Determination of Total Proteinogenic Amino Acids and Taurine by Pre-column Derivatization and UHPLC: Single Laboratory Validation, First Action Official MethodSM 2019.09
Authors: Joseph George , Varughese Asha , Daniel Ann 
Published: August 2020

Abstract

Background
A method has been developed and validated for selective, accurate, and precise determination of total proteinogenic amino acids and taurine from infant formula and adult/pediatric nutritional formulas (powders, ready-to-feed liquids, and liquid concentrates). The method was reviewed by the AOAC INTERNATIONAL SPIFAN Expert Review Panel (ERP) during the 133rd AOAC Annual Meeting and Expo on September 7, 2019 in Denver, CO, USA, and was recommended to First Action Official MethodsSM status.

Objective
The method involves protein hydrolysis to amino acids, a simple pre-column derivatization of amino acids and separation of derivatized amino acids by UHPLC. The quantification of amino acids is performed by multi-point calibration using norvaline as the internal standard. The analytical method is capable of quantitative determination for 22 proteinogenic amino acids, but cannot be used to quantitate tryptophan, which is destroyed during the acid hydrolysis step. Asparagine is determined as aspartic acid and glutamine as glutamic acid. Cystine and cysteine are converted to S-2-carboxyethylthiocysteine (CYSx) and the derivative is separated from the other amino acids. Citrulline, which is present in some matrixes, is separated from other amino acids and is not included in the method performance evaluation in the single-laboratory validation (SLV).

Method
The proposed method met all the performance requirement limits set in Standard Method Performance Requirement (SMPR®) 2014.013 for total proteinogenic amino acids and taurine. The correlation coefficient of multi-point calibration was not less than 0.999 for any amino acids at any point in the SLV study, confirming the validity of linear dynamic range (LDR) and linearity of the method. The individual amino acids in the chromatogram were identified by absolute retention time and relative retention time (RRT) with respect to the internal standard norvaline. There were no significant (S/N &lt;10) interferences from the reagents or byproducts of derivatization and targeted matrixes. The method demonstrated high selectivity.

Result
Accuracy of the method was validated using standard reference materials (SRMs; NIST SRM 1869 and 1849a) and spike recovery studies. The amino acid results in the SRMs were within the ranges of Reference Mass Fraction Values. The accuracy of the method was corroboratively validated by spike recovery studies. The average spike recovery ranged between 93 and 107% ensuring the accuracy of the method for amino acids and compliance with AOAC SMPR 2014.013.

Conclusions
Precision data of the method demonstrate that it meets the stakeholder requirements as per the SMPR. The mean RSDr for all the amino acids for 17 matrixes selected for the SLV were not more than 4%. The method is very sensitive and the LOQ can go down to approximately ten times lower than the SMPR requirements. The sensitivity of method is a direct reflection of its signal-to-noise ratio which ensures guaranteed method performance at the lower levels of amino acids in these matrixes.

Highlights
Taurine (aminoethane sulfonic acid), unlike the other amino acids, is a beta-sulfonic amino acid that is not used in protein synthesis but is found as a free amino acid in tissues. The acidic functional group (–COOH) in common amino acids is replaced with a sulfonic acid (–SO3H) group in taurine. The method offers baseline separation of citrulline which is an alpha amino acid generally present in infant formula and adult/pediatric nutritional products. The separation of citrulline eliminates the risk of interference of this compound with other amino acids. The method can also separate and quantitate hydroxyproline, an important component of collagen that is often used to quantitate collagen. The method is simple and does not include any proprietary chemicals or instruments, and it can be performed on any basic reverse phase UHPLC system with UV detection (1–6).