Quantitative analysis of multiple illicit drugs in preserved oral fluid by solid-phase extraction and liquid chromatography–tandem mass spectrometry

We present a validated method for the simultaneous analysis of basic drugs which comprises a sample clean-up step, using mixed-mode solid-phase extraction (SPE), followed by LC–MS/MS analysis. Deuterated analogues for all of the analytes of interest were used for quantitation. The applied HPLC gradient ensured the elution of all the drugs examined within 14 min and produced chromatographic peaks of acceptable symmetry. Selectivity of the method was achieved by a combination of retention time, and two precursor-product ion transitions for the non-deuterated analogues. Oral fluid was collected with the Intercept^®, a FDA approved sampling device that is used on a large scale in the US for workplace drug testing. However, this collection system contains some ingredients (stabilizers and preservatives) that can cause substantial interferences, e.g. ion suppression or enhancement during LC–MS/MS analysis, in the absence of suitable sample pre-treatment. The use of the SPE was demonstrated to be highly effective and led to significant decreases in the interferences. Extraction was found to be both reproducible and efficient with recoveries >76% for all of the analytes. Furthermore, the processed samples were demonstrated to be stable for 48 h, except for cocaine and benzoylecgonine, where a slight negative trend was observed, but did not compromise the quantitation. In all cases the method was linear over the range investigated (2–200 μg/L) with an excellent intra-assay and inter-assay precision (coefficients of variation <10% in most cases) for QC samples spiked at a concentration of 4, 12 and 100 μg/L. Limits of quantitation were estimated to be at 2 μg/L with limits of detection ranging from 0.2 to 0.5 μg/L, which meets the requirements of SAMHSA for oral fluid testing in the workplace. The method was subsequently applied to the analysis of Intercept^® samples collected at the roadside by the police, and to determine MDMA and MDA levels in oral fluid samples from a controlled study.     

血清中盐酸曲马多的膜式固相萃取及GC／MS／SIM测定

目的建立血清中盐酸曲马多的膜式固相萃取(SPE)气-质联用分析方法。方法1mL血清用2mL0.1mol/L磷酸盐缓冲液(pH6)稀释后,用含有C18和强酸型强阳离子交换基团的SPECC18AR/MP3固相萃取柱萃取,洗脱液为含2%氨水的乙酸乙酯;选择SKF525作为内标物,用GC/MS/SIM定量测定。结果在加标量为0.1、0.2和0.5μg/mL的血清样品中,盐酸曲马多的回收率分别为98.9%、92.5%和84.8%,5次测定的RSD分别为3.2%、8.7%和10.9%;线性范围为0.1～4μg/mL,多项式回归相关系数r2=0.9939;检出限为21ng/mL;同一根萃取柱,连续使用5次,没有出现堵塞和污染,回收率及RSD未见下降。结论本方法适合于法医毒物分析。     

Validation of an ELISA-based screening assay for the detection of amphetamine, MDMA and MDA in blood and oral fluid

The use of amphetamine and 'ecstasy' (MDMA) has increased exponentially in many European countries since the late nineties, leading to a rapid growth in the number of clinical and forensic analyses. Therefore, a rapid screening procedure for these substances in biological specimens has become an important part of routine toxicological analysis in forensic laboratories. The objective of this study was to evaluate the Cozart amphetamine enzyme-linked immunosorbent assay (ELISA) for the screening of plasma samples and oral fluid samples (collected with the Intercept device). Authentic plasma samples from drivers (n=360) were screened, using an 1:5-fold dilution. True positive, true negative, false positive and false negative results were determined relative to the in-house routine GC-MS analysis. Samples consisted of 144 amphetamine-only positives, 141MDMA/MDA-only positives, and 74 negatives when using the limit of quantitation as the cut-off level for confirmation (10 ng/mL). Using these results, receiver operating characteristic (ROC) curves were generated and optimal cut-off values for the screening assay were calculated. Analysis showed that the ELISA is able to predict the presence of either amphetamine or *MDMA/MDA (*MDMA as its metabolite MDA) in plasma samples with 98.3% sensitivity and 100% specificity at a cut-off value of 66.5 ng/mL d-amphetamine equivalents. A similar analysis was conducted on 216 oral fluid specimens collected from a controlled double blind study. Subjects received placebo or a high (100 mg) or low (75 mg) dose of MDMA. Oral fluid samples were collected at 1.5 and 5.5h after administration. Combined results of the analysis of the high and low dose oral fluid samples indicated a screening cut-off of 51 ng/mL d-amphetamine equivalents with both a sensitivity and specificity of 98.6% (using a LC-MS/MS confirmation cut-off of 10 ng/mL). In conclusion, these data indicate that the Cozart AMP EIA plates constitute a fast and accurate screening technique for the identification of amphetamine and MDMA/MDA positive plasma samples and oral fluid specimens (collected with Intercept. It should be emphasized that method validation should be performed for each type of biological matrix.     

Comparison of molecularly imprinted solid-phase extraction (MISPE) with classical solid-phase extraction (SPE) for the detection of benzodiazepines in post-mortem hair samples

This preliminary study compares the benzodiazepine results for 10 post-mortem scalp hair samples using a classical solid-phase extraction (SPE) and a molecularly imprinted solid-phase extraction (MISPE) system. The hair samples selected for testing were from drug-related deaths where a positive benzodiazepine blood result was obtained. Samples were decontaminated with 0.1% sodium dodecyl sulfate, distilled water and dichloromethane, incubated overnight in methanol/25% aqueous ammonium hydroxide (20:1), extracted by SPE or MISPE and subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Both extraction methods detected diazepam, nordiazepam, oxazepam, temazepam and nitrazepam in the samples. Diazepam was detected in a greater number of samples using MISPE due to both its lower limit of detection (LOD) and higher extraction recovery as a result of excellent molecular recognition of the template (diazepam) imparted by the imprinting process. The selective recognition of two diazepam analogues, nordiazepam and oxazepam, was demonstrated using MISPE since they were also detected in a greater number of samples. In contrast, another diazepam analogue, temazepam, was detected in a greater number of samples using SPE since the LOD using this extraction was lower than with MISPE. Nitrazepam was detected in one sample using both extraction methods. Overall the MISPE and SPE hair results were in good qualitative agreement. For the samples, where both extraction methods detected nordiazepam, temazepam and oxazepam, the concentrations were always higher for SPE. This is probably due to the MIP procedure producing extracts with fewer matrix interferences than the extracts produced using the classical SPE method. MISPE could be used as a complementary method to classical SPE for the analysis of benzodiazepine positive hair samples collected from chronic users.     

新型微流体液相色谱(micro-LC)串联四极杆质谱检测头发中5种苯二氮卓类药物

目的建立了新型微流体液相色谱(micro-LC)串联四级杆质谱检测头发中5种苯二氮卓类药物(三唑仑,α-羟基咪达唑仑,咪达唑仑,α-羟基阿普唑仑,普拉西泮)。方法采用液-液萃取法(LLE)提取毛发样品,微流体液相色谱串联四级杆质谱检测。结果在回归系数大于0.99的情况下,目标分析物的良好线性。精密度和萃取效率分别在2.7%~17.1%和66.5%~120.5%之间。每个分析物的检出限和定量限分别为0.001~0.08 pg·mg^-1和0.0125~0.25pg·mg^-1。结论本方法与传统的液相色谱串联质谱法(LC/MS-MS)相比灵敏度出高2-10倍。本研究展示了新型微流体分离系统串联质谱方法的实用性,并为法医毒理学领域关于头发样本中痕量药物检测提供了一种新的检测手段。     

Simultaneous determination of creatinine and uric acid in urine by liquid chromatography-tandem mass spectrometry with polarity switching electrospray ionization

A simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated to simultaneously determine creatinine (Cr) and uric acid (UA) levels as a confirmatory method for adulteration or dilution of urine. Centrifuged urine samples (10μL) were diluted with 390μL of distilled water. 30μL of internal standard solution (Cr-d(3), 5μg/mL) and 10μL of acetonitrile were added to 20μL aliquots of diluted urine samples and filtered. The samples (1μL) were introduced into LC-MS/MS with no further pretreatment. Cr and UA were separated on a multi-mode ODS column (Scherzo SM-C18, 75mm×2.0mm I.D., 3μm) and quantified by LC-MS/MS with polarity-switching electrospray ionization. Cr requires the positive-ion mode, whereas the negative-ion mode is required for the analysis of UA. The linear ranges were 1.0-300mg/dL for Cr and 0.5-300mg/dL for UA, with good determination coefficients (R(2)≥0.9988). The intra-day and inter-day precision of the analytes was within 13.0% and 14.4%, respectively. The intra-day and inter-day accuracy was -8.8 to 3.7% and -0.3 to 6.6%, respectively. The lower limits of detection (LLODs) were 0.3mg/dL for Cr and 0.07mg/dL for UA. The applicability of the developed method was examined by analyzing urine samples from suspected drug abusers (n=46).     

尿中氯胺酮及其代谢物盘鉴和GC/MS/SIM测定

目的 研究尿中氯胺酮(KET)及其代谢物去甲基氯胺酮(NKET)的盘鉴(Disk SPE)。方法 用含有化学键合C18和强酸型强阳离子交换(SCX)基团的萃取柱SPEC.C18 AR/MP3萃取,加入萃取柱前的尿样用0.1mol/L磷酸盐缓冲溶液(pH 6)稀释,洗脱溶剂为含2％(v/v)氨水的乙酸乙酯;以2,4,6-三硝基甲苯(TNT)为色谱内标,GC/MS/SIM检测。结果 在加标量为0.5μg/mL、2μg/mL和6μg/mL的控制尿样中,KET和NKET的平均回收率分别为91.5％和79.9％,6次测定的RSD均为8.7％;线性范围0.02-8μg/mL,线性相关系数分别为0.9819和0.9964;检出限(S/N=3)分别为6ng/mL和4ng/mL;总离子色谱图背景低,杂质少。同一根萃取柱重复使用8次以上未见性能下降;嫌疑尿样中检出KET和/或NKET,和常规的液液萃取结果相符。结论 该方法适用于尿中KET和NKET的同时测定。     

Screening and quantification of antipsychotic drugs in human brain tissue by liquid chromatography-tandem mass spectrometry: application to postmortem diagnostics of forensic interest

A quantitative LC-MS/MS method has been developed for the simultaneous determination of 17 antipsychotic drugs in human postmortem brain tissue. Sample preparation was performed using Hybrid Solid Phase Extraction-Precipitation technology for the removal of endogenous protein and phospholipid interferences. The chromatographic separation was performed for 16 min on a C8 column, which used a gradient elution of formate ammonium and acetonitrile, and a flow rate gradient. Triple quadrupole mass spectrometry was employed to generate tandem mass spectrometric (MS/MS) data of the target analytes to select the ion m/z signals. Quantitation of the analytes was performed by operating in the dynamic multiple reaction monitoring (dMRM) mode using an electrospray ionization interface. Calibration curves prepared in the spiked brain tissue were linear in the range 20-8000 ng/g (r(2)>0.993) for all drugs (except olanzapine). Within- and between-day coefficients of variation were lower than 25% for all drugs at the LOQ. The LOQ in the matrix ranged between 2 ng/g and 80 ng/g. The method was successfully applied to the unequivocal identification and accurate quantification of antipsychotic drugs in human postmortem brain tissues: therefore, this method can be used in forensic investigations.     

Determination of several psychiatric drugs in whole blood using capillary gas-liquid chromatography with nitrogen phosphorus detection: comparison of two solid phase extraction procedures

A simple and reliable gas chromatographic method with nitrogen-phosphorus detection without derivatization was developed for the detection of several psychiatric drugs in whole blood as part of systematic toxicological analyses (STA). Drugs included mirtazapine, chlorpromazine, methotrimeprazine (levomepromazine), clothiapine, olanzapine, clozapine, haloperidol, and thioridazine. All drugs were studied at concentrations of 100-2,000 microg/L, except haloperidol that was studied at concentrations of 400-8,000 microg/L. In order to select the best blood purification procedure and therefore increase the signal to noise ratio we have compared two solid-phase extraction (SPE) columns, Chem Elut and Bond Elut Certify, for their recovery, precision, sensitivity and matrix purification efficiency. Recoveries for these drugs using Chem Elut columns at 500 and 2,000 microg/L (2,000 and 8,000 microg/L for haloperidol) were in the range 21-65%, with intra-assay and inter-assay precisions of less than 17% and 19%, respectively. Limits of detection (LODs) and limits of quantitation (LOQs) for mirtazapine, chlorpromazine, methotrimeprazine, clothiapine, olanzapine, clozapine, and thioridazine ranged from 62 to 161 microg/L and from 205 to 531 microg/L, respectively. LOD and LOQ for haloperidol were 442 and 1,458 microg/L, respectively. Recoveries of these compounds using Bond Elut Certify columns at 500 and 2,000 microg/L (2,000 and 8,000 microg/L for haloperidol) were in the range 44-97%, with intra-assay and inter-assay precisions of less than 7% and 14%, respectively. LODs and LOQs for mirtazapine, chlorpromazine, methotrimeprazine, clothiapine, olanzapine, clozapine, and thioridazine ranged from 37 to 66 microg/L and from 122 to 218 microg/L, respectively. LOD and LOQ for haloperidol were 156 and 515 microg/L, respectively. Linearity was observed in the studied range for all compounds with r(2) values of >0.999. The use of the mixed-mode bonded-silica Bond Elut Certify columns showed advantages comparing with Chem Elut columns for the screening of these psychotropic agents such as higher recoveries, cleaner extracts, better sensitivity, better precision and less solvent consumption and subsequent disposal.     

LC-MS／MS测定尿液中可卡因及其代谢物苯甲酰爱康宁

目的建立尿液中可卡因(cocaine,COC)及其代谢物苯甲酰爱康宁(benzoylecgonine,BZE)的液相色谱-串联质谱分析方法。方法尿液经固相萃取后,用AllurePFP丙基柱分离,以V(甲醇):V(20mmol/L乙酸胺和0.1%甲酸的缓冲溶液)=80∶20为流动相,采用二级质谱多反应监测模式检测COC和BZE。按10mg/kg的剂量对豚鼠腹腔注射可卡因,给药后收集7d尿液。结果尿液中COC和BZE在2.0～100ng/mL质量浓度范围内线性关系良好(r=0.9995),最低检测限(LOD)为0.5ng/mL;回收率大于90%;日内和日间精密度均小于6%;豚鼠尿液中主要检测目标物是BZE,且BZE检测时限也较COC长。结论所建方法灵敏度高,选择性好,适用于尿液中可卡因和苯甲酰爱康宁的检测。     

SPE-HPLC/MS/MS检验体液中赛拉嗪及DMA

目的建立了一种高效液相色谱串联三重四极杆质谱同时测定体液中赛拉嗪及2,6-二甲基苯胺的分析方法。方法样品经HLB固相萃取柱提取净化,Waters Atlantis d C18色谱柱分离,正电离条件下进行选择监视扫描模式检测。结果方法的回收率为70.5%~79.8%,RSD为8.2%~10.5%。赛拉嗪及2,6-二甲基苯胺在血液和尿液中的检出限分别为0.4 ng/mL和0.3 ng/mL,定量限分别为1.2 ng/mL和1.0 ng/mL。结论本方法灵敏度高、特异性好、重现性好,适用于赛拉嗪中毒的血液和尿液检测。     

LC-MS/MS analysis of fentanyl and norfentanyl in a fatality due to application of multiple Durogesic transdermal therapeutic systems

Fentanyl is a potent synthetic narcotic analgesic administered in the form of a transdermal patch for the management of chronic pain. A 78-year-old woman with a history of cancer was found dead in bed. She was lying on her back. The external examination revealed 10 Durogesic transdermal therapeutic systems (100 microg/h fentanyl) on the body. Liquid-liquid extraction and liquid chromatography tandem mass spectrometry with electrospray source in positive ionization mode was applied for the quantitation of fentanyl and its major metabolite norfentanyl in the post-mortem samples. Fentanyl-d5 and norfentanyl-d5 were used as internal standards. Multiple reaction monitoring was used for specific detection. Calibration was performed by addition of standard solutions to drug-free matrix (blood, urine and liver) prior to extraction. The method showed good linearity for fentanyl and norfentanyl over a concentration range of 5-150 microg/L in reconstituted extracts with coefficients of determination equal or greater than 0.998. Percent mean within-day precision and accuracy of 0.9-1.0% and 99.4-101.1% for fentanyl and 2.0-4.5% and 93.1-101.0% for norfentanyl were obtained. Mean extraction recoveries varied between 95.5% and 100.3% for fentanyl and 39.2-57.4% for norfentanyl. The following fentanyl (norfentanyl) concentration in the post-mortem samples were measured; 28.6 microg/L (3.0 microg/L) in right and 28.2 microg/L (3.5 microg/L) in left subclavian blood, 21.3 microg/L (<2 microg/L) in right and 20.9 microg/L (<2 microg/L) in left femoral blood, 37.6 microg/L (4.2 microg/L) in right and 33.9 microg/L (4.4 microg/L) in left ventricular blood, 282.9 microg/L (121.2 microg/L) in urine, 688.2 microg/L in stomach contents, 122.5 microg/L (25.4 microg/L) in bile, 19.5 microg/L (< 2 microg/L) in vitreous humour, 203.0 microg/kg (26.6 microg/kg) in liver and 78.6 microg/kg (46.3 microg/kg) in kidney. We concluded that the woman's death was caused by acute intoxication with fentanyl. The manner of death was presumed to be suicide due to excessive administered Durogesic transdermal therapeutic systems.     

Screening for the presence of drugs in serum and urine using different separation modes of capillary electrophoresis

Capillary electrophoresis (CE) is a modern separation technique that has some distinct advantages for toxicological analysis, such as a high efficiency, fast analysis, flexibility, and complementary separation mechanisms to chromatographic methods. CE can be applied in various modes, which each have a different separation mechanism or selectivity. The most common mode is capillary zone electrophoresis (CZE), in which charged analytes migrate in a buffer under the influence of an electric field. In micellar electrokinetic chromatography (MEKC), micelles are added to the buffer which interact with the analytes. MEKC can also be used for the separation of neutral compounds. In non-aqueous CE (NACE), the aqueous buffer is replaced by a background of electrolytes in organic solvents. A sample that needs to be screened can easily be analyzed subsequently by these CE modes using the same instrumentation.The aim of the study was to develop procedures for the analysis of basic and acidic drugs in serum and urine using CZE, MEKC, and NACE. A test mixture that consisted of six basic and six acidic compounds was used to study the separation behavior of five CE methods. The results showed that three methods (based on CZE, MEKC, and NACE) were suitable for the analysis of basic compounds and three methods (based on CZE and MEKC) for the analysis of acidic compounds.For the extraction of analytes from serum and urine, a solid-phase extraction (SPE) and a liquid-liquid extraction (LLE) method were compared. Both SPE and LLE methods provided clean extracts after extraction of the basic compounds from serum and urine. The extracts of acidic compounds contained more matrix interferences, especially for urine. The SPE method had some advantages compared to LLE, as it lead to cleaner extracts and higher peaks, and as it elutes basic and acidic compounds in one fraction.The potentials and pitfalls of the various methods for screening purposes in analytical toxicology are discussed.     

Analysis of Delta9-tetrahydrocannabinol in oral fluid samples using solid-phase extraction and high-performance liquid chromatography-electrospray ionization mass spectrometry

An analytical method using solid-phase extraction (SPE) and high-performance liquid chromatography-mass spectrometry (LC-MS) has been developed and validated for the confirmation of Delta(9)-tetrahydrocannabinol (THC) in oral fluid samples. Oral fluid was extracted using Bond Elut LRC-Certify solid-phase extraction columns (10 cm(3), 300 mg) and elution performed with n-hexane/ethyl acetate. Quantitation made use of the selected ion-recording mode (SIR) using the most abundant characteristic ion [THC+H(+)], m/z 315.31 and the fragment ion, m/z 193.13 for confirmation, and m/z 318.00 for the protonated internal standard, [d(3)-THC+H(+)]. The method proved to be precise for THC, in terms of both intra-day and inter-day analyses, with coefficients of variation less than 10%, and the calculated extraction efficiencies for THC ranged from 76 to 83%. Calibration standards spiked with THC between 2 and 100 ng/mL showed a linear relationship (r(2)=0.999). The method presented was applied to the oral fluid samples taken from the volunteers during the largest music event in Portugal, named Rock in Rio-Lisboa. Oral fluid was collected from 40 persons by expectoration and with Salivette. In 55% of the samples obtained by expectorating, THC was detected with concentration ranges from 1033 to 6552 ng/mL and in 45% of cases THC was detected at concentrations between 51 and 937 ng/mL. However, using Salivette collection, 26 of the 40 cases had an undetectable THC.     

Determination of gamma-hydroxybutyrate in water and human urine by solid phase microextraction-gas chromatography/quadrupole ion trap spectrometry

A simple method of detection was developed for gamma-hydroxybutyrate (GHB). The method involves the derivatization of GHB using a hexyl-chloroformate procedure in aqueous media (such as water or urine), extraction of the derivatization product directly from the sample using solid-phase microextraction, and subsequent separation and detection with gas chromatography quadrupole ion trap mass spectrometry. The deuterated form of GHB (GHB-D6) is used as an internal standard for quantitation. The method was linear for GHB-spiked pure water samples from 2 to 150 microg/mL GHB with a detection limit of 0.2 microg/mL. Spiked urine samples showed linearity from 5 to 500 microg/mL GHB with a detection limit of 2 microg/mL. The SPME-GC/MS method is applied to actual case samples, and the results are compared to those values obtained using a conventional GC/MS method. Sensitivity and linearity are comparable to those seen using traditional methods of separation, yet the SPME method is superior due to the simplicity, speed of analysis, reduction in solvent waste, and ability to differentiate between GHB and gamma-butyrolactone (GBL).     

液相色谱-串联质谱法筛查鉴定203种毒品

目的建立203种毒品的液相色谱-串联质谱筛查鉴定方法。方法选用Accucore TM Phenyl/Hexyl苯基己基柱(100 mm×2.1 mm,2.6μm)为色谱柱,柱温50℃,以甲醇乙腈混合溶剂(体积比1:1,含0.1%甲酸和2 mmol/L甲酸铵)、水(含0.1%甲酸和2 mmol/L甲酸铵)作为流动相进行梯度洗脱,流速0.4 mL/min。质谱采用电喷雾正离子模式(ESI+)进行离子化,使用多反应监测(MRM)模式采集数据,总分析时间14 min。结果该方法实现了203种毒品的筛查鉴定分析,各目标物的方法检出限均为10 ng/mL。结论建立的筛查鉴定方法具有快速、准确、灵敏等优点,能够满足禁毒工作的日常需要。     

Performance characteristics of the Cozart RapiScan Oral Fluid Drug Testing System for opiates in comparison to ELISA and GC/MS following controlled codeine administration

Oral fluid is an interesting alternative matrix for drug testing in many environments, including law enforcement, workplace drug testing, and drug treatment facilities. Performance characteristics of the FDA-cleared, qualitative, Cozart RapiScan Opiate Oral Fluid Drug Testing System (Opiate Cozart RapiScan System or Opiate CRS) were compared to the semi-quantitative Cozart Microplate EIA Opiate Oral Fluid Kit (Opiate ELISA) and to gas chromatography/mass spectrometry (GC/MS). The following oral fluid opiate cutoffs were evaluated: the GC/MS limit of quantification (LOQ) of 2.5 mg/l; 15 microg/l currently used for oral fluid testing in the United Kingdom (UK); 30 microg/l (Opiate CRS cutoff); and 40 microg/l, the proposed Substance Abuse and Mental Health Services Administration (SAMHSA) cutoff. Subjects provided informed consent to participate in this IRB-approved research and resided on the closed research ward throughout the study. Three oral codeine doses of 60 mg/70 kg were administered over a 7-day period. After a 3-week break, subjects received three doses of 120 mg/70 kg within 7 days. Oral fluid specimens (N = 1273) were analyzed for codeine (COD), norcodeine (NCOD), morphine (MOR) and normorphine (NMOR) by GC/MS with an LOQ of 2.5 microg/l for all analytes. MOR and NMOR were not detected in any sample; 26.5% of the specimens were positive for COD and 13.7% for NCOD. Opiate CRS uses a preset, qualitative cutoff of 10 microg/l; this is equivalent to 30 microg/l in undiluted oral fluid as the oral fluid collection process involves a 1:3 dilution with buffer. Sensitivity, specificity, and efficiency of Opiate CRS compared to Opiate ELISA were 98.6, 98.1, and 98.2% at a 30 microg/l cutoff and 99.0, 96.2, and 96.6% at a 40 microg/l cutoff. Compared to the much lower GC/MS LOQ of 2.5 microg/l, sensitivity, specificity and efficiency were 66.8, 99.3 and 90.7%. Increasing the GC/MS cutoff to the current UK level yielded performance characteristics of 81.5% (sensitivity), 99.3% (specificity), and 95.4% (efficiency). Using a GC/MS cutoff identical to the preset Opiate CRS cutoff yielded sensitivity, specificity, and efficiency of 88.5, 99.2, and 97.5%, respectively. At the proposed SAMSHA confirmation cutoff of 40 microg/l, sensitivity increased with little change in specificity and efficiency (91.3% sensitivity, 98.9% specificity, and 97.5% efficiency). Oral fluid is a suitable matrix for detecting drugs of abuse. Opiate CRS, with a 30 microg/l cutoff, is sufficiently sensitive, specific and efficient for oral fluid opiate analysis, performing similarly to Opiate ELISA at the same cutoff, and having performance characteristics >91% when compared to GC/MS at the proposed SAMHSA cutoff.     

固相萃取/LC-MS/MS测定血液中的白藜芦醇

目的建立血液中白藜芦醇的固相萃取/LC-MS/MS方法。方法自制固相萃取柱,在长约13cm的酸性滴定管中,底部填入约0.02g玻璃棉,依次加入柱填料PSA 0.15g,Florisil 0.3g,Silica 0.45g。对样本进行固相萃取,采用LC-MS/MS方法进行检测,运用保留时间和多反应监测(MRM)方式对血液中白藜芦醇进行定性定量分析。结果回收率为95.66%～98.58%,最低检测限(LOD)为1.79μg/mL,线性范围2.5～20μg/mL;相关系数为0.9997。结论本文所建方法,适用于血液中白藜芦醇的检测。     

High prevalence of 6-acetylmorphine in morphine-positive oral fluid specimens

Identification of 6-acetylmorphine, a specific metabolite of heroin, is considered to be definitive evidence of heroin use. Although 6-acetylmorphine has been identified in oral fluid following controlled heroin administration, no prevalence data is available for oral fluid specimens collected in the workplace. We evaluated the prevalence of positive test results for 6-acetylmorphine in 77,218 oral fluid specimens collected over a 10-month period (January-October 2001) from private workplace testing programs. Specimens were analyzed by Intercept immunoassay (cutoff concentration=30 ng/ml) and confirmed by GC-MS-MS (cutoff concentrations=30 ng/ml for morphine and codeine, and 3 ng/ml for 6-acetylmorphine). Only morphine-positive oral fluid specimens were tested by GC-MS-MS for 6-acetylmorphine. A total of 48 confirmed positive morphine results were identified. An additional 107 specimens were confirmed for codeine only. Of the 48 morphine-positive specimens, 32 (66.7%) specimens were positive for 6-acetylmorphine. Mean concentrations (+/-S.E.M.) of morphine, 6-acetylmorphine and codeine in the 32 specimens were 755+/-201, 416+/-168 and 196+/-36 ng/ml, respectively. Concentrations of 6-acetylmorphine in oral fluid ranged from 3 to 4095 ng/ml. The mean ratio (+/-S.E.M.) of 6-acetylmorphine/morphine was 0.33+/-0.06. It is suggested that, based on controlled dose studies of heroin administration, ratios >1 of 6-acetylmorphine/morphine in oral fluid are consistent with heroin use within the last hour before specimen collection. The confirmation of 6-acetylmorphine in 66.7% of morphine-positive oral fluid specimens indicates that oral fluid testing for opioids may offer advantages over urine in workplace drug testing programs and in testing drugged drivers for recent heroin use.     

Determination of ethyl glucuronide in human hair by SPE and LC-MS/MS

A method for the sensitive and selective determination of ethyl glucuronide (EtG) in hair has been developed using solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Washed and cut hair segments were extracted by ultrasonication (3h, 50 degrees C) and the extracts were cleaned-up with aminopropyl SPE columns. LC-MS/MS analysis was performed using a polar-endcapped phenyl-hexyl-RP-phase with negative mode electrospray ionisation (ESI) using a triple quadrupole mass spectrometer (Sciex API 365) with a turboionspray source and post-column addition of acetonitrile for enhanced sensitivity. The MS/MS transitions monitored were m/z 221 -->75 for EtG and 226 -->75 for D(5)-EtG as an internal standard. The method was selective and sensitive, with a detection limit of 51 pg/mg hair at a signal-to-noise ratio of 3:1. The mean recovery was 96%, with an intra- and inter-day precision of less than 11.7% at a concentration of 200 pg/mg. The linearity was assessed in the range of 25-2000 pg/mg hair, with a correlation coefficient of 0.997. The method was successfully applied to 97 human hair samples which were taken at autopsies from persons with known alcoholism or were obtained from alcoholics who were hospitalized for ethanol withdrawal, from social drinkers and from children having not consumed any alcohol. Although, approximately two-third of the alcoholics showed EtG concentrations in hair of higher than 51 pg/mg (up to >4000 pg/mg), in one-third the EtG concentration was below the detection limit. However, only in one of five hair samples of "social drinkers", the EtG concentration was above the detection limit (51 pg/mg). No EtG has been detected in the hair of children. These investigations demonstrate that heavy alcohol consumption may be but not necessarily has to be detectable by EtG analysis in hair.