Campestrini J, Lecaillon J B, Godbillon J
Novartis Pharma, Drug Metabolism and Pharmacokinetics, Rueil-Malmaison, France.
J Chromatogr B Biomed Sci Appl. 1997 Dec 19;704(1-2):221-9. doi: 10.1016/s0378-4347(97)00425-8.
An automated high-performance liquid chromatography (HPLC) method for the determination of formoterol in human plasma with improved sensitivity has been developed and validated. Formoterol and CGP 47086, the internal standard, were extracted from plasma (1 ml) using a cation-exchange solid-phase extraction (SPE) cartridge. The compounds were eluted with pH 6 buffer solution-methanol (70:30, v/v) and the eluate was further diluted with water. An aliquot of the extract solution was injected and analyzed by HPLC. The extraction, dilution, injection and chromatographic analysis were combined and automated using the automate (ASPEC) system. The chromatographic separations were achieved on a 5 microm, Hypersil ODS analytical column (200 mm x 3 mm I.D.), using (pH 6 phosphate buffer, 0.035 M + 20 mg/l EDTA)-MeOH-CH3CN (70:25:5, v/v/v) as the mobile phase at a flow-rate of 0.4 ml/min. The analytes were detected with electrochemical detection at an operating potential of +0.63 V. Intra-day accuracy and precision were assessed from the relative recoveries of calibration/quality control plasma samples in the concentration range of 7.14 to 238 pmol/l of formoterol base. The accuracy over the entire concentration range varied from 81 to 105%, and the precision (C.V.) ranged from 3 to 14%. Inter-day accuracy and precision were assessed in the concentration range of 11.9 to 238 pmol/l of formoterol base in plasma. The accuracy over the entire concentration range varied from 98 to 109%, and precision ranged from 8 to 19%. At the limit of quantitation (LOQ) of 11.9 pmol/l for inter-day measurements, the recovery value was 109% and C.V. was 19%. As shown from intra-day accuracy and precision results, favorable conditions (a newly used column, a newly washed detector cell and moderate residual cell current level) allowed us to reach a LOQ of 7.14 pmol/l of formoterol base (3 pg/ml of formoterol fumarate dihydrate). Improvement of the limit of detection by a factor of about 10 was reached as compared to the previously described methods. The method has been applied for quantifying formoterol in plasma after 120 microg drug inhalation to volunteers. Formoterol was still measurable at 24 h post-dosing in most subjects and a slow elimination of formoterol from plasma beyond 6-8 h after inhalation was demonstrated for the first time thanks to the sensitivity of the method.
已开发并验证了一种用于测定人血浆中福莫特罗的自动化高效液相色谱（HPLC）方法，其灵敏度有所提高。使用阳离子交换固相萃取（SPE）柱从血浆（1 ml）中提取福莫特罗和内标物CGP 47086。用pH 6缓冲溶液 - 甲醇（70:30，v/v）洗脱化合物，并用纯水进一步稀释洗脱液。取一部分提取液进样并通过HPLC进行分析。提取、稀释、进样和色谱分析通过自动进样（ASPEC）系统进行组合和自动化操作。在5μm的Hypersil ODS分析柱（200 mm×3 mm内径）上进行色谱分离，以（pH 6磷酸盐缓冲液，0.035 M + 20 mg/l EDTA）-甲醇 - 乙腈（70:25:5，v/v/v）作为流动相，流速为0.4 ml/min。通过电化学检测在+0.63 V的工作电位下检测分析物。日内准确度和精密度通过校准/质量控制血浆样品在福莫特罗碱浓度范围为7.14至238 pmol/l时的相对回收率进行评估。整个浓度范围内的准确度在81%至105%之间变化，精密度（变异系数）在3%至14%之间。日间准确度和精密度在血浆中福莫特罗碱浓度范围为11.9至238 pmol/l时进行评估。整个浓度范围内的准确度在98%至109%之间变化，精密度在8%至19%之间。在日间测量的定量限（LOQ）为11.9 pmol/l时，回收率为109%，变异系数为19%。从日内准确度和精密度结果可以看出，在有利条件下（新使用的色谱柱、新清洗的检测池和适度的残留池电流水平），我们能够达到福莫特罗碱7.14 pmol/l（富马酸福莫特罗二水合物3 pg/ml）的定量限。与先前描述的方法相比，检测限提高了约10倍。该方法已应用于对志愿者吸入120μg药物后血浆中福莫特罗的定量分析。由于该方法的灵敏度，首次证明在大多数受试者给药后24小时仍可检测到福莫特罗，并且在吸入后6 - 8小时后福莫特罗从血浆中的消除缓慢。
