Interconversion of ephedrine and pseudoephedrine during chemical derivatization.

Gas chromatography-mass spectrometry (GC-MS) analysis after heptafluorobutyric anhydride (HFBA) derivatization was one of the published methods used for the quantification of ephedrine (EP) and pseudoephedrine (PE) in urine. This method allows the clear separation of the derivatized diastereoisomers on a methyl-silicone-based column. Recently the authors came across a human urine sample with apparently high levels (µg/ml) of EP and PE upon initial screening. However, duplicate analyses of this sample using the HFBA-GC-MS method revealed an unusual discrepancy in the estimated levels of EP and PE, with the area response ratios of EP/PE at around 29% on one occasion and around 57% on another. The same sample was re-analyzed for EP and PE using other techniques, including GC-MS after trimethylsilylation and ultra-high-performance liquid chromatography-tandem mass spectrometry. Surprisingly, the concentration of EP in the sample was determined to be at least two orders of magnitude less than what was observed with the HFBA-GC-MS method. A thorough investigation was then conducted, and the results showed that both substances could interconvert during HFBA derivatization. Similar diastereoisomeric conversion was also observed using other fluorinated acylating agents (e.g. pentafluoropropionic anhydride and trifluoroacetic anhydride). The extent of interconversion was correlated with the degree of fluorination of the acylating agents, with HFBA giving the highest conversion. This conversion has never been reported before. A mechanism for the interconversion was proposed. These findings indicated that fluorinated acylating agents should not be used for the unequivocal identification or quantification of EP and PE as the results obtained can be erroneous.

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 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 17ß-estradiol, 5(10)-estrene-3ß,17α-diol, 5α-estrane-3ß,17α-diol, 17α-ethyl-5α-estran-3α,17ß-diol, 17α-methyl-5α-androstan-3,17ß-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.

Doping control analysis of recombinant human erythropoietin, darbepoetin alfa and methoxy polyethylene glycol-epoetin beta in equine plasma by nano-liquid chromatography-tandem mass spectrometry.

Recombinant human erythropoietin (rhEPO), darbepoetin alfa (DPO) and methoxy polyethylene glycol-epoetin beta (PEG-EPO) are synthetic analogues of the endogenous hormone erythropoietin (EPO). These erythropoiesis-stimulating agents have the ability to stimulate the production of red blood cells and are commercially available for the treatment of anaemia in humans. These drugs are understood to have performance-enhancing effects on human athletes due to their stimulation of red blood cell production, thereby improving delivery of oxygen to the muscle tissues. Although their effect on horses has not been proven, these substances were thought to be similarly performance enhancing and have indeed been applied covertly to horses. As such, these protein-based drugs are prohibited by authorities in both human and equine sports. The method officially adopted by the International Olympic Committee (IOC) and World Anti Doping Agency (WADA) for the confirmation of rhEPO and/or DPO (rhEPO/DPO) in human urine is based on electrophoresis in combination with Western blotting. A shortcoming of the WADA method is the lack of definitive mass spectral data for the confirmation of a positive finding. Recently, a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the detection and confirmation of rhEPO/DPO in equine plasma was reported. However, we have not been successful in achieving the reported sensitivity. This paper presents a method for the detection and confirmation of rhEPO/DPO, as well as the newly released PEG-EPO, in equine plasma. The procedures involve immunoaffinity extraction using anti-rhEPO antibody-coated Dynabeads followed by trypsin digestion. The injected extract was further purified and concentrated using an on-line trap column in the nano-LC system. Detection and confirmation were achieved by monitoring a unique peptide segment of rhEPO/DPO/PEG-EPO using nano-liquid chromatography-tandem mass spectrometry equipped with a nanospray ionisation source operated in the selected reaction monitoring mode. rhEPO, DPO and PEG-EPO can be confirmed at 0.1, 0.2 and 1.0 ng/mL, respectively, in equine plasma.

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 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.

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 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.

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 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.

High-throughput screening of corticosteroids and basic drugs in horse urine by liquid chromatography-tandem mass spectrometry.

This paper describes two high-throughput liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods for the screening of two important classes of drugs in equine sports, namely corticosteroids and basic drugs, at low ppb levels in horse urine. The method utilized a high efficiency reversed-phase LC column (3.3 cm L x 2.1 mm i.d. with 3 microm particles) to provide fast turnaround times. The overall turnaround time for the corticosteroid screen was 5 min and that for the basic drug screen was 8 min, inclusive of post-run and equilibration times. Method specificity was assessed by analysing a total of 35 negative post-race horse urine samples. No interference from the matrices at the expected retention times of the targeted masses was observed. Inter-day precision for the screening of 19 corticosteroids and 48 basic drugs were evaluated by replicate analyses (n = 10) of a spiked sample on 4 consecutive days. The results demonstrated that both methods have acceptable precision to be used on a routine basis. The performance of these two methods on real samples was demonstrated by their applications to drug administration and positive post-race urine samples.

Screening of anabolic steroids in horse urine by liquid chromatography-tandem mass spectrometry.

Anabolic steroids have the capability of improving athletic performance and are banned substances in the Olympic games as well as in horseracing and equestrian competitions. The control of their abuse in racehorses is traditionally performed by detecting the presence of anabolic steroids and/or their metabolite(s) in urine samples using gas chromatography-mass spectrometry (GC-MS). However, this approach usually requires tedious sample processing and chemical derivatisation steps and could be very insensitive in detecting certain steroids. This paper describes a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method for the detection of anabolic steroids that are poorly covered by GC-MS. Enzyme-treated urine was processed by solid-phase extraction (SPE) using a Bond Elut Certify cartridge, followed by a base wash for further cleanup. Separation of the steroids was carried out on a reversed-phase DB-8 column using 0.1% acetic acid and methanol as the mobile phase in a gradient elution programme. The mass spectrometer for the detection of the steroids was operated in the positive electrospray ionisation (ESI) mode with multiple reaction monitoring (MRM). Urine samples fortified with 15 anabolic steroids (namely, androstadienone, 1-androstenedione, bolasterone, boldione, 4-estrenedione, gestrinone, methandrostenolone, methenolone, 17alpha-methyltestosterone, norbolethone, normethandrolone, oxandrolone, stenbolone, trenbolone and turinabol) at low ng/mL levels were consistently detected. No significant matrix interference was observed at the retention times of the targeted ion masses in blank urine samples. The method specificity, sensitivity, precision, recoveries, and the performance of the enzyme hydrolysis step were evaluated. The successful application of the method to analyse methenolone acetate administration urine samples demonstrated that the method could be effective in detecting anabolic steroids and their metabolites in horse urine.