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Green Analytical Method for Simultaneous Determination of Glucosamine and Calcium in Dietary Supplements by Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection

Green Analytical Method for Simultaneous Determination of Glucosamine and Calcium in Dietary... Hindawi Journal of Analytical Methods in Chemistry Volume 2023, Article ID 2765508, 10 pages https://doi.org/10.1155/2023/2765508 Research Article Green Analytical Method for Simultaneous Determination of Glucosamine and Calcium in Dietary Supplements by Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection 1 1,2 1 1,3 Yen Nhi Do, Thi Lan Phuong Kieu, Thi Huyen My Dang, Quang Huy Nguyen, 2 2 4 4 4 Thu Hien Dang, Cao Son Tran , Anh Phuong Vu, Thi Trang Do, Thi Ngan Nguyen, 4 1 1 1 Son Luong Dinh, Thi Minh Thu Nguyen , Thi Ngoc Mai Pham, Anh Quoc Hoang, 1 1 Bach Pham , and Thi Anh Huong Nguyen Faculty of Chemistry, University of Science, Vietnam National University, 19 Le Tanh Tong, Hanoi 10000, Vietnam National Institute for Food Control (NIFC), 65 Pham Tan Duat, Hanoi 10000, Vietnam Faculty of Pharmacy, University of Medicine and Pharmacy, Tai Nguyen University, 284 Luong Ngoc Quyen, Tai Nguyen 24000, Vietnam Poison Control Center, Bach Mai Hospital, 78 Giai Phong, Hanoi 10000, Vietnam Correspondence should be addressed to Bach Pham; phamgiabach@hus.edu.vn and Ti Anh Huong Nguyen; nguyenthianhhuong@hus.edu.vn Received 9 August 2022; Revised 25 September 2022; Accepted 24 November 2022; Published 31 January 2023 Academic Editor: Antony C. Calokerinos Copyright © 2023 Yen Nhi Do et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Te need for analytical methods that are fast, afordable, and ecologically friendly is expanding. Because of its low solvent consumption, minimal waste production, and speedy analysis, capillary electrophoresis is considered a “green” choice among analytical separation methods. With these “green” features, we have utilized the capillary electrophoresis method with capacitively 4 2+ coupled contactless conductivity detection (CE-C D) to simultaneously determine glucosamine and Ca in dietary supplements. Te CE analysis was performed in fused silica capillaries (50 μm inner diameter, 40 cm total length, 30 cm efective length), and the 2+ analytical time was around 5 min. After optimization, the CE conditions for selective determination of glucosamine and Ca were obtained, including a 10 mM tris (hydroxymethyl) aminomethane/acetic acid (Tris/Ace) bufer of pH 5.0 as the background electrolyte; separation voltage of 20 kV; and hydrodynamic injection (siphoning) at 25 cm height for 30 s. Te method illustrated 2 2+ good linearity over the concentration range of 5.00 to 200 mg/L of for glucosamine (R � 0.9994) and 1.00 to 100 mg/L for Ca 2 2+ (R � 0.9994). Under the optimum conditions, the detection limit of glucosamine was 1.00 mg/L, while that of Ca was 0.05 mg/L. 2+ Te validated method successfully analyzed glucosamine and Ca in seven dietary supplement samples. Te measured con- centrations were generally in line with the values of label claims and with cross-checking data from reference methods (HPLC and ICP-OES). can relieve osteoarthritis (OA) symptoms and restore ar- 1. Introduction ticular functions [2–5]. It also has many antiinfammatory Glucosamine (2-amino-2-deoxy-D-glucose) is an amino and antioxidative efects with minimal adverse impact on monosaccharide with essential roles in the biochemical human health [6–10]. Tus, glucosamine is suggested for synthesis of glycosylated proteins and lipids [1]. Intensive relieving pain and preventing or slowing the breakdown of studies have proved that the exogenous use of glucosamine cartilage in OA by the Osteoarthritis Research Society 2 Journal of Analytical Methods in Chemistry International (OARSI) [11]. Te European League Against the WaterPro RO system (Labconco Cooperation, Kansas, Rheumatism (EULAR) recommended glucosamine as MO, USA). a treatment for knee OA [12]. Te United States Food and Both glucosamine and calcium nitrate were purchased Drug Administration (FDA) also proposed glucosamine as from Merck KGaA (Germany). Te stock solutions of 2+ a dietary supplement used to manage and treat OA [13]. glucosamine (1000 mg/L) and Ca (1000 mg/L) and stan- Because of its benefts, glucosamine products are one of dard solutions of calibration curves were prepared with the most popular over-the-counter (OTC) dietary supple- deionized water in volumetric fasks and stored at 4 C before ments worldwide. Besides its use as a single supplement in further use. the form of glucosamine hydrochloride or glucosamine L-arginine (Arg), N-cyclohexyl-3-amino- sulfate, it can be mixed with other nutraceuticals, vitamins, propanesulfonic acid (CAPS), L-histidine (His), and tris and minerals. Among them, calcium is commonly combined (hydroxymethyl) aminomethane (Tris) used for background with glucosamine because of its crucial role in preventing electrolyte (BGE) and sample preparation were purchased osteoporosis [14]. Given that there are diferent combina- from Sigma–Aldrich (Singapore). Otherwise, acetic acid tions of glucosamine and other supplements, more than one (Ace) and trichloroacetic acid (TCA) were purchased from method is required to analyze these products. For example, Merck KgaA, Germany. Te BGEs were prepared by adding the most popular method for glucosamine analysis is Ace into Arg, Tris, His, and CAPS solutions, followed by pH reversed-phase high-performance liquid chromatography adjustment with acetic acid using a HI 2215 pH meter (RP-HPLC), coupled with various detectors like ultraviolet, (Hanna Instruments, Woonsocket, RI, USA). electrochemical, and mass spectrometry [15–19]. However, Fused silica capillaries (50μm i.d., 365μm OD) were this method is not suitable for analyzing minerals like purchased from BGB Analytik AG (Bockten, ¨ Switzerland). calcium, which have commonly been determined by in- Before use, the capillaries were conditioned by fushing with ductively coupled plasma mass spectrometry (ICP-MS) and 1 M NaOH solution, deionized water, and then the BGE. inductively coupled plasma optical emission spectrometry (ICP-OES), or fame atomic absorption spectrometry (F- 2.2. Sample Preparation. Dietary supplement samples, in- AAS). Due to the complexity of current methods for the cluding tablets, and hard capsules, were purchased from quantifcation of glucosamine products, there is a need to pharmacies in Hanoi, Vietnam. Te sample preparation develop a simple technique to simultaneously determine the procedure was described in previous studies [34, 35]. Briefy, amounts of glucosamine and other components in the food each sample was prepared with the contents of 20 whole supplement. tablets or capsules. Te tablets were ground into a fne Capillary electrophoresis (CE) can be considered a more powder using a ceramic mortar and pestle. Te hard capsules economical alternative for developing simple and cheap were opened to collect powder; the samples were ground if analytical techniques. CE instruments are coupled with necessary. Te sample was accurately weighed (about 0.5 g) various detection methods such as ultraviolet, laser-induced before being transferred into a 25-mL volumetric fask; 4% fuorescence, mass spectrometry, voltammetry, and TCA was added to the mark. Te fask was ultrasonicated for amperometry. CE coupled with capacitively coupled con- 30 min by an ultrasonic vibrator bath (BRANSON 521) and tactless conductivity detection (C D) has recently been centrifuged at 3000 rpm for 15 min. After centrifugation, the highlighted as an economical and efcient method for en- aqueous phases were collected and passed through a 0.45-μm vironmental monitoring, [20–22], food control [23–25], syringe flter. Te fltrated solution was transferred to forensics [26], and clinical analysis [27–29]. Te working a 25 mL volumetric fask and flled to the mark with 4% TCA. principles and applications of CE-C D have been reviewed Te solution could be diluted with deionized water (if by Hauser and Kuba´nˇ [30] and others [29, 31–33]. Tese necessary) before analysis by CE-C D. Concentrations of techniques have been applied to analyze inorganic, organic 2+ glucosamine and Ca were determined by using the ions, and biomolecules. Because of the ability to analyze the standard addition method. broad range of targets, we have developed a protocol to simultaneously quantify both glucosamine and calcium in dietary supplements by CE-C D. A simple procedure has 4 2.3. Instrument. Te in-house CE-C D instrument was pre- been reported, with cross-checking data using standard sented in the previous studies [30, 36, 37]. Generally, the high-performance liquid chromatography with a fuores- compact CE instrument was built in-house using high voltages cence detector (HPLC-FLD) and ICP-OES methods being of a maximum of 25 kV (UM25∗4, 12 V, Spellman, Pul- provided to prove the reliability of the CE-C D results. We borough, UK) and combined with a commercial C D detector also applied this protocol for the quality control of seven (ER815, eDAQ, Denistone East, NSW, Australia). Te system dietary supplements available in Vietnam. was operated with a 220-VAC power supply. Te silica capillary had a total length (L ) and efective length (L ) of 40 cm and tot ef 2. Materials and Methods 30 cm, respectively, and an inner diameter of 50μm. 2.1. Materials. All chemicals used were of analytical grade and were used as received without any further purifcation. 2.4. Method Development and Validation. Analytical pa- All solutions were prepared with deionized water of re- rameter optimization followed strategies in our earlier works −1, sistivity not less than 18.2 MΩ cm which was purifed by [26, 34, 35, 38, 39]. Overall, various factors of BGEs Journal of Analytical Methods in Chemistry 3 (composition, pH, and concentration), separation voltages, bufer when this compound passes the detector. Although all 2+ and injection conditions (siphoning height and injection BGE systems give a sharp peak of Ca , only the Tris/Ace time) were investigated to develop an efective CE-C D solution provides a high signal of glucosamine (Figure 1(a)). method for the simultaneous determination of glucosamine Other BGE solutions cause low peak signals for glucosamine, 2+ and Ca . A single analytical parameter was changed at leading to insufcient sensitivity. Tus, the BGE system a time while fxing all the others. Te background noise, containing Tris/Ace was selected because of the sufcient 2+ signal, and migration times of analytes were calculated from peak signal for Ca and glucosamine. the CE electropherograms to identify the optimized con- After selecting the BGE composition, the pH of the so- dition (Tables S1–S6). Because background noise may lution was further investigated (Figure 1(b)). Tis factor change during the recording of an electropherogram, the signifcantly infuences the glucosamine signal. In general, 2+ average noise was measured near the analyte peak regions. both peaks of Ca and glucosamine were far from the EOF For a reproducible and accurate measurement, the signal’s signals at all fve pH conditions (pH 6.5–4.5). Te high pH peak area was used in all this study’s steps. After comparing declines the migration time of both analyzes because of the rise the analytes’ signals, background noise, and retention time in the EOF mobility. At pH 6.0 and higher, the peak separation under diferent analytical conditions, the optimized CE-C D of glucosamine appears, possibly due to the conversion of glucosamine forms at diferent pHs. While β-anomer is conditions are summarized in Table S7. Based on optimized CE-C D conditions, the analytical dominant for nonprotonated neutral glucosamine, the pro- method was validated for linearity, detection and quantif- tonated glucosamine mainly exists as α-anomer [45–47]. Te cation limits, repeatability, and recovery by using the ana- two peaks of glucosamine are more resolved in higher pH lytical results of standard solutions and matrix-spike conditions. Two isomers of glucosamine also generate two samples. In pharmaceutical analysis, guidelines from the peaks in HPLC analysis; thus, the sum of the area of these two International Council for Harmonization (ICH) and peaks should be used to quantify the glucosamine [15, 48, 49]. EURACHEM are commonly used to calculate the limit of Te pH of 4.5 to 5.5 is suitable to accurately detect total detection (LOD) [40, 41]. Tey defne the LOD as the glucosamine by CE-C D since glucosamine generates a single concentration of analyte that generates a signal at least peak at these conditions, leading to a more straightforward 3 times the signal noise of the baseline (S/N � 3); for LOQ, calculation. All three pH conditions gave the stable baseline the S/N ratio equals 10. Tus, in this study, to determine the and the practical time for bulk analysis (Table S2). Te content 2+ LOD of each analyte (calcium or glucosamine), a standard of Ca is much lower than that of glucosamine in commercial 2+ solution was repeatedly diluted until its signal was 3 times supplements; thus, a condition that shows the best Ca signal higher than the background noise (S/N � 3). Te LOQ value should be chosen for the simultaneous determination of both of each analyte was measured by repeat dilution until the analytes in supplement samples. Among all pH conditions, the 2+ signal of the diluted solution was 10 times higher than the pH of 5.0 exhibited the highest peak area of Ca signals and background noise (S/N � 10). All LOD/LOQ measurements a single peak of glucosamine. Accordingly, a pH of 5.0 was were conducted under optimum conditions in Table S7. chosen for further studies. Cross-check analysis was performed for both sample types Tris concentration (from 8 to 20 mM) was optimized for 2+ (i.e., tablets and capsules). Glucosamine was analyzed with simultaneous determination of glucosamine and Ca HPLC coupled with a fuorescence (FLD) detector, while (Figure 1(c) and Table S3). Generally, the increase in Tris 2+ ICP-OES detected Ca at the National Institute for Food concentration shows a minor efect on the migration times 2+ Control (NIFC), Hanoi, Vietnam, according to methods of glucosamine and Ca . In the capillary tube, a high reported elsewhere [42, 43]. All measurements generally concentration of ions causes a change in the magnitude of followed the AOAC Ofcial Method 2016.14. Detailed in- the electric double layer; therefore, the sample moves un- formation about the reference methods was listed in Table S8. evenly, causing background noise, poor separation, and unbalanced peaks. Although high concentrations of Tris 3. Results and Discussion (≥10 mM) in BGE provide a stable baseline and balanced peaks, the intensity of the glucosamine peak declines at high 3.1. Optimization of CE-C D Analytical Conditions Tris concentration, decreasing the sensitivity. Tus, com- pared to other conditions, the 10 mM Tris/Ace electrolyte 3.1.1. Efects of Background Electrolytes. Te background solution was selected for further investigations because of electrolytes (BGEs) are one of the most critical factors in CE- both analytes’ stable baselines and balanced peaks. C D method development [44]. In this work, we investigated four standard BGE systems, including Arg/Ace, His/Ace, 3.1.2. Efects of Separation Voltages. Various applied volt- Tris/Ace, and CAPS/Ace (Figure 1(a), Table S1). Te initial concentrations of the Arg, His, Tris, and CAPS solutions ages from 10 to 25 kV were then investigated to evaluate their impacts on electropherograms of glucosamine and were 10 mM. All solutions were frst adjusted to a pH of 5.0 2+ by acetic acid. In four BGE systems, both glucosamine and Ca (Figure 2). Firstly, higher voltages resulted in shorter 2+ migration times and sharper peaks, but higher background Ca were eluted before the electroosmotic fow (EOF) signal, indicating that the two analytes have positive charges noise was observed. Voltages of 10 or 15 kV can lead to 2+ broadening peaks of Ca and glucosamine, which could under all studied conditions. Negative-going peaks of glu- cosamine show a conductivity reduction of the background cause false results when analyzing real sample matrices. 4 Journal of Analytical Methods in Chemistry 2+ 5 mV 5 mV 5 mV Ca 2+ 2+ Ca Ca Glucosamine Glucosamine 20 mM Glucosamine Tris/Ace pH 4.5 15 mM pH 5.0 EOF Arg/Ace pH 5.5 10 mM His/Ace pH 6.0 CAPS/Ace pH 6.5 8 mM 1.0 1.5 2.0 2.5 3.0 3.5 4.0 1.0 1.5 2.0 2.5 3.0 1.0 1.5 2.0 2.5 3.0 Migration time (min) Migration time (min) Migration time (min) (a) (b) (c) Figure 1: Optimization the BGE conditions for simultaneous determination of glucosamine and calcium. (a) Te efect of BGE com- positions. (b) Te efect of pH. (c) Te efect of Tris concentration. CE conditions: fused silica capillary (total length 40 cm, efective length 30 cm, ID 50μm); separation voltage +20 kV; hydrodynamic injection (siphoning at 25 cm in 30 s). Te selected conditions were highlighted in blue. 2+ Tese parameters control the sample amounts injected into Ca 5 mV the capillary, afecting the separation and detection ef- ciency. Peak areas are directly proportional to injection Glucosamine 10 kV times and siphoning heights. Although longer injection times and higher injection heights can lower detection limits, too large injected sample amounts may degrade the 15 kV peak shape and introduce elevated interferences. Consid- ering these facts, we injected samples at a siphoning height of 20 kV 25 cm in 30 s. 3.2. Method Validation 25 kV 3.2.1. Selectivity. Most commercial glucosamine supple- 0 1 2 3 4 5 ments combine chondroitin and methylsulfonylmethane Migration time (min) (MSM) to treat painful joints. Tus, the interference of chondroitin and MSM in the signals of glucosamine and Figure 2: Efects of separation voltages on the CE-C D analysis of 2+ Ca was investigated (Figure 4). Tere is no detectable glucosamine and calcium.BGE conditions: 10 mM Tris/Ace, pH 5.0, signal for chondroitin and MSM under experimental con- sample injection time of 30 s, sample injection height of 25 cm. Te selected condition was highlighted in blue. ditions. While a positive potential was applied to detect 2+ glucosamine and Ca (positive ions), chondroitin and MSM cannot form a positive charge under the studied condition. Moreover, a high separation voltage raises the background Chondroitin is a highly negative polysaccharide [50], and noise (Table S4). Te noise at +25 kV showed the highest MSM, an organosulfur compound, is considered chemically level, around 2.06 mV, while the background noise was only inert with a pKa of 28. Tus, it is apparent that we cannot 0.67 mV at +10 kV. On the other hand, the area of the peaks 2+ observe any signal of these two chemicals in the analyzing from glucosamine and Ca decreased at a high applied time (around 5 min). Moreover, the presence of chondroitin voltage (Table S4). Lastly, the high voltage (higher than and MSM showed no interference with the peak signals of 20 kV) could generate audible noise, especially in high 2+ glucosamine and Ca . Tus, the proposed method is humidity conditions in a tropical country like Vietnam. suitable for simultaneously determining glucosamine and Tus, it is recommended to use a low voltage in capillary 2+ Ca in commercial supplements. electrophoresis if possible for safety. Given these points, a separation voltage of 20 kV is optimal for simultaneously 2+ determining glucosamine and Ca . 3.2.2. Calibration Curves. A series of mixture standard solutions ranging from 5.00 mg/L to 200 mg/L and from 2+ 3.1.3. Efects of Injection Conditions. Next, hydrodynamic 1.00 mg/L to 100 mg/L of glucosamine and Ca , re- injection conditions, including injection times and si- spectively, were analyzed at optimized conditions to con- phoning heights, were studied (Figure 3, Tables S5 and S6). struct two calibration curves of two analytes (Figure 5). Te Journal of Analytical Methods in Chemistry 5 5 mV 5 mV 20 cm 20 s 25 s 25 cm 30 s 35 s 30 cm 0 1 2 3 4 0 1 2 3 4 Migration time (min) Migration time (min) (a) (b) Figure 3: Efects of injection conditions on the CE-C D analysis of glucosamine and calcium. (a) Injection time (siphoning at 25 cm). (b) Siphoning height (injection time 30 s). BGE conditions: 10 mM Tris/Ace electrolyte solution, pH of 5.0, separation voltage of +20 kV. Te selected conditions were highlighted in blue. 5 mV MSM Chondroitin 2+ Ca Glucosamine 2+ Ca +Glucosamine 2+ Ca 2+ Ca +Glucosamine+ Glucosamine MSM+Chondroitin 0 1 2 3 4 5 Migration time (min) Figure 4: Selective determination of glucosamine and calcium in the presence of chondroitin and MSM. Concentration of calcium was 10 mg/L while concentrations of glucosamine, chondroitin, and MSM were 100 mg/L. BGE conditions: 10 mM Tris/Ace electrolyte solution, pH of 5.0, separation voltage of +20 kV, sample injection time of 30 s, sample injection height of 25 cm. regression equations of the calibration curves represent analyte’s signal was 10 times higher than the background relationships between analyte concentrations (mg/L) and noise (Table S9). LOQs were estimated to be 3.30 mg/L for 2+ peak areas (mV.s). Good linearity was observed for both glucosamine and 0.17 mg/L for Ca . 2 2+ 2 glucosamine (R � 0.9994), and Ca (R � 0.9994) over the Te LOD of glucosamine obtained by our method was concentration ranges (Figure 5). Concentrations of analytes higher than that reported by Akamatsu and Mitsuhashi in analytical solutions (including real samples and standard (0.01 mg/L) [51]. However, their method required in-capillary addition samples) should be within the linear ranges. derivatization with o-phthalaldehyde, which may not be 2+ suitable for determining both Ca and glucosamine in that condition. Our LOD value was comparable to the LODs of 3.2.3. Limits of Detection and Quantifcation. Limits of other studies using CE and HPLC (0.25 to 1.0 mg/L) [52, 53]. 2+ detection (LODs) of glucosamine and Ca were determined Our method not only detects both analytes in a single run but using signal-to-noise ratios equal to 3 (S/N � 3). A standard also achieves low LOD and LOQ, which are compatible with solution of each analyte was repeatedly diluted until its signals pharmaceutical and nutraceutical analysis. were 3 times higher than the background noise measured near the peak area (S/N � 3) (Table S9). Te concentration of the analyte at S/N � 3 was recorded as the LOD value. LODs of 3.2.4. Repeatability. Standard solutions with three con- 2+ 2+ glucosamine and Ca were 1.00 and 0.05 mg/L, respectively. centration levels of glucosamine and Ca (20, 40, and For the LOQ calculation, we did a similar experiment until the 80 mg/L) were chosen for replication analysis (n � 5) for 6 Journal of Analytical Methods in Chemistry 2+ Ca 25 Glucosamine 200 S = 1.762 C + 3.226 S = 0.107 C + 0.219 peak Ca + peak glucosamine R = 0.9994 20 R = 0.9994 0 0 0 50 100 150 200 0 20 40 60 80 100 Concentration (mg/L) Concentration (mg/L) (a) (b) Figure 5: Calibration curves of glucosamine and calcium. BGE conditions: 10 mM Tris/Ace electrolyte solution, pH of 5.0, separation voltage of +20 kV, sample injection time of 30 s, sample injection height of 25 cm. 2+ repeatability evaluation (Table S4). For Ca , relative stan- and three hard capsules (Table 1 and Figure 6(a)). To verify if dard deviations (RSD) of peak areas ranged from 0.92% to signal peaks from samples were generated by glucosamine 2+ 2.16%. Otherwise, the RSD of the glucosamine peak areas and Ca , known amounts of standard solutions were added 2+ 4 ranged from 0.80% to 2.52%. Both glucosamine and Ca to the sample before analyzing by CE-C D (Figure 6(b)). Te migration times in standards had an RSD <2.5% at all intensities of both signals in the standard addition samples concentration levels. Tese method repeatability results increased compared to those in the samples. Te migration 2+ meet the standard precision criteria proposed by AOAC times of glucosamine and Ca peaks were similar between International for concentration ranges from 1 ppm (11%) to standard and standard addition samples. It is shown that the 100 ppm (5.3%) [54]. supplement additives have a minimal impact on the mi- gration time of both analytes. Tus, the content of glucos- 2+ amine and Ca in supplements could be determined using 3.2.5. Recovery. Recovery is an essential parameter in the standard addition method. Based on the peak area evaluating the method of accuracy. In this study, we used obtained and the standard addition equation, the content of 2+ a tablet sample without glucosamine and Ca (with no label calcium and glucosamine in the functional food samples will claim of these components and confrmed with reference be calculated. methods) to make matrix-spike samples. Tree spiking levels 2+ Te contents of Ca in the samples measured by our 2+ of glucosamine (40, 80, and 100 mg/L), and Ca (4, 8, and CE-C D method ranged from 7.82 to 55.43 mg per unit (e.g., 10 mg/L) were performed in triplicate. Te sample matrix tablet or capsule), with relative deviations compared to label (about 0.5 g) was spiked with a standard solution containing claims ranging from −7.62% to +5.20%. Te contents of 2+ glucosamine and Ca , equilibrated for at least 30 min at glucosamine ranged from 111.0 to 643.0 mg per unit, and room temperature, and treated according to the procedure measured-label deviations ranged from −5.26% to +8.10% 2+ described in Section 2.2. Recoveries of glucosamine and Ca (Table 1). Tere was an acceptable agreement between ranged from 96.9% to 102.0% and from 98.0% to 103.0%, measured and label claim contents because deviations are respectively (see Table S5). Tese recovery rates satisfy the less than ±10% in all samples. Te cross-checked analysis AOAC requirement for expected recovery at concentration was performed for all the samples (Table 1). Te relative levels from 10 ppm (80–100%) to 100 ppm (90–107%) [54], deviations between the results of CE and ICP-OES ranged indicating high accuracy with an insignifcant compound 2+ from −8.08% to +5.62% for Ca , and between CE and loss of our analytical procedure. HPLC, ranged from −5.93% to +6.47% for glucosamine. Good agreement between analyte contents measured by our 3.3. Method Application and Cross-Checked Results. Te method and the reference methods indicates that CE-C D validated method was applied to seven real samples collected could be an alternative tool for conventional methods such from pharmacies in Hanoi, Vietnam, including four tablets as HPLC and ICP-OES in specifc applications. S (mV.s) peak S (mV.s) peak Journal of Analytical Methods in Chemistry 7 2+ 4 Table 1: Determination of glucosamine and Ca in commercial dietary supplements from pharmacies in Vietnam, using CE-C D and HPLC, ICP-OES as the reference methods. 2+ Glucosamine Ca Types Label Sample no HPLC CE-HPLC Label 4 4 of pills CE-C D (mg/μ) (mg/ CE-LBL (%) CE-C D (mg/μ) ICP-OES (mg/μ) CE-LBL (%) CE-ICP (%) (mg/μ) (%) (mg/μ) μ) S1 Tablet 479.0 488.0 450.0 −1.84 6.44 26.30 25.00 24.90 5.20 5.62 S2 Tablet 494.0 457.0 464.0 8.10 6.47 41.90 40.00 44.80 4.75 −6.47 S3 Tablet 616.0 625.0 613.0 −1.44 0.49 36.60 35.00 36.90 4.57 −0.81 S4 Tablet 223.0 211.0 230.0 5.69 −3.04 48.50 50.00 47.50 −3.00 2.11 S5 Hard capsule 111.0 107.0 118.0 3.74 −5.93 49.40 48.00 47.70 2.92 3.56 S6 Hard capsule 324.0 342.0 344.0 −5.26 −5.81 7.82 8.00 7.80 −2.25 0.26 S7 Hard capsule 643.0 616.0 676.0 4.38 −4.88 55.43 60.00 60.30 −7.62 −8.08 CE-LBL: CE-label deviation the deviation of the result obtained with CE-C D from that indicated on the label; CE-LBL (%) � {(C − C )/C } × 100%. CE-HPLC: CE-HPLC deviation, CE-ICP : CE-ICP CE-C4D label label deviation; they are the deviation of the result obtained with CE-C D from that with the standard reference method (HPLC and ICP-OES); CE-HPLC (or CE-ICP) (%) � {(C − C )/C } - 100% mg/ CE-C4D reference reference μ : mg per unit (e.g., tablet or capsule). 8 Journal of Analytical Methods in Chemistry 2+ 10 mV Ca Glucosamine 2+ Ca S1 5 mV S2 Standard addition sample Glucosamine S3 S4 Sample S5 Standard S6 Blank S7 0 1 2 3 4 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Migration time (min) Migration time (min) (a) (b) Figure 6: Representative CE-C D electropherograms of (a) diferent supplement samples (Table 1). (b) Specifc peaks evaluated by blank, standard, sample, and standard addition sample. BGE conditions: 10 mM Tris/Ace electrolyte solution, pH of 5.0, separation voltage of +20 kV, sample injection time of 30 s, sample injection height of 25 cm. 4. Conclusions Supplementary Materials Te present study developed an analytical method for the Table S1. Te background noise, peak area, and migration 2+ 2+ simultaneous determination of glucosamine and Ca time of glucosamine and Ca at diferent BGE conditions. using CE-C D in commercial dietary supplements. Our Table S2: Te background noise, peak area, and migration 2+ method was demonstrated to be simple, fast, and cost- time of glucosamine and Ca at diferent pH conditions. efective with adequate validation parameters such as Table S3: Te background noise, peak area, and migration 2+ selectivity, linearity, detection limits, repeatability, and time of glucosamine and Ca at diferent Tris concentrations. 2+ recovery. Concentrations of glucosamine and Ca in Table S4: Te background noise, peak area, and migration 2+ supplement samples can be simultaneously determined in time of glucosamine and Ca at diferent seperation voltages. a single run analysis within fve min. Te validated Table S5: Te background noise, peak area, and migration 2+ method was applied to analyze samples, including tablets time of glucosamine and Ca at diferent injection time and hard capsules. Analytical results obtained by this CE- (siphoning at 25 cm). Table S6: Te background noise, peak 4 2+ C D method were in good agreement with labeling area, and migration time of glucosamine and Ca at diferent contents and those measured by reference methods (i.e., siphoning height (injection time 30 s). Table S7. Te optimal 2+ 4 HPLC-FLD for glucosamine and ICP-OES for Ca ). conditions of the CE-C D method for simultaneous de- 2+ Overall, our study has revealed another application of CE- termination of glucosamine and Ca . Table S8. Te optimal C D as an alternative green analytical tool for conven- conditions for determination of glucosamine by HPLC-FLD 2+ tional dietary supplement quality control methods, with and Ca by ICP-OES. Table S9. LOD and LOQ of glucos- 2+ the advantages of high efciencies, low sample and amine and Ca . Table S10. Te repeatability evaluation of electrolyte consumption, and minimal chemical waste. peak area (mV.s) and migration time (min) for simultaneous 2+ 4 Because one simple analytical procedure can be applied determination of glucosamine and Ca by CE-C D. Table for both analytes (organic compound and metal ion) with S11. Recoveries for simultaneous determination of glucos- 2+ 4 diferent properties, our method signifcantly reduced amine and Ca by CE-C D. (Supplementary Materials) analysis time and labor cost compared to using one conventional method for each analyte. Te proposed CE- 4 2+ References C D method for glucosamine and Ca analysis also provided reliable results compared to standard [1] N. R. Dostrovsky, T. E. Towheed, R. W. Hudson, and techniques. T. P. 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Green Analytical Method for Simultaneous Determination of Glucosamine and Calcium in Dietary Supplements by Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection

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10.1155/2023/2765508
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Hindawi Journal of Analytical Methods in Chemistry Volume 2023, Article ID 2765508, 10 pages https://doi.org/10.1155/2023/2765508 Research Article Green Analytical Method for Simultaneous Determination of Glucosamine and Calcium in Dietary Supplements by Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection 1 1,2 1 1,3 Yen Nhi Do, Thi Lan Phuong Kieu, Thi Huyen My Dang, Quang Huy Nguyen, 2 2 4 4 4 Thu Hien Dang, Cao Son Tran , Anh Phuong Vu, Thi Trang Do, Thi Ngan Nguyen, 4 1 1 1 Son Luong Dinh, Thi Minh Thu Nguyen , Thi Ngoc Mai Pham, Anh Quoc Hoang, 1 1 Bach Pham , and Thi Anh Huong Nguyen Faculty of Chemistry, University of Science, Vietnam National University, 19 Le Tanh Tong, Hanoi 10000, Vietnam National Institute for Food Control (NIFC), 65 Pham Tan Duat, Hanoi 10000, Vietnam Faculty of Pharmacy, University of Medicine and Pharmacy, Tai Nguyen University, 284 Luong Ngoc Quyen, Tai Nguyen 24000, Vietnam Poison Control Center, Bach Mai Hospital, 78 Giai Phong, Hanoi 10000, Vietnam Correspondence should be addressed to Bach Pham; phamgiabach@hus.edu.vn and Ti Anh Huong Nguyen; nguyenthianhhuong@hus.edu.vn Received 9 August 2022; Revised 25 September 2022; Accepted 24 November 2022; Published 31 January 2023 Academic Editor: Antony C. Calokerinos Copyright © 2023 Yen Nhi Do et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Te need for analytical methods that are fast, afordable, and ecologically friendly is expanding. Because of its low solvent consumption, minimal waste production, and speedy analysis, capillary electrophoresis is considered a “green” choice among analytical separation methods. With these “green” features, we have utilized the capillary electrophoresis method with capacitively 4 2+ coupled contactless conductivity detection (CE-C D) to simultaneously determine glucosamine and Ca in dietary supplements. Te CE analysis was performed in fused silica capillaries (50 μm inner diameter, 40 cm total length, 30 cm efective length), and the 2+ analytical time was around 5 min. After optimization, the CE conditions for selective determination of glucosamine and Ca were obtained, including a 10 mM tris (hydroxymethyl) aminomethane/acetic acid (Tris/Ace) bufer of pH 5.0 as the background electrolyte; separation voltage of 20 kV; and hydrodynamic injection (siphoning) at 25 cm height for 30 s. Te method illustrated 2 2+ good linearity over the concentration range of 5.00 to 200 mg/L of for glucosamine (R � 0.9994) and 1.00 to 100 mg/L for Ca 2 2+ (R � 0.9994). Under the optimum conditions, the detection limit of glucosamine was 1.00 mg/L, while that of Ca was 0.05 mg/L. 2+ Te validated method successfully analyzed glucosamine and Ca in seven dietary supplement samples. Te measured con- centrations were generally in line with the values of label claims and with cross-checking data from reference methods (HPLC and ICP-OES). can relieve osteoarthritis (OA) symptoms and restore ar- 1. Introduction ticular functions [2–5]. It also has many antiinfammatory Glucosamine (2-amino-2-deoxy-D-glucose) is an amino and antioxidative efects with minimal adverse impact on monosaccharide with essential roles in the biochemical human health [6–10]. Tus, glucosamine is suggested for synthesis of glycosylated proteins and lipids [1]. Intensive relieving pain and preventing or slowing the breakdown of studies have proved that the exogenous use of glucosamine cartilage in OA by the Osteoarthritis Research Society 2 Journal of Analytical Methods in Chemistry International (OARSI) [11]. Te European League Against the WaterPro RO system (Labconco Cooperation, Kansas, Rheumatism (EULAR) recommended glucosamine as MO, USA). a treatment for knee OA [12]. Te United States Food and Both glucosamine and calcium nitrate were purchased Drug Administration (FDA) also proposed glucosamine as from Merck KGaA (Germany). Te stock solutions of 2+ a dietary supplement used to manage and treat OA [13]. glucosamine (1000 mg/L) and Ca (1000 mg/L) and stan- Because of its benefts, glucosamine products are one of dard solutions of calibration curves were prepared with the most popular over-the-counter (OTC) dietary supple- deionized water in volumetric fasks and stored at 4 C before ments worldwide. Besides its use as a single supplement in further use. the form of glucosamine hydrochloride or glucosamine L-arginine (Arg), N-cyclohexyl-3-amino- sulfate, it can be mixed with other nutraceuticals, vitamins, propanesulfonic acid (CAPS), L-histidine (His), and tris and minerals. Among them, calcium is commonly combined (hydroxymethyl) aminomethane (Tris) used for background with glucosamine because of its crucial role in preventing electrolyte (BGE) and sample preparation were purchased osteoporosis [14]. Given that there are diferent combina- from Sigma–Aldrich (Singapore). Otherwise, acetic acid tions of glucosamine and other supplements, more than one (Ace) and trichloroacetic acid (TCA) were purchased from method is required to analyze these products. For example, Merck KgaA, Germany. Te BGEs were prepared by adding the most popular method for glucosamine analysis is Ace into Arg, Tris, His, and CAPS solutions, followed by pH reversed-phase high-performance liquid chromatography adjustment with acetic acid using a HI 2215 pH meter (RP-HPLC), coupled with various detectors like ultraviolet, (Hanna Instruments, Woonsocket, RI, USA). electrochemical, and mass spectrometry [15–19]. However, Fused silica capillaries (50μm i.d., 365μm OD) were this method is not suitable for analyzing minerals like purchased from BGB Analytik AG (Bockten, ¨ Switzerland). calcium, which have commonly been determined by in- Before use, the capillaries were conditioned by fushing with ductively coupled plasma mass spectrometry (ICP-MS) and 1 M NaOH solution, deionized water, and then the BGE. inductively coupled plasma optical emission spectrometry (ICP-OES), or fame atomic absorption spectrometry (F- 2.2. Sample Preparation. Dietary supplement samples, in- AAS). Due to the complexity of current methods for the cluding tablets, and hard capsules, were purchased from quantifcation of glucosamine products, there is a need to pharmacies in Hanoi, Vietnam. Te sample preparation develop a simple technique to simultaneously determine the procedure was described in previous studies [34, 35]. Briefy, amounts of glucosamine and other components in the food each sample was prepared with the contents of 20 whole supplement. tablets or capsules. Te tablets were ground into a fne Capillary electrophoresis (CE) can be considered a more powder using a ceramic mortar and pestle. Te hard capsules economical alternative for developing simple and cheap were opened to collect powder; the samples were ground if analytical techniques. CE instruments are coupled with necessary. Te sample was accurately weighed (about 0.5 g) various detection methods such as ultraviolet, laser-induced before being transferred into a 25-mL volumetric fask; 4% fuorescence, mass spectrometry, voltammetry, and TCA was added to the mark. Te fask was ultrasonicated for amperometry. CE coupled with capacitively coupled con- 30 min by an ultrasonic vibrator bath (BRANSON 521) and tactless conductivity detection (C D) has recently been centrifuged at 3000 rpm for 15 min. After centrifugation, the highlighted as an economical and efcient method for en- aqueous phases were collected and passed through a 0.45-μm vironmental monitoring, [20–22], food control [23–25], syringe flter. Te fltrated solution was transferred to forensics [26], and clinical analysis [27–29]. Te working a 25 mL volumetric fask and flled to the mark with 4% TCA. principles and applications of CE-C D have been reviewed Te solution could be diluted with deionized water (if by Hauser and Kuba´nˇ [30] and others [29, 31–33]. Tese necessary) before analysis by CE-C D. Concentrations of techniques have been applied to analyze inorganic, organic 2+ glucosamine and Ca were determined by using the ions, and biomolecules. Because of the ability to analyze the standard addition method. broad range of targets, we have developed a protocol to simultaneously quantify both glucosamine and calcium in dietary supplements by CE-C D. A simple procedure has 4 2.3. Instrument. Te in-house CE-C D instrument was pre- been reported, with cross-checking data using standard sented in the previous studies [30, 36, 37]. Generally, the high-performance liquid chromatography with a fuores- compact CE instrument was built in-house using high voltages cence detector (HPLC-FLD) and ICP-OES methods being of a maximum of 25 kV (UM25∗4, 12 V, Spellman, Pul- provided to prove the reliability of the CE-C D results. We borough, UK) and combined with a commercial C D detector also applied this protocol for the quality control of seven (ER815, eDAQ, Denistone East, NSW, Australia). Te system dietary supplements available in Vietnam. was operated with a 220-VAC power supply. Te silica capillary had a total length (L ) and efective length (L ) of 40 cm and tot ef 2. Materials and Methods 30 cm, respectively, and an inner diameter of 50μm. 2.1. Materials. All chemicals used were of analytical grade and were used as received without any further purifcation. 2.4. Method Development and Validation. Analytical pa- All solutions were prepared with deionized water of re- rameter optimization followed strategies in our earlier works −1, sistivity not less than 18.2 MΩ cm which was purifed by [26, 34, 35, 38, 39]. Overall, various factors of BGEs Journal of Analytical Methods in Chemistry 3 (composition, pH, and concentration), separation voltages, bufer when this compound passes the detector. Although all 2+ and injection conditions (siphoning height and injection BGE systems give a sharp peak of Ca , only the Tris/Ace time) were investigated to develop an efective CE-C D solution provides a high signal of glucosamine (Figure 1(a)). method for the simultaneous determination of glucosamine Other BGE solutions cause low peak signals for glucosamine, 2+ and Ca . A single analytical parameter was changed at leading to insufcient sensitivity. Tus, the BGE system a time while fxing all the others. Te background noise, containing Tris/Ace was selected because of the sufcient 2+ signal, and migration times of analytes were calculated from peak signal for Ca and glucosamine. the CE electropherograms to identify the optimized con- After selecting the BGE composition, the pH of the so- dition (Tables S1–S6). Because background noise may lution was further investigated (Figure 1(b)). Tis factor change during the recording of an electropherogram, the signifcantly infuences the glucosamine signal. In general, 2+ average noise was measured near the analyte peak regions. both peaks of Ca and glucosamine were far from the EOF For a reproducible and accurate measurement, the signal’s signals at all fve pH conditions (pH 6.5–4.5). Te high pH peak area was used in all this study’s steps. After comparing declines the migration time of both analyzes because of the rise the analytes’ signals, background noise, and retention time in the EOF mobility. At pH 6.0 and higher, the peak separation under diferent analytical conditions, the optimized CE-C D of glucosamine appears, possibly due to the conversion of glucosamine forms at diferent pHs. While β-anomer is conditions are summarized in Table S7. Based on optimized CE-C D conditions, the analytical dominant for nonprotonated neutral glucosamine, the pro- method was validated for linearity, detection and quantif- tonated glucosamine mainly exists as α-anomer [45–47]. Te cation limits, repeatability, and recovery by using the ana- two peaks of glucosamine are more resolved in higher pH lytical results of standard solutions and matrix-spike conditions. Two isomers of glucosamine also generate two samples. In pharmaceutical analysis, guidelines from the peaks in HPLC analysis; thus, the sum of the area of these two International Council for Harmonization (ICH) and peaks should be used to quantify the glucosamine [15, 48, 49]. EURACHEM are commonly used to calculate the limit of Te pH of 4.5 to 5.5 is suitable to accurately detect total detection (LOD) [40, 41]. Tey defne the LOD as the glucosamine by CE-C D since glucosamine generates a single concentration of analyte that generates a signal at least peak at these conditions, leading to a more straightforward 3 times the signal noise of the baseline (S/N � 3); for LOQ, calculation. All three pH conditions gave the stable baseline the S/N ratio equals 10. Tus, in this study, to determine the and the practical time for bulk analysis (Table S2). Te content 2+ LOD of each analyte (calcium or glucosamine), a standard of Ca is much lower than that of glucosamine in commercial 2+ solution was repeatedly diluted until its signal was 3 times supplements; thus, a condition that shows the best Ca signal higher than the background noise (S/N � 3). Te LOQ value should be chosen for the simultaneous determination of both of each analyte was measured by repeat dilution until the analytes in supplement samples. Among all pH conditions, the 2+ signal of the diluted solution was 10 times higher than the pH of 5.0 exhibited the highest peak area of Ca signals and background noise (S/N � 10). All LOD/LOQ measurements a single peak of glucosamine. Accordingly, a pH of 5.0 was were conducted under optimum conditions in Table S7. chosen for further studies. Cross-check analysis was performed for both sample types Tris concentration (from 8 to 20 mM) was optimized for 2+ (i.e., tablets and capsules). Glucosamine was analyzed with simultaneous determination of glucosamine and Ca HPLC coupled with a fuorescence (FLD) detector, while (Figure 1(c) and Table S3). Generally, the increase in Tris 2+ ICP-OES detected Ca at the National Institute for Food concentration shows a minor efect on the migration times 2+ Control (NIFC), Hanoi, Vietnam, according to methods of glucosamine and Ca . In the capillary tube, a high reported elsewhere [42, 43]. All measurements generally concentration of ions causes a change in the magnitude of followed the AOAC Ofcial Method 2016.14. Detailed in- the electric double layer; therefore, the sample moves un- formation about the reference methods was listed in Table S8. evenly, causing background noise, poor separation, and unbalanced peaks. Although high concentrations of Tris 3. Results and Discussion (≥10 mM) in BGE provide a stable baseline and balanced peaks, the intensity of the glucosamine peak declines at high 3.1. Optimization of CE-C D Analytical Conditions Tris concentration, decreasing the sensitivity. Tus, com- pared to other conditions, the 10 mM Tris/Ace electrolyte 3.1.1. Efects of Background Electrolytes. Te background solution was selected for further investigations because of electrolytes (BGEs) are one of the most critical factors in CE- both analytes’ stable baselines and balanced peaks. C D method development [44]. In this work, we investigated four standard BGE systems, including Arg/Ace, His/Ace, 3.1.2. Efects of Separation Voltages. Various applied volt- Tris/Ace, and CAPS/Ace (Figure 1(a), Table S1). Te initial concentrations of the Arg, His, Tris, and CAPS solutions ages from 10 to 25 kV were then investigated to evaluate their impacts on electropherograms of glucosamine and were 10 mM. All solutions were frst adjusted to a pH of 5.0 2+ by acetic acid. In four BGE systems, both glucosamine and Ca (Figure 2). Firstly, higher voltages resulted in shorter 2+ migration times and sharper peaks, but higher background Ca were eluted before the electroosmotic fow (EOF) signal, indicating that the two analytes have positive charges noise was observed. Voltages of 10 or 15 kV can lead to 2+ broadening peaks of Ca and glucosamine, which could under all studied conditions. Negative-going peaks of glu- cosamine show a conductivity reduction of the background cause false results when analyzing real sample matrices. 4 Journal of Analytical Methods in Chemistry 2+ 5 mV 5 mV 5 mV Ca 2+ 2+ Ca Ca Glucosamine Glucosamine 20 mM Glucosamine Tris/Ace pH 4.5 15 mM pH 5.0 EOF Arg/Ace pH 5.5 10 mM His/Ace pH 6.0 CAPS/Ace pH 6.5 8 mM 1.0 1.5 2.0 2.5 3.0 3.5 4.0 1.0 1.5 2.0 2.5 3.0 1.0 1.5 2.0 2.5 3.0 Migration time (min) Migration time (min) Migration time (min) (a) (b) (c) Figure 1: Optimization the BGE conditions for simultaneous determination of glucosamine and calcium. (a) Te efect of BGE com- positions. (b) Te efect of pH. (c) Te efect of Tris concentration. CE conditions: fused silica capillary (total length 40 cm, efective length 30 cm, ID 50μm); separation voltage +20 kV; hydrodynamic injection (siphoning at 25 cm in 30 s). Te selected conditions were highlighted in blue. 2+ Tese parameters control the sample amounts injected into Ca 5 mV the capillary, afecting the separation and detection ef- ciency. Peak areas are directly proportional to injection Glucosamine 10 kV times and siphoning heights. Although longer injection times and higher injection heights can lower detection limits, too large injected sample amounts may degrade the 15 kV peak shape and introduce elevated interferences. Consid- ering these facts, we injected samples at a siphoning height of 20 kV 25 cm in 30 s. 3.2. Method Validation 25 kV 3.2.1. Selectivity. Most commercial glucosamine supple- 0 1 2 3 4 5 ments combine chondroitin and methylsulfonylmethane Migration time (min) (MSM) to treat painful joints. Tus, the interference of chondroitin and MSM in the signals of glucosamine and Figure 2: Efects of separation voltages on the CE-C D analysis of 2+ Ca was investigated (Figure 4). Tere is no detectable glucosamine and calcium.BGE conditions: 10 mM Tris/Ace, pH 5.0, signal for chondroitin and MSM under experimental con- sample injection time of 30 s, sample injection height of 25 cm. Te selected condition was highlighted in blue. ditions. While a positive potential was applied to detect 2+ glucosamine and Ca (positive ions), chondroitin and MSM cannot form a positive charge under the studied condition. Moreover, a high separation voltage raises the background Chondroitin is a highly negative polysaccharide [50], and noise (Table S4). Te noise at +25 kV showed the highest MSM, an organosulfur compound, is considered chemically level, around 2.06 mV, while the background noise was only inert with a pKa of 28. Tus, it is apparent that we cannot 0.67 mV at +10 kV. On the other hand, the area of the peaks 2+ observe any signal of these two chemicals in the analyzing from glucosamine and Ca decreased at a high applied time (around 5 min). Moreover, the presence of chondroitin voltage (Table S4). Lastly, the high voltage (higher than and MSM showed no interference with the peak signals of 20 kV) could generate audible noise, especially in high 2+ glucosamine and Ca . Tus, the proposed method is humidity conditions in a tropical country like Vietnam. suitable for simultaneously determining glucosamine and Tus, it is recommended to use a low voltage in capillary 2+ Ca in commercial supplements. electrophoresis if possible for safety. Given these points, a separation voltage of 20 kV is optimal for simultaneously 2+ determining glucosamine and Ca . 3.2.2. Calibration Curves. A series of mixture standard solutions ranging from 5.00 mg/L to 200 mg/L and from 2+ 3.1.3. Efects of Injection Conditions. Next, hydrodynamic 1.00 mg/L to 100 mg/L of glucosamine and Ca , re- injection conditions, including injection times and si- spectively, were analyzed at optimized conditions to con- phoning heights, were studied (Figure 3, Tables S5 and S6). struct two calibration curves of two analytes (Figure 5). Te Journal of Analytical Methods in Chemistry 5 5 mV 5 mV 20 cm 20 s 25 s 25 cm 30 s 35 s 30 cm 0 1 2 3 4 0 1 2 3 4 Migration time (min) Migration time (min) (a) (b) Figure 3: Efects of injection conditions on the CE-C D analysis of glucosamine and calcium. (a) Injection time (siphoning at 25 cm). (b) Siphoning height (injection time 30 s). BGE conditions: 10 mM Tris/Ace electrolyte solution, pH of 5.0, separation voltage of +20 kV. Te selected conditions were highlighted in blue. 5 mV MSM Chondroitin 2+ Ca Glucosamine 2+ Ca +Glucosamine 2+ Ca 2+ Ca +Glucosamine+ Glucosamine MSM+Chondroitin 0 1 2 3 4 5 Migration time (min) Figure 4: Selective determination of glucosamine and calcium in the presence of chondroitin and MSM. Concentration of calcium was 10 mg/L while concentrations of glucosamine, chondroitin, and MSM were 100 mg/L. BGE conditions: 10 mM Tris/Ace electrolyte solution, pH of 5.0, separation voltage of +20 kV, sample injection time of 30 s, sample injection height of 25 cm. regression equations of the calibration curves represent analyte’s signal was 10 times higher than the background relationships between analyte concentrations (mg/L) and noise (Table S9). LOQs were estimated to be 3.30 mg/L for 2+ peak areas (mV.s). Good linearity was observed for both glucosamine and 0.17 mg/L for Ca . 2 2+ 2 glucosamine (R � 0.9994), and Ca (R � 0.9994) over the Te LOD of glucosamine obtained by our method was concentration ranges (Figure 5). Concentrations of analytes higher than that reported by Akamatsu and Mitsuhashi in analytical solutions (including real samples and standard (0.01 mg/L) [51]. However, their method required in-capillary addition samples) should be within the linear ranges. derivatization with o-phthalaldehyde, which may not be 2+ suitable for determining both Ca and glucosamine in that condition. Our LOD value was comparable to the LODs of 3.2.3. Limits of Detection and Quantifcation. Limits of other studies using CE and HPLC (0.25 to 1.0 mg/L) [52, 53]. 2+ detection (LODs) of glucosamine and Ca were determined Our method not only detects both analytes in a single run but using signal-to-noise ratios equal to 3 (S/N � 3). A standard also achieves low LOD and LOQ, which are compatible with solution of each analyte was repeatedly diluted until its signals pharmaceutical and nutraceutical analysis. were 3 times higher than the background noise measured near the peak area (S/N � 3) (Table S9). Te concentration of the analyte at S/N � 3 was recorded as the LOD value. LODs of 3.2.4. Repeatability. Standard solutions with three con- 2+ 2+ glucosamine and Ca were 1.00 and 0.05 mg/L, respectively. centration levels of glucosamine and Ca (20, 40, and For the LOQ calculation, we did a similar experiment until the 80 mg/L) were chosen for replication analysis (n � 5) for 6 Journal of Analytical Methods in Chemistry 2+ Ca 25 Glucosamine 200 S = 1.762 C + 3.226 S = 0.107 C + 0.219 peak Ca + peak glucosamine R = 0.9994 20 R = 0.9994 0 0 0 50 100 150 200 0 20 40 60 80 100 Concentration (mg/L) Concentration (mg/L) (a) (b) Figure 5: Calibration curves of glucosamine and calcium. BGE conditions: 10 mM Tris/Ace electrolyte solution, pH of 5.0, separation voltage of +20 kV, sample injection time of 30 s, sample injection height of 25 cm. 2+ repeatability evaluation (Table S4). For Ca , relative stan- and three hard capsules (Table 1 and Figure 6(a)). To verify if dard deviations (RSD) of peak areas ranged from 0.92% to signal peaks from samples were generated by glucosamine 2+ 2.16%. Otherwise, the RSD of the glucosamine peak areas and Ca , known amounts of standard solutions were added 2+ 4 ranged from 0.80% to 2.52%. Both glucosamine and Ca to the sample before analyzing by CE-C D (Figure 6(b)). Te migration times in standards had an RSD <2.5% at all intensities of both signals in the standard addition samples concentration levels. Tese method repeatability results increased compared to those in the samples. Te migration 2+ meet the standard precision criteria proposed by AOAC times of glucosamine and Ca peaks were similar between International for concentration ranges from 1 ppm (11%) to standard and standard addition samples. It is shown that the 100 ppm (5.3%) [54]. supplement additives have a minimal impact on the mi- gration time of both analytes. Tus, the content of glucos- 2+ amine and Ca in supplements could be determined using 3.2.5. Recovery. Recovery is an essential parameter in the standard addition method. Based on the peak area evaluating the method of accuracy. In this study, we used obtained and the standard addition equation, the content of 2+ a tablet sample without glucosamine and Ca (with no label calcium and glucosamine in the functional food samples will claim of these components and confrmed with reference be calculated. methods) to make matrix-spike samples. Tree spiking levels 2+ Te contents of Ca in the samples measured by our 2+ of glucosamine (40, 80, and 100 mg/L), and Ca (4, 8, and CE-C D method ranged from 7.82 to 55.43 mg per unit (e.g., 10 mg/L) were performed in triplicate. Te sample matrix tablet or capsule), with relative deviations compared to label (about 0.5 g) was spiked with a standard solution containing claims ranging from −7.62% to +5.20%. Te contents of 2+ glucosamine and Ca , equilibrated for at least 30 min at glucosamine ranged from 111.0 to 643.0 mg per unit, and room temperature, and treated according to the procedure measured-label deviations ranged from −5.26% to +8.10% 2+ described in Section 2.2. Recoveries of glucosamine and Ca (Table 1). Tere was an acceptable agreement between ranged from 96.9% to 102.0% and from 98.0% to 103.0%, measured and label claim contents because deviations are respectively (see Table S5). Tese recovery rates satisfy the less than ±10% in all samples. Te cross-checked analysis AOAC requirement for expected recovery at concentration was performed for all the samples (Table 1). Te relative levels from 10 ppm (80–100%) to 100 ppm (90–107%) [54], deviations between the results of CE and ICP-OES ranged indicating high accuracy with an insignifcant compound 2+ from −8.08% to +5.62% for Ca , and between CE and loss of our analytical procedure. HPLC, ranged from −5.93% to +6.47% for glucosamine. Good agreement between analyte contents measured by our 3.3. Method Application and Cross-Checked Results. Te method and the reference methods indicates that CE-C D validated method was applied to seven real samples collected could be an alternative tool for conventional methods such from pharmacies in Hanoi, Vietnam, including four tablets as HPLC and ICP-OES in specifc applications. S (mV.s) peak S (mV.s) peak Journal of Analytical Methods in Chemistry 7 2+ 4 Table 1: Determination of glucosamine and Ca in commercial dietary supplements from pharmacies in Vietnam, using CE-C D and HPLC, ICP-OES as the reference methods. 2+ Glucosamine Ca Types Label Sample no HPLC CE-HPLC Label 4 4 of pills CE-C D (mg/μ) (mg/ CE-LBL (%) CE-C D (mg/μ) ICP-OES (mg/μ) CE-LBL (%) CE-ICP (%) (mg/μ) (%) (mg/μ) μ) S1 Tablet 479.0 488.0 450.0 −1.84 6.44 26.30 25.00 24.90 5.20 5.62 S2 Tablet 494.0 457.0 464.0 8.10 6.47 41.90 40.00 44.80 4.75 −6.47 S3 Tablet 616.0 625.0 613.0 −1.44 0.49 36.60 35.00 36.90 4.57 −0.81 S4 Tablet 223.0 211.0 230.0 5.69 −3.04 48.50 50.00 47.50 −3.00 2.11 S5 Hard capsule 111.0 107.0 118.0 3.74 −5.93 49.40 48.00 47.70 2.92 3.56 S6 Hard capsule 324.0 342.0 344.0 −5.26 −5.81 7.82 8.00 7.80 −2.25 0.26 S7 Hard capsule 643.0 616.0 676.0 4.38 −4.88 55.43 60.00 60.30 −7.62 −8.08 CE-LBL: CE-label deviation the deviation of the result obtained with CE-C D from that indicated on the label; CE-LBL (%) � {(C − C )/C } × 100%. CE-HPLC: CE-HPLC deviation, CE-ICP : CE-ICP CE-C4D label label deviation; they are the deviation of the result obtained with CE-C D from that with the standard reference method (HPLC and ICP-OES); CE-HPLC (or CE-ICP) (%) � {(C − C )/C } - 100% mg/ CE-C4D reference reference μ : mg per unit (e.g., tablet or capsule). 8 Journal of Analytical Methods in Chemistry 2+ 10 mV Ca Glucosamine 2+ Ca S1 5 mV S2 Standard addition sample Glucosamine S3 S4 Sample S5 Standard S6 Blank S7 0 1 2 3 4 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Migration time (min) Migration time (min) (a) (b) Figure 6: Representative CE-C D electropherograms of (a) diferent supplement samples (Table 1). (b) Specifc peaks evaluated by blank, standard, sample, and standard addition sample. BGE conditions: 10 mM Tris/Ace electrolyte solution, pH of 5.0, separation voltage of +20 kV, sample injection time of 30 s, sample injection height of 25 cm. 4. Conclusions Supplementary Materials Te present study developed an analytical method for the Table S1. Te background noise, peak area, and migration 2+ 2+ simultaneous determination of glucosamine and Ca time of glucosamine and Ca at diferent BGE conditions. using CE-C D in commercial dietary supplements. Our Table S2: Te background noise, peak area, and migration 2+ method was demonstrated to be simple, fast, and cost- time of glucosamine and Ca at diferent pH conditions. efective with adequate validation parameters such as Table S3: Te background noise, peak area, and migration 2+ selectivity, linearity, detection limits, repeatability, and time of glucosamine and Ca at diferent Tris concentrations. 2+ recovery. Concentrations of glucosamine and Ca in Table S4: Te background noise, peak area, and migration 2+ supplement samples can be simultaneously determined in time of glucosamine and Ca at diferent seperation voltages. a single run analysis within fve min. Te validated Table S5: Te background noise, peak area, and migration 2+ method was applied to analyze samples, including tablets time of glucosamine and Ca at diferent injection time and hard capsules. Analytical results obtained by this CE- (siphoning at 25 cm). Table S6: Te background noise, peak 4 2+ C D method were in good agreement with labeling area, and migration time of glucosamine and Ca at diferent contents and those measured by reference methods (i.e., siphoning height (injection time 30 s). Table S7. Te optimal 2+ 4 HPLC-FLD for glucosamine and ICP-OES for Ca ). conditions of the CE-C D method for simultaneous de- 2+ Overall, our study has revealed another application of CE- termination of glucosamine and Ca . Table S8. Te optimal C D as an alternative green analytical tool for conven- conditions for determination of glucosamine by HPLC-FLD 2+ tional dietary supplement quality control methods, with and Ca by ICP-OES. Table S9. LOD and LOQ of glucos- 2+ the advantages of high efciencies, low sample and amine and Ca . Table S10. Te repeatability evaluation of electrolyte consumption, and minimal chemical waste. peak area (mV.s) and migration time (min) for simultaneous 2+ 4 Because one simple analytical procedure can be applied determination of glucosamine and Ca by CE-C D. Table for both analytes (organic compound and metal ion) with S11. Recoveries for simultaneous determination of glucos- 2+ 4 diferent properties, our method signifcantly reduced amine and Ca by CE-C D. (Supplementary Materials) analysis time and labor cost compared to using one conventional method for each analyte. Te proposed CE- 4 2+ References C D method for glucosamine and Ca analysis also provided reliable results compared to standard [1] N. R. Dostrovsky, T. E. Towheed, R. W. Hudson, and techniques. T. P. 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