seth K. Amponsah et al.   ADMET & DMPK 10(1) (2022) 27-62 

46  

Table 2. Continued... 
In

te
rm

e
d

ia
te

 

p
re

ci
si

o
n

 (
%

) 

n
.i

. 

<
8

 (
S

e
ru

m
) 

<
8

 (
U

ri
n

e
) 

<
3

.2
 

3
.7

-5
.8

 

3
.6

-6
.9

 

n
.i

. 

4
.6

-5
.1

 

<
6

.0
 

<
6

.0
 

<
6

.0
 

<
6

.0
 

<
6

.0
 

<
6

.0
 

<
6

.0
 

<
6

.0
 

2
.8

-3
.1

 

R
e

p
e

a
ta

b
il

it
y

 

(%
) 

n
.i

. 

2
.4

-1
0

.1
 

(S
e

ru
m

) 

2
.4

-1
0

.1
 

(U
ri

n
e

) 

<
2

.5
 

n
.i

.  

1
.5

-5
.3

 

4
.0

-4
.9

 

5
.9

 

n
.i

. 

n
.i

. 

4
.9

 

4
.9

 

4
.9

 

n
.i

. 

n
.i

. 

3
.5

-6
.0

 

LL
O

Q
 

(n
g

/m
L)

 

n
.i

. 

5
0

0
 

(S
e

ru
m

) 

5
0

0
 

(U
ri

n
e

) 

n
.i

. 

n
.i

.  

n
.i

. 

n
.i

.       

n
.i

. 

n
.i

. 

<
5

0
0

 

LO
D

 

(n
g

/m
L)

 

n
.i

. 

n
.i

.  

5
0

0
 

5
0

0
 

5
0

0
 

n
.i

. 

<
2

0
0

 

n
.i

.        

n
.i

. 

R
e

co
v

e
ry

 (
%

) 

n
.i

. 

>
8

5
 

(S
e

ru
m

) 

>
8

5
 

(U
ri

n
e

) 

9
7

-1
0

3
 

8
0

-9
0

 

8
0

-9
0

 

7
2

 

9
4

-9
8

.6
 

n
.i

.        

9
2

.8
-9

8
.4

 

y
-I

n
te

rc
e

p
t 

1
.9

2
 

n
.i

. 

n
.i

. 

-0
.1

2
6

 

n
.i

.  

0
.0

2
 

n
.i

. 

n
.i

. 

n
.i

. 

n
.i

. 

-1
.6

 

-0
.7

 

0
.3

 

n
.i

. 

n
.i

. 

n
.i

. 

S
lo

p
e

 

3
.2

1
 

n
.i

. 

n
.i

. 

0
.7

9
2

 

n
.i

.  

0
.9

2
 

n
.i

. 

n
.i

. 

n
.i

. 

n
.i

. 

1
6

.6
 

9
.5

 

7
.2

 

n
.i

. 

n
.i

. 

n
.i

. 

R
2
 

0
.9

9
9

6
 

0
.9

9
9

0
 

(S
e

ru

m
) 

0
.9

9
9

0
 

(U
ri

n
e

) 

0
.9

9
9

 

0
.9

8
 

n
.i

. 

0
.9

9
3

 

0
.9

5
 

n
.i

. 

n
.i

. 

n
.i

. 

0
.9

9
9

8
 

0
.9

9
9

7
 

0
.9

9
9

8
 

n
.i

. 

n
.i

. 

n
.i

. 

R
e

fe
re

n
ce

 r
a

n
g

e
 

(n
g

/m
L)

 

1
5

0
0

0
-2

5
0

0
0

b
)  

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

1
0

0
0

0
-1

6
0

0
0

c)
 

1
5

0
0

0
-2

5
0

0
0

b
)  

5
0

0
0

-1
0

0
0

0
b
 

1
5

0
0

0
-2

5
0

0
0

b
)  

5
0

0
0

-1
0

0
0

0
b
 

1
0

0
0

0
-1

6
0

0
0

c)
 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

n
.a

. 

n
.a

. 

1
5

0
0

0
-2

5
0

0
0

b
)  

R
a

n
g

e
 (

n
g

/m
L)

 

2
0

0
0

-6
4

0
0

0
 

5
0

0
-1

0
0

0
0

 

(S
e

ru
m

) 

5
0

0
-5

0
0

0
 

(U
ri

n
e

) 

2
7

0
0

-1
6

5
0

0
 

0
-2

0
0

0
0

 

0
-2

0
0

0
0

 

1
0

0
0

-6
4

0
0

0
 

1
0

0
0

-2
5

0
0

0
 

1
0

0
0

-

1
6

0
0

0
a

)  

       

2
5

0
0

-5
0

0
0

0
 

R
e

so
lu

ti
o

n
 

n
.i

. 

n
.i

.  

n
.i

. 

n
.i

.  

n
.i

. 

n
.i

. 

n
.i

.        

n
.i

. 

M
a

tr
ix

 

e
ff

e
ct

 

n
.i

. 

n
.i

.  

n
.i

. 

n
.i

.  

n
.i

. 

n
.i

. 

n
.i

.        

n
.i

. 

A
n

a
ly

te
 

A
m

ik
a

ci
n

 

G
e

n
ta

m
ic

in
 (

C
1

, 

C
1

a
, 

a
n

d
 C

2
) 

 

G
e

n
ta

m
ic

in
 

G
e

n
ta

m
ic

in
 

N
e

ti
lm

ic
in

 

A
m

ik
a

ci
n

 

T
o

b
ra

m
y

ci
n

 

A
m

ik
a

ci
n

 

T
o

b
ra

m
y

ci
n

 

N
e

ti
lm

ic
in

 

G
e

n
ta

m
ic

in
 C

1
 

G
e

n
ta

m
ic

in
 C

1
a

 

G
e

n
ta

m
ic

in
 C

2
 

D
ib

e
k

a
ci

n
 

S
is

o
m

ic
in

 

A
m

ik
a

ci
n

 

R
e

f.
 

[4
1

] 

[4
2

] 

 

[4
3

] 

[4
4

] 

 

[4
5

] 

[4
6

] 

[2
4

] 

       

[4
7

] 

 



ADMET & DMPK 10(1) (2022) 27-62 Bioanalysis of aminoglycozides by HPLC 

doi: http://dx.doi.org/10.5599/admet.1183 47 

Table 2. Continued... 
In

te
rm

e
d

ia
te

 

p
re

ci
si

o
n

 (
%

) 

2
.5

 

2
.8

 

3
.1

 

1
.9

 

2
.3

-2
.5

 

n
.i

. 

n
.i

. 

2
.0

-5
.0

 

1
.9

-5
.6

 

<
6

 

3
.0

1
-3

.5
0

 

<
4

.0
 

  

2
.3

4
-2

.5
8

 

6
.4

9
-8

.8
7

 

7
.2

2
-1

9
.7

6
 

1
0

.3
5

-1
4

.8
2

 

R
e

p
e

a
ta

b
il

it
y

 

(%
) 

n
.i

.    

2
.0

-2
.2

 

n
.i

. 

n
.i

. 

0
.8

-2
.9

 

1
.6

-3
.1

 

<
6

 

2
.6

7
-3

.0
2

 

<
4

.0
 

  

1
.2

1
-2

.7
5

 

6
.0

5
-6

.7
6

 

5
.1

3
-1

1
.6

6
 

6
.3

6
-7

.0
2

 

LL
O

Q
 

(n
g

/m
L)

 

n
.i

.    

n
.i

. 

n
.i

. 

n
.i

. 

n
.i

.  

n
.i

. 

n
.i

. 

n
.i

.   

n
.i

. 

n
.i

.   

LO
D

 

(n
g

/m
L)

 

n
.i

.    

3
0

0
 

3
0

0
 

3
0

0
 

n
.i

.  

2
0

0
0

 

5
0

0
 

5
0

0
 

  

1
0

0
 

2
0

 

8
0

 

8
0

 

R
e

co
v

e
ry

 (
%

) 

~
1

0
0

 

~
1

0
0

 

~
1

0
0

 

~
1

0
0

 

9
7

.5
-1

0
2

 

n
.i

. 

n
.i

. 

9
9

 

9
8

.5
 

8
0

 

1
0

0
 

1
0

3
.9

 

9
9

.5
 

1
0

1
.1

 

1
0

0
 

n
.i

.   

y
-I

n
te

rc
e

p
t 

0
.0

0
2

3
 

0
.0

0
8

5
 

0
.0

0
7

7
5

 

0
.0

1
1

7
 

-0
.0

5
7

 

0
.0

0
2

 

0
.0

0
3

 

-0
.0

0
6

 

-0
.0

4
 

-0
.0

2
5

8
 

0
.0

7
2

 

n
.i

.   

0
.0

8
6

 

1
2

.8
4

 

1
.5

 

4
.8

 

S
lo

p
e

 

0
.0

3
4

 

0
.1

0
1

 

0
.2

4
4

 

0
.0

8
2

 

0
.1

0
1

 

0
.1

3
6

 

0
.1

 

0
.9

7
9

 

0
.9

9
8

 

0
.0

4
7

6
 

0
.3

2
9

 

n
.i

.   

0
.1

3
6

 

3
1

1
.4

 

1
3

1
.6

 

1
2

4
.7

 

R
2
 

0
.9

9
9

7
 

0
.9

9
9

8
 

0
.9

9
9

7
 

0
.9

9
9

8
 

1
 

0
.9

9
9

 

0
.9

9
3

 

0
.9

9
9

 

0
.9

9
9

 

0
.9

9
9

7
 

0
.9

9
9

 

n
.i

.   

0
.9

9
9

 

0
.9

9
1

 

0
.9

9
5

 

0
.9

9
6

 

R
e

fe
re

n
ce

 r
a

n
g

e
 

(n
g

/m
L)

 

n
.a

. 

1
0

0
0

0
-1

6
0

0
0

c)
 

n
.a

. 

n
.a

. 

5
0

0
0

-1
0

0
0

0
b
 

n
.a

. 

1
0

0
0

0
-1

6
0

0
0

c)
 

1
5

0
0

0
-2

5
0

0
0

d
)  

n
.a

. 

1
0

0
0

0
-3

5
0

0
0

e
)  

1
0

0
0

0
-3

5
0

0
0

e
)  

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

1
0

0
0

0
-3

5
0

0
0

e
)  

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

R
a

n
g

e
 (

n
g

/m
L)

 

3
2

0
-2

2
8

0
0

 

1
7

0
-1

1
6

0
0

a
)  

1
0

0
-6

3
0

0
 

1
0

0
0

-3
0

0
0

 

1
8

5
0

-1
4

8
3

0
 

1
6

6
0

-1
3

3
3

0
 

1
6

3
0

-

1
3

0
6

0
a

)  

3
0

0
0

-5
0

0
0

0
 

5
0

0
-1

0
0

0
0

 

5
0

0
0

-5
0

0
0

0
 

5
0

0
0

-5
0

0
0

0
 

0
-5

0
0

0
0

 

  

5
0

0
0

-4
0

0
0

0
 

1
0

0
0

-2
0

0
0

0
 

  

R
e

so
lu

ti
o

n
 

n
.i

.    

n
.i

.   

n
.i

.  

n
.i

. 

n
.i

. 

n
.i

.   

.n
i 

n
.i

.   

M
a

tr
ix

 

e
ff

e
ct

 

n
.i

.    

n
.i

.   

n
.i

.  

n
.i

. 

n
.i

. 

n
.i

.   

n
.i

. 

n
.i

.   

A
n

a
ly

te
 

S
is

o
m

ic
in

 

N
e

ti
lm

ic
in

 

A
st

ro
m

ic
in

 

M
ic

ro
n

o
m

ic
in

 

T
o

b
ra

m
y

ci
n

 

S
is

o
m

ic
in

 

N
e

ti
lm

ic
in

 

K
a

n
a

m
y

ci
n

 

D
ib

e
k

a
ci

n
 

S
tr

e
p

to
m

y
ci

n
 

S
tr

e
p

to
m

y
ci

n
 

G
e

n
ta

m
ic

in
 C

1
 

G
e

n
ta

m
ic

in
 C

1
a

 

G
e

n
ta

m
ic

in
 C

2
 

S
tr

e
p

to
m

y
ci

n
 

G
e

n
ta

m
ic

in
 C

1
 

G
e

n
ta

m
ic

in
 C

1
a

 

G
e

n
ta

m
ic

in
 C

2
 

R
e

f.
 

[2
5

] 

   

[4
8

] 

  

[2
6

] 

 

[2
7

] 

[4
9

] 

[5
0

] 

  

[5
1

] 

[5
2

] 

  

 

http://dx.doi.org/10.5599/admet.1183


Seth K. Amponsah et al.   ADMET & DMPK 10(1) (2022) 27-62 

48  

Table 2. Continued... 
In

te
rm

e
d

ia
te

 

p
re

ci
si

o
n

 (
%

) 

4
.2

-4
.4

 

n
.i

.  

n
.i

. 

n
.i

. 

2
.6

-5
.5

 

(P
la

sm
a

) 

2
.9

-7
.5

 

(U
ri

n
e

) 

3
.2

-2
5

.1
 

2
.8

-3
.8

 

0
.1

-6
.6

 

2
.9

-5
.6

 

n
.i

. 

1
4

.2
3

 

6
.3

-7
.9

 

5
.7

-8
.9

 

5
.0

-5
.5

 

6
.0

-8
.9

 

R
e

p
e

a
ta

b
il

it
y

 

(%
) 

1
.8

-1
0

.9
 

7
.9

 

 

n
.i

. 

n
.i

. 

0
.5

-2
.5

 

(P
la

sm
a

) 

1
.0

-6
.0

 

(U
ri

n
e

) 

3
.9

-2
8

.4
 

1
.9

-3
.3

 

0
.1

-2
.1

 

1
.1

-4
.7

 

n
.i

. 

9
.2

2
 

6
.4

-8
.6

 

5
.9

-6
.0

 

5
.8

-7
.0

 

4
.3

-5
.8

 

LL
O

Q
 

(n
g

/m
L)

 

n
.i

. 

n
.i

.  

n
.i

. 

n
.i

. 

1
0

0
 

(p
la

sm
a

) 

5
0

 

(U
ri

n
e

) 

2
5

 

5
0

0
 

1
0

0
0

0
 

1
0

0
 

n
.i

. 

n
.i

. 

n
.i

.    

LO
D

 

(n
g

/m
L)

 

5
0

0
 

<
5

0
 

 

6
2

.5
 

5
0

 

n
.i

.  

n
.i

. 

5
0

0
-

7
5

0
0

0
 

 

2
5

 

6
0

0
 

1
0

0
 

<
5

0
 

   

R
e

co
v

e
ry

 (
%

) 

7
8

-8
1

 

9
1

.6
 

 

9
7

.5
 

9
4

-1
0

2
 

4
.9

±
0

.1
 

 

n
.i

. 

9
6

.9
 

9
2

.1
 

9
9

.5
-1

0
5

 

>
9

0
 

n
.i

. 

9
6

.8
 

9
9

 

9
7

.8
 

9
3

.9
 

y
-I

n
te

rc
e

p
t 

0
.0

0
1

3
 

-1
.4

0
0

0
 

(D
B

S
) 

-0
.0

1
4

0
 

(W
h

o
le

 

b
lo

o
d

) 

-0
.6

9
4

 

n
.i

. 

n
.i

.  

n
.i

. 

n
.i

.  

-4
2

4
0

4
 

n
.i

. 

n
.i

. 

-2
.5

 

-3
.7

 

-4
.7

 

-3
.3

 

S
lo

p
e

 

0
.0

3
5

4
 

0
.6

0
0

0
 

(D
B

S
) 

0
.6

6
9

0
 

(W
h

o
le

 

b
lo

o
d

) 

1
6

.7
5

3
 

n
.i

. 

n
.i

.  

n
.i

. 

n
.i

.  

2
4

5
0

2
0

9
 

n
.i

. 

n
.i

. 

1
1

8
.2

 

9
2

.9
 

1
2

0
.7

 

7
2

.7
 

R
2
 

0
.9

9
 

0
.9

9
5

0
 

(D
B

S
) 

0
.9

9
5

0
 

(W
h

o
le

 

b
lo

o
d

) 

0
.9

9
9

6
 

0
.9

8
9

 

n
.i

.  

n
.i

. 

n
.i

.  

0
.9

9
9

9
 

>
0

.9
9

0
 

0
.9

9
4

 

1
 

1
 

0
.9

9
9

9
 

0
.9

9
9

9
 

R
e

fe
re

n
ce

 r
a

n
g

e
 

(n
g

/m
L)

 

1
0

0
0

0
-1

6
0

0
0

c)
 

n
.a

. 

 

n
.a

. 

5
1

6
1

-1
0

3
2

3
f)
 

n
.a

. 

 

1
5

0
0

0
-2

5
0

0
0

b
)  

1
5

0
0

0
-2

5
0

0
0

b
)  

1
5

0
0

0
-2

5
0

0
0

b
)  

1
5

0
0

0
-2

5
0

0
0

b
)  

5
0

0
0

-1
0

0
0

0
b
 

1
0

0
0

0
-1

6
0

0
0

c)
 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

R
a

n
g

e
 (

n
g

/m
L)

 

5
0

0
-4

0
0

0
0

 

5
0

-5
0

0
0

 

(D
B

S
) 

5
0

-5
0

0
0

 

(W
h

o
le

 

b
lo

o
d

) 

5
0

0
-5

0
0

0
 

1
0

0
-5

0
0

0
a

)  

1
0

0
-1

0
0

0
0

0
 

 

2
5

-2
0

0
0

a
)  

5
0

0
-7

5
0

0
0

 

1
0

0
0

0
-

5
0

0
0

0
0

 

1
0

0
-2

0
0

0
a

)  

6
2

5
-8

0
0

0
0

 

n
.i

. 

2
0

0
-5

0
0

0
0

 

   

R
e

so
lu

ti
o

n
 

n
.i

. 

n
.i

.  

n
.i

. 

n
.i

. 

n
.i

.   

n
.i

.  

n
.i

. 

n
.i

. 

n
.i

. 

n
.i

.    

M
a

tr
ix

 

e
ff

e
ct

 

n
.i

. 

n
.i

.  

n
.i

. 

n
.i

. 

n
.i

.  

n
.i

. 

n
.i

.  

n
.i

. 

n
.i

. 

n
.i

. 

n
.i

.    

A
n

a
ly

te
 

N
e

ti
lm

ic
in

 

S
is

o
m

ic
in

 

 

S
is

o
m

ic
in

 

N
e

o
m

y
ci

n
 

Is
e

p
a

m
ic

in
 

 

A
m

ik
a

ci
n

 

A
m

ik
a

ci
n

 

 

A
m

ik
a

ci
n

 

G
e

n
ta

m
ic

in
 

N
e

ti
lm

ic
in

 

G
e

n
ta

m
ic

in
 C

1
 

G
e

n
ta

m
ic

in
 C

1
a

 

G
e

n
ta

m
ic

in
 C

2
 

G
e

n
ta

m
ic

in
 C

2
a

 

R
e

f.
 

[6
] 

[5
3

] 

 

[5
4

] 

[1
4

] 

[2
8

] 

 

[5
5

] 

[5
6

] 

 

[5
7

] 

[5
8

] 

[5
9

] 

[6
0

] 

   

 



ADMET & DMPK 10(1) (2022) 27-62 Bioanalysis of aminoglycozides by HPLC 

doi: http://dx.doi.org/10.5599/admet.1183 49 

Table 2. Continued... 
In

te
rm

e
d

ia
te

 

p
re

ci
si

o
n

 (
%

) 

0
.0

9
 

n
.i

. 

7
.4

 

3
.5

 

2
.4

 

2
.5

 

n
.i

.  

2
.1

6
-3

.6
1

 

2
.0

-7
.7

 

(P
la

sm
a

) 

1
2

-1
6

 

(U
ri

n
e

) 

4
.8

.2
0

1
3

 

8
.7

-1
2

 

(U
ri

n
e

) 

6
.3

.2
0

1
2

 

1
4

-1
6

 

(U
ri

n
e

) 

2
.3

7
 

4
.2

5
-6

.3
2

 

R
e

p
e

a
ta

b
il

it
y

 

(%
) 

0
.0

2
 

n
.i

. 

1
.9

-1
5

.0
 

4
.5

 

3
.5

 

3
.5

 

5
.1

-3
6

.8
 

7
.3

-3
6

.9
 

2
.4

1
-2

.6
8

 

1
.1

-1
1

 

(P
la

sm
a

) 

7
.8

-8
.1

 

(U
ri

n
e

) 

2
.1

-7
.7

 

6
.1

-1
0

 

(U
ri

n
e

) 

4
.2

.2
0

1
0

 

3
.1

-4
.2

 

(U
ri

n
e

) 

<
5

.8
 

2
.8

4
-5

.4
4

 

LL
O

Q
 

(n
g

/m
L)

 

n
.i

. 

1
5

 

1
0

0
 

1
0

 

  

1
2

 

8
 

n
.i

. 

7
0

 

 

1
0

0
 

 

1
0

0
 

  

2
5

0
 

LO
D

 

(n
g

/m
L)

 

7
8

 

0
.3

-

0
.4

 

n
.i

. 

n
.i

.   8
 

1
2

 

1
4

 

n
.i

.  

n
.i

.  

n
.i

.  

4
0

0
 

7
5

 

R
e

co
v

e
ry

 (
%

) 

n
.i

. 

7
8

-8
8

 

8
3

 

1
0

6
.7

 

9
5

.2
 

9
9

.1
 

7
7

.6
-9

6
.1

 

8
1

.6
-1

0
6

 

1
0

1
.1

-

1
0

5
.6

 

7
2

 

(P
la

sm
a

) 

9
8

 (
U

ri
n

e
) 

7
2

 
(P

la
sm

a
) 

9
8

 (
U

ri
n

e
) 

7
2

 

(P
la

sm
a

) 

9
8

 (
U

ri
n

e
) 

>
9

1
 

9
4

.3
 

y
-I

n
te

rc
e

p
t 

n
.i

. 

n
.i

. 

n
.i

. 

-0
.3

 

7
.7

 

1
3

 

3
7

.8
 

1
7

.2
 

n
.i

. 

0
 

(P
la

sm
a

) 

0
 (

U
ri

n
e

) 

0
 

(P
la

sm
a

) 

0
 (

U
ri

n
e

) 

0
 

(P
la

sm
a

) 

0
 (

U
ri

n
e

) 

-0
.0

4
9

8
 ±

 
0

.0
0

5
5

 

0
.2

4
6

5
 

S
lo

p
e

 

n
.i

. 

n
.i

. 

n
.i

. 

1
9

1
.8

 

2
2

9
.5

 

1
6

7
.1

 

0
.0

0
0

0
9

5
 

0
.0

0
0

3
4

 

n
.i

. 

3
4

0
 

(P
la

sm
a

) 

4
7

0
 

(U
ri

n
e

) 

1
8

3
 

(P
la

sm
a

) 

2
4

5
 

(U
ri

n
e

) 

1
6

9
 

(P
la

sm
a

) 

2
3

5
 

(U
ri

n
e

) 

0
.0

1
8

0
 ±

 
0

.0
0

0
2

 

0
.4

9
8

6
 

R
2
 

0
.9

9
9

 

>
0

.9
9

5
0

 

0
.9

9
 

0
.9

9
8

6
 

0
.9

9
8

7
 

9
9

6
5

 

0
.9

9
5

5
 

0
.9

9
0

5
 

0
.9

9
7

5
 

0
.9

9
9

0
 

(P
la

sm
a

) 

0
.9

9
9

0
 

(U
ri

n
e

) 

0
.9

9
6

0
 

(P
la

sm
a

) 

0
.9

9
8

0
 

(U
ri

n
e

) 

0
.9

9
8

0
 

(P
la

sm
a

) 

0
.9

9
8

0
 

(U
ri

n
e

) 

0
.9

9
8

 

0
.9

9
8

 

R
e

fe
re

n
ce

 r
a

n
g

e
 

(n
g

/m
L)

 

n
.a

. 

5
0

0
0

-1
0

0
0

0
b
 

n
.a

. 

5
1

6
1

-1
0

3
2

3
f  

1
0

0
0

0
-1

6
0

0
0

c)
 

n
.a

. 

1
0

0
0

0
-3

5
0

0
0

e
)  

n
.a

. 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

 

5
0

0
0

-1
0

0
0

0
b
 

 

5
0

0
0

-1
0

0
0

0
b
 

 

1
5

0
0

0
-2

5
0

0
0

b
)  

5
0

0
0

-1
0

0
0

0
b
 

R
a

n
g

e
 (

n
g

/m
L)

 

7
8

-1
0

0
0

0
 

1
5

-6
0

a
)  

1
0

0
-1

0
0

0
0

0
 

1
0

0
-1

0
0

0
 

  

0
-2

0
0

0
a

)  

 

2
0

0
-2

0
0

0
0

 

0
-5

0
0

0
0

 

 

0
-5

0
0

0
0

 

 

0
-5

0
0

0
0

 

 

4
0

0
0

-
2

0
8

0
0

a
)  

5
0

0
-1

0
0

0
0

 

R
e

so
lu

ti
o

n
 

n
.i

. 

n
.i

. 

n
.i

. 

n
.i

.   

n
.i

.  

n
.i

. 

n
.i

.      

n
.i

. 

n
.i

. 

M
a

tr
ix

 

e
ff

e
ct

 

n
.i

. 

n
.i

. 

n
.i

. 

n
.i

.   

n
.i

.  

n
.i

. 

n
.i

.      

n
.i

. 

n
.i

. 

A
n

a
ly

te
 

G
e

n
e

ti
ci

n
 

G
e

n
ta

m
ic

in
 

Is
e

p
a

m
ic

in
 

N
e

o
m

y
ci

n
 

N
e

ti
lm

ic
in

 

S
is

o
m

ic
in

 

S
tr

e
p

to
m

y
ci

n
 

D
ih

y
d

ro
st

re
p

to
m

y
ci

n
 

G
e

n
ta

m
ic

in
 

G
e

n
ta

m
ic

in
 C

1
 

 

G
e

n
ta

m
ic

in
 C

1
a

 

 

G
e

n
ta

m
ic

in
 C

 2
 

 

A
m

ik
a

ci
n

 

G
e

n
ta

m
ic

in
 

R
e

f.
 

[2
9

] 

[6
1

] 

[6
2

] 

[6
3

] 

  

[3
0

] 

 

[1
6

] 

[6
4

] 

     

[6
5

] 

[6
6

] 

 

http://dx.doi.org/10.5599/admet.1183


Seth K. Amponsah et al.   ADMET & DMPK 10(1) (2022) 27-62 

50  

Table 2. Continued... 
In

te
rm

e
d

ia
te

 

p
re

ci
si

o
n

 (
%

) 

<
5

.2
 

4
.0

-6
.0

 

n
.i

.      

6
.6

-7
.6

 

1
.1

 

 

<
9

 

6
.0

-1
5

.7
 

4
.6

6
-8

.9
9

 

   

R
e

p
e

a
ta

b
il

it
y

 

(%
) 

<
2

.1
 

2
.7

-5
.8

 

0
.5

-9
.4

 

6
.1

-1
1

.4
 

0
.5

-1
2

.7
 

0
.3

-1
1

.6
 

0
.3

-1
0

.2
 

0
.4

-9
.9

 

0
.2

8
-7

.9
4

 

1
  

<
9

 

3
.7

-1
6

.0
 

n
.i

.    

LL
O

Q
 

(n
g

/m
L)

 

n
.i

. 

1
5

0
 

1
0

0
 

     

1
0

0
 

n
.i

.  

2
0

0
0

 

1
 

2
0

0
 

   

LO
D

 

(n
g

/m
L)

 

2
3

0
 

1
0

0
 

n
.i

.      

n
.i

. 

3
0

0
 

 

5
0

 

0
.6

 

n
.i

.    

R
e

co
v

e
ry

 (
%

) 

>
9

9
 

9
3

-1
0

5
 

1
0

0
 

9
4

.4
-

1
0

2
.3

 

9
2

.4
-

1
0

4
.8

 

9
4

.5
-

1
0

1
.7

 

9
4

.5
-

1
0

4
.1

 

9
6

.0
-

1
0

1
.6

 

1
0

0
 

8
5

.5
 

(P
la

sm
a

) 

9
0

.9
 

(U
ri

n
e

) 

>
9

2
 

6
1

 

6
6

.6
 

   

y
-I

n
te

rc
e

p
t 

-0
.0

2
1

5
 ±

 

0
.0

0
4

0
 

0
.0

0
5

 

n
.i

.      

n
.i

. 

n
.i

.  

n
.i

. 

0
.0

8
3

0
3

 

 

0
.0

0
0

7
3

2
 

0
.0

0
0

2
2

8
 

0
.0

0
0

5
4

 

S
lo

p
e

 

0
.1

0
2

7
 ±

 

0
.0

0
1

1
 

0
.2

1
 

n
.i

.      

n
.i

. 

n
.i

.  

n
.i

. 

0
.0

0
3

1
0

1
 

 

0
.0

3
7

6
 

0
.0

3
8

5
 

0
.0

4
0

3
 

R
2
 

0
.9

9
9

9
 

0
.9

9
8

6
 

>
0

.9
9

3
0

 

     

0
.9

9
8

5
 

>
0

.9
9

9
2

 

(P
la

sm
a

) 

>
0

.9
9

2
 

(U
ri

n
e

) 

≤
0

.9
9

7
7

 

0
.9

9
9

5
 

 

0
.9

9
6

4
 

0
.9

9
6

1
 

0
.9

9
8

2
 

R
e

fe
re

n
ce

 r
a

n
g

e
 

(n
g

/m
L)

 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

1
5

0
0

0
-2

5
0

0
0

b
)  

5
0

0
0

-1
0

0
0

0
b
 

1
5

0
0

0
-2

5
0

0
0

d
)  

5
1

6
1

-1
0

3
2

3
f  

n
.a

. 

5
0

0
0

-1
0

0
0

0
b
 

5
1

6
1

-1
0

3
2

3
f  

5
0

0
0

-1
0

0
0

0
b
 

 

1
5

0
0

0
-2

5
0

0
0

b
)  

n
.a

. 

5
1

6
1

-1
0

3
2

3
f  

   

R
a

n
g

e
 (

n
g

/m
L)

 

9
3

0
-9

3
4

0
a

)  

0
-5

0
0

0
0

 

1
0

0
-5

0
0

0
a

)  

     

1
0

0
-5

0
0

0
0

 

1
0

0
0

-3
8

0
0

0
 

 

1
5

0
-2

0
0

0
0

a
)  

0
-2

0
0

 

2
0

0
-5

0
0

0
0

 

   

R
e

so
lu

ti
o

n
 

n
.i

. 

n
.i

. 

n
.i

.      

n
.i

. 

n
.i

.  

n
.i

. 

n
.i

. 

n
.i

.    

M
a

tr
ix

 

e
ff

e
ct

 

n
.i

. 

n
.i

. 

n
.i

.      

n
.i

. 

n
.i

.  

n
.i

. 

n
.i

. 

n
.i

.    

A
n

a
ly

te
 

T
o

b
ra

m
y

ci
n

 

T
o

b
ra

m
y

ci
n

 

A
m

ik
a

ci
n

 

G
e

n
ta

m
ic

in
 

K
a

n
a

m
y

ci
n

 

N
e

o
m

y
ci

n
 

P
a

ro
m

o
m

yc
in

 

T
o

b
ra

m
y

ci
n

 

N
e

o
m

y
ci

n
 

T
o

b
ra

m
y

ci
n

 

 

A
m

ik
a

ci
n

 

D
ih

y
d

ro
st

re
p

to
m

y
ci

n
 

N
e

o
m

y
ci

n
 

(N
e

o
 1

8
0

4
 A

7
) 

(N
e

o
 1

8
0

4
 A

8
) 

(N
e

o
 1

8
0

4
 A

9
) 

R
e

f.
 

[6
7

] 

[6
8

] 

[3
1

] 

     

[6
9

] 

[7
0

] 

 

[7
1

] 

[7
2

] 

[7
3

] 

   



ADMET & DMPK 10(1) (2022) 27-62 Bioanalysis of aminoglycozides by HPLC 

doi: http://dx.doi.org/10.5599/admet.1183 51 

Table 2. Continued... 
In

te
rm

e
d

ia
te

 

p
re

ci
si

o
n

 (
%

) 

 

n
.i

. 

8
.3

-1
1

.1
 

  

n
.i

. 

1
.7

-1
3

.0
 






 

5
-1

0
 

7
-8

 

6
-9

 

1
3

-1
7

 

6
-1

0
 

1
2

-1
7

 

1
0

-1
6

 

8
-1

2
 

1
0

-1
4

 

R
e

p
e

a
ta

b
il

it
y

 

(%
)  

n
.i

. 

5
.6

-1
1

.4
 

  

0
.9

 

7
.5

-1
0

.8
 






 

3
-6

 

4
-8

 

5
-9

 

8
-1

1
 

4
-8

 

7
-1

4
 

1
1

-1
4

 

6
-9

 

7
-1

3
 

LL
O

Q
 

(n
g

/m
L)

 

 

1
0

0
 

5
0

 

5
0

0
 

3
5

0
 

6
0

 

5
0

0
 

2
5

0
 

n
.i

.         

LO
D

 

(n
g

/m
L)

 

 

5
0

 

n
.i

. 

2
6

0
 

1
0

0
 

n
.i

. 

n
.i

. 

7
0

 

n
.i

.         

R
e

co
v

e
ry

 

(%
)  

9
5

.1
5

 

9
3

-1
0

5
 

1
0

0
.3

2
 

9
9

.9
8

 

≤
1

0
0

 

3
6

.1
±

 6
.1

 

>
9

9
 

7
6

-9
7

 

7
2

-1
1

3
 

8
1

-1
0

7
 

6
9

-9
7

 

7
8

-1
0

3
 

7
0

-9
4

 

6
2

-8
9

 

6
7

-9
2

 

7
1

-9
8

 

y
-I

n
te

rc
e

p
t 

0
.0

0
0

6
4

9
 

n
.i

. 

-0
.7

7
7

4
 

  

3
0

3
 

-0
.0

6
4

 

0
.0

0
1

2
 

±
0

.0
8

7
3

 

n
.i

.         

S
lo

p
e

 

0
.0

3
0

3
 

n
.i

. 

7
.1

8
6

-

8
.1

1
2

6
 

  

3
5

7
 

0
.0

8
 

0
.0

6
7

7
 

±
0

.0
1

1
8

 

n
.i

.         

R
2
 

0
.9

9
5

9
 

0
.9

9
1

2
 

0
.9

6
4

0

-0
.9

9
0

 

0
.9

9
9

8
 

0
.9

9
9

4
 

0
.9

9
 

1
 

0
.9

9
8

9
 

0
.9

9
9

8
 

0
.9

9
9

4
 

0
.9

9
9

 

0
.9

9
1

6
 

0
.9

9
8

5
 

0
.9

9
8

2
 

0
.9

9
8

1
 

0
.9

9
5

3
 

0
.9

9
0

6
 

R
e

fe
re

n
ce

 r
a

n
g

e
 

(n
g

/m
L)

 

 

1
5

0
0

0
-2

5
0

0
0

b
)  

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

5
1

6
1

-1
0

3
2

3
f  

1
5

0
0

0
-2

5
0

0
0

b
)  

n
.a

. 

5
0

0
0

-1
0

0
0

0
b
 

5
1

6
1

-1
0

3
2

3
f  

1
0

0
0

0
-3

5
0

0
0

e
)  

n
.a

. 

1
5

0
0

0
-2

5
0

0
0

b
)  

1
5

0
0

0
-2

5
0

0
0

d
)  

n
.a

. 

5
0

0
0

-1
0

0
0

0
b
 

1
4

4
0

0
0

-

2
1

0
0

0
0

h
)  

n
.a

. 

R
a

n
g

e
 (

n
g

/m
L)

 

 

5
0

-1
0

0
0

0
a

)  

5
0

-1
0

0
0

a
)  

8
0

0
-4

0
0

0
a

)  

5
0

0
-1

0
0

0
0

a
)  

6
0

-4
0

0
0

a
)  

5
0

0
-5

0
0

0
0

 

2
5

0
-2

0
0

0
0

 

n
.i

.         

R
e

so
lu

ti
o

n
 

 

n
.i

. 

n
.i

. 

1
.7

 

1
.4

 

n
.i

. 

n
.i

. 

n
.i

. 

n
.i

.         

M
a

tr
ix

 

e
ff

e
ct

 

 

n
.i

. 

n
.i

.   

n
.i

. 

n
.i

. 

n
.i

. 

n
.i

.         

A
n

a
ly

te
 

(N
e

o
 1

8
0

4
 B

4
) 

A
m

ik
a

ci
n

 

T
o

b
ra

m
y

ci
n

 

G
e

n
ta

m
ic

in
 

N
e

o
m

y
ci

n
 

A
m

ik
a

ci
n

 

Is
e

p
a

m
ic

in
 

T
o

b
ra

m
y

ci
n

 

N
e

o
m

y
ci

n
 

S
tr

e
p

to
m

y
ci

n
 

D
ih

y
d

ro
st

re
p

to
m

y
ci

n
 

A
m

ik
a

ci
n

 

K
a

n
a

m
y

ci
n

 

P
a

ro
n

o
m

y
ci

n
 

T
o

b
ra

m
y

ci
n

 

S
p

e
ct

in
o

m
y

ci
n

 

A
p

ra
m

y
ci

n
 

R
e

f.
 

 

[7
4

] 

[7
5

] 

  

[1
5

] 

[7
6

] 

[7
7

] 

[5
]         

http://dx.doi.org/10.5599/admet.1183


Seth K. Amponsah et al.   ADMET & DMPK 10(1) (2022) 27-62 

52  

Table 2. Continued... 
In

te
rm

e
d

ia
te

 

p
re

ci
si

o
n

 (
%

) 

9
-1

4
 

7
-1

0
 

8
-1

1
 

7
-1

0
 

<
5

.0
 

<
1

2
.1

0
 

 

<
3

.0
 

<
3

.0
 

<
3

.0
 

n
.i

. 

0
.3

3
1

-0
.7

8
4

 

1
.8

-1
2

.5
 

2
.0

-1
1

.0
 

2
.6

-1
0

.8
 

2
.8

-9
.3

 

0
.9

-8
.3

 

R
e

p
e

a
ta

b
il

it
y

 

(%
) 

9
-1

2
 

7
-9

 

4
-1

0
 

5
-8

 

<
5

.0
 

<
1

1
.5

6
 

 

<
4

.8
 

<
4

.8
 

<
4

.8
 

n
.i

. 

0
.5

7
6

-

0
.8

0
0

 

2
.0

-2
.8

 

1
.4

-3
.2

 

1
.6

-8
.5

 

1
.4

-4
.6

 

3
.2

-6
.9

 

LL
O

Q
 

(n
g

/m
L)

 

    

4
5

0
 

2
3

4
0

 

6
3

0
 

n
.i

.   

1
0

0
 

1
6

.3
 

n
.i

.    

5
0

 

LO
D

 

(n
g

/m
L)

 

    

1
0

0
 

5
9

0
 

3
2

0
 

1
8

 

2
1

 

5
5

 

n
.i

. 

5
.3

4
 

n
.i

.    

2
0

 

R
e

co
v

e
ry

 

(%
) 

7
8

-9
8

 

8
2

 1
0

7
 

7
6

-1
1

4
 

7
0

-1
0

5
 

9
9

.2
0

-

1
0

3
.1

7
 

8
5

.2
 

8
3

.6
 

9
9

.2
-

1
0

0
.3

 

9
9

.4
-

1
0

1
.2

 

9
9

.6
-

1
0

2
.4

 

9
1

.8
-

1
0

3
.6

 

9
8

.3
3

-

1
0

1
.7

4
 

1
0

5
.5

 

1
0

2
.9

 

1
0

0
 

1
0

5
.4

 

8
8

 

y
-I

n
te

rc
e

p
t 

    

-0
.2

8
1

5
 

n
.i

.  

1
.3

0
2

 

3
.0

2
6

 

1
.0

9
4

 

0
.0

0
7

5
1

 

1
.0

6
5

2
 

n
.i

.    

n
.i

. 

S
lo

p
e

 

    

1
.1

8
9

6
 

n
.i

.  

1
5

8
4

.0
6

 

2
1

8
5

.5
5

 

2
1

4
5

.9
2

 

0
.9

7
3

 

0
.0

2
4

7
 

±
 0

.0
0

3
 

n
.i

.    

n
.i

. 

R
2
 

0
.9

9
1

8
 

0
.9

9
9

5
 

0
.9

9
9

1
 

0
.9

9
6

7
 

0
.9

9
9

7
 

0
.9

9
8

 

0
.9

9
8

 

0
.9

9
9

8
 

0
.9

9
9

7
 

0
.9

9
9

7
 

n
.i

. 

0
.9

9
9

 

0
.9

9
9

9
 

0
.9

9
8

8
 

0
.9

9
9

2
 

0
.9

9
9

4
 

<
0

.9
9

9
0

 

R
e

fe
re

n
ce

 r
a

n
g

e
 

(n
g

/m
L)

 

n
.a

. 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

n
.a

. 

1
5

0
0

0
-2

5
0

0
0

b
)  

5
0

0
0

-1
0

0
0

0
b
 

1
5

0
0

0
-2

5
0

0
0

b
)  

1
0

0
0

0
-1

6
0

0
0

c)
 

n
.a

. 

1
5

0
0

0
-2

0
0

0
0

g
)  

5
0

0
0

-1
0

0
0

0
b
 

1
5

0
0

0
-2

5
0

0
0

b
)  

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

1
4

4
0

0
0

-

2
1

0
0

0
0

h
)  

R
a

n
g

e
 (

n
g

/m
L)

 

    

6
2

5
-1

5
0

0
0

 

n
.i

.  

2
5

-8
0

0
0

 

3
0

-7
4

0
0

0
 

5
0

-6
3

0
0

0
 

1
0

0
-4

5
9

0
0

 

2
0

-2
0

0
a

)  

3
0

0
-5

0
0

0
a

)  

1
0

0
0

-

1
0

0
0

0
0

 

1
0

0
0

-

1
0

0
0

0
0

 

1
0

0
0

-

1
0

0
0

0
0

 

5
-5

0
0

a
)  

R
e

so
lu

ti
o

n
 

    

n
.i

. 

n
.i

.  

n
.i

.   

n
.i

. 

n
.i

. 

n
.i

.    

n
.i

. 

M
a

tr
ix

 

e
ff

e
ct

 

    

n
.i

. 

n
.i

.  

n
.i

.   _
 

n
.i

. 

n
.i

.    

n
.i

. 

A
n

a
ly

te
 

H
y

g
ro

m
y

ci
n

 

G
e

n
ta

m
ic

in
 (

C
1

) 

G
e

n
ta

m
ic

in
 (

C
1

a
) 

G
e

n
ta

m
ic

in
 (

C
2

) 

Is
e

p
a

m
ic

in
 

A
m

ik
a

ci
n

 

G
e

n
ta

m
ic

in
 

A
m

ik
a

ci
n

 

N
e

ti
lm

ic
in

 

E
ti

m
ic

in
 

A
rb

e
k

a
ci

n
 

T
o

b
ra

m
y

ci
n

 

A
m

ik
a

ci
n

 

G
e

n
ta

m
ic

in
 C

1
 

G
e

n
ta

m
ic

in
 C

1
a

 

G
e

n
ta

m
ic

in
 C

2
 

S
p

e
ct

in
o

m
y

ci
n

 

R
e

f.
 

    

[7
8

] 

[7
9

] 

 

[3
2

] 

  

[3
3

] 

[8
0

] 

[8
1

] 

   

[3
4

] 

 



ADMET & DMPK 10(1) (2022) 27-62 Bioanalysis of aminoglycozides by HPLC 

doi: http://dx.doi.org/10.5599/admet.1183 53 

Table 2. Continued... 
In

te
rm

e
d

ia
te

 

p
re

ci
si

o
n

 (
%

) 

4
.3

-7
.5

 

1
.4

-1
1

.1
 

1
.2

-6
.7

 

1
.6

-6
.2

 

1
.8

-4
.7

 

1
.3

-6
.2

 

1
.4

-6
.5

 

4
.3

-5
.8

 

4
.7

-8
.3

 

2
.6

4
-5

.8
0

 

7
.0

-1
0

.4
 

3
.8

-5
.6

 

3
.8

-4
.9

 

≤
2

.3
 

R
2
, 

co
e

ff
ic

ie
n

t 
o

f 
co

rr
e

la
ti

o
n

; 
LO

D
, 

lim
it

 o
f 

d
e

te
ct

io
n

; 
LL

O
Q

, 
lo

w
e

r 
lim

it
 o

f 
q

u
a

n
ti

fi
ca

ti
o

n
; 

n
.i

.,
 n

o
t 

in
d

ic
a

te
d

; 
n

.a
.,

 n
o

t 
a

v
a

ila
b

le
; 

a
) c

a
lib

ra
ti

o
n

 r
a

n
g

e
 d

o
e

s 
n

o
t 

co
ve

r 
th

e
 t

h
e

ra
p

e
u

ti
c 

re
fe

re
n

ce
 r

a
n

g
e

; 
b

) s
o

u
rc

e
, 

[1
1

2
];

 
c)

S
o

u
rc

e
, 

[1
1

3
];

 d
) s

o
u

rc
e

, 
[1

1
4

];
 e

) s
o

u
rc

e
, 

[1
1

5
];

 f)
so

u
rc

e
, 

[1
1

6
] 

; 
g

) s
o

u
rc

e
, 

[1
1

7
];

 h
) s

o
u

rc
e

, 
[1

1
8

].
 

 

R
e

p
e

a
ta

b
il

it
y

 

(%
) 

4
.1

-9
.9

 

6
.0

-6
.6

 

4
.0

-5
.0

 

4
.8

-5
.7

 

3
.7

-6
.8

 

4
.3

-5
.1

 

4
.2

-6
.1

 

3
.8

-7
.6

 

2
.7

-6
.0

 

2
.1

2
-5

.0
7

 

5
.5

0
-1

1
.9

 

4
.7

.2
0

0
6

 

3
.6

-6
.6

 

≤
4

.3
 

LL
O

Q
 

(n
g

/m
L)

 

1
2

5
 

2
5

 

3
7

.5
 

1
2

5
 

1
2

5
 

5
0

 

5
0

 

1
2

5
 

1
2

5
 

n
.i

. 

1
2

5
0

 

5
0

0
 

5
0

0
 

5
 

LO
D

 

(n
g

/m
L)

 

5
0

 

1
5

 

1
5

 

5
0

 

5
0

 

3
0

 

2
0

 

5
0

 

5
0

 

5
0

 

6
2

5
 

n
.i

. 

n
.i

. 

n
.i

. 

R
e

co
v

e
ry

 

(%
) 

9
5

 

8
7

 

9
2

 

8
7

 

9
4

 

8
9

 

9
1

 

9
1

 

9
3

 

8
8

.0
2

-

1
0

2
.5

6
 

9
8

.9
-

1
1

3
.7

 

8
3

.1
-8

9
.7

 

8
2

.7
 

1
0

0
 

y
-I

n
te

rc
e

p
t 

         

1
8

9
7

5
2

8
 

n
.i

. 

n
.i

. 

n
.i

. 

n
.i

. 

S
lo

p
e

 

         

1
0

9
7

9
3

1
 

n
.i

. 

n
.i

. 

n
.i

. 

n
.i

. 

R
2
 

         1
 

0
.9

9
0

9
 

n
/i

 

0
.9

9
 

0
.9

9
7

 

R
e

fe
re

n
ce

 r
a

n
g

e
 

(n
g

/m
L)

 

5
0

0
0

-1
0

0
0

0
b
 

5
0

0
0

-1
0

0
0

0
b
 

1
5

0
0

0
-2

5
0

0
0

d
)  

n
.a

. 

n
.a

. 

1
0

0
0

0
-3

5
0

0
0

e
)  

n
.a

. 

1
5

0
0

0
-2

5
0

0
0

b
)  

5
1

6
1

-1
0

3
2

3
f  

1
5

0
0

0
-2

5
0

0
0

b
)  

5
1

6
1

-1
0

3
2

3
f  

1
5

0
0

0
-2

5
0

0
0

b
)  

1
5

0
0

0
-2

5
0

0
0

b
)  

n
.a

. 

R
a

n
g

e
 (

n
g

/m
L)

 

         

5
0

0
-1

0
0

0
0

a
)  

1
2

5
0

-

2
0

0
0

0
0

 

5
0

0
-1

0
0

0
0

0
 

5
0

0
-1

0
0

0
0

0
 

5
-1

0
0

0
 

R
e

so
lu

ti
o

n
 

         

n
.i

. 

n
.i

. 

n
.i

. 

n
.i

. 

n
.i

. 

M
a

tr
ix

 

e
ff

e
ct

 

         

n
.i

. 

-0
.1

0
 –

 

1
.3

3
 

-1
.1

 

-7
.6

 -
 

 -
8

.8
 

≤
1

2
 

A
n

a
ly

te
 

T
o

b
ra

m
y

ci
n

 

G
e

n
ta

m
ic

in
 

K
a

n
a

m
y

ci
n

 

H
y

g
ro

m
y

ci
n

 

A
p

ra
m

y
ci

n
 

S
tr

e
p

to
m

y
ci

n
 

D
ih

y
d

ro
st

re
p

to
m

y
ci

n
 

A
m

ik
a

ci
n

 

N
e

o
m

y
ci

n
 

A
m

ik
a

ci
n

 

N
e

o
m

y
ci

n
 

A
m

ik
a

ci
n

 

A
m

ik
a

ci
n

 

P
a

ro
m

o
m

yc
in

 

R
e

f.
 

         

[8
2

] 

[8
3

] 

[8
4

] 

[8
5

] 

[3
5

] 

http://dx.doi.org/10.5599/admet.1183


Seth K. Amponsah et al.   ADMET & DMPK 10(1) (2022) 27-62 

54  

Conclusion and outlook 

Despite reported nephrotoxic and ototoxic potentials of aminoglycosides, their use in clinical settings 

remains relevant. The current study sought to review bioanalytical methods (specifically liquid 

chromatography) used in the assay of aminoglycosides in biological matrices. In all, 71 articles were 

reviewed, and 66 of these articles reported the use of reversed-phase liquid chromatography as a 

bioanalytical method [7,119].  

The commonest sample treatment procedures adopted in the analysis of aminoglycosides using HPLC 

were protein precipitation (50 %) and solid phase extraction (39 %). Surprisingly, none of the current 

sample preparation methods was used by any of the articles reported in this review. It will be interesting if 

recent sample preparation methods such as solid-phase microextraction, micro-solid-phase extraction, 

dispersive micro-solid-phase extraction, magnetic solid-phase extraction, microextraction by packed 

sorbent, stir bar sorptive extraction, spin column extraction, liquid-phase microextraction, single-drop 

microextraction, hollow fiber liquid-phase microextraction, dispersive liquid-liquid microextraction, 

molecularly imprinted solid-phase extraction, and molecularly imprinted solid-phase micro-extraction [120-

122] could be applied for bioanalysis of aminoglycosides with the potential of improving method sensitivity 

and selectivity. 

Fluorescence (50 %), UV (24 %), and mass spectrometry (21 %) were the most adopted mode of 

detection in the assay of aminoglycosides, according to this review. Since mass spectrometry has been 

established as the detection mode of choice for bioanalysis of aminoglycoside using liquid chromatography 

in recent years, it is strongly recommended for use except in resource-challenged countries where 

fluorescence or UV detection methods can be applied after derivatization.  

There is the need to establish a therapeutic reference range for all the clinically reported 20 

aminoglycosides since the calibration range of analytical methods for the bioanalysis of aminoglycosides 

should cover such a range. It was quite surprising that some of the calibration range was outside the 

established therapeutic reference range. It is recommended that future liquid chromatography methods for 

the analysis of aminoglycosides should have calibration ranges covering established reference therapeutic 

ranges. 

Although this review is not a systematic one, the information provided is intended to serve as a 

comprehensive reference for related research that may involve the assay of aminoglycosides 

(pharmacokinetic or drug monitoring studies). 

Conflict of interest: The authors declare no conflict of interest. 

References 

[1] R. Hari, S. Taherunnisa, S.Y. Raut, S. Mutalik, K.B. Koteshwara. Challenges in the development of 
analytical test procedure for aminoglycosides: A critical review. J. Appl. Pharm. Sci 9 (2019) 145-152. 
https://doi.org/10.7324/JAPS.2019.91118. 

[2] K. Chan, W. Wang, K.R. Ledesma, T. Yin, V.H. Tam. A robust LC–MS/MS method for amikacin: 
application to cellular uptake and pharmacokinetic studies. Bioanalysis 12 (2020) 445-454. 
https://doi.org/10.4155/bio-2020-0007. 

[3] M.-P. Mingeot-Leclercq, Y. Glupczynski, P.M. Tulkens. Aminoglycosides: activity and resistance. 
Antimicrobial agents chemotherapy 43 (1999) 727-737. https://doi.org/10.1128/AAC.43.4.727. 

[4] L. Šoltés. Aminoglycoside antibiotics-two decades of their HPLC bioanalysis. Biomedical 
Chromatography 13 (1999) 3-10. https://doi.org/10.1002/(SICI)1099-0801(199902)13:1<3::AID-
BMC811>3.0.CO;2-T. 

https://doi.org/10.7324/JAPS.2019.91118
https://doi.org/10.4155/bio-2020-0007
https://doi.org/10.1128/AAC.43.4.727
https://doi.org/10.1002/(SICI)1099-0801(199902)13:1%3c3::AID-BMC811%3e3.0.CO;2-T
https://doi.org/10.1002/(SICI)1099-0801(199902)13:1%3c3::AID-BMC811%3e3.0.CO;2-T


ADMET & DMPK 10(1) (2022) 27-62 Bioanalysis of aminoglycozides by HPLC 

doi: http://dx.doi.org/10.5599/admet.1183 55 

[5] W.-x. Zhu, J.-z. Yang, W. Wei, Y.-f. Liu, S.-s. Zhang. Simultaneous determination of 13 aminoglycoside 
residues in foods of animal origin by liquid chromatography–electrospray ionization tandem mass 
spectrometry with two consecutive solid-phase extraction steps. Journal of Chromatography A 1207 
(2008) 29-37. https://doi.org/10.1016/j.chroma.2008.08.033. 

[6] S. Dionisotti, F. Bamonte, M. Gamba, E. Ongini. High-performance liquid chromatographic 
determination of netilmicin in guinea-pig and human serum by fluorodinitrobenzene derivatization 
with spectrophotometric detection. Journal of Chromatography B: Biomedical Sciences 434 (1988) 
169-176. https://doi.org/10.1016/0378-4347(88)80071-9. 

[7] A. Cabanes, Y. Cajal, I. Haro, J.G. Anton, F. Reig, M. Arboix. Gentamicin determination in biological 
fluids by HPLC, using tobramycin as internal standard. Journal of liquid chromatography 14 (1991) 
1989-2010. https://doi.org/10.1080/01483919108049669. 

[8] S.K. Amponsah, G.O. Adjei, C. Enweronu-Laryea, K.A. Bugyei, K. Hadji-Popovski, J.A.L. Kurtzhals, K. 
Kristensen. Population Pharmacokinetic Characteristics of Amikacin in Suspected Cases of Neonatal 
Sepsis in a Low-Resource African Setting: A Prospective Nonrandomized Single-Site Study. Curr Ther 
Res Clin Exp 84 (2017) e1-e6. https://doi.org/10.1016/j.curtheres.2017.01.001. 

[9] S.K. Amponsah, K.F. Opuni, K.A. Antwi, V.P. Kunkpeh. Effect of aminophylline on the 
pharmacokinetics of amikacin in Sprague-Dawley rats. J Infect Dev Ctries 13 (2019) 251-254. 
https://doi.org/10.3855/jidc.10514. 

[10] S.K. Amponsah, K.F.M. Opuni, A.A. Donkor. Animal model investigation suggests betamethasone 
alters the pharmacokinetics of amikacin. ADMET and DMPK 6 (2018) 279-283. http://dx.doi.org/-
10.5599/admet.613. 

[11] S.K. Banerjee, A. Wells, A. Dasgupta. Operator error: a critical determinant of false amikacin and 
tobramycin concentrations using fluorescence polarization immunoassay kits and TDX analyzer. 
Therapeutic drug monitoring 21 (1999) 540. https://doi.org/10.1097/00007691-199910000-00009. 

[12] S.K. Maitra, T.T. Yoshikawa, C.M. Steyn, L.B. Guze, M.C. Schotz. Amikacin assay in serum by high-
performance liquid chromatography. Antimicrobial Agents Chemotherapy 14 (1978) 880-885. 
https://doi.org/10.1128/AAC.14.6.880. 

[13] A. Marzo, L. Dal Bo. Chromatography as an analytical tool for selected antibiotic classes: a reappraisal 
addressed to pharmacokinetic applications. Journal of Chromatography A 812 (1998) 17-34. 
https://doi.org/10.1016/s0021-9673(98)00282-9. 

[14] V.K. Agarwal. High performance liquid chromatographic determination of neomycin in milk using a 
HISEP column. Journal of liquid chromatography 13 (1990) 2475-2487. https://doi.org/10.1080/-
01483919008049047. 

[15] G. Brajanoski, J. Hoogmartens, K. Allegaert, E. Adams. Determination of amikacin in cerebrospinal 
fluid by high-performance liquid chromatography with pulsed electrochemical detection. J 
Chromatogr B Analyt Technol Biomed Life Sci 867 (2008) 149-152. https://doi.org/10.1016/j.jchromb-
.2008.03.016. 

[16] A. Yusuf, S. Al-Rawithi, D. Raines, H. Frayha, A. Toonsi, I. Al-Mohsen, A. El-Yazigi. Simplified high-
performance liquid chromatographic method for the determination of gentamicin sulfate in a 
microsample of plasma: Comparison with fluorescence polarization immunoassay. Therapeutic drug 
monitoring 21 (1999) 647. https://doi.org/10.1097/00007691-199912000-00012. 

[17] Y. Al-Shamalii, Y.M. Ali, R.A. Al-Shamalii, M. Al-Melahi, F.R. Al-Shammari, A. Alsaber, W. Al-Adsani. 
Don't close the book on tocilizumab for the treatment of severe COVID-19 pneumonia- The jury is still 
out- The Kuwait experience. PLoS ONE 16 (2021). https://doi.org/10.1371/journal.pone.0254379.  

[18] R. Hari, S. Taherunnisa, S.Y. Raut, S. Mutalik, K.B. Koteshwara. Challenges in the development of 
analytical test procedure for aminoglycosides: A critical review. J. Appl. Pharm. Sci 9 (2019) 145-152. 
https://doi.org/10.7324/JAPS.2019.91118. 

[19] X. Zhang, J. Wang, Q. Wu, L. Li, Y. Wang, H. Yang. Determination of Kanamycin by High Performance 
Liquid Chromatography. Molecules 24 (2019). https://doi.org/10.3390/molecules24101902. 

http://dx.doi.org/10.5599/admet.1183
https://doi.org/10.1016/j.chroma.2008.08.033
https://doi.org/10.1016/0378-4347(88)80071-9
https://doi.org/10.1080/01483919108049669
https://doi.org/10.1016/j.curtheres.2017.01.001
https://doi.org/10.3855/jidc.10514
http://dx.doi.org/10.5599/admet.613
http://dx.doi.org/10.5599/admet.613
https://doi.org/10.1097/00007691-199910000-00009
https://doi.org/10.1128/AAC.14.6.880
https://doi.org/10.1016/s0021-9673(98)00282-9
https://doi.org/10.1080/01483919008049047
https://doi.org/10.1080/01483919008049047
https://doi.org/10.1016/j.jchromb.2008.03.016
https://doi.org/10.1016/j.jchromb.2008.03.016
https://doi.org/10.1097/00007691-199912000-00012
https://doi.org/10.1371/journal.pone.0254379
https://doi.org/10.7324/JAPS.2019.91118
https://doi.org/10.3390/molecules24101902


Seth K. Amponsah et al.   ADMET & DMPK 10(1) (2022) 27-62 

56  

[20] J.P. Anhalt. Assay of gentamicin in serum by high-pressure liquid chromatography. Antimicrobial 
Agents Chemotherapy 11 (1977) 651-655. https://doi.org/10.1128/AAC.11.4.651. 

[21] G.W. Peng, G.G. Jackson, W.L. Chiou. High-pressure liquid chromatographic assay of netilmicin in 
plasma. Antimicrobial Agents Chemotherapy 12 (1977) 707-709. https://doi.org/10.1128/AAC.12.6.-
707. 

[22] J.P. Anhalt, S.D. Brown. High-performance liquid-chromatographic assay of aminoglycoside 
antibiotics in serum. Clinical chemistry 24 (1978) 1940-1947. https://doi.org/10.1093/clinchem/24.-
11.1940. 

[23] S.-E. Bäck, I. Nilsson-Ehle, P. Nilsson-Ehle. Chemical assay, involving liquid chromatography, for 
aminoglycoside antibiotics in serum. Clinical chemistry 25 (1979) 1222-1225. https://doi.org/10.-
1093/clinchem/25.7.1222. 

[24] L. Essers. An automated high-performance liquid chromatographic method for the determination of 
aminoglycosides in serum using pre-column sample cleanup and derivatization. Journal of 
Chromatography B: Biomedical Sciences 305 (1984) 345-352. https://doi.org/10.1016/s0378-
4347(00)83348-4.  

[25] T. Kawamoto, I. Mashimo, S. Yamauchi, M. Watanabe. Determination of sisomicin, netilmicin, 
astromicin and micronomicin in serum by high-performance liquid chromatography. Journal of 
Chromatography B: Biomedical Sciences 305 (1984) 373-379. https://doi.org/10.1016/s0378-

4347(00)83351-4.  

[26] H. Kubo, Y. Kobayashi, T. Nishikawa. Rapid method for determination of kanamycin and dibekacin in 
serum by use of high-pressure liquid chromatography. Antimicrobial Agents Chemotherapy 28 (1985) 
521-523. https://doi.org/10.1128/aac.28.4.521. 

[27] N. Kurosawa, S. Kuribayashi, E. Owada, K. Ito, M. Nioka, M. Arakawa, R.J.J.o.C.B.B.S. Fukuda, 
Applications. Determination of streptomycin in serum by high-performance liquid chromatography. 
Journal of Chromatography B: Biomedical Sciences 343 (1985) 379-385. https://doi.org/10.1016/-
s0378-4347(00)84606-x. 

[28] J.A. Maloney, W.M. Awni. High-performance liquid chromatographic determination of isepamicin in 
plasma, urine and dialysate. Journal of Chromatography B: Biomedical Sciences 526 (1990) 487-496. 
https://doi.org/10.1016/s0378-4347(00)82530-x. 

[29] C. Bethune, T. Bui, M.L. Liu, M.A. Kay, R.J. Ho. Development of a high-performance liquid 
chromatographic assay for G418 sulfate (Geneticin). Antimicrob Agents Chemother 41 (1997) 661-
664. https://doi.org/10.1128/AAC.41.3.661. 

[30] G. Suhren, K. Knappstein. Detection of incurred dihydrostreptomycin residues in milk by liquid 
chromatography and preliminary confirmation methods. Analyst 123 (1998) 2797-2801. 
https://doi.org/10.1039/a805050c. 

[31] R. Oertel, V. Neumeister, W. Kirch. Hydrophilic interaction chromatography combined with tandem-
mass spectrometry to determine six aminoglycosides in serum. Journal of Chromatography A 1058 
(2004) 197-201. https://doi.org/10.1016/j.chroma.2004.08.158. 

[32] L. Zhang, J. Peng, J. Tang, B. Yuan, R. He, Y. Xiao. Description and validation of coupling high 
performance liquid chromatography with resonance Rayleigh scattering in aminoglycosides 
determination. Analytica chimica acta 706 (2011) 199-204. https://doi.org/10.1016/j.aca.-
2011.09.025. 

[33] A.R. Breaud, C.L. Henemyre-Harris, S. Schools, N. Emezienna, W. Clarke. Rapid quantification of the 
aminoglycoside arbekacin in serum using high performance liquid chromatography–tandem mass 
spectrometry. J Clinica Chimica Acta 418 (2013) 102-106. https://doi.org/10.1016/j.cca.2013.01.004. 

[34] J.B. Arsand, L. Jank, M.T. Martins, R.B. Hoff, F. Barreto, T.M. Pizzolato, C. Sirtori. Determination of 
aminoglycoside residues in milk and muscle based on a simple and fast extraction procedure 
followed by liquid chromatography coupled to tandem mass spectrometry and time of flight mass 
spectrometry. Talanta 154 (2016) 38-45. https://doi.org/10.1016/j.talanta.2016.03.045. 

https://doi.org/10.1128/AAC.11.4.651
https://doi.org/10.1128/AAC.12.6.707
https://doi.org/10.1128/AAC.12.6.707
https://doi.org/10.1093/clinchem/24.11.1940
https://doi.org/10.1093/clinchem/24.11.1940
https://doi.org/10.1093/clinchem/25.7.1222
https://doi.org/10.1093/clinchem/25.7.1222
https://doi.org/10.1016/s0378-4347(00)83348-4
https://doi.org/10.1016/s0378-4347(00)83348-4
https://doi.org/10.1016/s0378-4347(00)83351-4
https://doi.org/10.1016/s0378-4347(00)83351-4
https://doi.org/10.1128/aac.28.4.521
https://doi.org/10.1016/s0378-4347(00)84606-x
https://doi.org/10.1016/s0378-4347(00)84606-x
https://doi.org/10.1016/s0378-4347(00)82530-x
https://doi.org/10.1128/AAC.41.3.661
https://doi.org/10.1039/a805050c
https://doi.org/10.1016/j.chroma.2004.08.158
https://doi.org/10.1016/j.aca.2011.09.025
https://doi.org/10.1016/j.aca.2011.09.025
https://doi.org/10.1016/j.cca.2013.01.004
https://doi.org/10.1016/j.talanta.2016.03.045


ADMET & DMPK 10(1) (2022) 27-62 Bioanalysis of aminoglycozides by HPLC 

doi: http://dx.doi.org/10.5599/admet.1183 57 

[35] I.C. Roseboom, B. Thijssen, H. Rosing, J. Mbui, J.H. Beijnen, T.P. Dorlo. Highly sensitive UPLC-MS/MS 
method for the quantification of paromomycin in human plasma. Journal of pharmaceutical 
biomedical analysis 185 (2020) 113245. https://doi.org/10.1016/j.jpba.2020.113245. 

[36] S.K. Maitra, T.T. Yoshikawa, J.-L. Hansen, I. Nilsson-Ehle, W. Palin, M.-C. Schotz, L. Guze. Serum 
gentamicin assay by high-performance liquid chromatography. J Clinical chemistry 23 (1977) 2275-
2278. https://doi.org/10.1093/clinchem/23.12.2275. 

[37] D.B. Haughey, D.M. Janicke, M. Adelman, J.J. Schentag. High-pressure liquid chromatography analysis 
and single-dose disposition of tobramycin in human volunteers. Antimicrob Agents Chemother 17 
(1980) 649-653. https://doi.org/10.1128/aac.17.4.649. 

[38] D.M. Barends, J.S. van der Sandt, A. Hulshoff. Micro determination of gentamicin in serum by high-
performance liquid chromatography with ultraviolet detection. J Chromatogr 182 (1980) 201-210. 
https://doi.org/10.1016/s0378-4347(00)81624-2. 

[39] N.-E. Larsen, K. Marinelli, A.M. Heilesen. Determination of gentamicin in serum using liquid column 
chromatography. Journal of Chromatography B: Biomedical Sciences 221 (1980) 182-187.  

https://doi.org/10.1016/S0378-4347(00)81023-3.  

[40] S. Walker, P. Coates, Applications. High-performance liquid chromatographic method for 
determination of gentamicin in biological fluids. Journal of Chromatography B: Biomedical Sciences 
223 (1981) 131-138. https://doi.org/10.1016/s0378-4347(00)80075-4. 

[41] L.T. Wong, A.R. Beaubien, A.P. Pakuts, Applications. Determination of amikacin in microlitre 
quantities of biological fluids by high-performance liquid chromatography using 1-fluoro-2, 4-
dinitrobenzene derivatization. Journal of Chromatography B: Biomedical Sciences 231 (1982) 145-
154. https://doi.org/10.1016/s0378-4347(00)80518-6. 

[42] J. D'Souza, R.I. Ogilvie. Determination of gentamicin components C1a, C2 and C1 in plasma and urine 
by high-performance liquid chromatography. J Chromatogr 232 (1982) 212-218. https://doi.org/10.-
1016/s0378-4347(00)86029-6. 

[43] H. Kubo, T. Kinoshita, Y. Kobayashi, K. Tokunaga. Micro determination of gentamicin in serum by 
high-performance liquid chromatography. Journal of Chromatography B: Biomedical Sciences 227 

(1982) 244-248. https://doi.org/10.1016/S0378-4347(00)81624-2.  

[44] J. Marples, M. Oates. Serum gentamicin, netilmicin and tobramycin assays by high performance liquid 
chromatography. Journal of Antimicrobial Chemotherapy 10 (1982) 311-318. https://doi.org/10.1093-
/jac/10.4.311. 

[45] D. Barends, J. Blauw, M. Smits, A. Hulshoff. Determination of amikacin in serum by high-performance 
liquid chromatography with ultraviolet detection. Journal of Chromatography B: Biomedical Sciences 
Applications 276 (1983) 385-394. https://doi.org/10.1016/s0378-4347(00)85105-1.  

[46] P.M. Kabra, P.K. Bhatnagar, M.A. Nelson, J.H. Wall, L.J. Marton. Liquid-chromatographic 
determination of tobramycin in serum with spectrophotometric detection. J Clinical chemistry 29 
(1983) 672-674. https://doi.org/10.1093/jat/7.6.283. 

[47] P.M. Kabra, P.K. Bhatnager, M.A. Nelson. Liquid chromatographic determination of amikacin in serum 
with spectrophotometric detection. Journal of Chromatography B: Biomedical Sciences 307 (1984) 

224-229. https://doi.org/10.1016/S0378-4347(00)84093-1.  

[48] H. Kubo, T. Kinoshita, Y. Kobayashi, K. Tokunaga. Micro-scale method for determination of 
tobramycin in serum using high-performance liquid chromatography. Journal of liquid 
chromatography 7 (1984) 2219-2228. https://doi.org/10.1080/01483918408068871. 

[49] H. Kubo, Y. Kobayashi, T. Kinoshita. Fluorescence determination of streptomycin in serum by 
reversed-phase ion-pairing liquid chromatography. Analytical chemistry insights 58 (1986) 2653-
2655. https://doi.org/10.1021/ac00126a017. 

[50] G. Tamai, H. Imai, H. Yoshida. On-line deproteinization of serum sample for HPLC analysis of 
hydrophilic compounds and its application to gentamicin. Chromatographia 21 (1986) 519-522. 
https://doi.org/10.1007/BF02310539. 

http://dx.doi.org/10.5599/admet.1183
https://doi.org/10.1016/j.jpba.2020.113245
https://doi.org/10.1093/clinchem/23.12.2275
https://doi.org/10.1128/aac.17.4.649
https://doi.org/10.1016/s0378-4347(00)81624-2
https://doi.org/10.1016/S0378-4347(00)81023-3
https://doi.org/10.1016/s0378-4347(00)80075-4
https://doi.org/10.1016/s0378-4347(00)80518-6
https://doi.org/10.1016/s0378-4347(00)86029-6
https://doi.org/10.1016/s0378-4347(00)86029-6
https://doi.org/10.1016/S0378-4347(00)81624-2
https://doi.org/10.1093/jac/10.4.311
https://doi.org/10.1093/jac/10.4.311
https://doi.org/10.1016/s0378-4347(00)85105-1
https://doi.org/10.1093/jat/7.6.283
https://doi.org/10.1016/S0378-4347(00)84093-1
https://doi.org/10.1080/01483918408068871
https://doi.org/10.1021/ac00126a017
https://doi.org/10.1007/BF02310539


Seth K. Amponsah et al.   ADMET & DMPK 10(1) (2022) 27-62 

58  

[51] H. Kubo, H. Li, Y. Kobayashi, T. Kinoshita. Fluorometric determination of streptomycin in serum by 
high-performance liquid chromatography using mobile phase containing fluorogenic reagent. 
Analytical biochemistry 162 (1987) 219-223. https://doi.org/10.1016/0003-2697(87)90030-3. 

[52] R. Rumble, M. Roberts. High-performance liquid chromatographic assay of the major components of 
gentamicin in serum. Journal of Chromatography B: Biomedical Sciences 419 (1987) 408-413. 

https://doi.org/10.1016/0378-4347(87)80308-0. 

[53] T. Fujimoto, R. Tawa, S. Hirose. Fluorometric determination of sisomicin, an aminoglycoside 
antibiotic, in dried blood spots on filter paper by reversed-phase high-performance liquid 
chromatography with pre-column derivatization. Chemical pharmaceutical bulletin 36 (1988) 1571-
1574. https://doi.org/10.1248/cpb.36.1571. 

[54] H. Matsunaga, T. Fujimoto, R. Tawa, S. Hirose. An on-line cleanup procedure for large sample volume 
analysis of serum aminoglycoside antibiotics by reversed-phase high-performance liquid 
chromatography. Chemical J pharmaceutical Methods 36 (1988) 1565-1570. https://doi.org/10.-

1016/s0021-9673(98)00342-2.  

[55] B. Wichert, H. Schreier, H. Derendorf. Sensitive liquid chromatography assay for the determination of 
amikacin in human plasma. Journal of pharmaceutical biomedical analysis 9 (1991) 251-254. 
https://doi.org/10.1016/0731-7085(91)80154-2. 

[56] E.A. Papp, C.A. Knupp, R.H. Barbhaiya. High-performance liquid chromatographic assays for the 
quantification of amikacin in human plasma and urine. Journal of Chromatography B: Biomedical 
Sciences 574 (1992) 93-99. https://doi.org/10.1016/0378-4347(92)80102-V. 

[57] F. Sar, P. Leroy, A. Nicolas, P. Archimbault, G. Ambroggi. Determination of amikacin in dog plasma by 
reversed-phase ion-pairing liquid chromatography with post-column derivatization. Analytical letters 
25 (1992) 1235-1250. https://doi.org/10.1080/00032719208016125. 

[58] M.A. Fennell, C.E. Uboh, R.W. Sweeney, L.R. Soma. Gentamicin in tissue and whole milk: an improved 
method for extraction and cleanup of samples for quantitation on HPLC. Journal of Agricultural Food 
Chemistry 43 (1995) 1849-1852. https://doi.org/10.1021/jf00055a018. 

[59] M. Santos, E. Garcia, F. López, J. Lanao, A. Dominguez-Gil. Determination of netilmicin in plasma by 
HPLC. Journal of pharmaceutical biomedical analysis 13 (1995) 1059-1062. https://doi.org/10.1016/-
0731-7085(95)01331-E. 

[60] D. Stead, R. Richards. Sensitive fluorimetric determination of gentamicin sulfate in biological matrices 
using solid-phase extraction, pre-column derivatization with 9-fluorenylmethyl chloroformate and 
reversed-phase high-performance liquid chromatography. Journal of Chromatography B: Biomedical 
Sciences 675 (1996) 295-302. https://doi.org/10.1016/0378-4347(95)00355-x. 

[61] P.J. Kijak, J. Jackson, B. Shaikh. Determination of gentamicin in bovine milk using liquid 
chromatography with post-column derivatization and fluorescence detection. Journal of 
Chromatography B: Biomedical Sciences 691 (1997) 377-382. https://doi.org/10.1016/s0378-

4347(96)00445-8. 

[62] C.-c. Lin, E. Radwanski, C. Korduba, M. Affrime, M.N. Cayen. Pharmacokinetics of intramuscularly 
administered isepamicin in man. Chemotherapy 43 (1997) 86-93. https://doi.org/10.1159/-
000239541. 

[63] D. Stead, R. Richards, Applications. Sensitive high-performance liquid chromatographic assay for 
aminoglycosides in biological matrices enables the direct estimation of bacterial drug uptake. Journal 
of Chromatography B: Biomedical Sciences 693 (1997) 415-421. https://doi.org/10.1016/s0378-
4347(97)00032-7. 

[64] N. Isoherranen, S. Soback. Determination of gentamicins C1, C1a, and C2 in plasma and urine by 
HPLC. J Clinical chemistry 46 (2000) 837-842. https://doi.org/10.1093/clinchem/46.6.837. 

[65] C. Feng, S. Lin, H. Wu, S. Chen. Trace analysis of amikacin in human plasma by high-performance 
liquid chromatography. Chromatographia 53 (2001) S213-S217. https://doi.org/10.1007/BF024-
90330. 

https://doi.org/10.1016/0003-2697(87)90030-3
https://doi.org/10.1016/0378-4347(87)80308-0
https://doi.org/10.1248/cpb.36.1571
https://doi.org/10.1016/s0021-9673(98)00342-2
https://doi.org/10.1016/s0021-9673(98)00342-2
https://doi.org/10.1016/0731-7085(91)80154-2
https://doi.org/10.1016/0378-4347(92)80102-V
https://doi.org/10.1080/00032719208016125
https://doi.org/10.1021/jf00055a018
https://doi.org/10.1016/0731-7085(95)01331-E
https://doi.org/10.1016/0731-7085(95)01331-E
https://doi.org/10.1016/0378-4347(95)00355-x
https://doi.org/10.1016/s0378-4347(96)00445-8
https://doi.org/10.1016/s0378-4347(96)00445-8
https://doi.org/10.1159/000239541
https://doi.org/10.1159/000239541
https://doi.org/10.1016/s0378-4347(97)00032-7
https://doi.org/10.1016/s0378-4347(97)00032-7
https://doi.org/10.1093/clinchem/46.6.837
https://doi.org/10.1007/BF02490330
https://doi.org/10.1007/BF02490330


ADMET & DMPK 10(1) (2022) 27-62 Bioanalysis of aminoglycozides by HPLC 

doi: http://dx.doi.org/10.5599/admet.1183 59 

[66] A.I. Al-Amoud, B.J. Clark, H. Chrystyn. Determination of gentamicin in urine samples after inhalation 
by reversed-phase high-performance liquid chromatography using pre-column derivatisation with o-
phthalaldehyde. Journal of Chromatography B 769 (2002) 89-95. https://doi.org/10.1016/S1570-
0232(01)00636-5. 

[67] C.-H. Feng, S.-J. Lin, H.-L. Wu, S.-H. Chen. Trace analysis of tobramycin in human plasma by 
derivatization and high-performance liquid chromatography with ultraviolet detection. Journal of 
Chromatography B 780 (2002) 349-354. https://doi.org/10.1016/s1570-0232(02)00544-5. 

[68] B.G. Keevil, S.J. Lockhart, D.P. Cooper. Determination of tobramycin in serum using liquid 
chromatography–tandem mass spectrometry and comparison with a fluorescence polarisation assay. 
Journal of Chromatography B 794 (2003) 329-335. https://doi.org/10.1016/S1570-0232(03)00492-6. 

[69] R. Oertel, U. Renner, W. Kirch. Determination of neomycin by LC–tandem mass spectrometry using 
hydrophilic interaction chromatography. Journal of pharmaceutical biomedical analysis 35 (2004) 
633-638. https://doi.org/10.1016/j.jpba.2004.01.018. 

[70] N.C. Megoulas, M.A. Koupparis. Development and validation of a novel HPLC/ELSD method for the 
direct determination of tobramycin in pharmaceuticals, plasma, and urine. Anal Bioanal Chem 382 
(2005) 290-296. https://doi.org/10.1007/s00216-004-2948-8. 

[71] J.M. Serrano, M. Silva. Determination of amikacin in body fluid by high-performance liquid-
chromatography with chemiluminescence detection. Journal of Chromatography B 843 (2006) 20-24. 
https://doi.org/10.1016/j.jchromb.2006.05.016. 

[72] M. Cherlet, S. De Baere, P. De Backer. Quantitative determination of dihydrostreptomycin in bovine 
tissues and milk by liquid chromatography‐electrospray ionization‐tandem mass spectrometry. 
Journal of mass spectrometry 42 (2007) 647-656. https://doi.org/10.1002/jms.1194. 

[73] D.G. Mascher, C.P. Unger, H. Mascher. Determination of neomycin and bacitracin in human or rabbit 
serum by HPLC–MS/MS. Journal of pharmaceutical biomedical analysis 43 (2007) 691-700. 
https://doi.org/10.1016/j.jpba.2006.08.008. 

[74] A.A. Al-Majed. A new LC method for determination of some aminoglycoside antibiotics in dosage 
forms and human plasma using 7-fluoro-4-nitrobenz-2-oxa-1, 3-diazole as a fluorogenic pre-column 
label. Chromatographia 68 (2008) 927-934. https://doi.org/10.1365/s10337-008-0792-2. 

[75] M.E. Attema-de Jonge, J.M. Bekkers, H.M. Oudemans-van Straaten, R.W. Sparidans, E.J. Franssen. 
Simple and sensitive method for quantification of low tobramycin concentrations in human plasma 
using HPLC–MS/MS. Journal of Chromatography B 862 (2008) 257-262. https://doi.org/10.1016-
/j.jchromb.2007.12.008. 

[76] S. Hosokawa, K. Nakamura, Y. Fujita, R. Horiuchi, K. Yamamoto. Determination of isepamicin in 
human plasma by HPLC with fluorescence detection after derivatization using 6-aminoquinolyl-N-
hydroxysuccinimidyl-carbamate. Pharmaceutical Methods 31 (2008) 1866-1869. https://doi.org/10.-
1248/bpb.31.1866. 

[77] M. Mashat, H. Chrystyn, B. Clark, K. Assi. Development and validation of HPLC method for the 
determination of tobramycin in urine samples post-inhalation using pre-column derivatisation with 
fluorescein isothiocyanate. Journal of Chromatography B 869 (2008) 59-66. https://doi.org/10.1016-
/j.jchromb.2008.05.012. 

[78] X. Chang, J. Peng. LC Analysis of Isepamicin in Plasma Samples Post-Inhalation with Fluorescence 
Detection and Its Application to a Pharmacokinetic Study. Chromatographia 70 (2009) 1429-1433. 
https://doi.org/10.1365/s10337-009-1331-5. 

[79] L. Baietto, A. D’Avolio, F.G. De Rosa, S. Garazzino, M. Michelazzo, G. Ventimiglia, M. Siccardi, M. 
Simiele, M. Sciandra, G. Di Perri. Development and validation of a simultaneous extraction procedure 
for HPLC-MS quantification of daptomycin, amikacin, gentamicin, and rifampicin in human plasma. 
Analytical bioanalytical chemistry 396 (2010) 791-798. https://doi.org/10.1007/s00216-009-3263-1. 

[80] A.A. El-Zaher, M.A. Mahrouse. Utility of experimental Design in pre-column Derivatization for the 
Analysis of Tobramycin by HPLC-Fluorescence Detection: Application to Ophthalmic solution and 
Human plasma. Analytical chemistry insights 8 (2013) ACI. S11612. https://doi.org/10.4137/-
ACI.S11612. 

http://dx.doi.org/10.5599/admet.1183
https://doi.org/10.1016/S1570-0232(01)00636-5
https://doi.org/10.1016/S1570-0232(01)00636-5
https://doi.org/10.1016/s1570-0232(02)00544-5
https://doi.org/10.1016/S1570-0232(03)00492-6
https://doi.org/10.1016/j.jpba.2004.01.018
https://doi.org/10.1007/s00216-004-2948-8
https://doi.org/10.1016/j.jchromb.2006.05.016
https://doi.org/10.1002/jms.1194
https://doi.org/10.1016/j.jpba.2006.08.008
https://doi.org/10.1365/s10337-008-0792-2
https://doi.org/10.1016/j.jchromb.2007.12.008
https://doi.org/10.1016/j.jchromb.2007.12.008
https://doi.org/10.1248/bpb.31.1866
https://doi.org/10.1248/bpb.31.1866
https://doi.org/10.1016/j.jchromb.2008.05.012
https://doi.org/10.1016/j.jchromb.2008.05.012
https://doi.org/10.1365/s10337-009-1331-5
https://doi.org/10.1007/s00216-009-3263-1
https://doi.org/10.4137/ACI.S11612
https://doi.org/10.4137/ACI.S11612


Seth K. Amponsah et al.   ADMET & DMPK 10(1) (2022) 27-62 

60  

[81] Y. Bijleveld, T. de Haan, J. Toersche, S. Jorjani, J. van der Lee, F. Groenendaal, P. Dijk, A. van Heijst, 
A.W. Gavilanes, R. de Jonge. A simple quantitative method analysing amikacin, gentamicin, and 
vancomycin levels in human newborn plasma using ion-pair liquid chromatography/tandem mass 
spectrometry and its applicability to a clinical study. Journal of Chromatography B 951 (2014) 110-
118. https://doi.org/10.1016/j.jchromb.2014.01.035. 

[82] B. Chauhan, S. Jalalpure. Analysis of amikacin in human serum by UHPLC with fluorescence detector 
using chloro-formate reagent with glycine. Pharmaceutical Methods 7 (2016) 99-103. 
https://doi.org/10.5530/phm.2016.7.15. 

[83] M. Zu, J. Jiang, H. Zhao, S. Zhang, Y. Yan, S. Qiu, S. Yuan, J. Han, Y. Zhang, W. Guo. Rapid analysis of 
neomycin in cochlear perilymph of guinea pigs using disposable SPE cartridges and high performance 
liquid chromatography-tandem mass spectrometry. Journal of Chromatography B 1093 (2018) 52-59. 
https://doi.org/10.1016/j.jchromb.2018.06.055. 

[84] A.C.C. da Silva, L.d.L.F. Lizot, M.F. Bastiani, M.V. Antunes, N. Brucker, R. Linden. Ready for TDM: 
Simultaneous quantification of amikacin, vancomycin and creatinine in human plasma employing 
ultra-performance liquid chromatography-tandem mass spectrometry. Clinical biochemistry 70 
(2019) 39-45. https://doi.org/10.1016/j.clinbiochem.2019.06.011. 

[85] A.C.C. da Silva, L.d.L.F. Lizot, M.F. Bastiani, M.V. Antunes, N. Brucker, R. Linden. Dried plasma spots 
for therapeutic monitoring of amikacin: Validation of an UHPLC-MS/MS assay and pharmacokinetic 
application. Journal of pharmaceutical biomedical analysis 184 (2020) 113201. https://doi.org/10.-
1016/j.jpba.2020.113201. 

[86] J.-P. Antignac, K. de Wasch, F. Monteau, H. De Brabander, F. Andre, B. Le Bizec. The ion suppression 
phenomenon in liquid chromatography–mass spectrometry and its consequences in the field of 
residue analysis. Analytica Chimica Acta 529 (2005) 129-136. https://doi.org/10.1016/j.aca.2004.-
08.055. 

[87] C. Ghosh, C.P. Shinde, B.S. Chakraborty. Influence of ionization source design on matrix effects during 
LC–ESI-MS/MS analysis. Journal of Chromatography B 893 (2012) 193-200. https://doi.org/10.1016/-
j.jchromb.2012.03.012. 

[88] B. Matuszewski, M. Constanzer, C. Chavez-Eng. Strategies for the assessment of matrix effect in 
quantitative bioanalytical methods based on HPLC− MS/MS. Analytical chemistry insights 75 (2003) 
3019-3030. https://doi.org/10.1021/ac020361s. 

[89] P.J. Taylor. Matrix effects: the Achilles heel of quantitative high-performance liquid chromatography–
electrospray–tandem mass spectrometry. Clinical biochemistry 38 (2005) 328-334. https://doi.org-
/10.1016/j.clinbiochem.2004.11.007. 

[90] O.A. Ismaiel, T. Zhang, R.G. Jenkins, H.T. Karnes. Investigation of endogenous blood plasma 
phospholipids, cholesterol and glycerides that contribute to matrix effects in bioanalysis by liquid 
chromatography/mass spectrometry. Journal of Chromatography B 878 (2010) 3303-3316. 
https://doi.org/10.1016/j.jchromb.2010.10.012. 

[91] J.L. Little, M.F. Wempe, C.M. Buchanan. Liquid chromatography–mass spectrometry/mass 
spectrometry method development for drug metabolism studies: examining lipid matrix ionization 
effects in plasma. Journal of Chromatography B 833 (2006) 219-230. https://doi.org/10.1016/j.-
jchromb.2006.02.011. 

[92] M. Garcia. The effect of the mobile phase additives on sensitivity in the analysis of peptides and 
proteins by high-performance liquid chromatography–electrospray mass spectrometry. Journal of 
Chromatography B 825 (2005) 111-123. https://doi.org/10.1016/j.jchromb.2005.03.041. 

[93] H. Mei, Y. Hsieh, C. Nardo, X. Xu, S. Wang, K. Ng, W.A. Korfmacher. Investigation of matrix effects in 
bioanalytical high‐performance liquid chromatography/tandem mass spectrometric assays: 
application to drug discovery. Rapid Communications in Mass Spectrometry 17 (2003) 97-103. 
https://doi.org/10.1002/rcm.876. 

[94] W. Li, W. Jian, Y. Fu. Basic Sample Preparation Techniques in LC‐MS Bioanalysis: Protein Precipitation, 
Liquid–Liquid Extraction, and Solid‐Phase Extraction. Sample Preparation in LC‐MS Bioanalysis (2019) 
1-30. https://doi.org/10.1002/9781119274315.ch1. 

https://doi.org/10.1016/j.jchromb.2014.01.035
https://doi.org/10.5530/phm.2016.7.15
https://doi.org/10.1016/j.jchromb.2018.06.055
https://doi.org/10.1016/j.clinbiochem.2019.06.011
https://doi.org/10.1016/j.jpba.2020.113201
https://doi.org/10.1016/j.jpba.2020.113201
https://doi.org/10.1016/j.aca.2004.08.055
https://doi.org/10.1016/j.aca.2004.08.055
https://doi.org/10.1016/j.jchromb.2012.03.012
https://doi.org/10.1016/j.jchromb.2012.03.012
https://doi.org/10.1021/ac020361s
https://doi.org/10.1016/j.clinbiochem.2004.11.007
https://doi.org/10.1016/j.clinbiochem.2004.11.007
https://doi.org/10.1016/j.jchromb.2010.10.012
https://doi.org/10.1016/j.jchromb.2006.02.011
https://doi.org/10.1016/j.jchromb.2006.02.011
https://doi.org/10.1016/j.jchromb.2005.03.041
https://doi.org/10.1002/rcm.876
https://doi.org/10.1002/9781119274315.ch1


ADMET & DMPK 10(1) (2022) 27-62 Bioanalysis of aminoglycozides by HPLC 

doi: http://dx.doi.org/10.5599/admet.1183 61 

[95] A. Posyniak, J. Zmudzki, J. Niedzielska. Sample preparation for residue determination of gentamicin 
and neomycin by liquid chromatography. Journal of Chromatography A 914 (2001) 59-66. 
https://doi.org/10.1016/S0021-9673(00)00980-8. 

[96] V. Gupta, A.D.K.J. Jain, N. Gill, K. Guptan. Development and validation of HPLC method-a review. 
International research journal of pharmaceutical applied sciences 2 (2012) 17-25.  

[97] K.F.M. Opuni, J.A. Boadu, S.K. Amponsah, C.A. Okai. High performance liquid chromatography: A 
versatile tool for assaying antiepileptic drugs in biological matrices. Journal of Chromatography B 
1179 (2021) 122750. https://doi.org/10.1016/j.jchromb.2021.122750. 

[98] M. Charde, A. Welankiwar, J. Kumar. Method development by liquid chromatography with validation. 
International Journal of Pharmaceutical Chemistry 4 (2014) 57-61. https://doi.org/10.7439/ijpc.v4i1-
.65. 

[99] Y. Vidushi, B. Meenakshi. A review on HPLC method development and validation. Res J Life Sci, 
Bioinform, Pharm Chem Sci 2 (2017) 178. https://doi.org/10.26479/2017.0206.12. 

[100] D. Kowalczuk, R. Pietraś, B. Paw, A. Czerkies. Applying liquid chromatography with fluorescence 
detection to determine gentamicin. Pol. J. Environ. Stud 19 (2010) 587-591.  

[101] H. Itagaki. Fluorescence spectroscopy. Experimental Methods in Polymer Science, Academic Press, 
San Diego, CA (2000) 155-260.  

[102] R. LoBrutto, Y. Kazakevich, Reversed-phase HPLC, HPLC for Pharmaceutical Scientists, Wiley Online 
Library2007, pp. 139-195. 

[103] K.F.M. Opuni, M. Al-Majdoub, Y. Yefremova, R.F. El-Kased, C. Koy, M.O. Glocker. Mass spectrometric 
epitope mapping. Mass Spectrom Rev 37 (2018) 229-241. https://doi.org/10.1002/mas.21516. 

[104] M. Chen, H.H. Cai, F. Yang, D. Lin, P.H. Yang, J. Cai. Highly sensitive detection of chromium (III) ions by 
resonance Rayleigh scattering enhanced by gold nanoparticles. Spectrochim Acta A Mol Biomol 
Spectrosc 118 (2014) 776-781. https://doi.org/10.1016/j.saa.2013.09.058. 

[105] W.R. LaCourse, S.J. Modi. Microelectrode applications of pulsed electrochemical detection. 
Electroanalysis: An International Journal Devoted to Fundamental Practical Aspects of Electroanalysis 
17 (2005) 1141-1152. https://doi.org/10.1002/elan.200403233. 

[106] P.J. Vandeberg, D.C. Johnson. Pulsed electrochemical detection of cysteine, cystine, methionine, and 
glutathione at gold electrodes following their separation by liquid chromatography. Analytical 
Chemistry 65 (1993) 2713-2718. https://doi.org/10.1021/ac00068a002. 

[107] S.M. Moosavi, S. Ghassabian. Linearity of calibration curves for analytical methods: A review of 
criteria for assessment of method reliability. Calibration Validation of Analytical Methods—A 
Sampling of Current Approaches, IntechOpen Ltd.: London, UK (2018) 109-127.  

[108] A.M.d. Almeida, M.M. Castel-Branco, A. Falcao. Linear regression for calibration lines revisited: 
weighting schemes for bioanalytical methods. Journal of Chromatography B 774 (2002) 215-222. 
https://doi.org/10.1016/S1570-0232(02)00244-1. 

[109] L.C. Rodríguez, A.M.G. Campa Ta, C.J. Linares, M.R. Ceba. Estimation of performance characteristics 
of an analytical method using the data set of the calibration experiment. Analytical letters 26 (1993) 
1243-1258. https://doi.org/10.1080/00032719308019900. 

[110] G. Smith. European Medicines Agency guideline on bioanalytical method validation: what more is 
there to say? Bioanalysis 4 (2012) 865-868. https://doi.org/10.4155/bio.12.44. 

[111] L.V. Sonawane, B.N. Poul, S.V. Usnale, P.V. Waghmare, L.H. Surwase. Bioanalytical method validation 
and its pharmaceutical application-a review. Pharm Anal Acta 5 (2014) 2. 
https://doi.org/10.4172/2153-2435.1000288. 

[112] A. Dasgupta. Advances in antibiotic measurement. Adv Clin Chem 56 (2012) 75-104. 
https://doi.org/10.1016/b978-0-12-394317-0.00013-3. 

[113] P. Noone. Sisomicin, netilmicin and dibekacin. A review of their antibacterial activity and therapeutic 
use. Drugs 27 (1984) 548-578. https://doi.org/10.2165/00003495-198427060-00003. 

http://dx.doi.org/10.5599/admet.1183
https://doi.org/10.1016/S0021-9673(00)00980-8
https://doi.org/10.1016/j.jchromb.2021.122750
https://doi.org/10.7439/ijpc.v4i1.65
https://doi.org/10.7439/ijpc.v4i1.65
https://doi.org/10.26479/2017.0206.12
https://doi.org/10.1002/mas.21516
https://doi.org/10.1016/j.saa.2013.09.058
https://doi.org/10.1002/elan.200403233
https://doi.org/10.1021/ac00068a002
https://doi.org/10.1016/S1570-0232(02)00244-1
https://doi.org/10.1080/00032719308019900
https://doi.org/10.4155/bio.12.44
https://doi.org/10.4172/2153-2435.1000288
https://doi.org/10.1016/b978-0-12-394317-0.00013-3
https://doi.org/10.2165/00003495-198427060-00003


Seth K. Amponsah et al.   ADMET & DMPK 10(1) (2022) 27-62 

62  

[114] J.P. Hieber, J.D. Nelson. Reevaluation of kanamycin dosage in infants and children. Antimicrob Agents 
Chemother 9 (1976) 899-902. https://doi.org/10.1128/AAC.9.6.899. 

[115] Drugs.com. Streptomycin Dosage., 2021. 

[116] G.G. Duncan, C.F. Clancy, J.R. Wolgamot, B. Beidleman. NEOMYCIN: RESULTS OF CLINICAL USE IN TEN 
CASES. Journal of the American Medical Association 145 (1951) 75-80. 
https://doi.org/10.1001/jama.1951.02920200015005. 

[117] M. Urata, Y. Narita, D. Kadowaki, K. Tanoue, I. Tashiro, M. Fukunaga, A. Shibata, Y. Yoshida, K. 
Soejima, S. Hirata. Interaction of arbekacin with dialysis membrane. Renal Replacement Therapy 2 
(2016) 35. https://doi.org/10.1186/s41100-016-0045-z. 

[118] E. Novak, C.A. Schlagel, L.A. LeZotte, R.T. Pfeifer. The tolerance of high dose intravenous 
spectinomycin therapy in man. J Clin Pharmacol 14 (1974) 442-447. https://doi.org/10.1002/j.1552-
4604.1974.tb02326.x. 

[119] K.M. Krause, A.W. Serio, T.R. Kane, L.E. Connolly. Aminoglycosides: an overview. Cold Spring Harbor 
perspectives in medicine 6 (2016) a027029. https://doi.org/10.1101/cshperspect.a027029. 

[120] V. Alampanos, V. Samanidou. Current trends in green sample preparation before liquid 
chromatographic bioanalysis. Current Opinion in Green and Sustainable Chemistry 31 (2021) 100499. 
https://doi.org/10.1016/j.cogsc.2021.100499. 

[121] V. Alampanos, V. Samanidou, I. Papadoyannis. Trends in sample preparation for the hplc 
determination of penicillins in biofluids. Journal of Applied Bioanalysis 5 (2019) 9-17. 
https://doi.org/10.17145/jab.19.003. 

[122] K.D. Clark, C. Zhang, J.L. Anderson. Sample Preparation for Bioanalytical and Pharmaceutical Analysis. 
Anal Chem 88 (2016) 11262-11270. https://doi.org/10.1021/acs.analchem.6b02935. 

 

 

 

 

 

©2022 by the authors; licensee IAPC, Zagreb, Croatia. This article is an open-access article distributed under the terms and 

conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/)  

https://doi.org/10.1128/AAC.9.6.899
https://doi.org/10.1001/jama.1951.02920200015005
https://doi.org/10.1186/s41100-016-0045-z
https://doi.org/10.1002/j.1552-4604.1974.tb02326.x
https://doi.org/10.1002/j.1552-4604.1974.tb02326.x
https://doi.org/10.1101/cshperspect.a027029
https://doi.org/10.1016/j.cogsc.2021.100499
https://doi.org/10.17145/jab.19.003
https://doi.org/10.1021/acs.analchem.6b02935
http://creativecommons.org/licenses/by/3.0/