Sensitive liquid chromatographic/tandem mass spectrometric method for the determination of beclomethasone dipropionate and its metabolites in equine plasma and urine

Abstract

Beclomethasone dipropionate (BDP) is a potent pro-drug to beclomethasone (BOH) and is used in the treatment of chronic and acute respiratory disorders in the horse. The therapeutic dose of BDP (325 microg per horse) by inhalation results in very low plasma and urinary concentrations of BDP and its metabolites that pose a challenge to detection and confirmation by equine forensic laboratories. To solve this problem, a method involving the use of a liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) was developed for the detection, confirmation and quantification of the analytes in equine samples. Ammonium formate or acetate buffer added to LC mobile phase favored the formation of [M + H](+) ions from BDP and its metabolites, whereas formic acid led to the formation of sodium and potassium adduct ions ([M + Na](+), [M + K](+)) together with [M + H](+) ions. Acetonitrile, on the other hand, favored the formation of abundant solvent adduct ions [M + H + CH(3)CN](+) with the analytes under electrospray ionization (ESI) and atmospheric pressure chemical ionization conditions. In contrast, methanol formed much less solvent adduct ions than acetonitrile. The solvent adduct ions were thermally stable and could not be completely desolvated under the experimental conditions, but they were very fragile to collision-induced dissociation (CID). Interestingly, these solvent adduct ions were observed on a triple-quadrupole mass spectrometry but not on an ion trap instrument where helium used as a damping gas in the ion trap might cause the solvent adduct ions desolvated by collision. By CID studies on the [M + H](+) ions of BDP and its metabolites, their fragmentation paths were proposed. In equine plasma at ambient temperature over 2 h, BDP and B21P were hydrolyzed in part to B17P and BOH, respectively, but B17P was not hydrolyzed. Sodium fluoride added to equine plasma inhibited the hydrolysis of BDP and B21P. The matrix effect in ESI was evaluated in equine plasma and urine samples. The method involved the extraction of BDP and its metabolites from equine plasma and urine samples by methyl tert-butyl ether, resolution on a C(8) column with a mobile phase gradient consisting of methanol and ammonium formate (2 mmol l(-1), pH 3.4) and multiple reaction monitoring for the analytes on a triple-quadrupole mass spectrometer. The detection limit was 13 pg ml(-1) for BDP and B17P, 25 pg ml(-1) for BOH and 50 pg ml(-1) for B21P in plasma and 25 pg ml(-1) for BOH in urine. The method was successfully applied to the analysis of equine plasma and urine samples for the analytes following administration of BDP to horses by inhalation. B17P, the major and active metabolite of BDP, was detected and quantified in equine plasma up to 4 h post-administration by inhalation of a very low therapeutic dose (325 microg per horse) of BDP.

Quantitative, Highly Sensitive Liquid Chromatography–Tandem Mass Spectrometry Method for Detection of Synthetic Corticosteroids

“…Our method was developed specifically for screening of synthetic glucocorticoids in patients with symptoms of Cushing syndrome with low cortisol concentrations. Previously, methods for the detection of a limited number of steroids by MS have been reported (13)(14)(15)(16). The method described here provides a good screening tool for patients with unexplained cortisol suppression or suspected iatrogenic Cushing syndrome.…”

“…For endogenous steroids, methods based on liquid chromatography/electrospray tandem mass spectrometry have been developed. For synthetic steroids, assays have been developed specifically to analyze a single or only a few synthetic glucocorticoids and their metabolites (13)(14)(15)(16)(17). We therefore developed a liquid chromatography-tandem mass spectrometry (LC-MS/ MS) assay for the simultaneous detection and quantification of the 14 most frequently used synthetic corticosteroids in human serum, plasma, and urine and in tablet extracts.…”

Screening for anabolic steroids in doping analysis by liquid chromatography/electrospray ion trap mass spectrometry

“…To obtain maximum sensitivity from the complex MS/MS spectrum of 3OHSTAN, the sum of four abundant ions was used for identification. Product ions caused by loss of water were not selected as diagnostic ion as these fragments are highly unspecific (Guan et al, 2003;Deventer et al, 2005). Diagnostic ions are presented in Table 1.…”

The effect of alkylamine additives on the sensitivity of detection for paclitaxel and docetaxel and analysis in plasma of paclitaxel by liquid chromatography–tandem mass spectrometry

“…The formation of multiple molecular ions such as [M+H] + , [M+Na] + , [M+K] + and [M+NH 4 ] + rarely occurs in atmospheric pressure chemical ionization (APCI), which mostly involves proton transfer reactions (Niessen, 1999). The phenomenon, however, can be observed for many compounds in ESI, which involves the transfer of ions in the solution phase to the gas phase (Loo, 1995;Meng and Fenn, 1991;Guan et al, 2003). Since an increase in the number of ion species created will disperse the signal and make sensitive and reproducible quantitation difficult (Li et al, 2002), supression of the formation of multiple molecular ions in ESI needs to be addressed.…”