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dOI 10.11603/IJMMR.2413-6077.2019.2.10896

YOUDEN'S TEST FOR CHROMATOGRApHIC DETERMINATION  
OF ENALApRIL IN pHARMACEUTICALS

L.S. Logoyda
I. HORBACHEVSKY TERNOPIL NATIONAL MEDICAL UNIVERSITY, TERNOPIL, UKRAINE

Background. Robustness tests were firstly introduced for avoiding problems in interlaboratory studies and 
identifying the factors potentially responsible. A robustness test performing in late validation procedure involves 
the possibility that when the method is established not robust, it should be redeveloped and optimized. At this 
stage much effort has been made and money spent for optimization and validation, and therefore avoiding this 
would be great. 

Objective. The aim of the study was to consider the robustness of HPLC determination of enalapril (in tablets) 
by the Youden’s test. 

Methods. Youden’s test was chosen as an efficient method to assess the robustness among all analytical 
methods that is by means of an experiment design, which involved seven analytical parameters combined in 
eight tests. In previous studies, we evaluated the chromatographic method robustness to quantify enalapril (in 
tablets) by Youden’s test. 

Results. According to the Youden’s test criteria, HPLC method proved to be greatly robust regarding the 
enalapril content in introduction of variation of seven analytic parameters. The lowest variation in enalapril 
content was 0.91 %, when Grace Platinumр C8 EPS column (4.6 mm i.d. X 250 mm, 5 μm) was used. A holistic 
approach concerning simultaneous innovations in particle technology and instrument design was endeavored 
for the first time to meet and tackle the analytical laboratory issues. This was aimed at promoting success of 
analytical scientists as well as profitability and productiveness of business. 

 Conclusion. The Youden’s test has been proved to be an efficient and useful tool for evaluation of robustness 
of enalapril HPLC assay. 

KEY WORD: enalapril; high-performance liquid chromatography; robustness; quantitative analysis; 
Youden’s test.

Corresponding author: Liliya Logoyda, PhD, DSc, Department 
of Pharmaceutical Chemistry I. Horbachevsky Ternopil Na-
tional Medical University, 1 Maidan Voli, Ternopil, 46001, 
Ukraine. E-mail: logojda@tdmu.edu.ua

Introduction
Recently, Robustness testing is best known 

and most commonly used in the pharmaceutics 
because of the stringent regulations in the 
domain set by regulatory authorities that 
requires extensively validated methods. The-
refore most definitions and existing metho­
dologies, e.g. those from the ICH, are found in 
the field, as stated before. Though, this has no 
implications for robustness testing of analytical 
methods in other domains and therefore this 
guideline is not confined to pharmaceutical 
methods [1].

Evaluation of robustness of chromatographic 
method is a laborious, complex and straining 
process, taking into account a great number of 
analytical parameters considered while carrying 
out the test. Some authors consider specific 
analytical parameters presenting small varia-

tions in the nominal conditions; statistical 
analysis is made using the Student’s t-test or 
ANOVA test. Other alternative for evaluation of 
robustness of analytical methods is the 
Youden’s test. This test assesses not only the 
robustness of the method but also determines 
the each analytical parameter effect on final 
results. The main idea of the Youden’s test is 
not studying one alteration at time but 
introducing several changes all together in this 
way that the effects of individual chan ges can 
be determined [2, 3, 4].

Enalapril maleate is a maleate salt of ena-
lapril, the ethyl ester of a long-acting angiotensin 
converting enzyme inhibitor, enalaprilat. Ena-
lapril maleate is chemically defined as (S)­1­[N­
[1-(ethoxycarbonyl)-3-phenylpropyl]-L-alanyl]-
L­proline, (Z)­2­butenedioate salt (1:1). Enalapril, 
after hydrolysis to enalaprilat, inhibits angio-
tensin-converting enzyme (ACE) in humans and 
animals. ACE is a peptidyl dipeptidase that 
catalyzes the conversion of angiotensin I to the 

International Journal of Medicine and Medical Research 
2019, Volume 5, Issue 2, p. 83-87
copyright © 2019, TNMU, All Rights Reserved

L.S. Logoyda



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vasoconstrictor substance, angiotensin II. 
Angio tensin II also stimulates aldosterone 
secretion by the adrenal cortex. Enalapril in 
hypertension and heart failure beneficially 
effects primarily from suppression of the renin-
angiotensin-aldosterone system. Inhibition of 
ACE leads to decrease of plasma angiotensin II 
that results in decrease of vasopressor activity 
and decrease of aldosterone secretion [5].

The aim of the research was to determine 
the robustness of HPLC (High-Performance 
Liquid Chromatography) method for evaluation 
of enalapril by means of Youden’s test, and 
define the analytical parameters that have 
greater influence on the final analysis.

Methods
Enalapril maleate was presented by Farmak 

pharmaceuticals (Kiev, Ukraine). HPLC grade 
acetonitrile, sodium dihydrophosphate dihyd-
rate, phosphoric acid were got from Merck 
pharamaceticals.

Instrumentation and chromatographic 
conditions

Agilent 1260, Grace Platinumр C8 EPS 
column (4.6 mm i.d. X 250 mm, 5 μm). Chroma­
tographic separation was carried out at am-
bient temperature (22-25 °C). The compound 
was separated isocratically with a mobile phasе 
consisting of acetonitrile and buffer solution 
pH 2.2 (25/75, v/v), at a flow rate 2.0 mL/min 
with injection volume 50 μL. Column tempe­
rature was 50 °C. The effluent was monitored 
spectrophotometrically at a wavelength 
215 nm. 

Preparation of mobile phase
To prepare buffer solution pH 2.2.: 3.59 g of 

sodium dihydrophosphate dihydrate was 
dissolved in 1800 ml of water, the pH of the 
solution was fixed with phosphoric acid to the 
value (2.2±0.05), and then the volume of the 

solution with water R to 2000.0 ml was added 
and mixed.

Stock standard solutions
20 mg of the standard sample of enalapril 

maleate was dissolved in a solvent, added 
0.5 ml of a solution of enalaprilat with a concen-
tration of 0.4 mg/ml and 2.0 ml of enalapril 
diketopiperazine solution at a concentration of 
0.4 mg/ml was adjusted to a volume of 100.0 ml 
with the same solvent.

Procedures 
The standard solutions were prepared by 

dilution of the stock standard solution of mobile 
phase. Triplicate 50.0 μL injections were made 
for each concentration and chromatographed 
under the conditions described above. The peak 
area of each concentration was plotted against 
the corresponding concentration to obtain the 
calibration graph and regression equation was 
computed [6].

Results
The robustness assessment of HPLC me-

thod for enalapril quantitation was performed 
by the method suggested by Youdene Steiner. 
For the nominal values of the method, seven 
analytical parameters were chosen and minor 
va riations were induced. After, eight runs were 
completed in order to determine the effect of 
each parameter on the final result. The seven 
analytical parameters as well as the variations 
are presented in Table 1. The analytical circu-
mstances of the nominal values are defined by 
capi tal letters and of the small variation – by 
lowercase letters. 

The seven parameters and their respective 
variations were joined into eight assays or 
chromatographic runs randomly performed. 
The factorial combination of parameters for the 
Youden’s test is presented in Table 2. The re-
sults of the analyses are defined by the letters 

Table 1. Analytical parameters and variations for the robustness evaluation of HPLC method  
for enalapril quantitation

Parameter Nominal condition Variation
A/a Acetonitrile in mobile phase 25 – A 35 – a

B/b Buffer solution pH 2.2 in mobile phase
75 – B 65 – b

C/c pH of buffer solution in mobile phase
2.2 – C 2.7 – c

D/d Column temperature, °C 50 – D 40 – d
E/e Mobile phase flow rate, ml/min 2.0 – E 1.0 – e
F/f Column supplier Grace 

Platinumр 
C8 EPS

– F Nucleosil 
C18

– f

G/g Chromatograph model Agilent 1290 – G HP 1100 – g

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from s to z. Hence, when combination 1 was 
assessed, the result was s, for combination 2 
the result was t, and so on.

Three injections of each sam ple and stan_
dard solutions at the normal concentration 
were administered for each combination. A 
30-minute pause for system stabilization took 
place after alteration of chroma tographic co-
lumn or mobile phase composition. In each 
combination the assessed re sults were for a 
peak area, retention time (Rt), tailing factor (T), 
theoretical plates number (N) and captopril 
content. 

The following equation was used for eva-
luation of the effect of the column temperature 
on the final analyses results:

  Effect C/c=(s+u+w+y)/4–(t+v+x+z)/4Eq
The Youden’s test allows definite estab­

lishing of the parameters, which have a greater 
influence on the results of the analyses, and 
control more rigorously the eventual variations 
of these parameters that may arise during a 
routine analysis.

Discussion
In this research, the first trials were aimed 

to find optimal chromatographic conditions. 
The objective of the chromatographic method 
development was achievement of a peak tailing 
factor <1.5, retention time of between 4 and 5 

minutes in consort with well resolution [7-17]. 
In both equipment (Agilent 1290 and HP1100), 
the analyses of the robustness evaluation of 
chromatographic method were carried out 
simultaneously. The results were attained in 
eight runs to enalapril sample and standard 
solutions.

The effects of the parameter variations on 
the analysis results are presented in Table 3.

By means of the Youden’s test criteria, HPLC 
method proved to be significantly robust as 
regards the content of enalapril in case of intro-
duced variations of seven analytical parameters 
[18]. The lowest variation in enalapril content 
was 0.91 %, when column Grace Platinumр C8 
EPS column (4.6 mm i.d.×250 mm, 5 μm) was 
used.

A holistic approach concerning simultaneous 
innovations in particle technology and instru-
ment design was endeavored for the first time 
to meet and tackle the analytical laboratory 
issues. This was aimed at promoting success of 
analytical scientists as well as profitability and 
productiveness of business. The Platinum™ 
column advantage controlled silica exposure is 
the dissimilarity that makes Platinum™ columns 
unique. Instead of thorough covering of the 
silica with bonded phase to hide the silica, the 
exposure of the silica in Platinum™ columns is 
controlled to provide a dual mode separation 

Table 2. Factorial combination of the analytical parameters for robustness evaluation

Analytical parameter Factorial combination
Acetonitrile in mobile phase A A A A a a a a
Buffer solution pH 2.2 in mobile 
phase

B B b b B B b b

pH of buffer solution in mobile 
phase

C c C c C c C c

Column temperature D D d d d d D D
Mobile phase flow rate E e E e e E e E
Column supplier F f f F F f f F
Chromatograph model G g g G g G G g
Result s t u v w x y z

Table 3. Effects of the analytical parameters on content and retention time (Rt)  
for enalapril HPLC quantitation

Effect Content (%) Rt (min)
Acetonitrile in mobile phase 0.15 -0.26
Buffer solution pH 2.2 in mobile phase 0.16 -0.27
pH of buffer solution in mobile phase 0.12 0.05
Column temperature -0.05 0.05
Mobile phase flow rate -0.03 0.05
Column supplier 0.91 -2.05
Chromatograph model -0.04 0.11



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with both polar and non-polar sites exposed to 
the samples. This extends polar selectivity well 
beyond the other reversed-phase columns and 
gives separations that other columns cannot.

Conclusion
Youden’s test proved to be an efficient and 

useful tool for the robustness evaluation of 

HPLC method for assay of enalapril in pharma-
ceuticals. Therefore, Youden’s test can be 
successfully used for the ro bustness evaluation 
for validation process of analytical methods.

Funding
This research received no external funding.
Conflict of Interests
The author declares no conflict of interest.

юден тест хроматограФічного Визначення еналаприлу  
В лікарсЬких засоБах

Л.С. Логойда
ТЕРНОПІЛЬСЬКИЙ НАЦІОНАЛЬНИЙ МЕДИЧНИЙ УНІВЕРСИТЕТ ІМЕНІ І. Я. ГОРБАЧЕВСЬКОГО,  

ТЕРНОПІЛЬ, УКРАЇНА 

Вступ. Випробування на робасність спочатку були введені, щоб уникнути проблем у міжлабораторних 
дослідженнях та виявити потенційно відповідальні фактори. Виконання перевірки надійності в кінці 
процедури валідації передбачає ризик того, що, коли виявиться, що метод не є надійним, його слід 
переробити і оптимізувати. На цьому етапі вже було витрачено багато зусиль і грошей на оптимізацію 
і перевірку, і тому хочеться цього уникнути. 

Мета дослідження – визначити робасність хроматографічного визначення еналаприлу в 
таблетках з використанням тесту Юдена.

Методи дослідження. Ефективний метод оцінки надійності аналітичних методів за допомогою 
тесту Юдена шляхом розробки експерименту, який включає сім аналітичних параметрів, об'єднаних 
у восьми тестах. У дослідженнях ми оцінювали надійність хроматографічного методу для кількісного 
визначення еналаприлу в таблетках з використанням тесту Юдена.

Результати. Використовуючи критерії випробування Юдена, метод ВЕРХ показав високу надійність 
щодо вмісту еналаприлу при введенні варіації семи аналітичних параметрів. Найнижча зміна вмісту 
еналаприлу становила 0,91%, коли використовувалася колонка Grace Platinumr C8 EPS-колони (4,6 мм 
i.d. X 250 мм, 5 мкм). Вперше розроблено цілісний підхід, що передбачає одночасне впровадження інновацій 
у технології частинок та проектування приладів. Це було зроблено для того, щоб зробити вчених-
аналітиків більш успішними, а підприємства - більш прибутковими та продуктивними.

Висновки. Тест Юдена виявився ефективним і корисним інструментом для оцінки робастності 
для аналізу еналаприлу методом ВЕРХ.

КЛЮЧОВІ СЛОВА: еналаприл; високоефективна рідинна хроматографія; робасність; 
кількісний аналіз; Юден тест.

Відомості про автора
Логойда Лілія Святославівна – доктор фармацевтичних наук, доцент, завідувач кафедри 

фармацевтичної хімії Тернопільського національного медичного університету імені І.Я. Гор­
бачевського, Тернопіль, Україна.

Information about the author
Logoyda L.S. – PhD, DSc, Head of Department of Pharmaceutical Chemistry I. Horbachevsky Ternopil 

National Medical University, Ternopil, Ukraine.
ORCID 0000­0002­5252­8806, e­mail: logojda@tdmu.edu.ua

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