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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 unequivocally its prior use in horses is required. This study describes an effective screening method for filgrastim in equine plasma by enzyme-linked immunosorbant assays (ELISA), and a follow-up confirmatory method for the unequivocal identification of filgrastim by analysing its highly specific tryptic peptide (1)MTPLGPASSLPQSFLLK(17). Filgrastim was isolated from equine plasma by immunoaffinity purification. After trypsin digestion, the mixture was analysed by nano-liquid chromatography-tandem mass spectrometry (LC/MS/MS). Filgrastim could be detected and confirmed at 0.2ng/mL in equine plasma. The applicability of the ELISA screening method and the LC/MS/MS confirmation method was demonstrated by analysing post-administration plasma samples collected from horses having been co-administered with epoetin alfa as recombinant human erythropoietin (rhEPO) and filgrastim as rhG-CSF. rhEPO and filgrastim could be detected in plasma samples collected from horses for at least 57 and 101h respectively. To our knowledge, this is the first identification of filgrastim in post-administration samples from horses., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A selectable one-dimensional ((1)D) or two-dimensional ((2)D) gas chromatography-mass spectrometry (GC-MS) system coupled with flame ionization detector (FID) and olfactory detection port (ODP) was employed in this study to analyze perfume oil and fragrance in shower gel. A split/splitless (SSL) injector and a programmable temperature vaporization (PTV) injector are connected via a 2-way splitter of capillary flow technology (CFT) in this selectable (1)D/(2)D GC-MS/FID/ODP system to facilitate liquid sample injections and thermal desorption (TD) for stir bar sorptive extraction (SBSE) technique, respectively. The dual-linked injectors set-up enable the use of two different injector ports (one at a time) in single sequence run without having to relocate the (1)D capillary column from one inlet to another. Target analytes were separated in (1)D GC-MS/FID/ODP and followed by further separation of co-elution mixture from (1)D in (2)D GC-MS/FID/ODP in single injection without any instrumental reconfiguration. A (1)D/(2)D quantitative analysis method was developed and validated for its repeatability - tR; calculated linear retention indices (LRI); response ratio in both MS and FID signal, limit of detection (LOD), limit of quantitation (LOQ), as well as linearity over a concentration range. The method was successfully applied in quantitative analysis of perfume solution at different concentration level (RSD≤0.01%, n=5) and shower gel spiked with perfume at different dosages (RSD≤0.04%, n=5) with good recovery (96-103% for SSL injection; 94-107% for stir bar sorptive extraction-thermal desorption (SBSE-TD)., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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 equine sports, a method to definitely identify its prior use in horses is required. This study describes a method for the simultaneous detection of N-acetylated LKKTETQ and its metabolites in equine urine and plasma samples. The possible metabolites of N-acetylated LKKTETQ were first identified from in vitro studies. The parent peptide and its metabolites were isolated from equine urine or plasma by solid-phase extraction using ion-exchange cartridges, and analysed by liquid chromatography-mass spectrometry (LC/MS). These analytes were identified according to their LC retention times and relative abundances of the major product ions. The peptide N-acetylated LKKTETQ could be detected and confirmed at 0.02 ng/mL in equine plasma and 0.01 ng/mL in equine urine. This method was successful in confirming the presence of N-acetylated LKKTETQ and its metabolites in equine urine and plasma collected from horses administered with a single dose of TB-500 (containing 10mg of N-acetylated LKKTETQ). To our knowledge, this is the first identification of TB-500 and its metabolites in post-administration samples from horses., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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 anabolic steroids in horse urine by incorporating a chemical derivatisation step, using picolinic acid as the derivatisation reagent. The method involved solid-phase extraction (SPE) of both free and conjugated anabolic steroids in horse urine using a polymer-based SPE cartridge (Abs Elut Nexus). The conjugated steroids in the eluate were hydrolysed by methanolysis and the resulting extract was further cleaned up by liquid-liquid extraction. The resulting free steroids in the extract were derivatised with picolinic acid to form the corresponding picolinoyl esters and analysed by UHPLC/MS/MS in the positive ESI mode with selected-reaction-monitoring. Separation of the targeted steroids was performed on a C18 UHPLC column. The instrument turnaround time was 10.5 min inclusive of post-run equilibration. A total of thirty-three anabolic steroids (including 17β-estradiol, 5(10)-estrene-3β,17α-diol, 5α-estrane-3β,17α-diol, 17α-ethyl-5α-estran-3α,17β-diol, 17α-methyl-5α-androstan-3,17β-diols, androstanediols, nandrolone and testosterone) spiked in negative horse urine at the QC levels (ranging from 0.75 to 30 ng/mL) could be consistently detected. The intra-day and inter-day precisions (% RSD) for the peak area ratios were around 7-51% and around 1-72%, respectively. The intra-day and inter-day precisions (% RSD) for the relative retention times were both less than 1% for all analytes, except the inter-day precision for boldione at 1.2%. The extraction recoveries for all targets were not less than 48%. With exceptional separation achieved by the UHPLC system, matrix interferences were minimal at the expected retention times of the selected transitions. As detection was performed with an UHPLC system coupled to a fast-scanning triple quadrupole mass spectrometer, the method could easily be expanded to accommodate additional steroid targets. This method has been validated for recovery and precision, and could be used regularly for doping control testing of anabolic steroids in horse urine samples., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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 as doping agents. The author's laboratory has previously reported a method for the detection of bovine, porcine and human insulins, as well as the synthetic analogues Humalog (Lispro) and Novolog (Aspart) in equine plasma. This study describes a complementary method for the simultaneous detection of five exogenous insulins and their possible metabolites in equine urine. Insulins and their possible metabolites were isolated from equine urine by immunoaffinity purification, and analysed by nano liquid chromatography-tandem mass spectrometry (LC/MS/MS). Insulin and its analogues were detected and confirmed by comparing their retention times and major product ions. All five insulins (human insulin, Humalog, Novolog, bovine insulin and porcine insulin), which are exogenous in horse, could be detected and confirmed at 0.05ng/mL. This method was successfully applied to confirm the presence of human insulin in urine collected from horses up to 4h after having been administered a single low dose of recombinant human insulin (Humulin R, Eli Lilly). To our knowledge, this is the first identification of exogenous insulin in post-administration horse urine samples., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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 chromatographic separation of the targeted drugs were performed using respectively a polymeric extraction column (2 cm L x 2.1mm ID, 25 microm particle size) and a reversed-phase C18 LC column (3 cm L x 2.1mm ID, 3 microm particle size) with gradient elution to provide fast analysis time. The overall instrument turnaround time was 9.5 min, inclusive of post-run and equilibration time. Plasma samples fortified with 19 targeted drugs including narcotic analgesics, local anaesthetics, antipsychotics, bronchodilators, mucolytics, corticosteroids, sedative and tranquillisers at sub-parts per billion (ppb) to low parts per trillion (ppt) levels could be consistently detected. No significant matrix interference was observed at the expected retention times of the targeted ion transitions. Over 70% of the drugs studied gave detection limits at or below 100 pg/mL, with some detection limits reaching down to 19 pg/mL. The method had been validated for extraction recovery, precision and sensitivity, and a blockage study had also been carried out. This method is used regularly in the authors' laboratory to screen for the presence of targeted drugs in pre-race plasma samples from racehorses., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

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 insulins, as well as the synthetic analogues Humalog (Lilly) and Novolog (Novo Nordisk) in equine plasma. Insulins were isolated from equine plasma by immunoaffinity purification, followed by centrifugal filtration, and analysed by nano-liquid chromatography-tandem mass spectrometry (LC/MS/MS). Insulin and analogues were detected and confirmed by comparing their retention times and major product ions. All five insulins (human insulin, Humalog, Novolog, bovine insulin and porcine insulin), which are exogenous in the horse, could be detected and confirmed at 0.05ng/mL. This method was successful in confirming the presence of human insulin in plasma collected from horses up to 4h after having been administered a single low dose of recombinant human insulin (Humulin R, Eli Lilly). To our knowledge, this is the first identification of exogenous insulin from post-administration horse plasma samples.

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 ionization (+ESI) mode with selected reaction monitoring (SRM) scan function. Chromatographic separation of the targeted drugs was achieved using a reverse phase 3.3 cm L x 2.1 mm ID, 3 microm particle size LC column with gradient elution. Plasma samples fortified with 66 targeted drugs including betamethasone, boldione, capsaicin, flunisolide, gestrinone, gliclazide, 17alpha-hydroxyprogesterone hexanoate, isoflupredone and triamcinolone acetonide, etc. at low ppt to low ppb levels could be consistently detected. No significant matrix interference was observed at the retention time of the targeted ion transitions when blank plasma samples were analysed. The method has been validated for its extraction recoveries, precision and sensitivity, and is used regularly in the authors' laboratory to screen for the presence of these drugs in plasma samples from racehorses.

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 quantitative determinations. To comply with the new requirement, a protocol was established in the authors' laboratory in 2001. The protocol has since evolved based on our practical experience, and a refined version was adopted in 2004. This paper describes our approach in establishing the MU, as well as some other important considerations, for the quantification of threshold substances in biological samples as applied in the area of doping control for horses. The testing of threshold substances can be viewed as a compliance test (or testing to a specified limit). As such, it should only be necessary to establish the MU at the threshold level. The steps in a "Bottom-Up" approach adopted by us are similar to those described in the EURACHEM/CITAC guide, "Quantifying Uncertainty in Analytical Measurement". They involve first specifying the measurand, including the relationship between the measurand and the input quantities upon which it depends. This is followed by identifying all applicable uncertainty contributions using a "cause and effect" diagram. The magnitude of each uncertainty component is then calculated and converted to a standard uncertainty. A recovery study is also conducted to determine if the method bias is significant and whether a recovery (or correction) factor needs to be applied. All standard uncertainties with values greater than 30% of the largest one are then used to derive the combined standard uncertainty. Finally, an expanded uncertainty is calculated at 99% one-tailed confidence level by multiplying the standard uncertainty with an appropriate coverage factor (k). A sample is considered positive if the determined concentration of the threshold substance exceeds its threshold by the expanded uncertainty. In addition, other important considerations, which can have a significant impact on quantitative analyses, will be presented.

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 extraction (SPE) using a Bond Elut Certify cartridge, and analysed by LC-MS-MS in positive electrospray ionization (+ESI) and multiple reaction monitoring (MRM) mode. Liquid chromatography was performed using a short C(8) column (3.3 cm L x 2.1mm ID with 3 microm particles) to provide fast analysis time. The overall instrument turnaround time was 8 min, inclusive of post-run and equilibration time. No interference from the matrices at the expected retention times of the targeted masses was observed. Over 60% of the drugs studied gave limits of detection (LoD) at or below 25 pg/mL, with some LoDs reaching down to 0.5 pg/mL. The inter-day precision for the relative retention times ranged from 0.01 to 0.54%, and that for the relative peak area ratios (relative to the internal standard) ranged from 4 to 37%. The results indicated that the method has acceptable precision to be used on a day-to-day basis for qualitative identification.

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 remaining aqueous extract was re-adjusted to pH 6 and acidic drugs were recovered by liquid/liquid extraction. Detection was again achieved using LC-MRM but in the negative ESI mode. A total of 40 anabolic steroids and corticosteroids, and over 50 acidic drugs, including some cyclooxygenase-2 (COX-2) inhibitors, oxicams, anti-diabetics, sedatives, diuretics and Delta(9)-tetrahydro-11-norcannabinol-9-carboxylic acid, could be covered by the two LC-MS methods. Both methods utilized a high efficiency reversed-phase column (3.3 cm L x 2.1 mm I.D. with 3 microm particles) coupled with a fast-scanning triple-quadrupole mass spectrometer to achieve fast turnaround times. The overall turnaround times for both methods were 10 min, inclusive of post-run and equilibration times.