Reverse phase-high performance liquid chromatography: An alternative to expensive tandem mass spectrometry screening for amino acid profiling in dried blood spot in resource constrained diagnostic settings

Background: Altered patterns of amino acid profiles are observed in various pathological conditions including nutrition related disorders, cancer, diabetes, urea cycle defects, mitochondrial respiratory chain disorders, and aminoacidopathies. Aim and Objectives: To develop a cost effective Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method for the quantification of free amino acids in neonatal Dried Blood Spot (DBS) samples. Material and Methods: Free amino acids were extracted from neonatal DBS through elution with ice-cold methanol/2-mercaptoethanol. The extracted samples were then derivitized by using O-Pthalaldehyde (OPA)/2-mercaptoethanol, which resulted in the formation of OPA derivatives of aminoacids. These derivatives were quantified by RP-HPLC. The developed method was validated according to the International Conference on Harmonization ICH Q2 (R1) Guidelines and applied to clinically confirmed aminoacidopathies. Results: A good linearity was observed from 2.5 to 1000 μM. The between run imprecision (on two different days) though varied over a wide range for different amino-acids, the Coefficience of Variation (CV) was found to be less than 5% (range; 0.1 to 5% for different amino acids) and the recovery was found to be around 82-125% (for spiked amino acids). The quantified concentration of amino acids in plasma and DBS sample were found to be almost similar except for few amino acids like glycine, tyrosine, alanine, methionine and lysine which showed higher values in dried blood spot samples. DBS amino acid concentrations determined by RP-HPLC showed concordance to the concentrations obtained through Tandem Mass Spectrometry (TMS) of DBS/plasma. Reference intervals of amino acids were established in term neonates. The proposed method was applied to the analysis of samples obtained from confirmed aminoacidopathies. Conclusion: The present approach with DBS offers a valid alternative to the plasma/serum aminoacid profiling in HPLC or DBS in Tandem mass method, with advantages like lower sample volume, feasibility, affordability, improved specificity and can be attempted in a simple diagnostic setup with acceptable accuracy and precision.