Journal of chromatography. A.
Publisher:
Elsevier,
Frequency: Seventy eight no. a year, 2005-
Country: Netherlands
Language: English
Start Year:1993 -
ISSN:
0021-9673 (Print)
1873-3778 (Electronic)
0021-9673 (Linking)
1873-3778 (Electronic)
0021-9673 (Linking)
Impact Factor
4.1
2022
| NLM ID: | 9318488 |
| (DNLM): | SR0079068(s) |
| (OCoLC): | 29336194 |
| Coden: | JCRAEY |
| LCCN: | 94659073 |
| Classification: | W1 JO5845M |
Detection of seventy-two anabolic and androgenic steroids and/or their esters in horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry in multiplexed targeted MS2 mode and gas chromatography-tandem mass spectrometry. Anabolic and androgenic steroids (AAS) are banned substances in both human and equine sports. They are often administered intramuscularly to horses in esterified forms for the purpose of extending their time of action. The authors' laboratory has previously reported an UHPLC/HRMS method using quadrupole-Orbitrap mass spectrometer in full scan and parallel reaction monitoring (PRM) mode for the detection of 48 AAS and/or their esters in horse hair. However, two injections were required due to the long duty cycle time. In this paper, an UHPLC/HRMS method using multiplexed targeted MS mode was de...
Development and validation of an ultrahigh performance liquid chromatography-high resolution tandem mass spectrometry assay for nine toxic alkaloids from endophyte-infected pasture grasses in horse serum. Endophyte fungi (e.g. Epichloë ssp. and Neotyphodium ssp.) in symbiosis with pasture grasses (e.g. Festuca arundinacaea and Lolium perenne) can produce toxic alkaloids, which are suspected to be involved in equine diseases such as fescue toxicosis, ryegrass staggers, and equine fescue oedema. The aim of this study was, therefore, to develop and validate a quantification method for these and related alkaloids: ergocristine, ergocryptine, ergotamine, ergovaline, lolitrem B, lysergic acid, N-acetylloline, N-formylloline, peramine, and paxilline in horse serum. Horse serum samples (1.5mL) were wo...
Metabolic study of methylstenbolone in horses using liquid chromatography-high resolution mass spectrometry and gas chromatography-mass spectrometry. Methylstenbolone (2,17α-dimethyl-5α-androst-1-en-17β-ol-3-one) is a synthetic anabolic and androgenic steroid (AAS) sold as an oral 'nutritional supplement' under the brand names 'Ultradrol', 'M-Sten' and 'Methyl-Sten'. Like other AASs, methylstenbolone is a prohibited substance in both human and equine sports. This paper describes the studies of the in vitro and in vivo metabolism of methylstenbolone in horses using LC/HRMS, GC/MS and GC/MS/MS. Phase I in vitro metabolic study of methylstenbolone was performed using homogenised horse liver. Hydroxylation was the only biotransformation obse...
Detection and confirmation of α-cobratoxin in equine plasma by solid-phase extraction and liquid chromatography coupled to mass spectrometry. α-Cobratoxin (CTX) is a large peptide (71 amino acids) with strong analgesic effect and may be misused in sports such as horse racing. To prevent such misuse, a sensitive method is required for detection and confirmation of the toxin in equine samples. CTX was extracted from equine plasma using an optimized mixed-mode solid-phase extraction (SPE) procedure. Extracted CTX was reduced with dithiothreitol and alkylated with iodoacetamide, and then was digested by trypsin at 56°C for 30min. The digest was analysed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and trypt...
Liquid chromatography – high resolution mass spectrometry-based metabolomic approach for the detection of Continuous Erythropoiesis Receptor Activator effects in horse doping control. Erythropoiesis Stimulating Agents (ESAs) were developed for therapeutic purposes to stimulate red blood cell (RBC) production. Consequently, tissue oxygenation is enhanced as athlete's endurance and ESAs misuse now benefits doping. Our hypothesis is that most of ESAs should have similar mechanisms and thus have the same effects on metabolism. Studying the metabolome variations could allow suspecting the use of any ESAs with a single method by targeting their effects. In this objective, a metabolomic study was carried out on 3 thoroughbred horses with a single administration of 4.2μg/kg of Mir...
Detection of anabolic and androgenic steroids and/or their esters in horse hair using ultra-high performance liquid chromatography-high resolution mass spectrometry. Anabolic and androgenic steroids (AASs) are a class of prohibited substances banned in horseracing at all times. The common approach for controlling the misuse of AASs in equine sports is by detecting the presence of AASs and/or their metabolites in urine and blood samples using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). This approach, however, often falls short as the duration of effect for many AASs are longer than their detection time in both urine and blood. As a result, there is a high risk that such AASs could escape detection in the...
Screening of over 100 drugs in horse urine using automated on-line solid-phase extraction coupled to liquid chromatography-high resolution mass spectrometry for doping control. A fast method for the direct analysis of enzyme-hydrolysed horse urine using an automated on-line solid-phase extraction (SPE) coupled to a liquid-chromatography/high resolution mass spectrometer was developed. Over 100 drugs of diverse drug classes could be simultaneously detected in horse urine at sub to low parts per billion levels. Urine sample was first hydrolysed by β-glucuronidase to release conjugated drugs, followed by centrifugal filtration. The filtrate (1mL) was directly injected into an on-line SPE system consisting of a pre-column filter and a SPE cartridge column for the separa...
Simultaneous detection of recombinant growth hormones in equine plasma by liquid chromatography/high-resolution tandem mass spectrometry for doping control. Growth hormone (GH), or somatotropin, is a protein that may enhance physical performance and facilitate growth and wound healing. For this reason, growth hormones and their recombinant analogues are prohibited in human sports by the World Anti-Doping Agency (WADA) and in horseracing under Article 6 of the International Agreement on Breeding, Racing and Wagering published by the International Federation of Horse Racing Authorities (IFHA). Identifying the illicit use of GHs in both human athletes and racehorses is challenging, especially when differentiating between endogenous and exogenous GHs,...
Doping control analysis of 46 polar drugs in horse plasma and urine using a ‘dilute-and-shoot’ ultra high performance liquid chromatography-high resolution mass spectrometry approach. The high sensitivity of ultra high performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HRMS) allows the identification of many prohibited substances without pre-concentration, leading to the development of simple and fast 'dilute-and-shoot' methods for doping control for human and equine sports. While the detection of polar drugs in plasma and urine is difficult using liquid-liquid or solid-phase extraction as these substances are poorly extracted, the 'dilute-and-shoot' approach is plausible. This paper describes a 'dilute-and-shoot' UHPLC-HRMS screening me...
Doping control analysis of filgrastim in equine plasma and its application to a co-administration study of filgrastim and recombinant human erythropoietin in the horse. Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor regulating granulopoiesis. The recombinant human granulocyte colony-stimulating factor (rhG-CSF) is widely used for the treatment of granulopenia in humans. Filgrastim is a rhG-CSF analogue and is marketed under various brand names, including Neupogen(®) (Amgen), Imumax(®) (Abbott Laboratories), Neukine(®) (Intas Biopharmaceuticals) and others. It is banned in both human and equine sports owing to its potential for misuse. In order to control the abuse of filgrastim in equine sports, a method to identify unequivo...
Ultra high performance liquid chromatography/tandem mass spectrometry based identification of steroid esters in serum and plasma: an efficient strategy to detect natural steroids abuse in breeding and racing animals. During last decades, the use of natural steroids in racing and food producing animals for doping purposes has been flourishing. The endogenous or exogenous origin of these naturally occurring steroids has since remained a challenge for the different anti-doping laboratories. The administration of these substances to animals is usually made through an intra-muscular pathway with the steroid under its ester form for a higher bioavailability and a longer lasting effect. Detecting these steroid esters would provide an unequivocal proof of an exogenous administration of the considered naturally occ...
Doping control analysis of TB-500, a synthetic version of an active region of thymosin β₄, in equine urine and plasma by liquid chromatography-mass spectrometry. A veterinary preparation known as TB-500 and containing a synthetic version of the naturally occurring peptide LKKTETQ has emerged. The peptide segment (17)LKKTETQ(23) is the active site within the protein thymosin β(4) responsible for actin binding, cell migration and wound healing. The key ingredient of TB-500 is the peptide LKKTETQ with artificial acetylation of the N-terminus. TB-500 is claimed to promote endothelial cell differentiation, angiogenesis in dermal tissues, keratinocyte migration, collagen deposition and decrease inflammation. In order to control the misuse of TB-500 in equin...
Rapid screening of anabolic steroids in horse urine with ultra-high-performance liquid chromatography/tandem mass spectrometry after chemical derivatisation. Liquid chromatography/mass spectrometry (LC/MS) has been successfully applied to the detection of anabolic steroids in biological samples. However, the sensitive detection of saturated hydroxysteroids, such as androstanediols, by electrospray ionisation (ESI) is difficult because of their poor ability to ionise. In view of this, chemical derivatisation has been used to enhance the detection sensitivity of hydroxysteroids by LC/MS. This paper describes the development of a sensitive ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) method for the screening of a...
Simultaneous separation and determination of 16 testosterone and nandrolone esters in equine plasma using ultra high performance liquid chromatography-tandem mass spectrometry for doping control. The potential for using testosterone and nandrolone esters in racehorses to boost the biological concentrations of these steroids and enhance athletic performance is very compelling and should be seriously considered in formulating regulatory policies for doping control. In order to regulate the use of these esters in racehorses, a sensitive and validated method is needed. In this paper, we report such a method for simultaneous separation, screening, quantification and confirmation of 16 testosterone and nandrolone esters in equine plasma by ultra high performance liquid chromatography-tandem ...
Doping control analysis of insulin and its analogues in equine urine by liquid chromatography-tandem mass spectrometry. Insulin and its analogues have been banned in both human and equine sports owing to their potential for misuse. Insulin administration can increase muscle glycogen by utilising hyperinsulinaemic clamps prior to sports events or during the recovery phases, and increase muscle size by its chalonic action to inhibit protein breakdown. In order to control insulin abuse in equine sports, a method to effectively detect the use of insulins in horses is required. Besides the readily available human insulin and its synthetic analogues, structurally similar insulins from other species can also be used a...
Hydrophilic interaction chromatography of intact, soluble proteins. The separation of intact proteins by means of Hydrophilic Interaction Chromatography (HILIC) was demonstrated with human apoA-I, recombinant human apoM, and equine cytochrome C. Five different commercially available HILIC columns were compared. Using one of these columns, different glycosylated isoforms of apoM were separated from each other and from the aglyco-form.
The use of a simple backflush technology to improve sample throughput and system robustness in routine gas chromatography tandem mass spectrometry analysis of doping control samples. A simple, low cost system for the backflushing of capillary gas chromatography (GC) columns has been investigated and integrated into a method for the detection of anabolic steroids in equine urine. The modification to the method was simple to make and quick to setup and optimize. The use of backflushing technology was found to offer significant benefits in terms of sample throughput and improved system robustness.
Screening of drugs in equine plasma using automated on-line solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry. A rapid liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed for the simultaneous screening of 19 drugs of different classes in equine plasma using automated on-line solid-phase extraction (SPE) coupled with a triple quadrupole mass spectrometer. Plasma samples were first protein precipitated using acetonitrile. After centrifugation, the supernatant was directly injected into the on-line SPE system and analysed by a triple quadrupole LC-MS-MS in positive electrospray ionisation (+ESI) mode with selected reaction monitoring (SRM) scan function. On-line extraction and c...
Doping control analysis of insulin and its analogues in equine plasma by liquid chromatography-tandem mass spectrometry. Insulin administration can increase muscle glycogen by utilising hyperinsulinaemic clamps prior to sports events or during the recovery phases, and increase muscle size by its chalonic action to inhibit protein breakdown. In order to control insulin abuse in equine sports, a method to detect effectively the use of insulins in horses would be required. Besides the readily available human insulin and its synthetic analogues, structurally similar insulins from other species can also be used as doping agents. This study describes a method for the simultaneous detection of bovine, porcine and human...
Comprehensive screening of acidic and neutral drugs in equine plasma by liquid chromatography-tandem mass spectrometry. A multi-target high-throughput liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the detection of low ppt to low ppb levels of anabolic steroids, corticosteroids, anti-diabetics, and non-steroidal anti-inflammatory drugs (NSAIDs) in equine plasma was developed for the purpose of doping control. Plasma samples were first deproteinated by addition of trichloroacetic acid. Drugs were then extracted by solid-phase extraction (SPE) using Bond Elut Certify cartridges, and the extracts were analysed by a triple-quadrupole/linear ion trap LC-MS-MS instrument in positive electrospray...
A bottom-up approach in estimating the measurement uncertainty and other important considerations for quantitative analyses in drug testing for horses. Quantitative determination, particularly for threshold substances in biological samples, is much more demanding than qualitative identification. A proper assessment of any quantitative determination is the measurement uncertainty (MU) associated with the determined value. The International Standard ISO/IEC 17025, "General requirements for the competence of testing and calibration laboratories", has more prescriptive requirements on the MU than its superseded document, ISO/IEC Guide 25. Under the 2005 or 1999 versions of the new standard, an estimation of the MU is mandatory for all quantitativ...
High throughput screening of sub-ppb levels of basic drugs in equine plasma by liquid chromatography-tandem mass spectrometry. This paper describes a high throughput LC-MS-MS method for the screening of 75 basic drugs in equine plasma at sub-ppb levels. The test scope covers diversified classes of drugs including some alpha- and beta-blockers, alpha- and beta-agonists, antihypotensives, antihypertensives, analgesics, antiarrhythmics, antidepressants, antidiabetics, antipsychotics, antiulcers, anxiolytics, bronchodilators, CNS stimulants, decongestants, sedatives, tranquilizers and vasodilators. A plasma sample was first deproteinated by addition of trichloroacetic acid. Basic drugs were then extracted by solid-phase e...
Comprehensive screening of anabolic steroids, corticosteroids, and acidic drugs in horse urine by solid-phase extraction and liquid chromatography-mass spectrometry. This paper reports two highly efficient liquid chromatography-mass spectrometry (LC-MS) methods for the screening of anabolic steroids, corticosteroids, and acidic drugs for the purpose of doping control in equine sports. Sample extraction was performed using a mixed-mode C8-SCX solid-phase extraction (SPE) cartridge. The first eluted fraction (acidic/neutral fraction) was base-washed and the resulting organic extract was used for the screening of anabolic steroids and corticosteroids by LC-MS using multiple reaction monitoring (MRM) in the positive electrospray ionisation (ESI) mode. The rema...
Separation and detection of the isomeric equine conjugated estrogens, equilin sulfate and delta8,9-dehydroestrone sulfate, by liquid chromatography–electrospray-mass spectrometry using carbon-coated zirconia and porous graphitic carbon stationary phases. Equilin-3-sulfate and delta8,9-dehydroestrone-3-sulfate are two isomers found in equine conjugated estrogens that differ in structure only by the position of a double bond in the steroid B-ring. These geometric isomers were not resolved on a C18 column during the analysis of conjugated estrogen drug products by LC-MS using acetonitrile-ammonium acetate buffer as the mobile phase. While no separations of these two isomers were observed on C18 or other alkyl-bonded silica based phases using a variety of mobile phase conditions, partial separations were achieved on phenyl bonded silica phases wit...
Application of the restricted-access precolumn packing material alkyl-diol silica in a column-switching system for the determination of ketoprofen enantiomers in horse plasma. The group of LiChrospher ADS (alkyl-diol silica) sorbents that make part of a unique family of restricted-access materials, have been developed as special packings for precolumns used in the LC-integrated sample processing of biofluids. The advantage of these sorbents lies in the direct injection of untreated biological fluids, that is without sample clean-up, the elimination of the protein matrix with a quantitative recovery together with an on-column enrichment. The present method is based on previous work applying UV detection at 260 nm for ketoprofen determinations. Plasma samples introduc...
Determination of phase II drug metabolites in equine urine by micellar electrokinetic capillary chromatography. Micellar electrokinetic capillary chromatography (MECC) using diode array detection has been investigated for the determination of phase I and phase II metabolites of drugs in biofluids. Methods were optimised for the determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide, normorphine, meclofenamic acid and its metabolites in equine urine. Solid-phase extraction procedure were developed to concentrate and purify the analytes from spiked and post administration urines for MECC analysis. A simple on-line procedure for monitoring the kinetics of hydrolysis of morphine-glucuroni...
Detection of non-steroidal anti-inflammatory drugs in equine plasma and urine by gas chromatography-mass spectrometry. A gas chromatographic-mass spectrometric (GC-MS) procedure for the detection of seventeen non-steroidal anti-inflammatory drugs (NSAIDs) in equine plasma and urine samples is described. The extraction of the compounds from the biological matrix was performed at acidic pH (2-3) with diethyl ether. Ethereal extracts were washed with a saturated solution of sodium hydrogencarbonate (urine) or treated with a solid mixture of sodium carbonate and sodium hydrogencarbonate (plasma). The ethereal extracts were dried and derivatized by incubation at 60 degrees C with methyl iodide in acetone in the pre...
Effect of glycosylation on the heparin-binding capability of boar and stallion seminal plasma proteins. Boar and stallion seminal plasmas were fractionated using affinity chromatography on heparin-Sepharose. In both species, among other proteins, the heparin-binding (H+) and non-heparin-binding (H-) fractions each contained glycoforms of either porcine PSP-I or equine HSP-1 and HSP-2. However, porcine H+/PSP-I eluted as a monomeric protein, whereas H-/PSP-I formed a heterodimer with PSP-II, another major seminal plasma protein. On the other hand, the stallion proteins H+/HSP-1 and H+/HSP-2 eluted together as an aggregate of relative molecular mass (M(r)) 90,000, whereas H-/HSP-1 and H-/HSP-2 elu...