Guan Fuyu, Uboh Cornelius, Soma Lawrence, Hess Anne, Luo Yi, Tsang Deborah S
Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, New Bolton Center Campus, Kennett Square, PA 19348, USA.
J Mass Spectrom. 2003 Aug;38(8):823-38. doi: 10.1002/jms.495.
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.
二丙酸倍氯米松（BDP）是倍氯米松（BOH）的一种强效前体药物，用于治疗马匹的急慢性呼吸道疾病。吸入给药时，BDP的治疗剂量（每匹马325微克）会导致血浆和尿液中BDP及其代谢物的浓度极低，这给马法医实验室的检测和确认带来了挑战。为了解决这个问题，开发了一种液相色谱-串联质谱联用（LC/MS/MS）方法，用于检测、确认和定量马样本中的分析物。添加到液相色谱流动相中的甲酸铵或乙酸铵缓冲液有利于BDP及其代谢物形成M + H离子，而甲酸会导致形成钠和钾加合离子（M + Na、M + K）以及M + H离子。另一方面，乙腈有利于在电喷雾电离（ESI）和大气压化学电离条件下与分析物形成丰富的溶剂加合离子M + H + CH(3)CN。相比之下，甲醇形成的溶剂加合离子比乙腈少得多。这些溶剂加合离子热稳定性好，在实验条件下不能完全脱溶剂，但它们对碰撞诱导解离（CID）非常脆弱。有趣的是，这些溶剂加合离子在三重四极杆质谱仪上被观察到，但在离子阱仪器上未被观察到，在离子阱中用作阻尼气体的氦气可能会导致溶剂加合离子通过碰撞脱溶剂。通过对BDP及其代谢物的M + H离子进行CID研究，提出了它们的裂解途径。在环境温度下超过2小时的马血浆中，BDP和B21P分别部分水解为B17P和BOH，但B17P未水解。添加到马血浆中的氟化钠抑制了BDP和B21P的水解。在马血浆和尿液样本中评估了ESI中的基质效应。该方法包括用甲基叔丁基醚从马血浆和尿液样本中提取BDP及其代谢物，在C(8)柱上用由甲醇和甲酸铵（2 mmol l(-1)，pH 3.4）组成的流动相梯度进行分离，并在三重四极杆质谱仪上对分析物进行多反应监测。血浆中BDP和B17P的检测限为13 pg ml(-1)，BOH为25 pg ml(-1)，B21P为50 pg ml(-1)，尿液中BOH的检测限为25 pg ml(-1)。该方法成功应用于对吸入BDP后的马血浆和尿液样本中的分析物进行分析。吸入极低治疗剂量（每匹马325微克）的BDP后，在给药后长达4小时的马血浆中检测并定量了BDP的主要活性代谢物B17P。
