Identification and quantitation of xenobiotics by 1H NMR spectroscopy in poisoning cases

In order to analyse a wide range of xenobiotics and their metabolites present in biological fluids, NMR spectroscopy can be used. A large variety of xenobiotics (therapeutic agents, pesticides, solvents, alcohols) can be characterized and quantitated directly, without sample preparation. NMR investigations were applied to acute poisoning cases, involving drugs such as salicylates and valproic acid (VPA). In a salicylate poisoning case, the three major metabolites of acetylsalicylic acid have been detected in crude urine, and rapid identification of lysine revealed the origin of the intoxication, namely lysine acetylsalicylate (Aspegic). Valproic acid as its glucuronide was identified in urine samples from two poisoned patients. 1H NMR was also used to identify and quantitate paraquat (Gramoxone) in urine owing to its two aromatic signals at 8.49 and 9.02 ppm, in two acutely poisoned patients (183 and 93 mg/l). An intentional poisoning case with tetrahydrofuran (THF) was also investigated. Serum and urine samples were collected. THF was characterized by its resonances at 1.90 and 3.76 ppm, and quantified at 813 and 850 mg/l in the two biological fluids, respectively. Moreover, two other compounds were detected: lactate and gamma-hydroxybutyric acid (GHB). 1H NMR spectroscopic analysis of serum samples from three poisoned patients revealed methanol in one case and ethylene glycol in the two others. Moreover, in the same spectrum, the corresponding metabolites formate and glycolate were found. Compared with the reference chromatographic or spectrophotometric methods, requiring time-consuming extraction and/or derivatization steps, NMR spectroscopy allows the determination of many exogenous and endogenous compounds, without any pre-selection of the analytes.     

Determination of diquat in biological materials by electron spin resonance spectroscopy

An electron spin resonance (ESR) method already in use for the quantitative analysis of paraquat was applied to the analysis of diquat in blood, serum, urine, tissue homogenates and several drinks without purification of the samples. The diquat radical produced with ascorbic acid at alkaline pH was much more stable than that produced with the commonly used sodium dithionite. Radical decay in solutions covered with n-hexane was less than 5% after 60 min over a wide range of ascorbic acid concentrations. In 0.2 N NaOH solution 85% of the radicals was present even after 24 h. The limit of detection was 0.3 micrograms/ml and the required amount of sample was 0.1 ml. When both diquat and paraquat were present in a sample the diquat was first extracted with 1-butanol prior to the ESR measurement, because both species were converted to the radicals.     

Detection of dipiridamol in biological fluids

Acetone is proposed as an isolating agent for dipiridamol isolation from biological fluids. Purification of the isolates was performed with liquid-liquid extraction and colon chromatography with silasorb C-18 sorbent. The technique of dipiridamol detection in the blood and urine is described. The assays results are presented.     

Detection of furadan in biological fluids

Optimal conditions have been determined for furadan isolation from biological fluids by means of the mixture of solvents ethyl acetate-acetone in volume 1:1. Possible purification of the compound from coextractive substances of biomaterial on the column with silica gel L 100/160 mcm is demonstrated. IR-spectrophotometric methods and high performace liquid chromatography with a detector of photodiode matrix are proposed for identification and quantitation of furadan in blood and urine specimens.     

Identification and quantitation of 3,4-methylenedioxy-N-methylamphetamine (MDMA,ecstasy) in human urine by 1H NMR spectroscopy. Application to five cases of intoxication

Identification of 3,4-methylenedioxy-N-methylamphetamine (MDMA, ecstasy) in five cases of intoxication using nuclear magnetic resonance (NMR) spectroscopy of human urine is reported. A new water suppression technique PURGE (Presaturation Utilizing Relaxation Gradients and Echoes) was used. A calibration curve was obtained using spiked samples. The method gave a linear response (correlation coefficient of 0.992) over the range 0.01–1 mg/mL. Subsequently, quantitation of the amount of MDMA present in the samples was performed. The benefit and reliability of NMR investigations of human urine for cases of intoxication with MDMA are discussed.     

体液中微量士的宁的富集及同时检测

目的将分子印迹技术运用到法医毒物分析中,建立一种全新的测定体液中微量士的宁的方法。方法运用分子印迹原位聚合技术,在色谱柱中一步合成了对士的宁具有专属识别能力的分子印迹聚合物,以此聚合物为介质,装填一厘米的小柱,建立体液中微量士的宁的富集及同时检测方法。结果以士的宁分子印迹聚合物作为尿液和血浆中微量士的宁的富集和检测介质,方法的检出限为4.9ng,样品回收率均大于92%,相对标准偏差小于6.59%,工作曲线的线性相关系数分别为0.9991和0.9966。运用此方法对中毒家兔血浆和尿液中的士的宁进行了测定。结论建立的新方法可以实现对士的宁专属的、灵敏的检测,适合于法医学毒物分析运用。     

GC/MS研究海洛因代谢物在吸毒者体内的分布

应用GC／MS－SIM测定了海洛因代谢物吗啡在两例海洛因延缓死亡者的体内分布,并分析了收集到的7例案子的毛发（头发、腋毛和阴毛）中6-单乙酰吗啡和吗啡含量。生物检材和毛发经酸水解、提取、醋酸酐或双（三甲基硅基）三氟乙酰胺（BSTFA）衍生化,然后进行GC／MS-SIM分析。结果表明尿、胆汁和肝脏是判定死者是否曾使用海洛因的最佳检材;毛发分析与生物检材相比有其独特的优点,可提供数月甚至数年的用药情况。为公安司法机关打击吸毒、惩治犯罪提供更可靠、有效的证据。     

Identification and quantitation by 1H-NMR of metabolites in animal organs and tissues. An application of NMR spectroscopy in forensic science

1H-nuclear magnetic resonance (NMR) was applied to the study of metabolites in rat organs and tissues. From 1H-NMR spectra of D2O extracts of various organs and tissues, lactate, pyruvate, and some other substances were simultaneously identified and quantitated. On the basis of the results, the use of 1H-NMR for estimating post-mortem time was suggested.     

1H NMR spectroscopic identification of a glue sniffing biomarker

Organic solvent abuse typically involves sniffing organic solvents to experience the mind-altering conditions they induce. In Republic of Korea, organic solvent abuse is a serious social problem, especially among teenagers. Several studies have addressed the effects of organic solvent abuse on mind and body, but there are no simple methods by which such abuse can be positively identified. In this report, we describe a method for analyzing toluene metabolites (toluene is the main ingredient of glue) in glue-sniffers' urine using (1)H NMR spectroscopy. Toluene is a commonly used solvent in the rubber, paint, plastics, leather, printing, and chemical industries. Inhaled toluene is metabolized to hippuric acid in the liver and excreted in the urine. Hippuric acid is known as a good biomarker for biological monitoring of toluene exposure. We have scanned hippuric acid and other toluene metabolites by NMR spectroscopy and performed statistical multivariate analysis of the data. Based on this analysis, we sought to determine parameters by which glue-sniffing (toluene inhalation) behavior may be verified. We also demonstrate the use of a pattern recognition method for accurate and efficient analysis of NMR data. In comparison to conventional methods, such as mass spectroscopy coupled with liquid chromatography or gas chromatography, nuclear magnetic resonance spectroscopy has several advantages, including simple sample preparation, non-destructive sampling, accuracy, short acquisition time, and reproducibility in the determination of urinary hippuric acid.     

1H-NMR of human saliva. An application of NMR spectroscopy in forensic science

1H-NMR was applied to the study of metabolites in human saliva specimens. Acetate, lactate, ethanol, glucose and some other substances were simultaneously identified and quantitated from the 1H-NMR spectra of saliva specimens treated with D2O. These experiments demonstrated the value of 1H-NMR as an analytical method in the field of forensic science and clinical pathology and toxicology.     

Rapid presumptive "fingerprinting" of body fluids and materials by ATR FT-IR spectroscopy

Human body fluids and materials were evaluated using attenuated total reflectance Fourier transform infrared spectroscopy. Purified proteins, cosmetics, and foodstuffs were also assayed with the method. The results of this study show that the sampled fluids and materials vary in the fingerprint region and locations of the amide I peaks because of the secondary structure of the composite proteins although the C = O stretch is always present. The distinct 1016 cm(-1) peak serves as a signature for semen. The lipid-containing materials (e.g., fingerprints, earwax, tears, and skin) can also be easily separated from the aqueous materials because of the strong CH(3) asymmetric stretch of the former. Blood-saliva and blood-urine mixtures were also successfully differentiated using combinations of peaks. Crime scene investigators employing rapid, portable, or handheld infrared spectroscopic instruments may be able to reduce their need for invasive, destructive, and consumptive presumptive test reagents in evaluating trace evidence.     

Accidental potassium dichromate poisoning. Toxicokinetics of chromium by ICP-MS-CRC in biological fluids and in hair

Intoxications by chromium (Cr) compounds are very life threatening and often lethal. After oral ingestion of 2 or 3g of hexavalent Cr (Cr(VI)), gastrointestinal injury, but also hepatic and renal failure, often occurs which each leads to a fatal outcome in most patients. Cellular toxicity is associated with mitochondrial and lysosomal injury by biologically Cr(VI) reactive intermediates and reactive oxygen species. After Cr(VI) has been absorbed, there is not much that can be done except to control the main complications as the treatment is only symptomatic. The biotransformation of Cr(VI) to Cr(III) reduces the toxicity because the trivalent form does not cross cellular membranes as rapidly. In fact, more than 80% of Cr(VI) is cleared in urine as Cr(III). We report the case of a 58-year-old male patient who was admitted to hospital after accidental oral ingestion of a 30 g/L potassium dichromate (the estimated amount of ingested Cr is about 3g). ICP-MS equipped with a collision/reaction cell (CRC) and validated methods were used to monitor plasma (P), red blood cells (RBCs), urine (U) and hair chromium. For urine the results were expressed per gram of creatinine. After 7 days in the intensive care unit, the patient was discharged without renal or liver failure. P, RBC and U were monitored during 49 days. During this period Cr decreased respectively from 2088 μg/L to 5 μg/L, 631 μg/L to 129 μg/L and 3512 μg/g to 10 μg/g. The half-life was much shorter in P than in RBC as the poison was more quickly cleared from the P than from the RBC, suggesting a cellular trapping of the metal. Hair was collected 2 months after the intoxication. We report a very rare case of survival after accidental Cr poisoning which has an extremely poor prognosis and usually leads to rapid death. For the first time, this toxicokinetic study highlights a sequestration of chromium in the RBC and probably in all the cells.     

Determination of ibogaine and noribogaine in biological fluids and hair by LC-MS/MS after Tabernanthe iboga abuse Iboga alkaloids distribution in a drowning death case

Tabernanthe iboga belongs to the Apocynaceae family. In this study, we report the case of a 37-year-old black male working as a security agent in Paris and found dead naked on the beach in Gabon after consumption of iboga. Autopsy revealed a drowning fatality and a myocardial abnormality (myocardial bridging). Samples of blood, urine, bile, gastric content, liver, lungs, vitreous, spleen and hair were taken. Biological fluids were liquid-liquid extracted with saturated NH4Cl pH 9.5 and methylene chloride/isopropanol (95/5, v/v) in presence of clonazepam-d(4), used as internal standard. After decontamination with dichloromethane, hair was cut into small pieces then sonicated for 2h in saturated NH4Cl pH 9.5 before extraction by methylene chloride/isopropanol (95/5, v/v). After evaporation the residues were reconstituted in methanol/ACN/formate buffer pH 3, from which 10 microL were injected into an ODB Uptisphere C(18) column (150 mm x 2.1mm, 5 microm) and eluted with a gradient of acetonitrile and formate buffer delivered at a flow rate of 200 microL/min. A Quantum Ultra triple-quadrupole mass spectrometer was used for analyses. Ionization was achieved using electrospray in the positive ionization mode (ESI). For each compound, detection was related to three daughter ions (ibogaine: m/z 311.4-->122.1, 174.1 and 188.1; noribogaine: m/z 297.4-->122.1, 159.1 and 160.1; clonazepam-d(4): m/z 319.9-->218.1, 245.1 and 274.1). Ibogaine and noribogaine were detected in all autopsy samples. Hair segmentation was not possible as hair was very short and frizzy. Concentrations of 1.2 and 2.5 ng/mg, respectively were detected. Neither other licit or illicit drugs nor alcohol were found. The presence of ibogaine and noribogaine in all autopsy samples was consistent with the recent absorption of Tabernanthe iboga, which was assumed to be responsible of the drowning fatality. The history of exposure, regarding hair analysis, is discussed. LC-MS/MS appears to be the best method for analyzing complex and poorly volatile alkaloids in autopsy samples and particularly in hair, due to the presence of a nitrogen ring and the relatively low concentrations to be measured.     

高效液相色谱法测定水中的草甘膦

目的建立强极性除草剂草甘膦的高效液相检验方法;方法采用离子交换液相色谱法分离,经柱后衍生化以荧光检测器定性检测样品中草甘膦;结果经该方法检测草甘膦的最低检测限为5ng/ml(S/N≥3);结论该方法快速、灵敏、准确,为此类案件的检验提供了科学依据。     

Different assays for hingamine in the study of biological fluids

The authors present a method of hingamine identification in non-biological substances (tablets, powder, syringes) and biological fluids (blood, urine). Isolation was made with chloroform in pH 10. Identification was conducted with thin-layer chromatography, gas chromatography/mass-spectrometry, high-performance liquid chromatography, spectrophotometry in UV region. The quantity was estimated with spectrophotometry in UV region, high-performance liquid chromatography and high-performance thin-layer chromatography. The results of the three methods are comparable.     

GC/MS法测定人体血液和肾、肝组织中的丙泊酚

目的 采用气相色谱/质谱法检测人体血液和肾、肝组织中的丙泊酚.方法 心血和肾、肝组织中的丙泊酚经环己烷提取,采用气相色谱/质谱法,丙泊酚选择m/z 163、178,内标百里香酚选择m/z 150、135进行测定;并考察方法专属性、线性范围、定量线与检出限、回收率与精密度.结果 丙泊酚的保留时间为8.17min,与内标百里香酚分离良好;其在心血和肾、肝组织中的检测浓度线性关系良好,r值均大于0.992,最低检测质量浓度分别为0.05 μg/mL、0.10μg/g及0.05 μg/g,方法回收率90％ ～ 110％,日内、日间RSD均小于7％.结论 本文方法简便、灵敏、快速,其准确度、精密度和回收率均可满足生物检材中丙泊酚浓度的测定,可在相关研究及实践中选用.     

液相色谱-串联质谱法分析生物检材中的河豚毒素

目的建立血液、尿液以及肝中河豚毒素(tetrodotoxin,TTX)的液相色谱-串联质谱分析方法,并进行方法学验证。方法血液、尿液和肝用1%乙酸甲醇溶液去蛋白后,上清液用固相萃取法净化,LC-MS/MS检测。结果血液、尿液和肝中TTX检出限分别为2ng/mL、2ng/mL和4ng/g。血液和尿液在4～100ng/mL、肝在5～100ng/g的范围内线性关系良好,相关系数r≥0.9973;日内精密度和日间精密度均在12.80%以内;回收率大于47.2%。结论所建方法高效、灵敏、准确,可以为河豚毒素中毒的法医学鉴定、临床诊治以及食品安全的监控提供技术保障。