Jemal M, Ouyang Z, Chen B C, Teitz D
Bristol-Myers Squibb Pharmaceutical Research Institute, New Brunswick, NJ 08903-0191, USA.
Rapid Commun Mass Spectrom. 1999;13(11):1003-15. doi: 10.1002/(SICI)1097-0231(19990615)13:11<1003::AID-RCM597>3.0.CO;2-L.
A method for simultaneous quantitation of both the acid and lactone forms of atorvastatin, a new synthetic inhibitor of HMG-CoA reductase that is being marketed for the treatment of high serum cholesterol, and both the acid and lactone forms of its two biotransformation products, 2-hydroxyatorvastatin and 4-hydroxyatorvastatin, in human serum (a total of six analytes) by high-performance liquid chromatography with electrospray tandem mass spectrometry was developed and validated. A deuterium labeled analog was used as internal standard for each of the six analytes. Each point of the calibration standard curve, which ranged from 0.5 to 200 ng/mL, contained the six analytes at equal concentrations. Three groups of quality control (QC) samples were used. In the first group, combination QC samples contained all six analytes at equal concentrations. In the second group, acid-only QC samples contained only the acid forms (i.e. three analytes) at equal concentrations. In the third group, lactone-only QC samples contained only the lactone forms (i.e. three analytes) at equal concentrations. After adding the internal standard to 0.5 mL of each standard and the QC sample kept at 4 degrees C, the samples were acidified with sodium acetate buffer (pH 5.0) and then extracted with methyl tert-butyl ether. Detection was by positive ion electrospray tandem mass spectrometry using eight selected reaction monitoring channels. The acid compounds were stable in human serum at room temperature but the lactone compounds were unstable as they hydrolyzed rapidly to their respective acid forms. The conversion of the lactone compounds in both QC and post-dose human serum samples was nearly complete after 24 h at room temperature. The lactone compounds in serum could be stabilized by lowering the working temperature to 4 degrees C or lowering the serum pH to 6.0. The acid-only and the lactone-only QC samples showed that, under the sample processing conditions used, the degree of the hydrolysis of the lactone compounds or the lactonization of the acid compounds during the assay procedure was minimal (< 5%). The intra-day C.V., inter-day C.V. and the deviations from the nominal concentrations for all six analytes were within 15%, demonstrating good precision and accuracy. The required lower limit of quantitation (LLQ) of 0.5 ng/mL was achieved for each analyte.
开发并验证了一种通过高效液相色谱-电喷雾串联质谱法同时定量测定阿托伐他汀（一种新的用于治疗高血清胆固醇的HMG-CoA还原酶合成抑制剂）的酸形式和内酯形式及其两种生物转化产物2-羟基阿托伐他汀和4-羟基阿托伐他汀的酸形式和内酯形式（共六种分析物）在人血清中的方法。使用氘代标记类似物作为六种分析物中每种的内标。校准标准曲线范围为0.5至200 ng/mL，每个点包含等浓度的六种分析物。使用三组质量控制（QC）样品。在第一组中，组合QC样品包含等浓度的所有六种分析物。在第二组中，仅酸QC样品仅包含等浓度的酸形式（即三种分析物）。在第三组中，仅内酯QC样品仅包含等浓度的内酯形式（即三种分析物）。向0.5 mL保存在4℃的每种标准品和QC样品中加入内标后，用乙酸钠缓冲液（pH 5.0）酸化样品，然后用甲基叔丁基醚萃取。通过使用八个选定反应监测通道的正离子电喷雾串联质谱法进行检测。酸性化合物在室温下在人血清中稳定，但内酯化合物不稳定，因为它们会迅速水解为各自的酸形式。在室温下24小时后，QC和给药后人血清样品中内酯化合物的转化几乎完成。血清中的内酯化合物可通过将工作温度降至4℃或将血清pH降至6.0来稳定。仅酸和仅内酯QC样品表明，在所使用的样品处理条件下，测定过程中内酯化合物的水解程度或酸化合物的内酯化程度最小（<5%）。所有六种分析物的日内变异系数、日间变异系数和与标称浓度的偏差均在15%以内，表明具有良好的精密度和准确性。每种分析物均达到了所需的0.5 ng/mL的定量下限（LLQ）。
