固相萃取-高效液相色谱法测定血浆、尿液和唾液中1,4-苯二氮䓬类及其代谢物的方法验证
Validation of SPE-HPLC determination of 1,4-benzodiazepines and metabolites in blood plasma, urine, and saliva.
作者信息
Uddin Mohammad Nasir, Samanidou Victoria F, Papadoyannis Ioannis N
机构信息
Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece.
出版信息
J Sep Sci. 2008 Dec;31(21):3704-17. doi: 10.1002/jssc.200800342.
A simple, sensitive, selective, and reproducible RP-HPLC method with DAD detection at 240 nm was developed for the determination of six 1,4-benzodiazepines: bromazepam (BRZ), clonazepam (CLZ), diazepam (DZP), flunitrazepam (FNZ), lorazepam (LRZ), alprazolam (APZ); and two metabolites: alpha-hydroxyalprazolam (HALZ) and alpha-hydroxytriazolam (HTZL) in human plasma, urine, and saliva, using colchicine as internal standard, after SPE using Nexus Varian cartridges. Separation was performed on a Kromasil C(8) (250 mm x 5 mm, 5 microm) analytical column with a gradient mobile phase containing methanol, ACN and 0.05 M ammonium acetate. Linearity was held within the range 0.3-20.0 ng/microL, with coefficients of determination (r(2)) better than 0.997. The within- and between-day assay RSD at 2, 4, 8 ng/microL ranged from 0.03 to 4.7% and 0.5 to 7.0%, respectively in standards, from 1.3 to 7.9% and 3.3 to 7.3%, respectively in plasma, from 2.1 to 6.0% and 2.1 to 7.8%, respectively in urine and at 0.5, 1.0, 2.0 ng/microL ranged from 2.22 to 5.8% and 2.2 to 8.1%, respectively, in saliva. The mean relative recoveries were 96.3-108.6, 96.0-108.2, 94.3-107.1, 97.0-107.0% in within-day assay and 96.8-107.7, 94.6-107.6, 93.2-105.8, 96.0-108.6 in between-day assay for standard, plasma, urine, and saliva, respectively. The LOD and LOQ were 0.02-0.47 and 0.07-1.57 ng/microL, respectively.
建立了一种简单、灵敏、选择性好且可重现的反相高效液相色谱法，采用二极管阵列检测器在240 nm波长下检测，用于测定人血浆、尿液和唾液中的六种1,4 - 苯二氮䓬类药物：溴西泮（BRZ）、氯硝西泮（CLZ）、地西泮（DZP）、氟硝西泮（FNZ）、劳拉西泮（LRZ）、阿普唑仑（APZ）；以及两种代谢物：α - 羟基阿普唑仑（HALZ）和α - 羟基三唑仑（HTZL）。以秋水仙碱为内标，使用瓦里安Nexus柱进行固相萃取后进行分析。在Kromasil C(8)（250 mm×5 mm，5 µm）分析柱上进行分离，流动相为含甲醇、乙腈和0.05 M醋酸铵的梯度洗脱。线性范围为0.3 - 20.0 ng/µL，决定系数（r(2)）大于0.997。标准品中，2、4、8 ng/µL浓度下日内和日间测定的相对标准偏差（RSD）分别为0.03%至4.7%和0.5%至7.0%；血浆中分别为1.3%至7.9%和3.3%至7.3%；尿液中分别为2.1%至6.0%和2.1%至7.8%；唾液中0.5、1.0、2.0 ng/µL浓度下日内和日间测定的RSD分别为2.22%至5.8%和2.2%至8.1%。标准品、血浆、尿液和唾液日内测定的平均相对回收率分别为96.3 - 108.6%、96.0 - 108.2%、94.3 - 107.1%、97.0 - 107.0%，日间测定分别为96.8 - 107.7%、94.6 - 107.6%、93.2 - 105.8%、96.0 - 108.6%。检测限（LOD）和定量限（LOQ）分别为0.02 - 0.47 ng/µL和0.07 - 1.57 ng/µL。
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