LC-MS/MS法测定人血浆中利培酮和9-羟基利培酮的浓度及临床应用

目的：建立简单快速的液相串联质谱法（LC-MS/MS）测定人血浆中利培酮及其代谢物9-羟基利培酮的浓度。方法：血浆样本采用甲醇沉淀蛋白法处理,以地西泮为内标,采用ES Industries Sonoma C₁₈（2）色谱柱（100 mm×2.1 mm,3 μm）分离化合物;流动相包括A-50%甲醇水溶液（含6 mmol·L^-1乙酸铵）、B-0.1%甲酸乙腈溶液;梯度洗脱,总流速0.60 mL·min^-1;进样量10 μL;柱温30℃。采用正离子MRM模式扫描,电喷雾电离,利培酮、9-羟基利培酮及地西泮内标离子通道分别为m/z411.3→m/z191.1、m/z427.2→m/z207.0和m/z285.1→m/z193.1。依据《中国药典》（2015年版）通则中9012"生物样品定量分析方法验证指导原则"对所建方法进行验证。结果：利培酮在0.16~101.40 μg·L^-1的浓度范围内线性关系良好（r>0.999）,9-羟基利培酮在0.40~256.80 μg·L^-1浓度范围内线性关系良好（r>0.999）;在定量下限,低、中、高浓度批内与批间精密度的标准偏差（RSD）均小于10.9%,准确度符合要求;含分析物血浆样品在不同储存条件下结果稳定。该法已成功应用于临床样本治疗药物监测。运用此方法监测分析一千多份临床样本得出总利培酮临床实际测定浓度范围为13.30~93.22 μg·L^-1。结论：本研究建立了测定血浆中利培酮及其代谢物9-羟基利培酮浓度的快速、简便、实用的LC-MS/MS方法。利培酮临床实际测定浓度范围相比于神经精神药理学与药物精神病学协会专家组推荐治疗浓度范围更宽,且多次监测的患者中女性血药浓度相对于男性更趋于稳定。

Determination of plasma concentrations of risperidone and 9-hydroxyrisperidone by LC-MS/MS method and its clinical application

OBJECTIVE To develop a simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for monitoring the plasma concentrations of risperidone and its motabolite 9-hydroxyrisperidone.METHODS Sample preparation was performed by protein precipitation with methanol after addition of internal standard to 100 μL of plasma specimen.Chromatographic separation was performed on a ES Industries Sonoma C₁₈ (2) column (100 mm×2.1 mm,3 μm) using gradient elution.The mobile phase was methanol-water(1∶1,V/V) which contained 6 mmol·L^-1 ammonium acetate and acetonitrile with 0.1% formic acid.Analytes were detected through a triple quadrupole mass spectrometer that was operated in positive ion mode with electrospray ionization.Multiple reaction monitoring mode (MRM) was chosen for detection with the transitions of m/z 411.3→191.1 for risperidone,m/z 427.2→207.0 for 9-hydroxyrisperidone and m/z 285.1→193.1 for diazepam.RESULTS This method exhibited excellent linearity for all the analytes with regression coefficients higher than 0.999.The limit of quantification (LOQ) values for risperidone and 9-hydroxyrisperidone were 0.16 μg·L^-1and 0.40 μg·L^-1,respectively.The mean accuracy ranged from 97.40% to 115.00% and the RSD of precision were below 10.9%.Both the selectivity and matrix effect met the requirement.The sample could store at room temperature for 3 days,in refrigerator for one month.Samples could stay stable after freezing and thawing for 3 times.More than one thousand clinical samples were detected by LC-MS/MS to monitor clinical samples to conclude that the actual clinical concentration range of total risperidone was 13.30-93.22 μg·L^-1 in Chinese people.CONCLUSION The study has developed a simple and rapid LC-MS/MS method and is well validated.The method has been successfully applied to monitor the plasma concentrations of risperidone and 9-hydroxyrisperidone.The therapeutic drug monitoring results show that the actual clinical concentration range of total risperidone (13.30-93.22 μg·L^-1) is wider than the range recommended by Arbeitsgemeinschaft für Neuropsychopharmakologie und Pharmakopsychiatrie (AGNP) experts.And the variety of male plasma concentration is much bigger than female.Therefore the male plasma concentrations of risperidone and 9-hydroxyrisperidone should be monitored frequently.