Research Article: Development and validation of a novel high performance liquid chromatography-coupled with Corona charged aerosol detector method for quantification of glucosamine in dietary supplements

Date Published: May 6, 2019

Publisher: Public Library of Science

Author(s): Chhavi Asthana, Gregory M. Peterson, Madhur Shastri, Rahul P. Patel, Moonis Ali Khan.


Glucosamine dietary supplements are commonly used for the management of osteoarthritis (OA). However, clinical trials have reported varying outcomes with regard to joint function and disease progression. One of the possible reasons for variability in observed effects of glucosamine could be that, unlike prescription drugs, the quality of manufactured dietary supplements is not closely monitored in many countries. Therefore, there is the possibility that the actual amount of glucosamine present in a dietary supplement is different from that claimed on the label. The quality control of glucosamine supplements is further complicated by the unavailability of a simple and effective analytical method for the analysis of glucosamine. Therefore, the aim of this study was to develop a simple analytical method that could be easily adapted by the pharmaceutical industry for routine analysis of glucosamine.

To develop a novel high-performance liquid chromatography (HPLC) method for the quantification of glucosamine, and determine the amount of glucosamine present in a sample of dietary supplements commercially available in Australia and India.

Chromatographic separation of glucosamine was achieved using a zwitter-ionic hydrophilic interaction liquid chromatography column with a mobile phase consisting of 60% acetonitrile and 40% of 85 mM ammonium acetate, at a flow rate of 0.3 mL/min and column temperature 40°C. The developed method was validated for intra- and inter-day linearity, accuracy, precision, and reproducibility. The newly-developed method was subsequently used to analyse 12 glucosamine supplements.

The developed method was selective for glucosamine, which had a retention time of 5.9 min. The standard curve was linear with a correlation coefficient (r2) exceeding 0.99, over the range of 10–200 μg/mL for glucosamine. The relative standard deviations for intra- and inter-day accuracy, precision and reproducibility were all less than 4%. The amount of glucosamine determined in six Australian and six Indian glucosamine supplements ranged between 98.7–101.7% and 85.9–101.8% of the labelled values, respectively.

Unlike previous HPLC methods, this newly-developed HPLC technique does not require pre-derivatisation and can separate glucosamine from both hydrochloride and sulphate salts, and from other amino sugars, such as chondroitin sulphate present in dietary supplements. This simple and effective technique can be employed by analytical laboratories for the quality control of glucosamine dietary supplements.

The current study has developed a new analytical technique using HPLC-Corona CAD, which can analyse underivatised glucosamine hydrochloride and sulphate within 6 minutes. Using the novel assay, we confirmed that unlike the tested Australian dietary supplements, only half of the tested Indian products had a glucosamine content within ±10% of what was claimed on the label.

Partial Text

Osteoarthritis (OA) is a degenerative joint disease commonly affecting the population aged 45 years and above [1]. In OA, the articular cartilage, which is composed of water and substances such as proteoglycans and collagen, is progressively degenerated. Amino sugars, such as glucosamine and chondroitin, are two main components of the glycosaminoglycan that constitutes proteoglycans [2, 3]. Therefore, glucosamine is commonly used as a dietary supplement for the management of OA; approximately 27% patients in America [4], 13% in Canada [5], 6.2% in Ireland [6], 12.2% in Korea [7], 8.8% in France, 14.3% in Spain [8], 26.2% in India [9] and 22% in Australia use glucosamine supplements for the management of their OA [10].

We investigated the robustness of the method by determining the effect of deliberate changes to pH of ammonium acetate (6.57 ± 0.2), composition of ammonium acetate (85 ± 2 mM), composition of acetonitrile (60% ± 2%) and the column temperature (40 ± 5°C) on peak area of glucosamine (80 μg/mL of glucosamine reference standard, n = 3). These parameters were selected as per the recommended guidelines [33, 34].

The newly-developed method was applied to investigate the amount of glucosamine present in twelve different commercially available dietary supplements. Six supplements (capsule, tablet and liquid) were obtained from local pharmacies in Hobart, Tasmania, Australia and six supplements (tablet) were obtained from Bengaluru, Karnataka, India. The details of various brands of glucosamine supplements including brand name, type of formulation, salt form, amount claimed on the label, tablets were either film coated or uncoated are provided in Table 1. Three capsules and tablets were randomly selected and weighed individually, followed by calculation of the percent weight variation (%WV) using the following equation, %WV = (w/W) × 100, where ‘W’ is average weight of three randomly selected capsules or tablets and ‘w’ is the difference between the average weight and individual weight of capsule or tablet. The percentage weight variation calculated for capsules or tablets in each brand was less than 5% in all the brands as shown in Table 1, which is within the limit recommended by United States Pharmacopoeia (USP) [35].

Glucosamine capsules (n = 3) were weighed individually. The capsule shell was opened, and the content was emptied and weighed. The empty capsule shell was weighed, and its weight was added to its content weight to obtain a final weight value. This weight value was compared to the initial weight of the whole capsule to analyse any loss of capsule content during the process of emptying and weighing. The maximum percentage loss of capsule content was ≤0.02%. The capsule content containing either 750 mg or 1000 mg of glucosamine sulphate or hydrochloride was then mixed with either 15 or 20 mL of Milli-Q water to obtain a mixture expected to contain 50 mg/mL of glucosamine sulphate. Each sample was prepared in triplicate and analysed in triplicate.

A known concentration of glucosamine sulphate reference standard solution (500 μg/mL) was spiked into the capsule content (n = 2, containing glucosamine sulphate) that was emptied into a tube. A known concentration of glucosamine hydrochloride reference standard solution (500 μg/mL) was spiked into the crushed tablet (n = 2, containing glucosamine hydrochloride) and into the liquid (n = 2, containing glucosamine hydrochloride) content. Extraction of glucosamine from the spiked capsule, tablet and liquid content was performed as described above and a mixture containing 50 mg/mL of either glucosamine sulphate or glucosamine hydrochloride was obtained.

The stability of glucosamine hydrochloride and glucosamine sulphate was determined at 4°C and at room temperature for 48 hrs. The sample (10 mL) containing 200 μg/mL of glucosamine hydrochloride (n = 3) and 200 μg/mL of glucosamine sulphate (n = 3) was prepared and stored at 4°C and at room temperature. An aliquot (1 mL) was withdrawn at 0 (immediately after preparation), 2, 12, 24, 36 and 48 hours after the storage. The withdrawn samples were then analysed using the newly developed HPLC-Corona CAD method to determine the change in glucosamine concentration. Each sample was analysed in triplicate.

A calibration standard solution (n = 2) containing 80 μg/mL of either glucosamine hydrochloride or glucosamine sulphate reference standard was prepared. Glucosamine sample solutions, glucosamine spiked solutions and the reference standard were subjected to HPLC analysis. Each sample was prepared in triplicate and analysed in triplicate.

The linear regression equation obtained for glucosamine hydrochloride and glucosamine sulphate was y = 0.0464x + 0.1631 (Fig 4A) and y = 0.0299x + 0.1237 (Fig 4B), respectively (where y is the peak area corresponding to the concentration, x of glucosamine). The linearity of the method estimated using correlation coefficient (r2) was found to be greater than 0.99 for glucosamine hydrochloride and glucosamine sulphate. The assay performance results are shown in Table 2. The mean inter- and intra-day accuracy and precision % relative standard deviation (%RSD) for each of the tested composition of glucosamine hydrochloride and glucosamine sulphate (10, 80 and 200 μg/mL) were found to be less than 4%. The mean inter- and intra-day reproducibility % RSD for each of the tested composition of glucosamine hydrochloride and glucosamine sulphate (10, 80 and 200 μg/mL) were found to be less than 3% and 2%, respectively.

The mean percentage extraction recovery of glucosamine hydrochloride and glucosamine sulphate standard was 100.2% and 100.3%, respectively. The percentage extraction recovery of glucosamine recovered from the spiked glucosamine formulations (capsule, tablet and liquid) that were prepared with filtration was found to be in the range of 99.3% to 101.9% (Table 4).

Although commercially available samples were analysed within 2 hours of their extraction and preparation, this study investigated the stability of glucosamine for up to 48 hours at 4°C and room temperature. The results are shown in Table 5. The concentration of glucosamine at time 0 hours was considered as 100%. Glucosamine concentration in glucosamine hydrochloride and sulfate was found to be greater than 99% when stored at 4 and room temperature for 48 hours.

The newly-developed method was used to analyse 12 different glucosamine supplements containing either glucosamine hydrochloride or glucosamine sulphate with or without chondroitin sulphate. United States Pharmacopoeia/National Formulary suggests that the amount of glucosamine should not be less or more than 10% of the labelled amount of glucosamine [44]. The amount of glucosamine present in all the six tested supplements (100%) available in Australia was found to be within the limit of ± 5%, and only three out of six tested supplements (50%) available in India were found to be within the limit of ±10% of what was claimed on the label, while 50% of the tested Indian supplements contained less than 86% of the labelled amount of glucosamine (Table 6).

The method developed is simple and selective for the detection and quantification of glucosamine. The method was successfully applied to 12 different commercially available glucosamine supplements, with a significant reduction in the run time and increased resolution compared with previously reported analytical methods. The selectivity and simplicity of this method allows its application in manufacturing for the identification and monitoring of batch-to-batch consistency of commercially available glucosamine products.




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