Abstract
A RP-HPLC method with pre-column derivatization was developed and validated for the simultaneous quantification of carnosine (Carn), acetylcarnitine taurinate (AC-Tau), asparagine (Asn), potassium aspartate (Asp) and for the determination of phosphoserine (p-Ser) in new and commercial alimentary supplements. The effect of complex matrices was evaluated by the study of the amino acid derivatization reaction with 2,4-dinitrofluorobenzene (DNFB) both in standard and placebo solutions. The reaction was carried out for 20min at 70̊C in alkaline medium (pH10) for p-Ser analysis, whereas for 60min in the case of Carn, AC-Tau, Asn and Asp analysis. The adducts have been separated on a Discovery RP Amide C16 (250mm×4.6mm, i.d.) column using a mobile phase consisting of acetonitrile (ACN) and triethylammonium (TEA) phosphate buffer (pH 3, 0.05M) under gradient elution conditions at a flow-rate of 0.8mL/min. Detection was set at λ=360nm. The validation parameters such as linearity, sensitivity, accuracy, precision and specificity were found to be highly satisfactory. Linear responses were observed by placebo solutions (determination coefficient ≤0.9996). Intra-day precision (relative standard deviation, RSD) was ≤1.06% for corrected peak area and ≤0.99% for retention times (tR) without significant differences between intra- and inter-day data. Recovery studies showed good results for all examined compounds (from 97.7% to 101.5%) with RSD ranging from 0.5% to 1.3%). The high stability of derivatized compound solutions at room temperature means an undoubted advantage of the method allowing the simultaneous preparation of a large number of samples and consecutive chromatographic analyses by the use of an autosampler. The developed method can be considered suitable for the quality control of new and commercial products.
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