High-performance Liquid Chromatography-positive Ion Research Articles

Kynurenine to tryptophan ratio as a biomarker of acute stress in fish

The aim of this study was to determine the kynurenine (KYN) to tryptophan (TRP) ratio (KTR) in fish tissue to assess its usefulness as a biomarker of acute stress. Laboratory held rainbow trout (Oncorhynchus mykiss) were subjected to an acute stressor and KYN, TRP and cortisol were measured in liver and brain tissues at 4- and 48-h post-stress. The analytical method used to determine our analytes was based on lyophilization, and liquid-solid extraction followed by isotope dilution high-performance liquid chromatography positive ion electrospray tandem mass spectrometry. The [KYN]/[TRP] ratio (KTR) was greater in fish liver and brain in the 48-h post-stress exposure group (n = 8) relative to controls (n = 8, p < 0.05); a similar increase was not observed in fish in the 4-h post-stress exposure group. Hepatic and brain cortisol levels were also elevated in fish from both stress-induced groups relative to their respective controls implying that cortisol responded more quickly to the stressful stimulus than KYN and TRP. Our results suggest that the KTR is a promising acute stress diagnostic biomarker in fish. Efforts are ongoing to assess whether the KTR can be used as a biomarker for chronic stress in fish exposed to aquatic contaminants and other environmental stressors and if similar assessments can be made on tissues collected via non-lethal approaches.

Liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry method for the quantification of pregabalin in human plasma

A sensitive high-performance liquid chromatography positive ion atmospheric pressure chemical ionization tandem mass spectrometry method was developed and validated for the quantification of pregabalin in human plasma. Following liquid–liquid extraction, the analyte was separated using an isocratic mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M+H] + ions, m/ z 160–142 for pregabalin and m/ z 482–258 for the internal standard. The assay exhibited a linear dynamic range of 1–10,000 ng/mL for pregabalin in human plasma. The lower limit of quantification was 1 ng/mL with a relative standard deviation of less than 11.4%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 4.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic studies.