Accurate Quantification of Plasma Amino Acids Achieved through HPLC–MS/MS: A Reliable Method for Clinical and Research Applications

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Researchers have developed a robust method utilizing high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) to accurately quantify amino acids in plasma, making it a valuable tool for clinical diagnostics and research in amino acid metabolism.

Scientists at General Diagnostics International have developed a reliable and straightforward method for quantifying amino acids in plasma using high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) technology. The study, published in the journal Chromatographia, describes the optimized approach that utilizes a small sample size and a labeled internal standard for accurate quantification (1). The method employs a gradient method with water, formic acid, and methanol as the mobile phase to achieve clear separation. The results demonstrate excellent correlation coefficients, linear responses, and satisfactory precision and recovery rates for all measured amino acids, validating the effectiveness of the developed approach.

Accurate quantification of amino acids in plasma is of utmost importance for various clinical and research applications. To address this need, researchers at General Diagnostics International in India have established a robust and dependable method using HPLC–MS/MS technology.

The developed approach involves the use of a small sample size and a labeled internal standard to ensure accurate quantification of plasma amino acids. A gradient method with water, formic acid, and methanol as the mobile phase was optimized to achieve clear separation of the amino acids of interest.

Using a commercial mass spectrometer, the team utilized optimized multiple reaction monitoring (MRM) to detect and quantify the amino acids. Remarkably, all measured amino acids exhibited excellent coefficients of correlation ranging from 0.91 to 0.99, indicating strong linear responses within their respective reference ranges.

The precision of the method was assessed and reported by calculating the intra-day and inter-day coefficients of variation (CV %), which ranged from 3.29% to 11.73% and 5.04% to 12.48%, respectively. These values reflect the satisfactory repeatability and reproducibility of the method.


Furthermore, the recovery rates for the amino acids ranged from 92.1% to 108.2%, further validating the accuracy and reliability of the developed approach for quantifying plasma amino acids.

This study presents a robust and efficient method for the quantification of plasma amino acids using HPLC–MS/MS technology. The optimized approach offers accurate measurements, satisfactory precision, and excellent recovery rates, making it a valuable tool for clinical and research applications.

The establishment of this reliable method for quantifying plasma amino acids opens up new avenues for studying amino acid metabolism, diagnosing metabolic disorders, and monitoring treatment responses in various clinical conditions.


(1) Panaskar, S. N.; Singh, S. K. Quantification of Amino Acids in Plasma by High-Performance Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS). Chromatographia 2023. DOI: