Solid‐phase extraction of small biologically active peptides on cartridges and microelution 96‐well plates from human urine

Abstract

Currently liquid chromatography - mass spectrometry (LC-MS) analysis after solid-phase extraction (SPE) on weak cation-exchange cartridges is a method of choice for anti-doping analysis of small bioactive peptides such as growth hormone releasing peptides (GHRPs), desmoporessin, LHRH, and TB-500 short fragment. Dilution of urine samples with phosphate buffer for pH adjustment and SPE on weak cation exchange microelution plates was tested as a means to increase throughput of this analysis. Dilution using 200 mM phosphate buffer provides good buffering capacity without affecting the peptides recoveries. SPE on microelution plates was performed on Waters Positive Pressure-96 Processor with subsequent evaporation of eluates in nitrogen flow. Though the use of smaller sample volume decreases the pre-concentration factor and increases the limits of detection of 5 out of 17 detected peptides, the recovery, linearity, and reproducibility of the microelution extraction were comparable with cartridge SPE. The effectiveness of protocols was confirmed by analysis of urine samples containing ipamorelin, and GHRP-6 and its metabolites. SPE after urine sample dilution with buffer can be used for faster sample preparation. The use of microelution plates decreases consumption of solvents and allows processing of up to 96 samples simultaneously. Cartridge SPE with manual рН adjustment remains the best option for confirmation. Copyright © 2015 John Wiley & Sons, Ltd.

Detection of bioactive peptides including gonadotrophin‐releasing factors (GnRHs) in horse urine using ultra‐high performance liquid chromatography–high resolution mass spectrometry (UHPLC/HRMS)

“…Comprehensive detection methods of small peptides in both human and equine urine and blood samples using liquid chromatography mass spectrometry (LC/MS) have been reported recently. [13][14][15][16][17][18][19][20][21][22][23] Solidphase extraction (SPE) is a commonly adopted technique for sample clean-up and pre-concentration of peptidic drugs from biological matrices. In 2012, the authors' laboratory first published a method on the detection of a peptide, N-acetylated Leu-Lys-Lys-Thr-Glu-Thr-Gln (also known as TB-500) and its metabolites in equine urine and plasma using ion-exchange cartridges followed by analysis using liquid chromatography-high resolution mass spectrometry (LC/HRMS).…”

“…16 Similar approaches of using mixed mode SPE cartridges to cover different classes of peptides have also been reported by other laboratories. [17][18][19][20] Dilute-and-shoot is another promising approach for the detection of bioactive peptides in the past few years, however, high sensitivity instrumentation would be required. Thomas et al has reported a direct urine injection method using two-dimensional LC coupled to ion mobility mass spectrometry (IM/MS) for the detection of 18 peptides (<2 kDa each) in human urine.…”

Identification of gonadotropin‐releasing hormone metabolites in greyhound urine

“…A wide range of MS‐based methods for the detection of intact GnRH in urine have been reported including affinity isolation techniques based on antibodies or aptamers combined with nanoLC–MS or capillary electrophoresis‐mass spectrometry (CE‐MS), mixed‐mode solid‐phase extraction (SPE) followed by LC–MS and direct injection of urine into liquid chromatography‐ion mobility‐mass spectrometry (LC‐IM‐MS) . In vitro metabolites of GnRH were identified in human serum by LC–MS following SPE .…”

Detection and in vitro metabolism of the confiscated peptides BPC 157 and MGF R23H

“…Since no method has been reported for detection of BPC 157, it was tested in the GHRF peptide screening method currently used in several laboratories . The method, which uses 0.5–2.0 mL of urine, extracts peptides using a weak cation exchange solid‐phase extraction (WCX SPE) step followed by detection using HRAM LC–MS or LC–MS/MS.…”