13C4-secobarbital as the internal standard for the quantitative determination of secobarbital--a critical evaluation

In this study, 13C4-secobarbital was used as an exemplar compound to illustrate the mechanism based on which the effectiveness of a proposed internal standard (IS) could be evaluated. A deuterated analog, 2H5-secobarbital, was also studied in parallel for comparison purposes. Well-established solid-phase extraction and methylation procedures were used prior to the GC/MS measurement step. The contribution of the intensity of an ion designated for the analyte (secobarbital) by the proposed IS, and similarly, the contribution of the intensity of an ion designated for the IS by the analyte--a phenomenon termed "cross-contribution"-were evaluated based on a "direct measurement" procedure in which equimolar amounts of the analyte and the IS were used to generate intensity data. These intensity data were then used as the basis for the calculation of "cross-contribution" (in percentages) of ions designated for the analyte and the IS. Cross-contribution data were compared with the linearity data resulting from two series of standards containing 25 to 9600 ng/mL secobarbital using two sets of quantitation ion pairs--m/z 196/200 and 195/199 with 13C4-secobarbital as the IS and m/z 196/201 and 195/200 with 2HS-secobarbital as the IS. 13C4-secobarbital was found to be much less problematic and thus can serve as a very effective IS. Cross-contribution data alone cannot fully explain the observed differences resulting from the use of these two ISs; further systematic study is needed to provide better understanding of the underlying interference mechanism.     

Isotopic analogs as internal standards for quantitative analyses by GC/MS--evaluation of cross-contribution to ions designated for the analyte and the isotopic internal standard

Isotopic analogs of the analytes are currently preferred internal standards (IS) for quantitative analyses of drugs and their metabolites in biological matrices by GC/MS procedures. Contributions of the analyte and the IS to the intensities of ions designated for the IS and the analyte, respectively--an undesirable phenomenon termed "cross-contribution"--greatly weakens the effectiveness of this approach. The cross-contribution phenomenon has been, in the past, evaluated by a "direct measurement" approach, in which intensities of interested ions were measured in two separate experiments using equal quantities of the analyte and the IS. Alternate procedures that may generate improved results are hereby studied. For the "improved direct measurement" approach, ion intensity data derived from the previously reported direct measurement procedure are first normalized before being used to calculate the extent of cross-contribution. An "internal standard" approach is also developed, in which a set amount of a third compound is incorporated into these two separate experiments, thus allowing corrections of ion intensity data that are imbedded with variations inherent to separate experiments. Finally, a "standard addition" approach, involving a series "addition" of "standards", generates multiple data points; thus, providing a mechanism to validate the resulting cross-contribution data. Secobarbital/(2)H(5)-secobarbital and secobarbital/(13)C(4)-secobarbital pairs are adapted as the exemplar systems for this study.     

Analytical approach to determine ticlopidine in post-mortem blood

A new and sensitive method to determine ticlopidine in whole human blood using proadifen as the internal standard (IS) is described. The analyte and IS were extracted by solid-phase extraction using Oasis HLB cartridges, and the extracts were analyzed by gas chromatography-electron impact ionisation-mass spectrometry (GC/EI-MS). Calibration curves were established daily in spiked blood samples using a non-linear calibration model, between 0.01 and 4.5 microg/mL. The correlation coefficients were higher than 0.995. Precision and accuracy fulfilled the internationally accepted criteria (coefficients of variation were less than 9%, and the measured concentrations were within +/-7% of the true value). Limits of detection and quantitation were respectively, 3 and 10 ng/mL. No interfering substances were detected by analysis of 10 blank blood samples of different origin. Mean recovery, calculated at three concentration levels, was 71%. Because of its simplicity and speed, the proposed method can be applied in the determination of this inhibitor of platelet aggregation in whole blood samples, and is suitable for application in toxicology routine analysis.     

Enantiomeric Identification of Pregabalin by GC‐MS via Methylation and S‐TPC Chiral Derivatization

Pregabalin is a Schedule V controlled substance which is defined as the (S) enantiomer of 3‐(aminomethyl)‐5‐methylhexanoic acid. It is used legitimately to treat neuropathy in patients with diabetes as well as for epilepsy and fibromyalgia. Pregabalin is an amino acid and an amphoteric compound, which makes it difficult to analyze using the conventional GC‐MS instrumentation found in most forensic drug analysis laboratories. Problems associated with the traditional GC‐MS analysis of pregabalin include selective solubility, ring closure to the corresponding lactam in the GC injection port and/or the MS transfer line and difficulty with chiral derivatization due to the presence of a carboxylic acid moiety. Here, we show that these challenges can be overcome by methylating (capping) the carboxylic acid portion of the pregabalin molecule and converting to the corresponding methyl ester. Once the methyl ester is synthesized, chiral derivatization at the amine can be achieved to identify the controlled (S) enantiomer of pregabalin via GC‐MS.     

Performance of the Emit® II Plus 6-Acetylmorphine Assay on the Viva-E® analyzer

We evaluated the performance of Emit(?) II Plus 6-Acetylmorphine Assay for human urine screening on the Viva-E(?) analyzer. Precision was evaluated using the cutoff and ±25% controls. Recovery and linearity were studied by spiking 6-acetylmorphine (6-AM) into human urine pools. Accuracy was evaluated using urine specimens and the results were compared to those from GC/MS. Cross-reactivity with structurally related drugs was assessed at different cross-reactant concentrations. Interferences were assessed in the presence of 7.5 and 12.5 ng/mL of 6-AM. The qualitative repeatability coefficients of variation (CV's) ranged from 0.3% to 0.4% and the within-lab CV's ranged from 2.0% to 2.2%. In analyte units (ng/mL), the repeatability CV's ranged from 1.3% to 2.2% and the within-lab CV's ranged from 2.6% to 4.3%. The limit of detection of the assay was found to be 2.1 ng/mL. Recovery was within 15% of expected value. Linearity was 2.1-20 ng/mL. Method comparison showed 99% agreement with GC/MS. The assay had minimal cross-reactivity with morphine, morphine-3-glucuronide, morphine-6-glucuronide and other opioids. No interference was observed with endogenous interferences and structurally unrelated drugs. The assay correctly classified CAP survey samples. The Emit(?) II Plus 6-Acetylmorphine Assay will be a suitable screening method for urine specimens in both qualitative and semi-quantitative analyses.     

Quantitation of atenolol, metoprolol, and propranolol in postmortem human fluid and tissue specimens via LC/APCI-MS

Hypertension is a growing medical concern in the United States. With the number of Americans suffering from hypertension increasing, the use of antihypertensives such as beta-blockers is increasing as well. In fact, three beta-blockers - atenolol, metoprolol and propranolol - were among the 200 most prescribed medications in the United States in 2003. Pilots that successfully manage their hypertension can remain certified to fly. The Federal Aviation Administration currently designates approximately 8% of active pilots as "hypertensive with medication". The Civil Aerospace Medical Institute (CAMI) performs toxicological evaluation on victims of fatal aviation accidents. At CAMI beta-blockers are analyzed using gas chromatography with mass spectrometric detection. We have, however, recently developed a liquid chromatography with mass spectrometric detection (LC/MS) method for the simultaneous quantitation of three commonly prescribed beta-blockers, atenolol, metoprolol and propranolol. One advantage of our LC/MS method is the specificity provided by an ion trap MS. Utilizing an ion trap MS, we were able to conduct MS/MS and MS/MS/MS on each analyte. This method also eliminates the time-consuming and costly derivitization step necessary during GC/MS analysis. Additionally, by utilizing this novel method, any concerns about beta-blocker metabolite and/or sample matrix interference are eliminated. The limits of detection for this method ranged from 0.39 to 0.78 ng/mL and the linear dynamic range was generally 1.6-3200 ng/mL. The extraction efficiencies for each analyte ranged from 58% to 82%. This method was successfully applied to postmortem fluid and tissue specimens obtained from victims of three separate aviation accidents.     

Postmortem determination of the biological distribution of sufentanil and midazolam after an acute intoxication

A case is presented of a death caused by self-injection of sufentanil and midazolam. Biological fluids and tissues were analyzed for midazolam by high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) and for sufentanil by GC/MS. Midazolam was extracted from basified fluids or tissues homogenated with n-butyl chloride and analyzed by HPLC by using a phosphate buffer: acetonitrile (60:40) mobile phase on a mu-Bondapak C18 column at 240 nm. Sufentanil was extracted from basified fluids and tissue homogenates with hexane:ethanol (19:1). GC/MS methodology for both compounds consisted of chromatographic separation on a 15-m by 0.25-mm inside diameter (ID) DB-5 (1.0-micron-thick film) bonded phase fused silica capillary column with helium carrier (29 cm/s) splitless injection at 260 degrees C; column 200 degrees C (0.8 min) 10 degrees C/min to 270 degrees C; and electron ionization and multiple ion detection for midazolam (m/z 310), methaqualone (IS, m/z 235), sufentanil (m/z 289), and fentanyl (IS, m/z 245). Sufentanil concentrations were: blood 1.1 ng/mL, urine 1.3 ng/mL, vitreous humor 1.2 ng/mL, liver 1.75 ng/g, and kidney 5.5 ng/g. Midazolam concentrations were: blood 50 ng/mL, urine 300 ng/mL, liver 930 ng/g, and kidney 290 ng/g. Cause of death was attributed to an acute sufentanil/midazolam intoxication and manner of death a suicide.     

芬氟拉明和苯丙胺类兴奋剂的固相微萃取

用固相微萃取技术从血中提取芬氟拉明、苯丙胺和甲基苯丙胺。在 70℃条件下用 10 0 μm聚二甲基硅氧烷萃取头吸附 15min。重氢甲基苯丙胺作内标 ,采用柱前衍生化的进样方式 ,气质联用仪测定。选择离子m /z2 6 8(芬氟拉明 )、m/z2 40 (苯丙胺 )、m /z2 5 4(甲基苯丙胺 )和m/z2 5 8(重氢甲基苯丙胺 ,内标 )的峰面积比定量。血中检测浓度可达 0 0 1～ 0 0 3μg/g。通过解剖例中芬氟拉明的实际测定 ,证明这是一个从血液中提取分析苯丙胺类衍生物的快速准确的方法     

毛细管气相色谱法测定酱油中4-甲基咪唑的含量

目的建立毛细管气相色谱法定量分析酱油中有毒成分4-甲基咪唑的方法。方法酱油样品在层析柱中用二氯甲烷洗脱,洗脱液浓缩后加入N,N-二甲基苯胺作为内标,采用DB-FFAP毛细管柱分离样品,氮磷检测器测定4-甲基咪唑含量。结果方法线性范围为4.9~1.5×102μg/L;检测限为0.16μg/L;标准加入0.0102mg和0.0602mg 4-甲基咪唑的平均回收率为97.25%和99.44%。结论本文方法具有操作简便、快速、准确等优点,可用于检验酱油中的4-甲基咪唑。     

三种羟亚胺分析方法的比较

目的建立羟亚胺及氯胺酮定性和定量分析方法。方法分别使用气相色谱质谱联用法(GC/MS)、液相色谱质谱联用法(LC/MS)和液相色谱紫外法(LC/UV)分析羟亚胺,考察各方法的特点及适用范围。结果采用GC/MS法分析时,进样口的高温会导致部分羟亚胺转化为氯胺酮。LC/MS及LC/UV分析则不存在干扰,羟亚胺和氯胺酮的线性范围分别为3.0～300 ng/mL(LC/MS)、0.02～1.00mg/mL(LC/UV);最低检测限分别为1.0ng/mL(LC/MS)、5.0μg/mL(LC/UV)。结论 GC/MS法仅可确定样品中羟亚胺的存在,不能确认是否含有氯胺酮。LC/MS和LC/UV法可分别用于痕量和常量羟亚胺和氯胺酮定性和定量分析。     

固相萃取-GC/MS法检测生物检材中的百草枯

目的采用固相萃取-气相色谱/质谱分析方法检测血液、尿液和脏器组织中的百草枯。方法人血液、尿液和猪肺组织样品经三氯乙酸去除蛋白后,取上清用十二烷基三甲基溴化铵和十二烷基硫酸钠处理过的C18小柱提取,提取物用硼氢化钠在碱性条件下还原,产物用气相色谱/质谱法分析,外标法定量。结果生物检材中百草枯回收率为78%～87%,最低检出限为0.1μg/mL,在0.5～1mg/mL范围内线性关系良好,可对实际案例检材进行定量检测。结论本文固相萃取-气相色谱/质谱分析方法能满足中毒生物检材检验及临床毒物检验需要。     

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。     

Effective injection in pulsed splitless mode for impurity profiling of methamphetamine crystal by GC or GC/MS

Gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) are commonly used for the impurity profiling of illegal drugs. For the impurity profiling of methamphetamine, it is very important to obtain information about impurities related to the manufacturing route and the precursor chemicals [B. Remberg, A.H. Stead, Drug characterization/impurity profiling, with special focus on methamphetamine: recent work of the United Nations International Drug Control Programme, Bull. Narcotics LI (1999) 97-117 ]. There are many artifact impurities arising from the preparation of samples and conditions of GC. Moreover, some impurities pose a barrier to the statistical processing of methamphetamine profiling. We investigated capillary GC analysis using pulsed splitless (PS/L) injection to minimize the thermal decomposition of impurities at the injection port and improve the transfer of samples into the column. We confirmed that the optimal conditions of PS/L-mode are 230 degrees C (injection temperature), 50 psi (pulsed pressure) and 1.1 min (pulsed time) for the methamphetamine profiling. Based on the impurity profiles of 48 methamphetamine crystals in PS/L-mode, we can achieve very easy handling and obtained, good results.     

Positive identification of the principal component of a white powder as scopolamine by quantitative one-dimensional and two-dimensional NMR techniques

An unidentified white powder collected as evidence in an intelligence investigation was characterized exclusively by nuclear magnetic resonance (NMR) analysis. A small fraction of the powder dissolved in D2O was subjected to a series of one- and two-dimensional techniques which were used to elucidate the molecular structure of the powder's major component and positively identify it as the scopolamine biotoxin. Quantitative one-dimensional experiments identified individual proton and carbon atom sites, and conventional 14N spectroscopy detected a single nitrogen atom site. Heteronuclear single quantum coherence data correlated all protons to their directly bonded carbon atom, and together with the quantitative spectra, were used to determine the number of protons directly bonded to each carbon atom. The presence of a methyl, carboxyl, and a benzyl group was also identified from these data. Correlation spectroscopy detected a three proton and a nine proton JH,H network, representing a CH2CH moiety and seven carbon atom ring, respectively. These five elements were assembled into an almost complete molecular structure by using long-range, J-coupled, 1H-13C pairs detected by heteronuclear multiple bond correlation (HMBC) spectroscopy and 1H-1H dipolar-coupled pairs found from nuclear Overhauser effect spectroscopy (NOESY) data. Additional oxygen atom sites were inferred from 1H-13C correlation intensities in the HMBC spectra along with 1H and 13C chemical shift values, or directly from NOESY correlations. Only a single oxygen atom site could not be inferred from NMR data, but its presence was inferred from comparisons to target analyte structures to complete the structure of the scopolamine molecule. To confirm these results, an ethanol/H2O solution of the powder was analyzed by direct infusion into an ion trap mass spectrometer. A prominent base signal was observed at m/z 304.1 amu, corresponding to the protonated molecular ion of scopolamine. Subsequently, the ion was selected and subjected to collision-induced dissociation, producing characteristic major MS/MS fragments at m/z 138.1 and 156.1. Comparisons of 1H and 13C chemical shift values and JH,H values measured from our NMR data were found to agree very favorably with previously reported values for scopolamine in D2O.     

Butalbital and Driving Impairment

Butalbital (Fiorinal^®), used in the treatment of migraines and muscle pain, is the most commonly encountered barbiturate in impaired driving cases. It has central nervous system (CNS) depressant properties, including sedation, drowsiness, and feelings of intoxication, which can contribute to driving impairment. Twenty‐six driving under the influence cases are reviewed including results from field sobriety tests and toxicology testing. Blood samples were screened using enzyme multiplied immunoassay technique immunoassay, and the presence of butalbital was confirmed and quantified using gas chromatography/mass spectrometry, gas chromatography with flame ionization detection, or gas chromatography nitrogen/phosphorus detection. Butalbital concentrations ranged from 1.0 to 30.2 mg/L, with a mean and median of 16.0 mg/L. General impairment indicators in these cases included horizontal and vertical nystagmus, lack of convergence, poor motor coordination, and balance and speech problems, which are common to CNS depressant intoxication, similar to that associated with alcohol. These findings indicate the importance of toxicological testing for butalbital in cases where CNS depressants are indicated.     

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.     

Attempted murder with pancuronium

A nurse was accused of attempting to murder her anesthesiologist husband on two occasions by administering to him a neuromuscular blocking agent. In both episodes, urine specimens were obtained from the victim shortly after the suspected assaults. The samples were initially tested fluorometrically using Rose Bengal dye as a pairing agent. Both were presumptively positive for pancuronium. Confirmation of these results was achieved by pairing the drug with potassium iodide, extracting the complex, and submitting the extract to thin-layer chromatography (TLC) cleanup, elution at the appropriate retardation factor (Rf), and, finally, gas chromatography/mass spectrometry (GC/MS) analysis in the selected-ion monitoring mode. The two quaternary amines of pancuronium appear to undergo pyrolytic N-demethylation in the injection port to yield an entity amenable to capillary column gas chromatography. The mass spectrum of the compound consists of a base peak of m/z 322, with additional fragments of 292, 323, 338, and 397 m/z, each of which was monitored. The confirmed positive findings were instrumental in adjudicating the case.     

Analysis of Explosives by GC‐UV

A mixture of explosives was analyzed by gas chromatography (GC) linked to ultraviolet (UV) spectrophotometry that enabled detection in the range of 178–330 nm. The gas‐phase UV spectra of 2,4,6‐trinitrotoluene (TNT), 2,4‐dinitrotoluene (DNT), ethylene glycol dinitrate (EGDN), glycerine trinitrate (NG, nitroglycerine), triacetone triperoxide (TATP), and pentaerythritol tetranitrate (PETN) were successfully recorded. The most interesting aspect of the current application is that it enabled simultaneous detection of both the target analyte and its decomposition products. At suitable elevated temperatures of the transfer line between the GC instrument and the UV detector, a partial decomposition was accomplished. Detection was made in real time and resulted in overlaid spectra of the mother compound and its decomposition product. Hence, the presented approach added another level to the qualitative identification of the explosives in comparison with traditional methods that relies only on the detection of the target analyte. As expected, the decomposition product of EGDN, NG, and PETN was NO, while TATP degraded to acetone. DNT and TNT did not exhibit any decomposition at the temperatures used.     

A validated SPME-GC-MS method for simultaneous quantification of club drugs in human urine

A solid-phase microextraction-gas chromatographic-mass spectrometric (SPME-GC-MS) method has been developed and validated for measuring four club drugs in human urine. These drugs include gamma-hydroxybutyrate (GHB), ketamine (KET), methamphetamine (MAMP), and methylenedioxymethamphetamine (MDMA). These drugs are referred to as 'club drugs' because of their prevalence at parties and raves. Deuterium labeled internal standards for each of the four drugs was included in the assay to aid in quantitation. The drugs were spiked into human urine and derivatized using pyridine and hexylchloroformate to make them suitable for GC-MS analysis. The SPME conditions of extraction time/temperature and desorption time/temperature were optimized to yield the highest peak area for each of the four drugs. The final SPME parameters included a 90 degrees C extraction for 20min with a 1min desorption in the GC injector at 225 degrees C using a splitless injection. All SPME work was done using a 100microm PDMS fiber by Supelco. The ratio of pyridine to hexylchloroformate for derivatization was also optimized. The GC separation was carried out on a VF-5ht column by Varian (30m, 0.25mm i.d., 0.10microm film thickness) using a temperature program of 150-270 degrees C at 10 degrees C/min. The instrument used was a ThermoFinnigan Trace GC-Polaris Q interfaced with a LEAP CombiPal autosampler. The data was collected by using extracted ion chromatograms of marker m/z values for each drug from the total ion chromatograms (TIC) (full scan mode). Calibration curves with R(2)>0.99 were generated each day using the peak area ratios (peak area drug/peak area internal standard) versus concentration. The validated method resulted in intra-day and inter-day precision (% R.S.D.) of less than 15% and a % error of less than 15% for four concentrations in the range of 0.05-20microg/mL (MAMP) and 0.10-20microg/mL (GHB, KET, and MDMA). This method has the advantage of an easy sample preparation with acceptable accuracy and precision for the simultaneous quantification of these four drugs of abuse and shows no interference from the urine matrix.     

Simultaneous quantitation of delta-9-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in serum by GC/MS using deuterated internal standards and its application to a smoking study and forensic cases.

A new procedure for the simultaneous detection of delta-9-tetrahydrocannabinol (THC) and its major metabolite, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in serum has been evaluated. The method combines rapid, efficient, solid-phase extraction and simple derivatization by methylation. Analysis and quantitation is performed by gas chromatography/mass spectrometry (GC/MS) using deuterated cannabinoids as internal standards (IS). Reproducibility and sensitivity of the method are good. The procedure is applied to serum specimens collected from a smoking study with 24 volunteers and 212 forensic cases. Results are interpreted based upon the current knowledge about THC metabolism and pharmacokinetics.