










































 Global Journal of Education and Allied  

Research (GJEAR) 
Volume.13, Number 12; December-2022; 

ISSN: 2837-3707 | Impact Factor: 6.79 

https://zapjournals.com/Journals/index.php/gjear  

Published By: Zendo Academic Publishing 

 

 

pg. 18 

CHROMATOGRAPHIC INSIGHTS: UPLC-TUV ANALYSIS OF GLUCOSAMINE 

AND CHONDROITIN IN DIETARY PRODUCTS 

 
1Dr. Nguyen Thi Linh Tran, 2Prof. Le Minh Duc, 3Dr. Pham Khanh Nguyen and 4Prof. 

Nguyen Van Hoang  

 

Article Info  Abstract 

Keywords: Osteoarthritis, 

glucosamine, chondroitin 

sulfate, joint health, symptom 

management. 

 Osteoarthritis (OA) is a prevalent chronic disease characterized by 

irreversible joint tissue damage, impacting cartilage, synovial 

membrane, and subchondral bone. This study explores the therapeutic 

potential of two compounds, glucosamine and chondroitin sulfate, in 

managing OA symptoms. Glucosamine, a vital component of chitin and 

mucopolysaccharides, is known for its role in connective tissue and 

cartilage formation. The safety profile of glucosamine, as demonstrated 

by research from the U.S. National Institutes of Health (NIH) and other 

sources, highlights its advantages over non-steroidal anti-inflammatory 

drugs (NSAIDs), with significantly fewer side effects observed. 

Furthermore, glucosamine does not interfere with glucose metabolism. 

Chondroitin sulfate, a sulfated glycosaminoglycan, is widely used in 

OA patient management. Its multifaceted effects on chondrocyte 

survival, modulation of cartilage matrix balance, and reduction of 

proinflammatory factors underscore its potential benefits. In 

subchondral bone osteoblasts, chondroitin sulfate contributes to 

enhanced OPG/RANKL ratio, indicative of improved bone health. This 

review consolidates evidence on the efficacy and safety of glucosamine 

and chondroitin sulfate in OA management, shedding light on their 

roles in mitigating joint degeneration and inflammation. These 

compounds offer promising avenues for OA treatment, warranting 

further research and clinical exploration. 
 

 

1. Introduction  

Osteoarthritis is a chronic disease considered a global organ failure and characterized by irremediable damage to 

significant tissues of the joint, namely cartilage, synovial membrane, and subchondral bone. The disease 

 
1,2,3,4 CanTho University of Medicine and Pharmacy, 179 Nguyen Van Cu Street, An Khanh Ward, Ninh Kieu 

District, Can Tho City, Vietnam  

 

https://zapjournals.com/Journals/index.php/gjear


Global Research Journal of Management and Social Sciences (GRJMSS) Vol. 13 (12) 
 

pg. 19 

pathogenesis was observed, including articular cartilage loss, synovial membrane inflammation, and subchondral 

bone alteration (Martel-Pelletier, 2005).  

Glucosamine, 2-amino-2-deoxy-D-glucose, is an essential part of chitin and mucopolysaccharides. Large 

complexes of negatively charged carbohydrate chains are incorporated into mucous secretions, connective tissue, 

ligaments, and cartilage. Glucosamine is also a widely used compound for reducing symptoms of osteoarthritis. 

According to U.S. National Institutes of Health (NIH) and other scientists' research, side effects of glucosamine 

were significantly less common than non-steroidal antiinflammatory drugs (NSAIDs), significantly less than one-

third compared to ibuprofen (a commonly used of this group of drugs). Its safety was proved to be acceptable 

under using conditions and does not affect glucose metabolism (Anderson, 2005; Stanley, 2004). Chondroitin 

sulfate is a sulfated glycosaminoglycan which is a long, unbranched chain made from a repeating disaccharide 

structure of glucuronic acid and N-acetyl galactosamine residues. It is also one of the most widely used 

compounds in managing osteoarthritis patients. Chondroitin sulfate has been shown to change the death process 

of a chondrocyte, improving the anabolic/catabolic balance of the extracellular cartilage matrix, reducing some 

proinflammatory and catabolic factors and, in subchondral bone osteoblasts, increasing the ratio of OPG/ RANKL 

(Martel-Pelletier, 2005).  

Moreover, the long-term administration of oral CS is proved to be safe, well-tolerated, and fully indicated to 

control the pain symptoms and increase the mobility of knee osteoarthritis patients (Uebelhart, 2008). Ultra-

performance liquid chromatography (UPLC) has become one of the most frequently applied approaches in fast 

chromatographic separations (Liang, 2010).  

Hydrophilic interaction liquid chromatography (HILIC) uses traditional polar stationary phases, commonly 

applied to determinate polar compounds, such as pharmaceuticals, biopharmaceuticals, organic contaminants in 

environmental samples, and food components in foods (Redón, 2020). Pre-column derivatization is an efficient 

method that produces stable and highly fluorescent derivatives for analysis, and the most commonly used 

derivatization reagent for this purpose is 9fluorenylmethyloxycarbonyl chloride (FMOC-Cl) (Jámbor, 2009; 

Molnár-Perl, 2011). However, these methods all take a long time and have complex processing.   

There have been many methods to quantify glucosamine and chondroitin on different subjects by many techniques 

such as electrophoresis, high-performance liquid chromatography combined with different detectors (HPLC-RID, 

UV, PDA, FD), MS/MS) (Harmita, 2017; Huang, 2011; Kosman, 2017). In the United States Pharmacopoeia 

(USP, 40), glucosamine and chondroitin are quantified separately with two methods, PDA probe high-

performance liquid chromatography, and potentiometric titration, respectively. While in the British 

Pharmacopoeia (BP, 2016), quantification of glucosamine and chondroitin by potentiometric titration (USP, 40). 

According to Vietnam Pharmacopoeia V (VMPH, 2018), glucosamine is determined by liquid chromatography 

with UV probe, aminopropylsilyl silica gel column, but chondroitin still does not have a process for quantification. 

However, these methods all take a long time and have complex processing. Therefore, this study aims to establish 

a quantitative and straightforward glucosamine chondroitin process, less time analysis, by UPLC-TUV and pre-

column derivatization with FMOC-Cl.  

2. Materials and Methods   

2.1. Chemicals and materials  

The reference samples of glucosamine hydrochloride (≥ 99%) and chondroitin sulfate (≥ 99%) were purchased 

from Sigma  

(St. Louis, MO, USA). Fluorenylmethyloxycarbonyl chloride derivatization reagents were purchased from 

Sigma-Aldrich (St. Louis, MO, USA). Methanol, acetonitrile, and HPLC-grade water were acquired from 

Honeywell (North Carolina, USA). Ammonium acetate, acid formic, sodium hydroxide, sodium borate was 



Global Research Journal of Management and Social Sciences (GRJMSS) Vol. 13 (12) 
 

pg. 20 

purchased from Merck (Darmstadt, Germany). The tablets contain two active ingredients: glucosamine and 

chondroitin.  

2.2. Instrumentation  

A UPLC Acquity H-Class system (Waters Corporation, Massachusetts, USA) equipped with a TUV detector. 

2.3. Methods  

According to Qian et al. (2013) and the structures and polarities of analytes, chondroitin was quantified by HILIC 

column, and glucosamine was determined by UPLC-TUV with a C18 column after derivatization with 9-

fluorenylmethyloxycarbonyl chloride (Michel, 2004; Qian, 2013). The chromatographic parameters, including 

elution solvents, mobile phase ratios, detective wavelengths, flow rates, and column temperatures, were 

investigated. Meanwhile, the FMOC-Cl derivatization conditions have optimized reaction temperatures (30 °C, 

50 °C, and 70 °C).   

Preparation of stock solutions. Primary stock solutions of glucosamine and chondroitin are prepared with 

methanol at 1000 μg/mL. Working solutions for analysis and validation are suitably diluted from stock 

solutions.   

Preparation of reagent solutions. FMOC-Cl is dissolved in acetonitrile at 1500 μg/mL.  

Methods validation. According to ICH guidelines, the proposed method was validated for selectivity, linearity, 

the limit of detection (LOD), quantification (LOQ), precision, and accuracy. 

3. Results and Discussion  

3.1. Methods development  

The high polarity of the determined compounds made it challenging to optimize chromatographic separation 

(Huang, 2006; Václavíková, 2015). On the other hand, the molecular weight of glucosamine and chondroitin is 

also a problem for HPLC quantification. Establishing and optimizing the analysis process, therefore, becomes 

much more necessary. According to Government Chemist (2012), chondroitin determination has several typical 

approaches, such as high-performance liquid (HPLC) methods based on size exclusion and using amine column 

or ion exchange HPLC. Meanwhile, glucosamine also has been researched by an extensive range of methods like 

gas chromatography, liquid chromatography-tandem mass spectral, and one of the most effective methods that 

have been reported is high- performance chromatography method involving derivatization with FMOC-Cl 

(Huang, 2006).  

3.1.1. Chondroitin quantification  

The survey results showed that aliquots of the process samples (10 μL) were injected into column-a BEH HILIC 

column (2.1 × 100 mm, 1.7 μm) with mobile phase, a 50:50 mixture of acetonitrile and 5 mM ammonium acetate 

(pH 8), was delivered at 0.25 mL/min and samples were detected at a wavelength of 255 nm. This mobile phase 

mixture brings back an exemplary chromatogram of chondroitin, which is an excellent and symmetrical peak.   



Global Research Journal of Management and Social Sciences (GRJMSS) Vol. 13 (12) 
 

pg. 21 

 
  

Figure 1. Chondroitin chromatogram (Mobile phase: acetonitrile: 5 mM ammonium acetate (pH 8) (50:50))  

 
  

Figure 2. Glucosamine chromatogram under optimal conditions 

3.1.2. Glucosamine determination  

The following factors were investigated: reaction temperature of derivatization, quantitative wavelength, and 

mobile phase ratio, respectively. Depending on the stability of glucosamine-FMOC-Cl over time and a result 

shown on the chromatogram, reaction temperature has been chosen. The maximum absorption peak of the derived 

glucosamine is 259 nm; conducting a survey, at 255 nm, the derived peak signal is good, with little background 

noise, so the quantitation wavelength is 255 nm. The chosen mobile phase was also accepted as a reasonable and 

symmetrical peak requirement.  

 The optimal chromatographic conditions for glucosamine were selected based on the research of Huang (2006), 

the investigation factor of derivatization and quantification has been chosen. The results showed the most potential 

condition is using the indirect method of pre-column derivatization by hydrolysis with alkaline agents and FMOC-

Cl reagent at the reaction temperature of 50 °C, quantified on the TUV probe UPLC system with the Acquity 

UPLC BEH C18 column (2.1 × 100mm, 1.7 μm); detection wavelength 255 nm; flow rate 0.25 mL/min; sample 

injection volume 10 μL. Mobile phase A is acetonitrile; mobile phase B is water. The mobile phase ratio was 

adjusted according to the gradient elution program [time (minutes)/%A] as follows 0-2.5/35,  

2.5-4/60.  



Global Research Journal of Management and Social Sciences (GRJMSS) Vol. 13 (12) 
 

pg. 22 

 
  

Figure 3.  Chromatogram (1) reactive solvent, (2) unreacted glucosamine sample (3) sample solvent, (4) mobile 

phase sample 

3.2. System suitability testing  

System suitability testing was assessed by analyzing six replicates of glucosamine - FMOC-Cl standard at 25 

g/mL and chondroitin standard at 300 µg/mL. The results in table 1 show that the RSD% of the retention time 

and the peak area are both less than 2%. The chromatographic parameters are within the acceptable range: from 

0.8 to 1.5 for asymmetry factor (As) and higher than 1.5 for resolution (Rs). Consequently, instrumentation quality 

is acceptable for research.  

Table 1. System suitability 

testing  

     

  tR  S  As  Rs  k’  

Glucosamine  Mean  2.24  17437.5  1.45  3.8  1.49  

RSD%  0.4  0.21  1.23  1.2  0.86  

Chondroitin  Mean  0.69  1400.2  1.39  2.5  0.75  

RSD%  1.58  0.32  1.45  0.87  0.67  

3.3. Selectivity  

Figure 3-5 shows that sample chromatograms had peaks of glucosamine-FMOC-Cl and chondroitin with retention 

times equivalent to the prominent peaks in the standard chromatograms. No significant interference from mobile 

phase, solvent, underivatized glucosamine, and reaction solvent was observed at the retention time of analytes. 

The procedure for quantifying glucosamine and chondroitin meets the requirements for selectivity.  

 



Global Research Journal of Management and Social Sciences (GRJMSS) Vol. 13 (12) 
 

pg. 23 

Figure 4. Chromatogram of glucosamine-FMOC-Cl in the standard sample (1), testing sample (2), and spiked 

sample (3) 

 
  

Figure 5. Chromatogram of mobile phase (1), solvent (2), testing sample (3), chondroitin standard sample (4), 

spiked sample (5)  

3.4. Linearity  

The investigation of linearity is over the concentration range of 10-100 µg/mL for Glucosamine- FMOC-Cl and 

50-1000 µg/mL for chondroitin. The correlation coefficients during the validation were 0.999 and 0.998, 

respectively.  

Table 2. Linearity of glucosamine-FMOC-Cl and 

chondroitin  

  Linearity 

range 

(µg/mL)  

Regression equations, 

r2  

Glucosamin- 

FMOC-Cl  

10-100  y = 668.46x , r2 = 

0.999  

Chondroitin  50-1000  y = 6.42x, r2 = 0.998  

3.5. Precision  

The method's precision was verified by evaluating the intra-day and inter-day precisions. The relative standard 

deviation (%RSD) was selected to measure precision. The intra-day precision was examined by analyzing six 

samples in a single day, while the inter-day precision was determined by analyzing six samples each day for three 

days. The precision results were shown in Table 3, indicating that the overall intra- and inter-day variations 

(%RSD) were less than 2% (1.50% and 0,79%, respectively). 

Table 3. Intra-day and Inter-day precision of glucosamine-

FMOC-Cl (n = 6)  

   

Intra-day   Inter-day    

Sample  
Peak area 

(mAu.s)  
Content (mg)  

Peak area 

(mAu.s)  

  Content 

(mg)  

 

Day 1  Day 2  Day 3  Day 1  Day 2  Day 3  

1  11948  491.653  11948  11651  11472  491.65  477.94  469.75  

2  11657  478.256  11657  11439  11401  478.25  468.25  466.51  



Global Research Journal of Management and Social Sciences (GRJMSS) Vol. 13 (12) 
 

pg. 24 

3  11586  475.140  11586  11526  11484  475.14  472.24  470.31  

4  11429  467.662  11429  11541  11462  467.66  472.93  469.28  

5  11634  477.321  11634  11554  11446  477.32  473.52  468.57  

6  11539  472.959  11539  11521  11479  472.96  471.99  470.06  

Mean  11632.17  477.165  11542.72    473.01   

RSD%  1.50  1.685  0.79    0.88   

 

3.6. Accuracy   

Spiked samples evaluated the accuracy at three concentrations of 80, 100, 120 g/mL for glucosamine-FMOC-Cl 

and chondroitin. Each concentration was analyzed in duplicate three times. The mean recoveries of glucosamine-

FMOC-Cl and chondroitin are 101.5% (RSD = 1.07%) and 100.59% (RSD = 0.34%), respectively, which meet 

the requirement of ICH guidelines. The results are shown in Table 4. The validation parameters followed the ICH 

(2005) criteria, making this study potentially a widely used procedure for determining glucosamine and 

chondroitin. 

Table 4. Accuracy of glucosamine-FMOC-Cl and 

chondroitin (n = 6)   

 

  Concentration 

μg/mL  

Recovery 

(%)  

RSD %  

Glucosamine- FMOC-

Cl  

80  100.67  2.7  

100  101.78  0.25  

120  102.05  0.19  

Chondroitin  80  100.57  0.32  

100  100.05  0.59  

120  101.15  0.11  

  

3.7. Application  

The quantitative procedure after good validation was applied to quantify glucosamine and chondroitin in two 

tablets containing glucosamine and chondroitin simultaneously. The quantitative results are presented in Table 5, 

which shows that two samples do not meet the content requirement. This result brings back a reminder of the 

necessity to have the quantification process for the quality control of the functional foods sold in the market.  

  

Table 5. Application study  

Producer  Content of analytes (%)*  

Glucosamine  Chondroitin  

Company 

A  

99.7 ± 0.88  99.5 ± 1.18  

Company 

B  

31.67 ± 0.28  79.9 ± 0.87  

*: The mean values ± SD (n = 3)  



Global Research Journal of Management and Social Sciences (GRJMSS) Vol. 13 (12) 
 

pg. 25 

4. Conclusion  

A procedure for glucosamine quantification by pre-column derivatization with FMOC-Cl reagent on UPLC 

Acquity H-Class Waters system - TUV probe, with C18 (2.1 × 100 mm, 1.7 μm), mobile phase consisting of 

ACN – water with gradient elution program and a process to quantify chondroitin by UPLC/TUV system, with 

BEH HILIC column (2.1 × 100 mm, 1.7 μm), mobile phase consisting 50:50 ACN – 5 mM ammonium acetate 

(pH 8) were researched. Both procedures were validated according to ICH guidelines to fully meet the 

requirements of a quantitative analytical procedure, including selectivity, linearity, accuracy, and precision. 

Experiment for quantifying glucosamine and chondroitin in dietary supplements was proved to be a potential 

application and pharmaceuticals.   

Acknowledgment   

An Acknowledgements section is optional and may recognize those individuals who provided help during the 

research and preparation of the manuscript.   

Conflict of interest. The authors declare that there are no conflicts of interest regarding the publication of this 

paper.  

ORCID ID   

Ngoc-Van Thi Nguyen: https://orcid.org/0000-0002-7397-4071  

Kim-Ngan Huynh Nguyen: https://orcid.org/0000-0001-9914-9224    

References   

Government Chemist (2012). A review of methods available for the determination of chondroitin sulpahte in 

supplement. Middlesex, TW11 0LY, pp1-5.  

Anderson, J.W., Nicolosi, R.J. & Borzelleca, J.F. (2005). Glucosamine effects in humans: a review of effects on 

glucose metabolism, side effects, safety considerations and efficacy. Food and Chemical Toxicology, 

43(2), 187-201.  

British Pharmacopoeia (BP, 2016). 1091-1093.  

Harmita, H., Jatmika, C. & Nugraha, M.I. (2017). Determination of levels of glucosamine hydrochloride and 

chondroitin sulfate in mixtures in tablet and cream forms using high-performance liquid chromatography 

with fluorescence. International Journal of Applied Pharmaceutics, 9, 144-149.  

Huang, T.M., Deng, C.H., Chen, N.Z., Liu, Z. & Duan, G.L. (2006). High performance liquid chromatography 

for the determination of glucosamine sulfate in human plasma after derivatization with 9‐

fluorenylmethyl chloroformate. Journal of Separation Science, 29(15), 2296-2302.  

Huang, R., Pomin, V.H. & Sharp, J.S. (2011). LC-MS n analysis of isomeric chondroitin sulfate oligosaccharides 

using a chemical derivatization strategy. Journal of the American Society for Mass Spectrometry, 22(9), 

1577-87.  

Guideline, I.C.H. (2005). Validation of Analytical Procedures: Text and Methodology. Q2 (R1), 1(20), 05.  

Jámbor, A. & Molnár-Perl, I. (2009). Quantitation of amino acids in plasma by high performance liquid 

chromatography:   Simultaneous deproteinization and derivatization with 9-fluorenylmethyloxycarbonyl 

chloride. Journal of Chromatography A, 1216(34), 6218-6223.  

https://orcid.org/0000-0002-7397-4071
https://orcid.org/0000-0002-7397-4071
https://orcid.org/0000-0002-7397-4071
https://orcid.org/0000-0002-7397-4071
https://orcid.org/0000-0002-7397-4071
https://orcid.org/0000-0002-7397-4071
https://orcid.org/0000-0002-7397-4071
https://orcid.org/0000-0002-7397-4071
https://orcid.org/0000-0002-7397-4071
https://orcid.org/0000-0001-9914-9224
https://orcid.org/0000-0001-9914-9224
https://orcid.org/0000-0001-9914-9224
https://orcid.org/0000-0001-9914-9224
https://orcid.org/0000-0001-9914-9224
https://orcid.org/0000-0001-9914-9224
https://orcid.org/0000-0001-9914-9224
https://orcid.org/0000-0001-9914-9224
https://orcid.org/0000-0001-9914-9224


Global Research Journal of Management and Social Sciences (GRJMSS) Vol. 13 (12) 
 

pg. 26 

Kosman, V.M., Karlina, M.N., Pozharitskaya, O.N., Shikov, A.N. & Makarov, V.G. (2017). HPLC determination 

of glucosamine hydrochloride and chondroitin sulfate, weakly absorbing in the near UV region, in various 

buffer media. Journal of Analytical Chemistry, 72(8), 879-885.  

Liang, X., Zhang, L., Zhang, X., Dai, W., Li, H., Hu, L. & Zhang, W. (2010). Qualitative and quantitative analysis 

of traditional Chinese medicine Niu Huang Jie Du Pill using ultra performance liquid chromatography 

coupled with tunable UV detector and rapid resolution liquid chromatography coupled with time-of-flight 

tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis, 51(3), 565-571.  

Martel-Pelletier, J., Lajeunesse, D. & Pelletier, J.P. (2005). Etiopathogenesis of osteoarthritis, In: Koopman, W.J. 

& Moreland, L.W. (eds.), Arthritis & Allied Conditions. A Textbook of Rheumatology, Lippincott, 

Williams & Wilkins: Baltimore, pp. 21992226.  

Michel, G., Pojasek, K., Li, Y., Sulea, T., Linhardt, R. J., Raman, R. & Cygler, M. (2004). The structure of 

chondroitin B lyase complexed with glycosaminoglycan oligosaccharides unravels a calcium-dependent 

catalytic machinery. Journal of Biological Chemistry, 279(31), 32882-32896.  

Molnár-Perl, I. (2011). Advancement in the derivatizations of the amino groups with the o-phthaldehyde-thiol and 

with the 9- fluorenylmethyloxycarbonyl chloride reagents. Journal of Chromatography B, 879(17-18), 

1241-1269.  

Qian, S., Zhang, Q., Wang, Y., Lee, B., Betageri, G. V., Chow, M. S., ... & Zuo, Z. (2013). Bioavailability 

enhancement of glucosamine hydrochloride by chitosan. International Journal of Pharmaceutics, 455(1-

2), 365-373.  

Redón, L., Subirats, X. & Rosés, M. (2020). HILIC characterization: Estimation of phase volumes and 

composition for a zwitterionic column. Analytica Chimica Acta, 1130, 39-48.  

Stanley, R. & Mohler, M.D. (2004). Prompt treatment may minimize knee problems and their interference with 

flight duties– and daily routines. Human Factors & Aviation Medicine, 51(4), 1-7.  

Uebelhart, D. (2008). Clinical review of chondroitin sulfate in osteoarthritis. Osteoarthritis and Cartilage, 16, 

S19-S21.  United States Pharmacopoeia (USP, 40). 7019-1021.  

Václavíková, E. & Kvasnička, F. (2015). Quality control of chondroitin sulphate used in dietary supplements. 

Czech Journal of Food Sciences, 33(2), 165-173.  

Vietnam Ministry of Public Health (VMPH, 2018). Vietnam Pharmacopoeia V, Medical Publishing House, 454-

455.  

 


