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

Abstract

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 μg/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.

Optimization of Melanin Production by Streptomyces antibioticus NRRL B-1701 Using Arthrospira (Spirulina) platensis Residues Hydrolysates as Low-Cost L-tyrosine Supplement

“…HPLC is the most commonly used method for amino acid quantification due to its high sensitivity, specificity, and ability to separate amino acids in complex matrices. For example, Eid et al [ 48 ] used HPLC-UV for the simultaneous quantification of multiple underivatized amino acids in dietary supplements, including isoleucine, leucine, lysine, threonine, histidine, valine, methionine, phenylalanine, tryptophan, and tyrosine. Thus, HPLC is a suitable method for L-tyrosine quantification.…”

Simultaneous determination of selected ionophoric coccidiostats and amino acids in feed premixes using high‐performance liquid chromatography‐ultraviolet detection method

“…The derivatization was performed with 6% vanillin solution in methanol (0.35 mL/min) in the presence of 4% sulfuric acid in methanol (0.35 mL/min), and the coil was maintained at 110 • C. The derivatives of ionophores were then detected at 𝜆 520 nm [26]. Regarding the reported method for the determination of KLEU, VAL, and TRP in dietary supplements, it was based also on the HPLC-UV technique using a gradient elution of the mobile phase consisting of acetonitrile and phosphate buffer (pH 7.4, 10 mM) at 225 nm at room temperature [38]. Statistical analysis of the results using Student's t-test and Variance Ratio F-test [42] revealed no significant difference between the performance of the developed and reference methods regarding accuracy and precision, respectively (Table 2).…”

“…Few methods have been described for the chromatographic determination of underivatized AAs in different samples [36][37][38]. There are currently no EU AAs MRL set in feed premix as it is considered a nutritional feed additive [39].…”

A comprehensive review on recent trends in amino acids detection through analytical techniques

“…A UV-visible detector was used. The reported RT of L-lysine, L-threonine, L-His, L-valine, L-methionine, L-isoleucine, L-leucine, L-Tyr, L-phenylalanine, and L-Tryp was in the range of 2.63-28.50 min [20]. Ridwan et al reported the development of an RP-HPLC technique for the isolation and detection of L-arginine and L-citrulline in watermelons.…”

“…A UV‐visible detector was used. The reported RT of L‐lysine, L‐threonine, L‐His, L‐valine, L‐methionine, L‐isoleucine, L‐leucine, L‐Tyr, L‐phenylalanine, and L‐Tryp was in the range of 2.63–28.50 min [20]. Ridwan et al.…”