A rapid acetonitrile (ACN)-based extraction method has been developed that reproducibly depletes high abundance and high molecular weight proteins from serum prior to mass spectrometric analysis. A nanoflow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) multiple reaction monitoring (MRM) method for 57 high to medium abundance serum proteins was used to characterise the ACN-depleted fraction after tryptic digestion. Of the 57 targeted proteins 29 were detected and albumin, the most abundant protein in serum and plasma, was identified as the 20th most abundant protein in the extract. The combination of ACN depletion and one-dimensional nano-LC/MS/MS enabled the detection of the low abundance serum protein, insulin-like growth factor-I (IGF-I), which has a serum concentration in the region of 100 ng/mL. One-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the depleted serum showed no bands corresponding to proteins of molecular mass over 75 kDa after extraction, demonstrating the efficiency of the method for the depletion of high molecular weight proteins. Total protein analysis of the ACN extracts showed that approximately 99.6% of all protein is removed from the serum. The ACN-depletion strategy offers a viable alternative to the immunochemistry-based protein-depletion techniques commonly used for removing high abundance proteins from serum prior to MS-based proteomic analyses.

A detailed understanding of equine drug metabolism is important for detection of drug abuse in horseracing and also in veterinary drug development and practice. To date, however, no comprehensive review of equine drug metabolism has been published. The majority of literature regarding equine drug metabolite profiles is derived from sports drug detection research and is generally targeted at detecting marker metabolites of drug abuse. However, the bulk of the literature on equine drug metabolism enzymology is derived from veterinary studies aimed at determining the molecular basis of metabolism. In this article, the phase 1 and 2 metabolisms of seven of the most important classes of drugs monitored in horseracing are reviewed, including: anabolic-androgenic steroids (AAS), β₂ -agonists, stimulants, sedatives/tranquilizers, local anesthetics, non-steroidal anti-inflammatory analgesics (NSAIDS)/cyclooxygenase-2 (COX-2) inhibitors, and opioid analgesics. A summary of the literature relating to the enzymology of drug metabolism in this species is also be presented. The future of equine drug metabolism in the area of doping research will be influenced by several factors, including: a possible move towards the increased use of blood and other alternative testing matrices; the development of assays based on intact drug conjugates; the increasing threat of 'designer' and herbal- based products; advances in the use of in vitro technologies; the increased use of liquid-chromatography/high-resolution mass spectrometry; and the possibility of screening using 'omics' approaches. Also, the recent cloning of a range of equine cytochrome P450 (CYP) enzymes opens up the potential for carrying out more detailed mechanistic pharmacological and toxicological veterinary studies.

The presence and metabolism of endogenous steroid hormones in meat-producing animals has been the subject of much research over the past 40 years. While significant data are available, no comprehensive review has yet been performed. Species considered in this review are bovine, porcine, ovine, equine, caprine and cervine, while steroid hormones include the androgenic-anabolic steroids testosterone, nandrolone and boldenone, as well as their precursors and metabolites. Information on endogenous steroid hormone concentrations is primarily useful in two ways: (1) in relation to pathological versus 'normal' physiology and (2) in relation to the detection of the illegal abuse of these hormones in residue surveillance programmes. Since the major focus of this review is on the detection of steroids abuse in animal production, the information gathered to date is used to guide future research. A major deficiency in much of the existing published literature is the lack of standardization and formal validation of experimental approach. Key articles are cited that highlight the huge variation in reported steroid concentrations that can result when samples are analysed by different laboratories under different conditions. These deficiencies are in most cases so fundamental that it is difficult to make reliable comparisons between data sets and hence it is currently impossible to recommend definitive detection strategies. Standardization of the experimental approach would need to involve common experimental protocols and collaboratively validated analytical methods. In particular, standardization would need to cover everything from the demographic of the animal population studied, the method of sample collection and storage (especially the need to sample live versus slaughter sampling since the two methods of surveillance have very different requirements, particularly temporally), sample preparation technique (including mode of extraction, hydrolysis and derivatization), the end-point analytical detection technique, validation protocols, and the statistical methods applied to the resulting data. Although efforts are already underway (at HFL and LABERCA) to produce more definitive data and promote communication among the scientific community on this issue, the convening of a formal European Union working party is recommended.

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Insulin-like growth factor-I (IGF-I) is a known biomarker of recombinant human growth hormone (rhGH) abuse, and is also used clinically to confirm acromegaly. The protein leucine-rich alpha-2-glycoprotein (LRG) was recently identified as a putative biomarker of rhGH administration. The combination of an ACN depletion method and a 5-min ultra-high-performance liquid chromatography/tandem mass spectrometry (uHPLC/MS/MS)-based selected reaction monitoring (SRM) assay detected both IGF-I and LRG at endogenous concentrations. Four eight-point standard addition curves of IGF-I (16-2000 ng/mL) demonstrated good linearity (r(2) = 0.9991 and coefficients of variance (CVs) <13%). Serum samples from two rhGH administrations were extracted and their uHPLC/MS/MS-derived IGF-I concentrations correlated well against immunochemistry-derived values. Combining IGF-I and LRG data improved the separation of treated and placebo states compared with IGF-I alone, further strengthening the hypothesis that LRG is a biomarker of rhGH administration. Artificial neural networks (ANNs) analysis of the LRG and IGF-I data demonstrated an improved model over that developed using IGF-I alone, with a predictive accuracy of 97%, specificity of 96% and sensitivity of 100%. Receiver operator characteristic (ROC) analysis gave an AUC value of 0.98. This study demonstrates the first large scale and high throughput uHPLC/MS/MS-based quantitation of a medium abundance protein (IGF-I) in human serum. Furthermore, the data we have presented for the quantitative analysis of IGF-I suggest that, in this case, monitoring a single SRM transition to a trypsin peptide surrogate is a valid approach to protein quantitation by LC/MS/MS.