Challenge
A premier biotech was looking to design and implement a multi-analyte (21) LC-MS/MS method for Quantitative Amino Acid Analysis in human plasma.
Quantitating amino acids in human plasma relies on selective, reliable, and accurate bioanalytical methods and biomolecular assay development. Not to mention, amino acids are present in varying concentrations in complex biological matrices and have strong polarities and low molecular weights.
Bioanalytical scientists employ several detection methods such as fluorescence, ultraviolet, electrochemical, and mass spectrometry for determining amino acids and capillary electrophoresis or HPLC for separation. Ultraviolet and fluorescence techniques usually need a derivatization step. This step improves the detection sensitivity and separation capabilities. However, sample preparation is a tedious process and requires longer analytical time.
In addition to balancing multiple analytes, we needed to tackle the low molecular weight of these analytes. Another major challenge our scientists faced was the high endogenous levels of these amino acids in the biological matrix. Our team was tsked with quantifying these amino acids present in varying concentrations and strong polarities utilizing selective, reliable, and accurate bioanalytical methods.
Recent technological efforts have enabled tandem mass spectrometry detection of underivatized amino acids. Tandem mass spectrometry offers high sensitivity and selectivity and has a simpler sample preparation protocol. That said, quantitating underivatized amino acids when conducting an analysis by UHPLC is still challenging, given the variable effects from biological matrices and poor retention times.