Simple and Sensitive Quantification of Ghrelin Hormone in Human Plasma Using SBSE-HPLC/DAD-MS

Abstract

During a decade and more since its discovery, the emerging physiological roles of ghrelin in mammalian are increasingly being introduced, proposing a critical need for its quantification in biological milieu. Here in, a simple and sensitive single-step method for extraction and quantification of ghrelin in human plasma was developed and validated using stir bar sorptive extraction (SBSE) followed by high-performance liquid chromatography (HPLC) with diode array detection (DAD) coupled to mass spectrometry (MS). Influential parameters of SBSE procedure were optimized including extraction and desorption times of 45 and 30 min, respectively; pH of 4; no addition of salt. The sum of peak heights of three most intense selected ions in mass spectrum (844, 1125 and 1686 m/z) related to 4-, 3- and 2-fold-charged ions of ghrelin was used for quantification. Validation parameters containing linear dynamic range, limit of quantification and limit of detection were 0.02-80, 0.02 and 0.007 µg L-1, respectively, and calculated relative standard deviation for peak heights was 6.5% (0.7 µg L-1 standard solution). Mean recovery for ghrelin in spiked plasma samples was 96% ± 3. The efficiency of the SBSE-HPLC/DAD-MS procedure was proved by analysis of plasma samples from obese patients undergoing gastric plication surgery. The suggested methodology would contribute to simple and fast analysis of ghrelin levels in obesity and related diseases and also biochemical cycles in which gherlin is present.