LC-MS/MS Quantitative Amino Acid Analysis

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.

Solution

Our veteran scientists chose to adopt the technologically advanced tandem mass spectrometry detection of underivatized amino acids instead of the traditional fluorescence, ultraviolet, electrochemical, and mass spectrometry approaches.

We prepared all samples for the amino acid assay per FDA guidelines. Here are the steps our team followed for the quantitative amino acid analysis:

Developed two distinct LC-MS/MS methods to analyze 17 and 5 (out of 21) amino acids in human plasma, respectively
Separated these amino acids through reverse-phase liquid chromatography, which offered peak symmetry and better retention for all analytes
Ensured minimal matrix effects and high accuracy by using a cocktail of stable labeled internal standard, canonical amino acid mix for all analytes of interest

Performed GLP LC-MS/MS method validation to quantitate these amino acids in small volume (75 µL) of human plasma
The amino acids were detected using in-source fragmentation and tandem mass spectrometry (LC-MS/MS)
Achieved acceptable intra-run and inter-run accuracy and precision at five different concentration levels for quality controls
Completed data acquisition with Scienx’s Analyst v1.7, and data processing with Watson LIMS™ v7.6

Outcome

NorthEast BioLab has successfully developed and validated a robust and transferable LC-MS/MS method for analyzing 21 amino acids in biological matrices. We can apply the finding of our amino acid analysis by HPLC method in numerous investigations requiring amino acid quantification as a biomarker. It can thus help our sponsors explore new pathways and treatments that involve amino acids.

Some salient features our combined LC MS method for 21 amino acids, include:

Critical evaluation of several parameters, including calibration range, accuracy and precision, analyte carryover, dilution integrity, matrix effect, and analyte recovery
The analyte response at the QC_LLQ was ≥ 5 times the analyte response of the zero calibrators, demonstrating greater sensitivity of the LC-MS/MS method

Intra-run and inter-run accuracy and precision (%CV) range for QC_Low, QC_Mid, and QC_High met the batch acceptance criteria, indicating that LC-MS amino acid analysis provides reliable, accurate, and reproducible results for target amino acids
The development of a reproducible LC-MS/MS-based bioanalytical method for simultaneous quantitation of L-Aspartic Acid, Glycine, DL-Alanine, L-Asparagine, L-Tryptophan, L-Phenylalanine, DL-Arginine, L-Isoleucine, DL-Leucine, L-(+)-Lysine, L-Proline, DL Serine, DL-Tyrosine, L-Valine, Trans-4-hydroxy-L-Proline, DL-5-Hydroxy-DL-Lysine, L-Histidine in human plasma

Our current amino acid analysis services using the LC-MS/MS method can safely provide reliable, accurate, and reproducible results for your targeted amino acids. The internally labeled standards help reduce the matrix effects, supporting the precise quantitation of these crucial amino acids in biological matrices.