Development of a second-tier method for C4, C5 and C2 acylcarnitine analysis in plasma.

INTRODUCTION: Acylcarnitines are typically analyzed using either a flow injection analysis (FIA) method or liquid chromatography-mass spectrometry (LC-MS/MS) methods. The FIA method is a fast, efficient method, however it does not have the capability to separate compounds with the same molecular weight. These isobaric interferences can be removed by chromatographic separation with LC-MS/MS. In this study, we aimed to develop and optimize a qualitative LC-MS/MS method to separate the isobaric interferences for two-, four- and five-carbon acylcarnitines. METHODS: The samples were first prepared by acylcarnitine derivatization with butanolic HCl. The developed LC-MS/MS method is a combination of isocratic and gradient elution used to separate acylcarnitines. Multiple reaction monitoring was used for determination of precursor and product ions for each acylcarnitine species as well as known interferences used in our study. We used this method to analyze quality assurance and patient samples with elevated two-, four- and five-carbon acylcarnitines. RESULTS: Butyryl- and isobutyrylcarnitines as well as valeryl- and isovalerylcarnitines were successfully separated using the developed method. This method was able also to separate and distinguish acetylcarnitine from glutamate interference that has been causing overestimation of acetylcarnitine. In patients, the dominant five-carbon acylcarnitine was found to be isovalerylcarnitine. We confirmed that the majority of analyzed patient samples had additional carnitine adducts present but not valerylcarnitine. Butyryl- and isobutyrylcarnitines, in variable ratios, were present in every patient sample. CONCLUSION: We developed a qualitative LC-MS/MS method for butyl-ester derivatized acylcarnitines, which can be used as a second-tier method for diagnosis and monitoring of various inborn errors of metabolism in our hospital network.

Analysis of serum tranexamic acid in patients undergoing open heart surgery.

BACKGROUND: Tranexamic acid is a drug used during open cardiac surgery to prevent blood loss. The blood levels of 10-100 µg/mL are reported to be in the therapeutic range and higher levels are linked to increased incidence of adverse effects. The aim of this study was to optimize and validate an LC-MS/MS method for serum tranexamic acid and measure its levels in patients from the DEPOSITION Pilot trial in order to prove the concept that topical administration will yield lower serum concentration. METHODS: The method development was carried out in several steps including sample preparation, and optimization of chromatography and tandem mass spectrometry parameters. Method validation including day-to-day precision with 4 QC levels, limit of detection, sample stability, carryover, and concentration-signal linearity was carried out. Ninety patient samples were analyzed using the validated method. RESULTS: Fast and efficient LC-MS/MS method for analysis of tranexamic acid in serum was developed. The run time was 7 min with the total time of one hour including the sample preparation. The method precision was acceptable (%CV = 10.5-12.6%) with no sample carryover observed. The matrix effect on the analytical sensitivity was negligible and the lower limit of detection was 0.5 µg/mL. The difference in the mean adjusted concentrations between topical (45 patients) and intravenous (45 patients) groups was statistically significant (0.1154 µg/mL/kg vs. 0.2542 µg/mL/kg, p < 0.0001) CONCLUSIONS: Rapid and simple LC-MS/MS method for analysis of tranexamic acid was optimized and validated. The laboratory has played a crucial role in proving the concept that topical administration yields significantly lower systemic levels of tranexamic acid, and thus decreases the risk of adverse outcomes in patients undergoing open cardiac surgery.