Validated analytical workflow for quantifying proline metabolism related amino acids in HaCaT cells with LC-Q-TOF-MS.

It is known that altered amino acid metabolism can influence immunological responses, thus studying the molecular mechanisms underlying these changes requires analytical methods for reliably determining the quantities of individual analytes. To address the ongoing need for quantitative assessment of amino acid composition, a validated method for determining their quantities was proposed. This study presents the development and validation of a novel method for the quantitative analysis of selected amino acids using high-performance liquid chromatography coupled with a quadrupole time-of-flight (Q-TOF) mass spectrometer equipped with an electrospray ionization source. The method has been satisfactorily validated in terms of linearity, range, precision, accuracy, and stability. Its applicability to biological matrices was demonstrated by the successful quantification of amino acids in HaCaT cell lysates. Owing to the use of a robust, state-of-the-art analytical platform based on Q-TOF technology, which enables high-resolution mass detection, the method is readily adaptable for the inclusion of additional analytes and can be extended to other complex biological samples. The applicability of the method for the simultaneous determination of selected amino acids was demonstrated, and the method successfully applied to the HaCaT cell lysates.