Míguez-Framil Martha, Moreda-Piñeiro Antonio, Bermejo-Barrera Pilar, López Patricia, Tabernero María Jesús, Bermejo Ana María
Department of Analytical Chemistry, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain.
Anal Chem. 2007 Nov 15;79(22):8564-70. doi: 10.1021/ac071337n. Epub 2007 Oct 23.
The use of ultrasound energy for accelerating the pronase E enzymatic hydrolysis of human hair for extracting illicit drugs has been novelty tested. The enzymatic extracts obtained after 30 min of sonication in an ultrasonic water bath were subjected to an optimized solid-phase extraction process, which involved a solution of 2.0% (v/v) acetic acid in methanol as eluting solution and concentration by N2 stream evaporation. A gas chromatography/mass spectrometry method was used to separate and determine cocaine, benzoylecgonine, codeine, morphine, and 6-monoacethylmorphine in 20 min. Variables affecting ultrasound-assisted pronase E hydrolysis such as hydrolysis temperature, hydrolysis time, enzyme concentration, catalyzer (1,4-dithiothreitol) concentration, ionic strength, pH, and ultrasound frequency were simultaneously evaluated by a Plackett-Burman design 2(8) PBD of resolution III. The most statistically significant variables were ionic strength and pH, which means that analyte extraction is mainly attributed to pronase E activity. The optimization or evaluation of all the factors has led to an accelerated pronase E hydrolysis of human hair, which can be completed in 30 min. Results have been found to be statistically similar to those obtained with conventional pronase E hydrolysis. The accelerated method was finally applied to several human hair samples from multidrug abusers.
已对使用超声能量加速胰凝乳蛋白酶E对人发进行酶解以提取非法药物进行了创新性测试。在超声水浴中超声处理30分钟后获得的酶提取物经过优化的固相萃取过程，该过程使用2.0%（v/v）乙酸甲醇溶液作为洗脱液，并通过氮气气流蒸发进行浓缩。采用气相色谱/质谱法在20分钟内分离并测定可卡因、苯甲酰芽子碱、可待因、吗啡和6-单乙酰吗啡。通过分辨率为III的Plackett-Burman设计2(8)PBD同时评估影响超声辅助胰凝乳蛋白酶E水解的变量，如水解温度、水解时间、酶浓度、催化剂（1,4-二硫苏糖醇）浓度、离子强度、pH值和超声频率。统计学上最显著的变量是离子强度和pH值，这意味着分析物的提取主要归因于胰凝乳蛋白酶E的活性。对所有因素的优化或评估导致了人发中胰凝乳蛋白酶E水解的加速，可在30分钟内完成。结果在统计学上与传统胰凝乳蛋白酶E水解获得的结果相似。该加速方法最终应用于来自多药滥用者的几个人发样本。
