Kapron James T, Pace Ellen, Van Pelt Colleen K, Henion Jack
Advion BioSciences, Inc., 15 Catherwood Rd, Ithaca, NY 14850, USA.
Rapid Commun Mass Spectrom. 2003;17(18):2019-26. doi: 10.1002/rcm.1145.
An automated chip-based infusion nanoelectrospray ionization (nanoESI) platform was used to demonstrate reproducible quantitation of drug molecules from biological matrices. Three sample preparation strategies were explored including protein precipitation of plasma with acetonitrile, de-salting of the plasma, and a combination of protein precipitation with subsequent de-salting of the dried and reconstituted extract. The best results were obtained when fortified human plasma samples containing midazolam were precipitated with acetonitrile containing alprazolam as the internal standard (IS). The supernatant was concentrated to dryness, reconstituted in aqueous acid, and de-salted by automated reversed-phase solid-phase extraction (SPE) prior to infusion nanoESI-MS/MS. Analyses employed a triple quadrupole mass spectrometer operated in selected reaction monitoring (SRM) mode. Each sample was infused for approximately 10 s and the resulting ion current profiles were integrated. Area ratios were used for regression analysis of standard samples (1.5-500 ng/mL). Quality control samples (3, 250, and 400 ng/mL) in five replicates from three different analysis days demonstrated intra-assay precision (< or =16%), inter-assay precision (< or =5%), and overall accuracy (+/-9% deviation). Infusion reproducibility of the assay was established by analyzing extracts after storage for 24 h at ambient temperature. Control plasma samples from six different sources probed the potential utility of this technique for the analysis of clinical samples. At the lower limit of quantitation (LLQ), variability and mean overall accuracy were < or =13% CV and +/-3% deviation, respectively, while at the upper limit of quantitation (ULQ) variability and mean overall accuracy were < or =9% CV and +/-9% deviation, respectively. Inter-chip variability was established by determining standard sample extracts across five different chips (< or =12% CV). Throughput for the assay was 55 s per sample, although this time may be shortened to 40 s per sample with recent improvements in the automated nanoESI system. No contamination or carryover was observed using this promising automated nanoESI-MS/MS platform.
使用基于芯片的自动化输注纳米电喷雾电离（nanoESI）平台来证明对生物基质中药物分子进行可重复定量。探索了三种样品制备策略，包括用乙腈沉淀血浆中的蛋白质、血浆脱盐，以及蛋白质沉淀与后续对干燥并复溶的提取物进行脱盐相结合。当含有咪达唑仑的加标人血浆样品用含有阿普唑仑作为内标（IS）的乙腈沉淀时，获得了最佳结果。将上清液浓缩至干，在酸性水溶液中复溶，并在进行输注nanoESI-MS/MS之前通过自动反相固相萃取（SPE）进行脱盐。分析采用在选择反应监测（SRM）模式下运行的三重四极杆质谱仪。每个样品输注约10秒，并对所得离子电流曲线进行积分。面积比用于标准样品（1.5 - 500 ng/mL）的回归分析。来自三个不同分析日的五个重复的质量控制样品（3、250和400 ng/mL）显示批内精密度（≤16%）、批间精密度（≤5%）和总体准确度（偏差±9%）。通过分析在室温下储存24小时后的提取物来确定该测定法的输注重现性。来自六个不同来源的对照血浆样品探究了该技术用于临床样品分析的潜在效用。在定量下限（LLQ），变异度和平均总体准确度分别为≤13% CV和偏差±3%，而在定量上限（ULQ），变异度和平均总体准确度分别为≤9% CV和偏差±9%。通过测定五个不同芯片上的标准样品提取物来确定芯片间变异度（≤12% CV）。该测定法的通量为每个样品55秒，不过随着自动化纳米电喷雾系统的最新改进，这个时间可能会缩短至每个样品40秒。使用这个有前景的自动化nanoESI-MS/MS平台未观察到污染或残留。
