P. Teale

Gradient capillary electrochromatography of drug mixtures with UV and electrospray ionisation mass spectrometric detection

Abstract A simple method for forming reproducible mobile phase gradients with capillary electrochromatography (CEC) has been demonstrated. It allows short columns and low ionic strength buffers to be used and it lends itself to mass spectrometric detection. Mixtures of benzodiazepines, corticosteroids and thiazide diuretic drugs were separated using UV absorbance and electrospray ionisation mass spectrometric (ESI-MS) detection. Separations were performed on fused-silica capillary columns (33–50 cm×50–75 μm I.D.) packed with CEC Hypersil ODS (3 μm) and Apex ODS (3 μm) particles. A stainless steel tee connector was used to interface the CEC column with samples and mobile phase delivered from a HPLC autosampler and pump. A voltage (30 kV) was applied to the tee connector causing electrokinetic transfer of samples and mobile phase into the CEC column. Pre-conditioning of the CEC column with mobile phase of low organic content allowed dilute samples to be stacked at the head of the column prior to elution.

Development of a gas chromatographic—mass spectrometric method using multiple analytes for the confirmatory analysis of anabolic steroid residues in horse urine : II. Detection of administration of 19-nortestosterone phenylpropionate to equine male castrates and fillies

Esters of 19-nortestosterone form an important group of anabolic preparations used in veterinary practice. Based upon results from detailed metabolic studies for 19-nortestosterone in the horse, a method to confirm the administration of anabolic preparations of this steroid to castrated male horses and fillies is described; the method is based upon the use of multiple analytes. Following administration of the anabolic preparations, solid-phase extraction of urinary conjugates and the separation of the conjugate groups prior to hydrolysis allow for the determination of specific metabolites conjugated with either glucuronic acid or sulphate. Following hydrolysis of the conjugates, purification of the free neutral steroids on thin-layer chromatography, derivatisation and gas chromatographic-mass spectrometric analysis, the presence of the major metabolites, estrane-3,17 alpha-diol in the glucuronic acid fraction and 19-nortestosterone and two isomers of estrane-3,17-diol in the sulphate fraction, could be confirmed for 17-18 days after administration of Nandrolin (19-nortestosterone phenylpropionate).

Improved capillary gas-chromatographic-mass spectrometric method for the determination of anabolic steroid and corticosteroid metabolites in horse urine using on-column injection with high-boiling solvents

When analysing large numbers of samples containing high-boiling components by capillary column gas chromatography, the analysis time using hexane as solvent in the splitless mode is prohibitive. In order to reduce the analysis time for the steroid TMS and MO-TMS derivatives but yet maintain a solvent effect, the use of solvents with high boiling-points has been investigated. The use of solvents with high boiling-points has now been extended to on-column injection without any loss in resolution or adverse effects on column lifetime.

Comparison of the use of mass spectrometry and methylene unit values in the determination of the stereochemistry of estranediol, the major urinary metabolite of 19-nortestosterone in the horse.

The stereochemistry of an isomer of 5-estrane-3,17 alpha-diol, the major metabolite of 19-nortestosterone in horse urine has been established by the use of methylene unit (MU) values. The empirical MU values of the bis-trimethylsilyl (TMS) derivatives of the eight available isomers of 5-androstane-3,17-diol and four isomers of 5-estrane-3,17 beta-diol were determined by capillary gas chromatography using three different columns. From this data the theoretical MU values for the bis-TMS derivatives of the four 5-estrane-3,17 alpha-diol isomers were predicted. Comparison of the experimentally determined MU value of the urinary metabolite with those of the theoretical values established the correct stereochemistry of the steroid. This method has been compared with the use of gas chromatography-mas spectrometry in the determination of the stereochemistry of unknown metabolites.

Some applications of chromatography to steroid analysis in the horse

Since 1974 an extensive research programme has been carried out in this laboratory in order to develop methods to detect and confirm the administration of both anabolic steroids and corticosteroids to the horse. Success has been achieved through the application of results obtained from detailed studies on metabolism and from the determination of enaogenous steroid profiles. Chromatography, in a number of different forms, has played a vital role in these studies and, where necessary, essential chromatographic techniques have been incorporated in routine screening and confirmatory analysis procedures.

On-line immunoaffinity chromatography—high-performance liquid chromatography—mass spectrometry for the determination of dexamethasone

The determination of the synthetic corticosteroid dexamethasone in equine urine is described using coupled on-line immunoaffinity chromatography–high-performance liquid chromatography (IAC–HPLC) with detection by UV or mass spectrometry. The limit of detection for dexamethasone with UV detection was 30 µg l–1 for a 10 ml urine sample. Atmospheric pressure chemical ionization mass spectrometry, with single ion monitoring, gave a limit of detection of 0.1 µg l–1 for dexamethasone. The determination of dexamethasone by on-line IAC–HPLC–MS has been demonstrated in post-administration equine urine samples.

Analysis of endogenous human endothelin peptides by high-performance liquid chromatography and mass spectrometry

Summary: The three isoforms of both endothelin (ET) and big ET are not readily distinguishable by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) alone. We have previously used high-performance liquid chromatography (HPLC) linked with RIA to determine ET isoforms in various tissue extracts, and cell supernatants. Our aim was to confirm that the isoforms of ET secreted by human umbilical vein endothelial cells (HUVECs) are ET-1 and big ET-1, using HPLC linked to electrospray mass spectrometry (ESI-MS). Authentic synthetic peptide standards were used to obtain the specific ions for ET isoforms and to calculate their retention times on the HPLC system. ET-1, [Met7]-sulfoxy ET-1, ET-2, ET-3, and big ET-1 could all be separated by HPLC and by specific ions on ESI-MS. Pooled supernatant (500 ml) from 50 cultures of HUVECs was applied first to an anti-C-terminal ET-1 immunoaffinity column and then to an anti-C-terminal big ET-1 immunoaffinity column. The eluates were solid phase-extracted and then injected onto an HPLC system linked to ESI-MS. Peaks of mass intensity at the specific ions and retention times corresponding to ET-1, [Met7]-sulfoxy ET-1, and big ET-1 were found in these eluates. However, no signal was present for ET-2 or ET-3. These data confirm that HUVECs in culture produce ET-1, [Met7]-sulfoxy ET-1, and big ET-1.