Electrophoretic Separation of Alginic Sodium Diester and Sodium Hexametaphosphate in Chondroitin Sulfate that Interfere with the Cetylpyridinium Chloride Titration Assay

Abstract

The most commonly used chondroitin sulfate (CS) assay method is cetylpyridinium chloride (CPC) titration. Cellulose acetate membrane electrophoresis (CAME) is the technique used for detection of impurities in the U.S. Pharmacopeia's CS monograph. Because CPC titration is a relatively nonspecific quantitative technique, the apparent amount of CS as determined by CPC titration alone may not reflect the true amount of CS due to possible interference with the CPC assay by impurities that contain CPC titratable functional groups. When CAME is used in conjunction with CPC titration, certain non-CS and adulterants can be visualized and estimated, and a true value for CS can be assigned once the presence of these non-CS impurities has been ruled out. This study examines conjunct application of CPC and CAME in ascertaining CS assay and purity in the presence of certain adulterants. These include propylene glycol alginate sulfate sodium, known in commerce as alginic sodium diester (ASD), and Zero One (Z1), a water-soluble agent newly reported in the CS marketplace and subsequently identified as sodium hexametaphosphate. ASD, Z1, and CS are similar in physical appearance and solubility in water and ethanol. They are also titratable anions and form ionic pairs with CPC, therefore interfering with the CPC titration assay for CS CAME separates these adulterants from each other and from CS by differences in their electrophoretic mobility. CAME is able to detect these impurities in CS at levels as low as 0.66% by weight. Although it is recommended that a method for detecting impurities (e.g., CAME) be used in cormbination with relatively nonspecific assay methods such as CPC titration, this is seldom done in practice. Assay results for CS derived fromn CPC titration may, therefore, be misleading, leaving the CS supply chain vulnerable to adulteration. In this study, the authors investigated ASD and Z1 adulteration of CS and developed an electrophoretic separation of these adulterants in CS and procedures to isolate ASD from CS matrixes containing these adulterants. The authors describe in this paper utilization of an orthogonal approach to establish the identity of Z1 as sodium hexametaphosphate and to confirm the identity of ASD, including ethanol fractionation, FTIR spectroscopy, differential scanning calorimetry, and NMR spectroscopy. The authors suggest that CAME is a cost-effective and easy to use methodfor detecting certain impurities in CS raw ingredients and recommend that CPC and CAME be used in combination by QC laboratories as a means of effectively deterring the practice of adulterating CS raw materials with the known adulterants ASD and Z1 and/or other non-chondroitin substances that can be separated from CSby CAME and that exhibit CPC titration behavior similar to CS.

Chondroitin Sulfate Safety and Quality

“…Besides the presence of natural contamination, finished CS preparations may also be adulterated by the voluntary addition of cheaper polysaccharides and organic derivatives unable to be determined with usual analytical controls utilized in the nutraceutical industry (Figure 7). In fact, CS is well known to be one of the most adulterated supplements in the market, a widespread and probably underestimated problem, abetted by the lack of industry-wide specific and sensitive quality control analytical methods with several of the adulterated agents going undetected [6,39].…”

“…The application of specific analytical methods such as eHPLC (enzymatic HPLC), HPSEC (high-performance size-exclusion chromatography) and electrophoresis (Figure 4 and see below for analytical methods) on commercially available nutraceuticals has demonstrated the poor quality of many of these products in the global market showing a lower concentration of CS than specified on the label as well as their intentionally adulteration [39,41,42]. The adulteration is obtained with molecules that may interfere with aspecific widely-used analytical methods of CS quantitation, such as the CPC titration and carbazole assay, to give an artificially inflated estimate of CS concentration [6].…”

Chondroitin sodium sulfate – impurities and standardization problems (literature review)

“…Для анализа примесей ХС используют капиллярный электрофорез и электрофорез в геле. Электрофорез обладает высокой селективностью, позволяя определить присутствие ряда примесей 3-й группы, каррагинанов, альгината пропиленгликоля и гексаметафосфата натрия, некоторых примесей 1-й группы (ДС) [6,7,[63][64][65]. Метод обладает высокой чувствительностью (определение примесей до 0,1 %) и часто используется в исследовательских работах.…”

“…Метод позволил определить присутствие ДС и сверхсульфатированного ХС в субстанциях гепарина. Идентификацию проводили по расположению сигнала протона N-ацетильной группы полисахаридов в области около 1,9-2,5 м. д., которые характеризуют присутствие в сырье контаминантных примесей [6,31].…”

“…Выявлены такие проблемы, как снижение уровня очистки субстанций, увеличение содержания различных примесей, включая присутствие других ГАГ, искусственное разбавление субстанций ХС растительными полисахаридами, другими химическими соединениями и т. д. [5][6][7][8].…”