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, respectively, and calculated relative standard deviation for peak heights was 6.5% (0.7 µg L 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.

Recent developments in liquid chromatography–mass spectrometry analyses of ghrelin and related peptides

“…As an example, Sidibé et al () observed the molecular ion of (M + 7H) 7+ as the most intense peak at pH of around 2, whereas a mobile phase pH of about 5 displayed the highest signal intensity for the [M + 4H] 4+ protonated molecule in the ESI‐MS spectrum. During our previous studies, we observed average masses of 1686.2, 1125.2 and 844.3 m / z , related to the [M + 2H] 2+ , [M + 3H] 3+ and [M + 4H] 4+ molecular ions of human ghrelin, using ESI‐ion trap mass spectrometry (Figure ; Eslami et al , ).…”

“…After vigorous shaking, the mixture was centrifuged at 4°C/36,000 g for 10 min, and clear supernatant was collected. During our previous study, we also used this reagent for precipitation of plasma proteins, using volume proportions of 1:1:4 for acetonitrile–sulfosalicylic acid (100 g L −1 )–plasma (Eslami et al , ).…”

“…Our results demonstrated that the methodology was capable of efficiently extracting and pre‐concentrating ghrelin from plasma samples and also simplified the complex medium to a great extent. This would dramatically assist quantification of ghrelin as well as extending the useful life of the chromatographic column (Eslami et al , ).…”

Interpol review of toxicology 2016–2019

“…Determination of dimethyl trisulfide in rabbit blood [ 471 ], Ghrelin hormone (aka. hunger hormone) [ 472 ], losartan and valsartan in human plasma [ 473 ] were reported by different research groups. The aforementioned examples made use of commercially available stir bars.…”

SBSE(EG-Silicone)-UFLC-MS/MS and HPLC-Flu Analysis of Norharman in Passiflora edulis Rinds

“…The levels of the factors were chosen based on the Gerstel's instructions for stir bar users and also considering other publications about SBSE. 10,[27][28][29]34 Experiments were conducted to evaluate the influence and interaction of five selected factors, at three levels each: pH (4, 7 and 10), time of extraction (30, 90 and 120 min), percentage of methanol (0.50, 2.75 and 5.00%), percentage of NaCl (0, 15 and 30%) and total sample volume (3.0, 6.5 and 10.0 mL). The pH level was adjusted to keep it at 4, using a solution of HCl (0.0001 mol L -1 ); and at pH = 10 during the extraction using a buffer solution of NH 4 OH/NH 4 Cl (pH = 11).…”