Abstract
The reports on the probable beneficial impact of tyrosine (TYR) supplementation on performance enhancement have contributed to the growth of interest in TYR in equestrian sports field and related industries, such as the manufacture of dietary and nutritional supplements. In this study, the first attempt to the assessment of horses exposure to TYR during nutritional supplementation was demonstrated by quantification of unbound TYR and a total free amino acids (AAs), and subsequent determination of the ratio of TYR to a total free AAs content in equestrian supplement samples. Within the scope of this study, a rapid and sensitive LC–MS/MS-based method for the determination of unbound TYR and a simple spectrophotometric protocol for the quantification of a total free AAs have been developed and validated according to the international guidelines for bioanalytical methods. For sample preparation, an ultrasound-assisted solid-liquid extraction (USLE; for solid samples) and ultrasound-assisted liquid-liquid extraction (ULLE; for liquid samples) have been optimized. The comprehensive approach on the simultaneous assessment of matrix effect, recovery, process efficiency, accuracy, precision, curve factor and internal standard association demonstrated satisfactory validation parameters for LC–MS/MS-based procedure, such as global matrix effect in the range of 89.9–91.9%, good linearity (R2 > 0.9929), limit of detection (0.6 ng/mL) and recovery within 89.2–108.0%. For spectrophotometric method, limit of detection was 1100 ng/mL, recovery ranged from 103.2 to 108.8%, both, intra- and inter-day accuracy was within 89.5–108.7%, and both, intra- and inter-assay precision was below 8.5%. In view of the satisfactory validation parameters obtained for both methods, the procedures can be utilized for routine analysis of dietary and nutritional supplements.
Published Version
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