Development and validation of a generic method for quantification of collagen in food supplement tablets using liquid chromatography coupled with time-of-flight mass spectrometry

Abstract

A generic method for the quantification of type II collagen in protein-based dietary supplements is described. This quantitative analysis was conducted using liquid chromatography-electrospray ionization-time-of-flight mass spectrometry (LC-ESI-TOF MS). Compared to classical methods with the use of isotope-labeled standards, our method includes, for the first time, the quantification of hydroxyproline using histidine as an internal standard. Separation of the analytes was performed on a Phenomenex Synergi 4 μm Fusion-RP 80 Ǻ column (150 × 2.0 mm, 4.0 μm) with a mobile phase made of 10 mM ammonium formate in water (A) and 10 mM ammonium formate in methanol (B). The assay was fully validated according to FDA guidelines, and the method exhibited sufficient specificity, accuracy, and precision. Intra- and inter-batch accuracy, determined as a deviation between nominal and measured values, ranged from -4.8 to 9.1% and from 0.9 to 6.4 %, respectively. All analytes (hydroxyproline and histidine) at three concentration levels showed extraction recoveries from 89 to 98 %. The method was successfully applied to protein-based dietary supplements of the pharmaceutical industry.

Intra-Laboratory Validation of Alpha-Galactosidase Activity Measurement in Dietary Supplements

“…The choice of correct statistical procedures for data exploration and the interpretation of results are important keys for the proper assessment of method trueness. Assay optimization and the pre-validation step are important to determine how a range of matrix and sample elements, as well as assay conditions, can affect assay performance [35][36][37][38][39][43][44][45].…”

“…In order to study the simultaneous variation of the factors on the considered responses, a multivariate approach using an experimental design by Plackett-Burman was employed [35,36,39]. A fractional factorial Plackett-Burman design was used to screen and evaluate the significant variables that could influence the enzymatic assay method, because this model does not explain the interaction among various variables [36,37,39,44].…”

“…To ensure that a new process can produce reliable results, all laboratories should guarantee that the analytical method's performance meets the requirements during all steps of the validation [31,[35][36][37]. Assay corroboration provides an assurance of reliability during normal use and is referred to for documented evidence that the method does what it is intended to do [31,32,[38][39][40].…”

“…A well-defined substrate with adequate lot-to-lot uniformity should be used [19,31,32]. Validation of enzyme assays should document assay specificity, sensitivity, variability, and assay linearity [26,27,29,35].…”

Bioanalytical Method for the Determination of Hydroxyproline in Mouse Kidney by High-Performance Liquid Chromatography with Tandem Mass Spectrometric Detection

“…[3][4][5] To measure the concentrations of Hyp in biological samples or food, various methods have been developed, such as highperformance liquid chromatography (HPLC) with derivatization for fluorescence detection and high-performance liquid chromatography with tandem mass spectrometric detection (LC/ MS/MS). [6][7][8][9][10][11][12][13] We have developed an analytical method for the determination of urinary Hyp in rat. This method involves separating the Hyp in hydrolyzed urine by column-switching HPLC and detecting it with electrogenerated chemiluminescence (ECL) using tris(2,2-bipyridyl)ruthenium(II), utilizing an HPLC-ECL system developed in our laboratories.…”