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Pre-race dosing of horses with alkalinising agents to manipulate performance has been evident in racing worldwide for over 30 years. To regulate the use of alkalinising agents, racing authorities adopted thresholds for total plasma carbon dioxide (TCO 2 ) in racehorses. Traditionally, racing laboratories have measured plasma TCO 2 using ion selective electrode (ISE) technology, with the Association of Official Racing Chemists (AORC) approving the use of only three ISE instruments for measurement. Because of the manufacture and support of these instruments ceasing, racing laboratories have explored alternative techniques to measure plasma TCO 2 . In this study, headspace gas chromatography mass spectrometry (HSGCMS) with fully automated sample preparation was investigated as an alternative technique to ISE. Sample preparation was carried out online on a Gerstel robot, where plasma was aspirated directly from sealed vacutainer tubes before further treatment and headspace injection into a GCMS. The method was successfully cross validated against a Beckman Unicel DxC®600, meeting all criteria stipulated in the AORC cross-validation protocol. The method achieved an accuracy of 99.8%, within-run relative standard deviation of 0.22% and interday reproducibility of 0.04 mM, all significant improvements on the authors ISE method. A population study was also conducted to ensure the plasma TCO 2 threshold, established with ISE methodology, did not change with the developed HSGCMS method. The concentrations and standard deviations for the two methods were almost identical, HSGCMS mean 30.62 mM, standard deviation 1.65 mM, and ISE 30.65 and 1.55 mM. The results indicate that the fully automated HSGCMS method is suitable for measurement of equine plasma TCO 2 for regulatory purposes., (© 2022 John Wiley & Sons Ltd.)

The doping of greyhound dogs with testosterone is done in an attempt to improve their athletic performance, but such doping cannot easily be confirmed, especially in male dogs owing to the natural presence of endogenous testosterone. As testosterone is usually administered as its esters, their direct detection in hair would provide confirmatory evidence of the administration of a pharmaceutical product. This article demonstrates that the use of a liquid chromatography-high resolution mass spectrometry method with heated electrospray ionisation (HESI) combined with the use of amino solid-phase extraction (SPE) cartridges for sample clean-up, is suitable for the sensitive determination of propionate, phenyl propionate, isocaproate, decanoate, and enanthate esters of testosterone in greyhound hair. The method is linear over the range, 0.1 μg/kg-10 μg/kg, for all the testosterone esters analysed. The limits of detection (LOD) are 0.05 μg/kg for testosterone phenyl propionate, isocaproate, and decanoate, 0.025 μg/kg for testosterone propionate, and 0.25 μg/kg for testosterone enanthate. This method was applied to hair samples collected from male greyhounds before and after a single administration of a product containing several testosterone esters, each of which could be detected up to 100 days post-administration. The study also demonstrates that tail hair is the specimen of choice for the analysis of testosterone in dog hair and that washing of dogs does not impact the analysis of testosterone esters in hair. This method may be useful in racing regulation for the detection of illegitimate use of testosterone in all species., (Copyright © 2017 John Wiley & Sons, Ltd.)

Zolpidem is a non-benzodiazepine hypnotic that has been implicated in both drug-facilitated sexual assault and drink spiking. Detection of the drug in urine is extremely difficult because of its extensive metabolism. A method is presented for the detection and quantitation of zolpidem carboxylic acid (ZCA), the major urinary metabolite of zolpidem. The metabolite was extracted from urine at pH 4.5-5.0 with chloroform/isopropanol alkylated with ethyl iodide and identified by gas chromatography-mass spectrometry in the selected ion-monitoring mode. Following a single ingestion of 10 mg zopidem, ZCA is detected for up to 72 h. The limit of detection is 2 ng/mL, with an overall recovery of 80%. Using this procedure, zolpidem was identified in two cases of alleged drug-facilitated sexual assault.

Protopine was extracted from Fumaria officinalis and purified by column chromatography. Urine samples were collected from horses and a human volunteer that had been administered either F. officinalis or protopine free base. Plant and urine samples were acetylated and analysed by GCMS after solid-phase extraction (SPE). The urinary metabolites of protopine were identified as 4,6,7,13-tetrahydro-9,10-dihydroxy-5-methyl-benzo[e]-l,3-benzodioxolo [4,5-1][2] benzazecin-12(5H)-one, 4,6,7,13-tetrahydro-10-hydroxy-9-methoxy-5-methyl-benzo[e]-1,3-benzodioxolo[4,5-1][2] benzazecin-12(5H)-one and 4,6,7,13-tetrahydro-9-hydroxy-10-methoxy-5-methyl-benzo[e]-1,3-benzodioxolo[4,5-l][2] benzazecin-12(5H)-one, chelianthifoline, isochelianthifoline and 2-O-desmethylchelianthifoline. The metabolic formation of the tetrahydroprotoberberines by closure of the bridge across N5 and C13 is rate limited and protopine-like metabolites accumulate only when the route is overloaded. Metabolism was qualitatively similar in the horse and human.

The influence of sampling variables on the concentration of the dopamine metabolites 3-methoxytyramine (3MT), dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) was examined in equine urine. A logarithmic transformation of the data for all horses gave distribution which approximated the normal distributions for each metabolite. The mean urinary concentration of 3 MT in horses was 214 ng/mL and the application of a threshold with a probability of 1 in 10,000 gave an actionable level of 4 microg/mL. Environmental variables were not forensically significant in determining the population distribution. HVA was not found to be a reliable indicator of dopamine or levodopa administration.

Comprehensive two-dimensional gas chromatography (GC x GC) now occupies a niche within the GC technology regime. The technique is undeniably unique in the manner in which the experiment is conducted, the way results are presented and the interpretive opportunities offered. For the 1000th volume of this journal it is appropriate to expand upon these features, and review the progress made in GC x GC to date. Firstly, brief general comment is made on multidimensional procedures, and to review key aspects of GC x GC. The use of the targeted multidimensional GC method allows absolute retentions in the second dimension of a GC x GC experiment to be estimated, and also offers a novel way to obtain enhanced response for resolved solutes. Then, to illustrate the utility of the technique, the application of GC x GC to the screening of drugs and their metabolites in biological fluids is described using prolintane metabolites in canine urine as an example, with samples taken at four time intervals after administration. This example illustrates the first application of GC x GC in the field of forensic toxicology, an area traditionally dominated by GC-MS. Most drug compounds were found to be retained on the 0.8-m second column for a greater time than the modulation period (3 s) used for initial analysis, under the conditions described. Hence a 0.4-m D2 BPX50 (50% phenyl methyl polysilphenylene) column was then used throughout, with most compounds retained less than 4 s. For the standard drug mixture, three overlapping drugs on the first dimension column (BPX5) were subsequently baseline resolved on the BPX50 column. For prolintane administration samples, the parent drug and metabolites could be effectively resolved from background matrix peaks. Likewise a 23-drug spike standard in horse urine blank gave acceptable resolution of the drugs from matrix peaks.

Objective: To compare physiologic and analgesic effects of morphine when given by IV constant-rate infusion or by IM injection to dogs undergoing laparotomy and to determine pharmacokinetics of morphine in dogs following IV constant-rate infusion., Design: Prospective randomized controlled trial., Animals: 20 dogs., Procedure: Dogs undergoing laparotomy were treated with morphine beginning at the time of anesthetic induction. Morphine was administered by IV infusion (0.12 mg/kg/h [0.05 mg/lb/h] of body weight) or by IM injection (1 mg/kg [0.45 mg/lb]) at induction and extubation and every 4 hours thereafter. Treatments continued for 24 hours after extubation., Results: Blood gas values did not indicate clinically significant respiratory depression in either group, and degree of analgesia (determined as the University of Melbourne Pain Scale score) and incidence of adverse effects (panting, vomiting, defecation, and dysphoria) were not significantly different between groups. Dogs in both groups had significant decreases in mean heart rate, rectal temperature, and serum sodium and potassium concentrations, compared with preoperative values. Mean +/- SEM total body clearance of morphine was 68 +/- 6 ml/min/kg (31 +/- 3 ml/min/lb). Mean steady-state serum morphine concentration in dogs receiving morphine by constant-rate infusion was 30 +/- 2 ng/ml., Conclusions and Clinical Relevance: Results indicated that administration of morphine as a constant-rate IV infusion at a dose of 0.12 mg/kg/h induced effects similar to those obtained with administration at a dose of 1 mg/kg, IM, every 4 hours in dogs undergoing laparotomy. Panting was attributed to an opioid-induced resetting of the hypothalamic temperature set point, rather than respiratory depression.

A procedure for the quantification of leukotriene B4 (LTB4) in synovial fluid has been developed using gas chromatography/tandem mass spectrometry based on selected reaction monitoring of the elimination of t-butyldimethylsilanol from the ions of m/z 431 and 438 in the negative ion chemical ionization mass spectra of the di-t-butyldimethylsilyl/pentafluorobenzyl derivatives of leukotriene B4 and the internal standard (2H8)leukotriene B4. The detection limit (approximately 10 pg ml-1) is sufficiently low to permit determination of LTB4 concentration in the synovial fluid of patients with various arthropathies. Single-stage mass spectrometry was found not to be selective enough to permit quantification of LTB4 in synovial fluid.

Indole 3-acetic acid (IAA) was analyzed in apple, orange, and prune tissue by GC-MS by monitoring the protonated molecular ion of its methyl ester at mass to charge ratio (m/z) 190 together with the major fragment ion at m/z 130 and the corresponding ions from the methyl esters of either [(2)H(4)]IAA (m/z 194, 134) or [(2)H(5)]IAA (m/z 195, 135). Abscisic acid (ABA) was analyzed by monitoring the major fragment ions of its methyl ester at m/z 261 and m/z 247 and the corresponding ions from the methyl ester of [(2)H(3)]ABA (m/z 264, 250). Detection limits for IAA and ABA were 1 and 10 picograms, respectively using standards and 1 nanogram per gram dry weight for both phytohormones in plant tissue.

Plasma and synovial fluid concentrations of biphenylacetic acid were determined following application of 3 g of 3% biphenylacetic acid gel to one knee of patients suffering from rheumatoid arthritis. The mean peak plasma concentration was 34 ng/ml. Synovial fluid concentrations tended to follow plasma concentrations but at a somewhat lower level, the mean peak synovial fluid concentration was 21 ng/ml. The average ratio of synovial fluid AUC (0-24 h) to plasma AUC (0-24 h) was 0.58, r = 0.97. Where patients had bilateral effusions, the concentration in the ipsilateral knee at each time point examined was not significantly different to that in the contralateral knee, suggesting that absorption was initially into the plasma and subsequently into the synovium.