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). 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