Optimization of extraction procedure for quantification of succinate in biological samples by gas chromatography — mass spectrometry method

Abstract

Introduction. It is well known that ischemia/reperfusion injury induces cellular damage, which is accompanied by a variety of metabolic alterations. Several studies indicate that accumulation of mitochondrial metabolite succinate (citric acid cycle intermediate) during ischemia controls reperfusion injury through mitochondrial reactive oxygen production [1]. Gas chromatography coupled with mass spectrometry (GC-MS) is one of the technologies widely used for qualitative and quantitative analysis of small molecules. The aim of this study was to optimize extraction procedure and GC-MS method for quantification of succinate in biological samples after ischemia/reperfusion injury. Materials and methods. Warm renal ischemia was induced by the application of microvascular clips around renal artery. At the end of ischemia (after 30 or 40 min.) the clips were removed and reperfusion was performed for 30 minutes. Mitochondria from rat’s kidneys were isolated by differential centrifugation. Biological samples with mitochondria were lyophilized and were prepared for chemical analysis. The analytical procedures for quantification of succinic acid consisted of three main steps: extraction of succinic acid (the solvent was tetrahydrofuran), derivatization (trimethylsilylation with N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA)) of succinic acid to more stable and volatile compound and GC-MS separation, identification and quantification of the derivatized compound. Two different extraction procedures were chosen. Samples were evaporated to dryness with a gentle stream of nitrogen, other samples have not been evaporated. The all samples are capped, wrapped with paraffin tape, and heated at 70 ºC for 3 hours to convert the targeted analytes to their trimethylsilyl derivatives. The evaluation of the GC-MS method has been performed by full scan (total ion current — TIC) using internal and external standards. The reaserch was don [...].