Validation of a high-performance thin-layer chromatography/densitometry method for the quantitative determination of...

A quantitative densitometric high-performance thin-layer chromatography (HPTLC) method was developed for the determination of glucosamine in a dietary supplement containing dried extracts of the main plants traditionally used for rheumatic disorders. The HPTLC method was chosen in order to circumvent the tedious and time-consuming sample preparation steps necessarily performed before using HPLC methods when analysing complex matrixes. Glucosamine was separated from the plant extracts on a silica gel 60 F(254) HPTLC plate using a saturated mixture of 2-propanol-ethyl acetate-ammonia solution (8%) (10:10:10, v/v/v). The plates were developed vertically up to a distance of 80 mm. For visualization, the plate was dipped into a modified anisaldehyde reagent and heated at 120 degrees C for 30 min in a drying oven. Glucosamine appeared as brownish-red chromatographic zones on a colourless background. Densitometric quantification was performed at lambda = 415 nm by reflectance scanning. The HPTLC method was successfully validated by applying the novel validation protocol proposed by a commission of the "Société Française des Sciences et Techniques Pharmaceutiques" (SFSTP). In the pre-validation phase, the appropriate response function was determined, while in the validation phase the method showed good performance thereby fulfilling its objective of quantifying accurately. The relative standard deviations for repeatability and intermediate precision were between 4.9 and 8.6%. Moreover, the method was found to be accurate, as the two-sided 95% beta-expectation tolerance interval did not exceed the acceptance limits of 85 and 115% on the whole analytical range (800-1,200 ng of glucosamine).