Development of a harmonised method for the profiling of amphetamines: IV. Optimisation of sample preparation

The suitability of liquid-liquid extraction (LLE) and solid-phase extraction (SPE) for the preparation of impurity extracts intended for gas chromatographic profiling analyses of amphetamine were evaluated. Both techniques were optimised with respect to the extraction of selected target compounds by use of full factorial designs in which the variables affecting the performance were evaluated. Test samples consisted of amphetamine synthesised by the Leuckart reaction, by reductive amination of benzyl methyl ketone and by the nitrostyrene route. The performance of LLE and SPE were comparable in terms of repeatability and recovery of the target compounds. LLE was considered the better choice for the present harmonised amphetamine profiling method due to the lack of information on the long-term stability of SPE columns.     

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。     

全血中扎来普隆两种萃取方法的比较

目的建立血样中扎来普隆的液液萃取和固相萃取方法。方法液液萃取方法是在碱性条件下用二氯甲烷提取。固相萃取是采用OasisHLB固相萃取柱提取,萃取液均用GC/NPD检验,并对其进行比较。结果血样中扎来普隆固相萃取平均回收率达89%以上,液液萃取平均回收率为84%,GC/NPD最低检出限为2×10-2μg.mL-1。结论两种方法均能满足实际检案的需求,而固相萃取方法在减少仪器损耗和内源性杂质干扰方面具有较大的优势。     

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。     

生物检材中甲基苯丙胺及苯丙胺的分析研究进展

对近几年国内外22篇有关生物检材中甲基苯丙胺及苯丙胺测定的文献进行了综述。介绍了血、尿、毛发等生物检材的收集与预处理方法,比较了生物检材中甲基苯丙胺及苯丙胺的液-液萃取(LLE)、固相萃取(SPE)、固相微萃取(SPME)和顶空固相微萃取(HS-SPME)等提取方法,以及内标的选取、不同的衍生化方法和包括免疫、GC/MS、GC/NPD、GC/ECD、GC/FID、HPLC、HPCE在内的各种检测方法。最后,对分析结果的评定进行了讨论。     

Preparation and usage of a new solid phase micro-extraction membrane

Liquid-liquid or solid phase extraction methods are widely used for isolating analytes from urine, blood and other samples. But the preparation procedures of the samples are laborious, intensive, and costly. In addition, the organic solvents used are toxic to both the human body and the environment. An accurate, simple and rapid method for analysis of some compounds is required for forensic, judicial, and clinical purposes. Solid phase micro-extraction membrane (SPMEM) is a new, simple and solventless preparation technique. It integrates sampling, extraction and concentration into a single step and has the advantages of both the solid phase micro-extraction (SPME) and membrane separation. In this paper, a new kind of membrane used for the solid phase micro-extraction was prepared with amide compounds. The extraction conditions such as adsorption time, desorption solvents, methods and time are studied and optimized. The dichlorvos in the blood, morphine and phenobarbital in the urine were perfectly separated by using this solid phase micro-extraction membrane, and were tested by gas chromatograph-mass spectrometer (GC-MS). All the data were acquired in scan mode except that of morphine which was obtained in a selected ion monitoring (SIM) mode. Ions used for identification were those with m/z 57, 115, 162, 215, 285.     

气相色谱-串联质谱法分析尿和血中除草剂百草枯

目的建立尿和血中百草枯的离子交换固相萃取-气相色谱-串联质谱分析方法。方法尿样加内标乙基百草枯,用732阳离子交换树脂提取;血样加内标乙基百草枯,用三氯乙酸凝聚蛋白质后取上清液用732阳离子交换树脂提取。提取物用硼氢化钠在水溶液中碱性条件下还原,还原物用有机溶剂提取进行气相色谱-串联质谱法分析。结果尿和血中百草枯的提取率分别为76％和74％,检测限分别为2ng／mL和10ng／mL,尿添加百草枯100ng／mL和血添加百草枯500ng／mL水平的回收率分别为99．6±5．6％和99．3±7．6％（Mean±CV）。结论本文建立的分析方法灵敏度高,能够满足中毒致死案件检验及临床毒物检验的需要。     

体液中常见滥用药物的系统筛选分析

本文建立了体液中常见滥用药物的筛选分析体系.尿液或血液经固相萃取(SPE)或液提取(LLE)后,直接用GC/NPD分析或经TFA、BSTFA衍生化后用GC/MS分析.方法适用于同时分析甲基苯丙胺、MDMA、度冷丁、去甲度冷丁、曲马多、美沙酮、EDDP、可卡因、苯甲酰芽子碱、可待因、安定、氯丙嗪、吗啡、单乙酰吗啡等十四种常见滥用药物及代谢物.SPE法和LLE法回收率分别为66～102%和50～86%,最低检出限为2-5ng/ml尿.涉毒案件的鉴定应用表明该分析方法简便、快速、可靠.     

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.     

Contribution of thermal desorption and liquid-liquid extraction for identification and profiling of impurities in methamphetamine by gas chromatography-mass spectrometry

Impurity profiling of methamphetamine (MA) using thermal desorption (TD) and gas chromatography-mass spectrometry (GC-MS) was examined. Using TD/GC-MS, impurities were extracted and separated under various conditions. Optimal chromatograms were obtained when a 20 mg MA sample was extracted at 120 degrees C for 3 min using a TD instrument, followed by separation of the extracts using a non-polar capillary column coated with (5%phenyl)-methylpolysiloxane. MA samples from nine different batches were analyzed under optimized conditions. Compounds related to the structure of MA, such as benzaldehyde, benzyl alcohol, amphetamine, cis- and trans-1,2-dimethyl-3-phenylaziridine, dimethylamphetamine, and N-acetylephedrine, were detected in the chromatograms without any laborious extraction procedure. Compounds such as ethanol, diethyl ether, and acetic acid, which are considered reagents and solvents for MA synthesis, were also detected in some of the chromatograms. The numbers and intensities of the peaks detected were different among the samples. Impurity profiling of MA using TD was compared with that using liquid-liquid extraction (LLE). Better reproducibility of peak areas was obtained using LLE, whereas higher intensities and numbers of peaks were detected using TD. Solvents were extracted more effectively using TD. The nine batches of MA were classified using both extraction procedures. The nine batches were divided roughly into two groups using data from LLE. Subsequently, the groups were classified in detail using data from TD. TD can be used to provide supplemental information for LLE, and the combination of these extraction methods can be helpful for impurity profiling of MA.     

尿液中丁丙诺啡的提取方法

目的建立尿液中丁丙诺啡提取方法。方法尿液加pH 7.0缓冲液后,用三氯甲烷进行液相萃取或加pH 10.8缓冲液后,用401有机担体作吸附剂、三氯甲烷作洗脱剂进行固相萃取,GC/NPD法检测。结果尿液中丁丙诺啡液相萃取萃取率可达86.6%以上,固相萃取可达83.0%以上。结论两种提取方法萃取率高,可用于尿液中丁丙诺啡的提取。     

Simultaneous separation of different types of amphetamine and piperazine designer drugs by capillary electrophoresis with a chiral selector

The recent emergence of a new class of piperazine-type compounds has brought about the need for laboratory screening methods for both seized drugs and toxicological samples. These piperazine compounds, which include 1-benzylpiperazine (BZP) and 1-(3-trifluoromethylphenyl)piperazine (TFMPP), exhibit comparable physiological effects and can be substituted for the classic amphetamine-type drugs. We have optimized a chiral capillary electrophoresis (CE) separation that detects a set of 6 piperazine and 4 chiral amphetamine compounds in under 23 min using a 200 mM phosphate buffer at a pH = 2.8 with 20 mM hydroxypropyl- beta-cyclodextrin (HPbeta3CD). In addition to the above compounds, a series of "clandestine" BZP diHCl samples were also analyzed using this method to assess the ruggedness of the procedure. The novel CE separation was tailored to simultaneously detect these piperzine compounds in addition to amphetamine-type drugs. Distinct migration time and UV-spectral data were obtained for all compounds of interest.     

Simultaneous analyses of cocaine, cocaethylene, and their possible metabolic and pyrolytic products

A method was developed for simultaneously analyzing cocaine (COC), benzoylecgonine (BZE), norbenzoylecgonine (BNE), norcocaine (NCOC), ecgonine (ECG), ecgonine methyl ester (EME), m-hydroxybenzoylecgonine (HBZE), anhydroecgonine methyl ester (AEME), cocaethylene (CE), norcocaethylene (NCE), and ecgonine ethyl ester (EEE) in blood, urine, and muscle. Available deuterated analogs of these analytes were used as internal standards. Proteins from blood and muscle homogenate were precipitated with cold acetonitrile. After the removal of acetonitrile by evaporation, the supernatants and urine were subjected to solid-phase extraction. The eluted analytes were converted to their hydrochloride salts and derivatized with pentafluoropropionic anhydride and 2,2,3,3,3-pentafluoro-1-propanol. The derivatized products were analyzed by a gas chromatograph (GC)/mass spectrometer by selected ion monitoring. The limit of detection (LOD) for COC, BZE, NCOC, EME, CE, NCE, and EEE was 2ng/ml, while the LODs for BNE, ECG, HBZE, and AEME were 25, 640, 50, and 13 ng/ml, respectively. This method was successfully applied in analyzing 13 case samples from aviation accident pilot fatalities and motor vehicle operators. AEME concentrations found in the 13 samples were consistent with those produced solely by the GC inlet pyrolysis of COC controls in blood. Anhydroecgonine cannot be used as a marker for the abuse of COC by smoking because it is also pyrolytically produced from COC metabolites on the GC inlet. The developed method can be effectively adopted for analyzing COC and related compounds in urine, blood, and muscle by a single extraction with increased sensitivity through formation of hydrochloride salts and using a one-step derivatization.     

Detection of drugs using XAD-2 resin. I:Choice of resin, chromatographic conditions, and recovery studies

Amberlite XAD-2, a nonionic polystyrene divinylbenzene resin, was first used for the analysis of drugs in urine and a number of reports have described the development at optimal conditions for extraction, including type of resin columns, pH conditions, and eluting solvents. XAD-4 and XAD-7 resins were compared to the similarly structured XAD-2 resin and no significant advantage over the XAD-2 resin for drug screening was observed. A quantity of 5 to 6 g of resin was found to have sufficient capacity for the extraction of 200 ml of pentobarbital solution (1 mg/100ml). A column flow rate of approximately 15 ml/min (gravitational flow) was sufficient for analysis and slower rates were not more efficient. A mixture of ethyl acetate and 1,2-dichloroethane (3:2) was found to give best overall recovery (66 to 94%) of drugs, the resulting extracts being reasonably free of interfering substances. A pH value of 8.5 is recommended as optimum for comprehensive analysis of acidic and basic drugs. Recovery studies were conducted on spiked samples to determine drug losses occuring during various steps in the XAD-2 extraction procedure for four acidic (amobarbital, secobarbital, pentobarbital, and phenobarbital) and four basic (morphine, codeine, meperidine, and methadone) drugs. A relatively small amount (0 to 5%) of the drugs was not adsorbed by the resin and amounts varying from 6 to 40% failed to be desorbed by the eluting solvent. Additional losses occurred during the removal and analysis of TLC spots. Recovery of drugs from aqueous solutions analyzed with the XAD-2 resin were compared to recoveries reported in the literature with other XAD-2 resin methods for the extraction of drugs from urine. Recovery of phenobarbital, morphine, and codeine improved by 4 to 23% while recoveries of amobarbital, pentobarbital, secobarbital, methadone, and meperidine were 4 to 28% less efficient when compared to literature data.     

Fast confirmation of 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) in urine by LC/MS/MS using negative atmospheric-pressure chemical ionisation (APCI)

A fast method using automated solid-phase extraction (SPE) and short-column liquid-chromatography coupled to tandem mass-spectrometry (LC/MS/MS) with negative atmospheric-pressure chemical ionisation (APCI) has been developed for the confirmation of 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) in urine samples. This highly specific method which combines chromatographic separation and MS/MS-analysis can be used for the confirmation of positive immunoassay results with a NIDA cut-off of 15ng/ml. The conjugates of THC-COOH were hydrolysed prior to SPE, and a standard SPE was performed using C18-SPE columns. No derivatisation of the extracts was needed as in GC/MS analysis, and the LC run-time was 6.5min by gradient elution with a retention time of 2.4min. Linearity of calibration was obtained in the range between 0 and 500ng/ml (correlation coefficient R(2)=0.998). Using linear regression (0-50ng/ml) the limit of detection (LOD) was 2.0ng/ml and the limit of quantitation (LOQ) was 5.1ng/ml; day-to-day reproducibility and precision were tested at 15 and 250ng/ml and were 13.4ng/ml+/-3.3% and 255.8ng/ml+/-4.5%, respectively.     

Analysis of ketamine and norketamine in urine by automatic solid-phase extraction (SPE) and positive ion chemical ionization-gas chromatography-mass spectrometry (PCI-GC-MS)

Ketamine (KT) is widely abused for hallucination and also misused as a "date-rape" drug in recent years. An analytical method using positive ion chemical ionization-gas chromatography-mass spectrometry (PCI-GC-MS) with an automatic solid-phase extraction (SPE) apparatus was studied for the determination of KT and its major metabolite, norketamine (NK), in urine. Six ketamine suspected urine samples were provided by the police. For the research of KT metabolism, KT was administered to SD rats by i.p. at a single dose of 5, 10 and 20mg/kg, respectively, and urine samples were collected 24, 48 and 72 h after administration. For the detection of KT and NK, urine samples were extracted on an automatic SPE apparatus (RapidTrace, Zymark) with mixed mode type cartridge, Drug-Clean (200 mg, Alltech). The identification of KT and NK was by PCI-GC-MS. m/z238 (M+1), 220 for KT, m/z 224 (M+1), 207 for NK and m/z307 (M+1) for Cocaine-D(3) as internal standard were extracted from the full-scan mass spectrum and the underlined ions were used for quantitation. Extracted calibration curves were linear from 50 to 1000 ng/mL for KT and NK with correlation coefficients exceeding 0.99. The limit of detection (LOD) was 25 ng/mL for KT and NK. The limit of quantitation (LOQ) was 50 ng/mL for KT and NK. The recoveries of KT and NK at three different concentrations (86, 430 and 860 ng/mL) were 53.1 to 79.7% and 45.7 to 83.0%, respectively. The intra- and inter-day run precisions (CV) for KT and NK were less than 15.0%, and the accuracies (bias) for KT and NK were also less than 15% at the three different concentration levels (86, 430 and 860 ng/mL). The analytical method was also applied to real six KT suspected urine specimens and KT administered rat urines, and the concentrations of KT and NK were determined. Dehydronorketamine (DHNK) was also confirmed in these urine samples, however the concentration of DHNK was not calculated. SPE is simple, and needs less organic solvent than liquid-liquid extraction (LLE), and PCI-GC-MS can offer both qualitative and quantitative information for urinalysis of KT in forensic analysis.     

A single-column procedure on Bond Elut Certify for systematic toxicological analysis of drugs in plasma and urine.

A single-column solid-phase extraction procedure was developed for the screening of acidic, neutral, and basic drugs from plasma. The recoveries of all 25 tested drugs exceeded 82%. After the plasma had been diluted with phosphate buffer (pH 6.0), the drugs were extracted using a single Bond Elut Certify column. The acidic and most of the neutral drugs were eluted by acetone/chloroform (1:1) and the basic drugs were eluted by 2% ammoniated ethyl acetate. Some neutral drugs appeared in both fractions. The two fractions were collected separately and evaporated until approximately 100 microL of solvent remained in the tube. Both fractions were analyzed separately on a gas chromatograph equipped with a wide-bore capillary column and a flame ionization detector. The procedure could also be used for urine samples.     

Enantiomeric separation of methamphetamine and related analogs by capillary zone electrophoresis: intelligence study in routine methamphetamine seizures

A method for simultaneous enantiomeric separation of ephedrine, pseudoephedrine, and methamphetamine (MA) in a single run by simple capillary zone electrophoresis (CZE) with beta-cyclodextrin as a chiral selector is described. The effects of the buffer pH, phosphate concentration, beta-cyclodextrin concentration, voltage and temperature on the peak resolution were examined. Good enantiomeric resolution was attained for each analyte under our optimized conditions: 15 mM beta-cyclodextrin, 300 mM NaH2PO4 at pH 2.5 with an uncoated capillary (64.5 cm x 50 microm), applied potential at 20 kV and temperature at 30 degrees C. Ultraviolet (UV) detection at a fixed wavelength (200 nm) was employed using a diode array detector. Using phentermine as an internal standard, migration times for all analytes are reproducible within 0.16% for intra-day and 0.6% for inter-day runs. Application of this method to the analysis of confiscated drugs is discussed.     

固相萃取／LC-MS／MS测定尿液中吗啡类药物

目的 建立尿液中吗啡类药物的固相萃取／LC—MS／MS方法。方法采用OASIS MCX3cc（60mg）固相萃取柱进行提取,应用LC—MS／MS方法进行检测,运用保留时间和MRM方式对尿液中吗啡类药物及其代谢物进行定性定量分析。结果磷酸盐缓冲液pH4．0时,海洛因、6-MAM、可待因、吗啡、M3G的固相萃取回收率分别达64．33％-70．21％,96．95％～117．57％,83．60％～123．63％,68．82％～91．03％,94．64％～107．33％;最低检测限（LOD）分别为5、10、5、5、2pg,线性范围0．005～10μg／mL;相关系数分别为0．9998、0．9958、0．9992、0．9994、0．9997。结论本文所建方法,适用于尿液中吗啡类药物的分析。     

Developing a quantitative extraction technique for determining the organic additives in smokeless handgun powder.

Compositional analysis of the organic additives in smokeless handgun powder can provide forensic information to associate known and questioned samples. A reliable method for the quantitative extraction of smokeless powder additives would strengthen these measurements. To achieve quantitative recovery, both supercritical fluid extraction (SFE) and ultrasonic solvent extraction (USE) were evaluated as candidate techniques. Following a detailed evaluation of the solvent choice, the recovery of spiked additive compounds, and the effect of the powder matrix, a reliable USE technique was developed. When quantitative USE recovery of the target analytes, nitroglycerin (NG), diphenylamine (DPA), and ethyl centralite (EC), is coupled with additive measurement by micellar capillary electrophoresis (CE), compositional information can be obtained in less than 1 h.