Underivatized Amino Acid Chromatographic Separation: Optimized Conditions for HPLC-UV Simultaneous Quantification of Isoleucine, Leucine, Lysine, Threonine, Histidine, Valine, Methionine, and in Supplements

Amino acids (AAs) are considered as the building blocks of life. Unlike nonessential AAs, the human body cannot synthesize essential AAs and should be supplied in food or dietary supplements. The aim of the work is simultaneous HPLC-UV determination of 10 structurally related AAs without pre-or postderivatization in powdered dietary supplements (PDSs). This was challenging, especially because PDS has no standardized procedures for its quality control. HPLC-UV chromatograms of the 10 AAs were recorded using a gradient elution of the mobile phase on a CLC-C18 column at 225 nm. The elution started with 100% of phosphate buffer (pH 7.4, 10 mM) for 10 min; then, the concentration of acetonitrile increased linearly to reach 50% for another 15 min at room temperature. Good separation was achieved within a 25 min run time without pre-or postderivatization. The method was carefully validated according to the ICH guidelines over the linearity range of 100-200, 50-200, 20-150, 50-400, 20-250, 75- 175, 50-250, 50-250, 50-300, and 5-100 mu g/mL for L-lysine, L-threonine, L-histidine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, and L-tryptophan, respectively, with mean recoveries ranges between 98.91 and 100.77. The method was found to be precise, and the relative standard deviation (RSD) was found to be between 0.28 and 1.92 with recoveries between 97.91 and 101.11. The method was found to be robust that resists deliberate changes in pH, flow rate, and mobile-phase percentages. It was successfully applied for the analysis of PDSs. The proposed method could be very useful for the quality control of the 10 structurally related AAs during their synthesis and for testing raw materials and pharmaceutical preparations.

Automated summary

Amino acids are essential building blocks of life. This study aimed to develop a new HPLC-UV method for the simultaneous determination of 10 structurally related amino acids in powdered dietary supplements. The method was carefully validated and found to be accurate and reliable for quality control purposes.