Development of a novel analytical approach combining the quantification of amino acids, organic acids and glucose using HPLC-UV-Vis and HPLC-MS with screening viaNMR

Abstract

A simple, rapid, sensitive and selective procedure based on the combination of HPLC-UV-Vis and HPLC-MS has been developed and single laboratory partially validated for the determination of a set of 13 analytes present in a commercially available IVF medium utilising small sample volumes (20–30 μL). The composition fingerprint of the complex sample obtained by NMR spectroscopy in 11 minutes provided identification based on a screening of the metabolomic profile. HPLC-MS allowed the glucose–sodium adduct to be measured accurately and the working and linear ranges achieved were 0.028–0.389 mmol L⁻¹ with a detection limit of 13 μM. HPLC-UV-Vis allowed accurate concentrations of pyruvic and lactic acids with linear ranges over 0.005–0.1 mmol L⁻¹ with a limit of detection of 28 μM for pyruvic acid to be determined in 8 minutes, while lactic acid presented a linear range over 0.1–2 mmol L⁻¹ with a limit of detection of 1.2 mM possible. The use of HPLC-UV-Vis allowed the chromatographic separation of 8 amino acids (aspartate, glutamate, serine, glycine, asparagine, glutamine, alanine, and proline), the dipeptide alanyl-glutamine and taurine previous to a chemical derivatization, providing a total run time of 40 minutes. The method was partially validated to show a linear range of 0.028–0.280 mmol L⁻¹ with detection limits ranging between 1 and 30 μM. Development of the analytical approach provided determination and quantification of a set of 13 analytes from a very complex sample. Although well established analytical techniques were used here, combinatory methodologies were partially validated for the first time to this purpose. The novelty of the combination of techniques relies on a screening tool and a strategy to the future evaluation and an improved assessment of human embryo viability.