Gold nanoparticle antibody conjugates for use in competitive lateral flow assays.

Gold nanoparticles (GNPs) are widely used in a variety of biomedical diagnostic assays and for imaging. Their popularity stems from key properties such as their low toxicity and high extinction coefficients, as well as straightforward synthesis methods that allow GNPs to be produced quickly and inexpensively. Here we describe the use of GNPs for visual detection in a lateral flow assay using benzodiazepine affinity assay to illustrate the methods.

The use of in vitro technologies and high-resolution/accurate-mass LC-MS to screen for metabolites of 'designer' steroids in the equine.

Detection of androgenic-anabolic steroid abuse in equine sports requires knowledge of the drug's metabolism in order to target appropriate metabolites, especially where urine is the matrix of choice. Studying 'designer' steroid metabolism is problematic since it is difficult to obtain ethical approval for in vivo metabolism studies due to a lack of toxicological data. In this study, the equine in vitro metabolism of eight steroids available for purchase on the Internet is reported; including androsta-1,4,6-triene-3,17-dione, 4-chloro,17α-methyl-androsta-1,4-diene-3,17ß-diol, estra-4,9-diene-3,17-dione, 4-hydroxyandrostenedione, 20-hydroxyecdysone, 11-keto-androstenedione, 17α-methyldrostanolone, and tetrahydrogestrinone. In order to allow for retrospective analysis of sample testing data, the use of a high-resolution (HR) accurate-mass Thermo LTQ-Orbitrap liquid chromatography-mass spectrometry (LC-MS) instrument was employed for metabolite identification of underivatized sample extracts. The full scan LC-HRMS Orbitrap data were complimented by LC-HRMS/MS and gas-chromatography-mass spectrometry (GC-MS) experiments in order to provide fragmentation information and to ascertain whether GC-MS was capable of detecting any metabolite not detected by LC-HRMS. With the exception of 20-hydroxyecdysone, all compounds were found to be metabolized by equine liver S9 and/or microsomes. With the exception of 17α-methyldrostanolone, which produced metabolites that could only be detected by GC-MS, the metabolites of all other compounds could be identified using LC-HRMS, thus allowing retrospective analysis of previously acquired full-scan data resulting from routine equine drug testing screens. In summary, while in vitro techniques do not serve as a replacement for more definitive in vivo studies in all situations, their use does offer an alternative in situations where it would not be ethical to administer untested drugs to animals.

Analysis of methyloxime derivatives of intact esters of testosterone and boldenone in equine plasma using ultra high performance liquid chromatography tandem mass spectrometry.

Analysis of equine plasma samples to detect the abuse of anabolic steroids can be complicated when the parent steroid is endogenous to the animal. Anabolic steroids are usually administered intramuscularly as synthetic esters and therefore detection of the exogenous esters provides unequivocal proof of illegal administration. An ultra high performance liquid chromatography tandem mass spectrometric (UPLC-MSMS) method for the analysis of esters of testosterone (propionate, phenylpropionate, isocaproate, and decanoate) and boldenone (undecylenate) in equine plasma has been developed. Esters were extracted from equine plasma using a mixture of hexane and ethyl acetate and treated with methoxyamine hydrochloride to form methyloxime derivatives. Metenolone enanthate was used as an internal standard. After chromatographic separation, the derivatized steroid esters were quantified using selected reaction monitoring (SRM). The limit of detection for all of the steroid esters, based on a signal to noise ratio (S/N) of 3:1, was 1-3 pg/mL. The lower limit of quantification (LLOQ) for the all of the steroid esters was 5 pg/mL when 2 mL of plasma was extracted. Recovery of the steroid esters was 85-97% for all esters except for testosterone decanoate which was recovered at 62%. The intra-day coefficient of variation (CV) for the analysis of plasma quality control (QC) samples was less than 9.2% at 40 pg/mL and less than 6.0% at 400 pg/mL. The developed assay was used to successfully confirm the presence of intact testosterone esters in equine plasma samples following intramuscular injection of Durateston® (mixed testosterone esters).

Drug metabolism in the horse: a review.

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), ß2 -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.

Multiplexed LC-MS/MS analysis of horse plasma proteins to study doping in sport.

The development of protein biomarkers for the indirect detection of doping in horse is a potential solution to doping threats such as gene and protein doping. A method for biomarker candidate discovery in horse plasma is presented using targeted analysis of proteotypic peptides from horse proteins. These peptides were first identified in a novel list of the abundant proteins in horse plasma. To monitor these peptides, an LC-MS/MS method using multiple reaction monitoring was developed to study the quantity of 49 proteins in horse plasma in a single run. The method was optimised and validated, and then applied to a population of race-horses to study protein variance within a population. The method was finally applied to longitudinal time courses of horse plasma collected after administration of an anabolic steroid to demonstrate utility for hypothesis-driven discovery of doping biomarker candidates.

A proteomic approach combining MS and bioinformatic analysis for the detection and identification of biomarkers of administration of exogenous human growth hormone in humans.

An integrated MS-based proteomic approach is described that combines MALDI-MS and LC-MS with artificial neural networks for the identification of protein and peptide biomarkers associated with recombinant human growth hormone (rhGH) administration. Serum from exercised males administered with rhGH or placebo was analysed using ELISA to determine insulin-like growth factor-I concentrations. Diluted serum from rhGH- and placebo-treated subjects was analysed for protein biomarkers by MALDI-MS, whereas LC-MS was used to analyse tryptically digested ACN-depleted serum extracts for peptide biomarkers. Ion intensities and m/z values were used as inputs to artificial neural networks to classify samples into rhGH- and placebo-treated groups. Six protein ions (MALDI-MS) correctly classified 96% of samples into their respective groups, with a sensitivity of 91% (20 of 22 rhGH treated) and specificity of 100% (24 of 24 controls). Six peptide ions (LC-MS) were also identified and correctly classified 93% of samples with a sensitivity of 90% (19 of 21 rhGH treated) and a specificity of 95% (20 of 21 controls). The peptide biomarker ion with the highest significance was sequenced using LC-MS/MS and database searching and found to be associated with leucine-rich α-2-glycoprotein.

Biomarkers: unrealized potential in sports doping analysis.

The fight against doping in sport using analytical chemistry is a mature area with a history of approximately 100 years in horse racing and at least 40 years in human sport. Over that period, the techniques used and the breadth of coverage have developed significantly. These improvements in the testing methods have been matched by the increased sophistication of the methods, drugs and therapies available to the cheat and, as a result, testing has been a reactive process constantly adapting to meet new threats. Following the inception of the World Anti-Doping Agency, research into the methods and technologies available for human doping control have received coordinated funding on an international basis. The area of biomarker research has been a major beneficiary of this funding. The aim of this article is to review recent developments in the application of biomarkers to doping control and to assess the impact this could make in the future.

Enrichment of low molecular weight serum proteins using acetonitrile precipitation for mass spectrometry based proteomic analysis.

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.