Mazhar S H R A, Chrystyn H
School of Pharmacy and Institute of Pharmaceutical Innovation, School of Life Sciences, University of Bradford, Bradford BD71DP, UK.
J Pharm Biomed Anal. 2009 Sep 8;50(2):175-82. doi: 10.1016/j.jpba.2009.04.006. Epub 2009 Apr 11.
A new reversed phase high performance liquid chromatography (HPLC) method with fluorescence detection and two solid phase extraction (SPE) methods have been developed, optimised and validated for determining salbutamol in human urine after an inhalation. SPE methodology for unchanged salbutamol (USAL) and salbutamol plus its metabolites (USALMET) concentrations in urine has been developed using terbutaline as the internal standard. Confirm HCX cartridges were used for USAL and Oasis HLB for USALMET. Calibration lines of salbutamol urine standards were linear over the range 25-300 microg/L with mean (RSD) r(2) values of 0.9983 (0.06%) for USAL and 0.9976 (0.202%) for USALMET. The HPLC method was accurate (mean bias -0.40% for USAL and 0.46% for USALMET) and precise (mean RSD 5.0% for USAL and 2.90% for USALMET). The calculated LOD and LOQ for salbutamol using a 1 mL urine sample were 4.0 and 12.12 microg/L for USAL, and 4.80 and 14.56 microg/L for USALMET, respectively. The mean (RSD) SPE recoveries of salbutamol were 90.82% (2.32%) for USAL and 91.54% (2.96%) for USALMET. Both HPLC and SPE methods were applied to quantify unchanged and metabolised salbutamol excreted in urine after the inhalation of 200 microg salbutamol from metered dose inhalers (MDIs) by 14 healthy volunteers. Charcoal slurries were also ingested to prevent gastro-intestinal absorption. Urine samples were collected at 30 min post-inhalation and then pooled for the next 24h. All urine concentrations were within the sensitive portion of the assay. The volunteer study revealed that following inhalation from an MDI about 20% of the nominal dose is deposited into the lungs and 46% is delivered to the systemic circulation. The results confirm the application, sensitivity, reliability and robustness of the HPLC and SPE methods for urinary pharmacokinetic studies after salbutamol inhalations using therapeutic doses.
已开发、优化并验证了一种用于测定吸入后人体尿液中沙丁胺醇的新型反相高效液相色谱（HPLC）荧光检测法以及两种固相萃取（SPE）方法。已使用特布他林作为内标物开发了用于测定尿液中未变化的沙丁胺醇（USAL）和沙丁胺醇及其代谢物（USALMET）浓度的SPE方法。对于USAL使用Confirm HCX柱，对于USALMET使用Oasis HLB柱。沙丁胺醇尿液标准品的校准曲线在25 - 300μg/L范围内呈线性，USAL的平均（相对标准偏差）r²值为0.9983（0.06%），USALMET的平均（相对标准偏差）r²值为0.9976（0.202%）。HPLC方法准确（USAL的平均偏差为 - 0.40%，USALMET的平均偏差为0.46%）且精密度高（USAL的平均相对标准偏差为5.0%，USALMET的平均相对标准偏差为2.90%）。使用1 mL尿液样品计算出的沙丁胺醇的检测限（LOD）和定量限（LOQ），USAL分别为4.0和12.12μg/L，USALMET分别为4.80和14.56μg/L。沙丁胺醇的平均（相对标准偏差）SPE回收率，USAL为90.82%（2.32%），USALMET为91.54%（2.96%）。HPLC和SPE方法均应用于对14名健康志愿者吸入200μg沙丁胺醇计量吸入器（MDI）后尿液中排泄的未变化和代谢的沙丁胺醇进行定量。还摄入了活性炭浆液以防止胃肠道吸收。在吸入后30分钟收集尿液样品，然后在接下来的24小时内合并。所有尿液浓度均在分析方法的灵敏范围内。志愿者研究表明，从MDI吸入后，约20%的标称剂量沉积到肺部，46%进入体循环。结果证实了HPLC和SPE方法在使用治疗剂量吸入沙丁胺醇后进行尿液药代动力学研究中的应用、灵敏度、可靠性和稳健性。
