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.     

Identification and quantitation of gamma-hydroxybutyrate (NaGHB) by nuclear magnetic resonance spectroscopy

The most common means of identification of gamma-hydroxybutyrate (NaGHB) involves using Fourier transform infrared spectroscopy (FTIR) or gas chromatography-mass spectrometry (GC-MS) of a suitable derivative. However, these methods may be complicated by possible shifts in chemical equilibrium between gamma-hydroxybutyric acid (GHB), GHB salts and the precursor lactone, gamma-butyrolactone (GBL). This paper addresses the technique of proton and carbon nuclear magnetic resonance spectroscopy (1H and 13C NMR) for the direct and accurate identification of GHB and GBL. The application of 1H NMR for GHB quantitation is also discussed.     

Apparent effects of glyphosate on alkaloid production in coca plants grown in Colombia

During the routine analysis of coca leaf material from South America, alkaloids in Erythroxylum coca var. ipadu (ECVI) leaf samples from fields suspected of being treated with glyphosate were compared with those from non-treated E. coca var. ipadu and Erythroxylum novogranatense var. novogranatense (ENVN) plants. Cocaine levels in leaf tissue from non-treated ECVI and ENVN were 0.53+/-0.08% and 0.64+/-0.08% (w/w), respectively, whereas leaves from treated plants were nearly devoid of cocaine. Further analysis demonstrated the presence of several previously undescribed N-nortropane alkaloids, several of which were tentatively identified. The results suggest that applications of glyphosate to coca plants can have dramatic effects on the quantity and quality of alkaloids produced by surviving or subsequent leaves. The analytical data presented will be of value to forensic chemists who encounter illicit cocaine preparations containing alkaloids produced from coca plants treated with glyphosate.     

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.     

A modest proposal to clarify the status of coca in the United Nations conventions

This essay surveys the status of coca in the United Nations Conventions, explains why it is confusing, how a few changes would eliminate some of the sources of conflict and help organize and control licit coca markets in the Andes. The current disorganized and weakly controlled legal coca market in Peru has been analyzed to demonstrate its deficiencies and to illustrate possible improvements in international drug control policies.     

14C analyses quantify time lag between coca leaf harvest and street-level seizure of cocaine

Measurements were made on the natural abundance (14)C content (Δ(14)C) of cocaine specimens seized between 2003 and 2009. The objective of this study was to determine the extent to which Δ(14)C analyses could quantify the "age" of recent cocaine seizures. Here "age" of a seized cocaine specimen is defined as the time period between when a coca leaf was harvested in South America and its seizure as cocaine at either the international or domestic street levels. Based on Δ(14)C analyses of seizure specimens, there were no statistically significant differences in the ages of domestic cocaine HCl and cocaine base specimens seized on the streets in different locations across the United States. Between 2007 and 2009, the average age of a street-level cocaine seizure in the United States was 24.6±1.1 months. Cocaine shipment seizures that were in excess of 150 kg during this time period had an average age of 18.2±1.4 months, whereas smaller shipment seizures were significantly older with an average age of 22.3±0.6 months. Analyses of the largest cocaine shipment seizures suggested that these seizures were composed of specimens with different ages, possibly representing accumulations over as much as a 31-month period.     

Identification of sinicuichi alkaloids in human serum after intoxication caused by oral intake of a Heimia salicifolia extract

A 26-year-old male came to hospital around midnight complaining about muscle pain of the extremities as well as the tongue and slightly raised temperature. He reported the intake of an unknown amount of sinicuichi tea he had fermented over 24 h by adding yeast and sugar. The patient was treated with Vomex A (dimenhydrinate) and released from hospital the following afternoon. A blood sample taken shortly after submission and a small amount of the used plant material were available for analysis. Herbal drugs are widely used as stimulants as a legal alternative to illegal psychoactive drugs or in traditional context. Among many others like Sassafras officinalis, Salvia divinorum or Ephedra, Heimia salicifolia ("sinicuichi"), a species of the lythraceae family, is available via several online shops. Brewed up or fermented and consumed, the so-called sinicuichi tea may cause exhilarating feelings and an alteration of awareness accompanied by bradycardia, relaxation of the muscles and a pleasant faintness. Therefore Sinicuichi brew and heimia leaves are widely used for medication by the natives of Central and South America. After liquid extraction with acetone five different alkaloids were detected in the plant material by LC-MS/MS operated in the Q1 scan mode applying a TurboIonSpray source. Subsequently, Product Ion Spectra were recorded and after confirming the molecular formula by determining the accurate masses, possible structures of H. salicifolia alkaloids were assigned. The information of the Product Ion Spectra was then used to set up a sensitive multiple reaction monitoring (MRM) method. Applying the MRM method to the patient's serum sample after alkaline liquid-liquid extraction all of the five heimia alkaloids detected in the plant material were also detected qualitatively in the serum extract, confirming the ingestion.     

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.     

乌头中剧毒生物碱的提取分离

目的从乌头属植物中提取分离剧毒生物碱乌头碱、中乌头碱、次乌头碱;方法酸性水溶液提取,碱化后中性氧化铝柱层析,IR、EI-MS确认结构;结果利用该方法提取以上3种生物碱得到的回收率与碱性苯溶液法相似。结论该方法所用试剂成本低、毒性小,适用于制备乌头碱、中乌头碱、次乌头碱对照品。     

Preparation of carboxyhemoglobin standards and calculation of spectrophotometric quantitation constants

A method was developed for the preparation of carboxyhemoglobin (COHB) standards, which were stable for more than four months with the prepared control remaining within acceptable limits during this time. A mathematical equation was developed to more accurately determine the constants A and B used in the equation COHB% = 100[(C - B)/(A - B)], where B = 0% COHB peak ratio at 540 nm and 579 nm; A = 100% COHB peak ratio at 540 nm and 579 nm; and C = the peak ratio at 540 nm and 579 nm for the blood being analyzed. The following equations were developed to calculate A and B: B = Pavg - (P) [(Pavg - Navg)/(P - N)]; A = B + (Pavg - Navg)/(P - N), Pavg = average peak ratio 540/579 for the positive standard run on the spectrophotometer; P = average decimal concentration measured on the CO-OXIMETER for the positive standard; Navg = average peak ratio 540/579 for the negative standard; N = average decimal concentration measured on the CO-OXIMETER for the negative standard. The new equations provided results consistent with those obtained from a CO-OXIMETER.     

Run-specific limits of detection and quantitation for STR-based DNA testing

STR-based DNA profiling is an exceptionally sensitive analytical technique that is often used to obtain results at the very limits of its sensitivity. The challenge of reliably distinguishing between signal and noise in such situations is one that has been rigorously addressed in numerous other analytical disciplines. However, an inability to determine accurately the height of electropherogram baselines has caused forensic DNA profiling laboratories to utilize alternative approaches. Minimum thresholds established during laboratory validation studies have become the de facto standard for distinguishing between reliable signal and noise/technical artifacts. These minimum peak height thresholds generally fail to consider variability in the sensitivity of instruments, reagents, and the skill of human analysts involved in the DNA profiling process over the course of time. Software (BatchExtract) made publicly available by the National Center for Biotechnology Information now provides an alternative means of establishing limits of detection and quantitation that is more consistent with those used in other analytical disciplines. We have used that software to determine the height of each data collection point for each dye along a control sample's electropherogram trace. These values were then used to determine a limit of detection (the average amount of background noise plus three standard deviations) and a limit of quantitation (the average amount of background noise plus 10 standard deviations) for each control sample. Analyses of the electropherogram data associated with the positive, negative, and reagent blank controls included in 50 different capillary electrophoresis runs validate that this approach could be used to determine run-specific thresholds objectively for use in forensic DNA casework.     

乌头属植物生物碱高效液相色谱研究

目的建立高效液相色谱法测定多种乌头属植物生物碱的方法。方法XTerraTMRP185μm,(250mm×4.6mm)色谱柱;流动相为乙腈∶30mmol/L碳酸氢铵梯度洗脱;流速1.0ml/min;检测波长233nm。结果该方法可成功应用于5种乌头属植物12个样品中生物碱成分分析。结论本研究结果提示乌头属不同种植物中生物碱成分及含量差异显著,因此为确保该类中药材用药安全,提高临床疗效,必须建立严格的药材质量控制体系。     

Rapid and sensitive quantitation of cyanide in blood and its application to fire victims.

We developed a head-space method for the determination of blood cyanide by gas chromatography with electron-capture detection. In this technique, a reaction precolumn packed with chloramine-T was used for the conversion of hydrogen cyanide into cyanogen chloride. Since the reaction precolumn eliminated the necessity of trapping hydrogen cyanide from biological samples, blood cyanide could be analyzed quickly by acidification only. Using this method, blood cyanide levels of fire victims were determined at autopsy. The serum values of cyanide ranged from 0.11 micrograms/ml to 18.12 micrograms/ml. However, a significantly higher cyanide content was detected in the left ventricular blood than in the right. This indicates that death was caused by the fire and suggests that the collecting point of the blood sample is an important factor in the determination of inhaled cyanide. There was a positive correlation between blood cyanide and carboxyhemoglobin contents.     

Separation and quantitation of optical isomers of methylamphetamine samples by capillary electrophoresis

A capillary electrophoretic (CE) procedure using an electrolyte modified with hydroxypropyl-β-cyclodextrin has been used for the separation and quantitation of (S)- and (R)-methylamphetamine in fifteen laboratory prepared samples and ten `street' samples. Excellent separation of (S)- and (R)-methylamphetamine was achieved and the levels of methylamphetamine in the samples were in good agreement with the levels determined by a separate CE procedure previously developed in our laboratory. Both CE systems showed the presence of impurities in some of the samples suggesting that CE could be used in profiling illicit methylamphetamine seizures. Gas chromatography-mass spectrometry (GC-MS) analysis of two of the samples identified one of the impurities as N-formyl methylamphetamine, however, no conclusive proof for the presence of this compound in the electropherograms was available. The chloride and sulphate content of the ten `street' samples was also determined by CE.     

Isolation of alkaloids and glycosides from tissue following enzymic digestion

Four enzymic digestion methods have been evaluated for their efficiency in releasing certain alkaloids and glycosides from spiked liver tissue. Enzymic digestion gives better recoveries of all the plant poisons studied than those obtained by conventional methods. A flow diagram for the enzymic methods of drug isolation and quantitation by HPLC is presented. Enzymic digestion for the release of glycosides is reported for the first time. It is concluded that papain digestion is the most suitable method for the analysis of broad spectrum of compounds of forensic and clinical importance.     

The quantitation of psilocybin in hallucinogenic mushrooms using high performance liquid chromatography

A method using high performance liquid chromatography (HPLC) with an acetonitrile, water, and phosphoric acid mobile phase and a bonded cyano-amino-type polar phase column has been developed for the rapid, selective, and accurate quantitation of psilocybin in dry mushroom material. A simple one-step procedure is used for the quantitative extraction of psilocybin in under 60 min. The 267:254 nm absorbance ratio is used as a check on peak purity for the psilocybin response.     

A contribution to the improvement of accuracy in the quantitation of THC.

The accuracy of a quantitative analysis is highly dependent on the quality of the reference standard. Although reference standards are more and more supplied with a certificate, laboratories may feel the need for additional acceptance testing. In general, confirmation of the purity of many solid reference substances can be obtained by a number of simple tests. However, verification of the true content of reference solutions may be complicated. A number of problems with the THC quantitation caused our interest for a verification method for the THC reference solution. The quantitation of THC is performed by gas chromatography with flame ionisation detector. The effective carbon number concept was used to predict GC/FID response factors. Equations and data are presented to calculate theoretical response ratios of cannabinoids. The experimental data for CBD and CBN were in excellent agreement with the theoretical ones. The paper shows that the response factors of CBD and/or CBN can be used for the calculation of the THC content of either reference solutions or cannabis samples.