Sensitive determination of midazolam and propofol in human plasma by GC–MS/MS

Forensic Toxicology - A sensitive method was developed for the simultaneous determination of midazolam and propofol in human plasma samples via modified quick, easy, cheap, effective, rugged, and...     

In vivo metabolism of the new synthetic cannabinoid APINAC in rats by GC–MS and LC–QTOF-MS

The recent appearance of APINAC (AKB-57, ACBL(N)-018, adamantan-1-yl 1-pentyl-1H-indazole-3-carboxylate) in the market of the so-called novel psychoactive substances resulted in the need of defining its characteristics and searching its metabolites for subsequent detection in biological samples. The structure of the APINAC molecule has great similarity to the molecules of other synthetic cannabinoids. Here we report on the in vivo metabolism of APINAC using rats as an experimental model. Rat urine samples were analyzed by using gas chromatography–mass spectrometry and liquid chromatography–high resolution mass spectrometry. Data were acquired via time-of-flight mass scan, followed by Auto MS and triggered product ion scans. The predominant metabolic pathway for APINAC was ester hydrolysis yielding a wide variety of N-pentylindazole-3-carboxylic acid metabolites and 1-adamantanol metabolites. Ten metabolites for APINAC were identified, with the majority generated by hydroxylation, carbonylation, and carboxylation with or without glucuronidation. Therefore, in vivo metabolic profiles in rats were generated for APINAC. N-Pentylindazole-3-carboxylic acid, hydroxylated N-pentylindazole-3-carboxylic acid, and 1-adamantanol are likely the best targets to incorporate into analytical screening methods for drugs analysis. The presented mass spectra and retention time data may be useful for detection of these compounds in human urine.     

Identification and quantification of mepirapim and acetyl fentanyl in authentic human whole blood and urine samples by GC–MS/MS and LC–MS/MS

Purpose

We encountered a curious case in which two male subjects self-administered mepirapim plus acetyl fentanyl by different routes, i.e., intravenously and by inhalation. We have thus established a detailed procedure for quantification of mepirapim and acetyl fentanyl in whole blood and urine specimens using gas chromatography (GC)–tandem mass spectrometry (MS/MS).

Methods

The GC–MS/MS method was validated for linearity, extraction recovery, accuracy, and precision. Liquid chromatography–MS/MS was also used for identification of the target compounds.

Results

Good linearity and reproducibility were achieved in the range of 20–1000 ng/g for both target compounds in both matrices. The concentrations of mepirapim in heart whole blood, femoral vein whole blood, and urine of the deceased individual with administration by intravenous injection were 593, 567, and 527 ng/g, respectively; those of acetyl fentanyl were 155, 125, and 126 ng/g, respectively. The mepirapim and acetyl fentanyl concentrations in the urine specimen of the surviving individual who had administered them by inhalation were 4900 and 570 ng/g, respectively.

Conclusions

To our knowledge, with the exception of a brief mention of a mepirapim concentration in a serum sample in emergency medicine, there are no reported data on the identification and quantification of mepirapim in biological samples. Mepirapim is a new synthetic cannabinoid. The concentration profiles of unchanged mepirapim in whole blood and urine were quite different and unique. A detailed clarification of such uniqueness is under way in our laboratory.

     

Reliable determination of cyanide,thiocyanate and azide in human whole blood by GC–MS,and its application in NAGINATA–GC–MS screening

Purpose

Cyanide, its metabolite thiocyanate and azide in human biological fluids are commonly analyzed by gas chromatography–mass spectrometry (GC–MS) after derivatization with pentafluorobenzyl bromide using extractive alkylation. However, the reported methods have some drawbacks. We examined each step of these reported methods and attempted to establish a more reliable method to determine the levels of the above compounds in human whole blood. We also examined the applicability of the established method to NAGINATA–GC–MS screening.

Methods

The deproteinization method, internal standard (IS), the cause of column damage, and the effect of the addition of ascorbic acid were examined, and the best procedure was selected. The obtained data, including mass specta, retention times and calibration curves were registered to the database of NAGINATA software.

Results

The analysis of cyanide in whole blood was possible only when the blood was deproteinized with trichloroacetic acid. A high recovery of thiocyanate and azide was obtained without the deproteinization step. K¹³C¹⁵N (for cyanide) and tribromobenzene (for thiocyanate and azide) were selected as ISs. The column damage caused by the phase transfer catalyst was successfully eliminated by passing the catalyst containing solution through an ethyl benzoic sulfonic silica gel column. By these improvements, a more reliable determination method was established. All anions were rapidly identified using NAGINATA software, and the approximate concentration of each compound in whole blood was obtained at the same time.

Conclusions

Because NAGINATA–GC–MS screening can rapidly identify these poisons without using toxic compounds as reference standards, it should be useful in forensic and emergency medicine laboratories.

     

VOCs as fingerprints for the chemical profiling of hashish samples analyzed by HS-SPME/GC–MS and multivariate statistical tools

Purpose

The statistical evaluation of the chemical profile of seized hashish samples is a valuable tool to aid the estimation of the route through which the material has reached the dealers’ market.

Methods

In this study, the complete volatile organic compound (VOC) emission profiles of 48 seized hashish samples have been analyzed by means of headspace solid-phase microextraction/gas chromatography–mass spectrometry and evaluated with chemometric tools; multivariate statistical analyses, both hierarchical cluster analysis and principal component analysis (PCA) methods have been performed on the results to assess the existence of possible patterns throughout the samples.

Results

The total VOC emission profiles sharply distributed the samples in clusters based on their batches of origin; this trend was also clearly shown in the PCA plot, in which samples coming from the same seizure were grouped together. The Δ⁹-tetrahydrocannabinol (THC) content analysis did not show a relevant trend in terms of lot of origin of the samples.

Conclusions

The evaluation of the VOCs released into the headspace traced a much more complete chemical profiling of the samples, as compared to the analysis of cannabinoids only, or the THC titration. The multivariate statistical analyses were very useful to estimate the origin of the seized material.

     

Detection and quantification of psychotropic drug etaqualone in human hair using GC–MS/MS

In this study, sensitive analytical procedure for detection and quantification of etaqualone in human hair samples using gas chromatography tandem mass spectrometry (GC–MS/MS) was newly established, and applied it to authentic human samples obtained from an abuser. In this method, the hair samples were treated with hydrochloric acid and then extracted with ethyl ether. The ether layer was dried in a warm water bath, and the residue was reconstituted in ethyl acetate, followed by GC–MS/MS analysis. Multiple reaction monitoring (MRM) mode was used for data collection, and quantitative analysis was performed using internal standard method. Good linear relationship within the concentration range of 1–100 pg/mg were obtained in calibrators for the hair samples showing its correlation coefficient value was 0.9993. The lower limit of quantitation in this study was 1 pg/mg and the recovery rate examined ranged from 100.4% to 108.5%. The intra-day precision and accuracy were less than 5.0% and 5.8%, respectively. The inter-day precision and accuracy were lower than 6.4% and 4.6%, respectively. Using this established method, etaqualone could be detected in the hair sample obtained from a suspected user to be level of 65.2 pg/mg. It should be expected that the method established in this study would contribute to rapid detection and identification of psychotropic drug etaqualone among multiple fields including forensic investigation, clinical application and of course public health matters.     

Synovial fluid as an alternative specimen for quantification of drugs of abuse by GC–MS

Forensic Toxicology -     

A GC–MS method for the determination of furanylfentanyl and ocfentanil in whole blood with full validation

Purpose

Fentanyl analogues are popular in recent years among drug addicts and have been related to many overdoses and deaths worldwide. Furanylfentanyl, ocfentanil, acetylfentanyl and butyrfentanyl are among the most common of these drugs. Methods for the determination of furanylfentanyl and ocfentanil by gas chromatography–mass spectrometry (GC–MS) in biological samples do not exist, and therefore, their development would be extremely useful for routine toxicological analysis.

Methods

A GC–MS method was developed and fully validated for the determination of furanylfentanyl and ocfentanil in whole blood. This method was also suitable for the determination of acetylfentanyl and butyrfentanyl. The method included solid-phase extraction after protein precipitation using acetonitrile, and it was applied during the toxicological investigation of forensic cases. Methadone-d₃ was used as internal standard for the quantification of the analytes.

Results

The limit of detection and limit of quantification values were 0.30 and 1.0 ng/mL for furanylfentanyl and ocfentanil and 0.15 and 0.50 ng/mL for acetylfentanyl and butyrfentanyl, respectively. The calibration curves were linear (R²?≥?0.993) from 1.00 to 100 ng/mL for furanylfentanyl and ocfentanil and from 0.50 to 50.0 ng/mL for acetylfentanyl and butyrfentanyl. The recoveries were not lower than 85%, while accuracies and precisions were not greater than 6.0% (% error) and 8.0% (% relative standard deviation), respectively, for all four fentanyl analogues.

Conclusions

The developed method is the first one in the literature for the detection of furanylfentanyl and ocfentanil in biological fluids by GC–MS, and it provides very high sensitivity comparable to that by liquid chromatography–tandem mass spectrometry.

     

Development of a preparation method to produce a single sample that can be applied to both LC–MS/MS and GC–MS for the screening of postmortem specimens

Simple and efficient extraction methods have been developed for the screening of a wide array of drugs in postmortem autopsy specimens. Acidic and basic compounds were targeted with two extraction methods that can be applied to both GC–MS and LC–MS/MS instrumentation. The extractions were achieved by utilizing lipid-removal and solid-phase extraction cartridges while carefully monitoring the pH of the samples to ensure the adequate removal of interfering substances like lipids and amino acid derivatives. These methods were applied to actual autopsy cases, with 94 and 124 compounds detected by GC–MS and LC–MS/MS, respectively. The developed methods could easily be incorporated into a forensic laboratory’s daily routine for screening many different compounds from postmortem samples.     

Simple and rapid analysis of amatoxins using UPLC–MS–MS

Amatoxins, such as α-amanitin and β-amanitin, are highly toxic bicyclic octapeptides present in Amanita mushrooms. We present a simple and rapid sensitive determination of α-amanitin, β-amanitin, and phalloidin in body fluids (serum, plasma, and urine) using an ultraperformance liquid chromatography (UPLC)–tandem mass spectrometry (MS–MS) system. After extraction from serum, plasma, and urine samples by an Oasis HLB column, the three compounds were subjected to an UPLC–MS–MS system with an electrospray ionization interface. All three compounds were completely separated within 4.5 min. The calibration curves for serum, plasma, and urine samples showed good linearities in the range of 2–420 ng/ml, using virginiamycin B as internal standard. Their detection limits were as low as 0.5–1.5 ng/ml. The present method was validated; the coefficients of variation (CV) were: intraday for serum, plasma, and urine samples, less than 9.9, 13.2, and 11.5 %, respectively; interday for serum, plasma, and urine samples, less than 15.0, 10.4, and 15.3 %, respectively. The influence of matrix effects, especially in urine, was observed in human and rat samples; however, the difference in matrix effects between individuals were low. Therefore, each calibration curve was prepared for each biological specimen type. Furthermore, we succeeded in determining the compounds in rat urine samples, obtained 6 or 24 h after intraperitoneal administration. The present method is applicable in forensic and clinical toxicology because of its simplicity and rapidness with high sensitivity.     

Rapid and simultaneous extraction of acidic and basic drugs from human whole blood for reliable semi-quantitative NAGINATA drug screening by GC–MS

We present a rapid procedure for simultaneous extraction of a wide range of acidic and basic drugs from whole blood samples for reliable semi-quantitative NAGINATA drug screening by gas chromatography–mass spectrometry (GC–MS). To extract a wide range of drugs, the partition/extraction procedure used for the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) method was employed as the initial step. Various procedures were tested as the second step for the removal of whole blood impurities, including the use of primary secondary amine and C₁₈ for the dispersive solid-phase extraction of the QuEChERS method, four kinds of silica-based C₁₈ columns, alumina columns, and protein–lipid removal filter cartridges (Captiva ND Lipids). Subsequent GC–MS screening used the NAGINATA software with a constructed calibration-locking database for detection of acidic and basic drugs; drug detection ability, accuracy of tentative concentrations, and drug recoveries were examined and compared. We also examined the applicability of our established method in an actual forensic case. Our results showed that the number of drugs detectable at low concentrations was greatly increased by the use of the partition/extraction procedure of the QuEChERS method as the initial step and the protein–lipid removal filter cartridge as the second step. These combined steps provided notably clean extracts. Recoveries carefully measured with each reference standard were largely more than 60 %, and tentative concentrations obtained by the established screening method without reference standards were in the range of 48–310 % of the expected values for 65 acidic and basic drugs. Therefore, relatively reliable semi-quantitative values were obtained at the screening step without the need for each reference standard. We also experienced significant time savings for the extraction and in obtaining tentative concentrations at the screening step in an actual forensic case, indicating that the method is useful for rapid diagnosis of drug intoxication. Our proposed method should prove useful for semi-quantitative screening of a wide range of drugs and poisons in whole blood samples in clinical and forensic cases.     

Quantitative determination of taxine B in body fluids by LC–MS–MS

A new specific and sensitive LC–MS–MS method for the detection of taxine B and isotaxine B, the main toxic pseudo-alkaloids from yew (Taxus sp.), in biological samples (blood, urine, gastric content) was developed. Biological samples were prepared for LC–MS–MS by means of solid-phase extraction (SPE) procedure and yielded a recovery of 86%. Chromatographic separation was achieved using an RP₁₈ column. Detection of taxine B and isotaxine B was performed using multiple reaction monitoring with m/z 584.2 as precursor ion, i.e. [M+H]⁺, of both isomers and m/z 194.3 and m/z 107.1 as product ions after collision-induced dissociation. Docetaxel was applied as internal standard. The method was fully validated for the analysis of blood samples. Linearity was proven in the range from 0.1–500 ng/g. The limit of detection and the limit of quantitation are 0.4 and 2 ng/g, respectively. The method was applied to the determination of taxine B and isotaxine B in four fatal cases (two humans, two horses) with suspected yew intoxication. Blood levels were 105, 168, 174 and 212 ng/g.     

Application of the QuEChERS procedure for analysis of Δ<Superscript>9</Superscript>-tetrahydrocannabinol and its metabolites in authentic whole blood samples by GC–MS/MS

Purpose

Analysis of drugs and their metabolites in biofluids usually demands the application of sample preparation methods that allow for full isolation of analyzed substances from the matrix. The purpose of this study was to develop a method using the QuEChERS procedure for analysis of Δ⁹-tetrahydrocannabinol (THC), 11-hydroxy-Δ⁹-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol (11-COOH-THC).

Methods

THC, 11-OH-THC and 11-COOH-THC were quantified in whole blood samples using QuEChERS extraction and gas chromatography–tandem mass spectrometry.

Results

The described method is characterized by good linearity, very low detection limits and satisfactory inter- and intraday precisions for THC, 11-OH-THC and 11-COOH-THC. The applicability of the procedure was confirmed using authentic whole blood samples collected from 30 persons suspected of driving under the influence of drugs.

Conclusions

The application of QuEChERS extraction described herein is a simple and convenient method for the routine analysis of THC, 11-OH-THC and 11-COOH-THC in whole blood samples from living and deceased humans. To our knowledge, this paper is the first academic report describing the QuEChERS extraction of THC and its metabolites from whole blood specimens.

     

A validated method for quantitation of psilocin in plasma by LC–MS/MS and study of stability

A liquid chromatography-electrospray ionization/tandem mass spectrometry method for the quantitation of psilocin in plasma is presented. Sample workup was performed with mixed-mode solid-phase extraction using ascorbic acid and nitrogen for drying to protect the unstable analyte. Calibration curves were linear from 2 to 100?ng/mL, and no selectivity problems occurred. The limit of detection was 0.1?ng/mL, and the limit of quantitation was 0.34?ng/mL. Recovery was >86% and matrix effects were <110%. Both were reproducible. Interday and intraday precisions at different concentrations were 1.5-4.3% relative standard deviation, bias within ±9%. Processed samples were stable in the autosampler for at least 26?h. Furthermore, the stability of psilocin in blood stored at different temperatures over various periods of time was investigated. Samples stored at room temperature showed a continuous decrease of analyte leading to a loss of about 90% after 1?week. Storage in the fridge improved sample stability significantly. Freezing of blood samples led to a not reproducible loss of psilocin.     

Furanylfentanyl in whole blood measured by GC–MS/MS after QuEChERS extraction in a fatal case

Forensic Toxicology - The purpose of this work is the determination of the probable cause of a person’s death on the basis of analytical results obtained from the deceased’s blood...     

Postmortem distribution of α-pyrrolidinovalerophenone and its metabolite in body fluids and solid tissues in a fatal poisoning case measured by LC–MS–MS with the standard addition method

We experienced an autopsy case, in which the cause of death was judged as α-pyrrolidinovalerophenone (α-PVP) poisoning. Other drugs or poisons that could have caused the death were not detected by our screening using gas chromatography–mass spectrometry. The deceased was a 41-year-old man. The postmortem interval was about 40 h. Cardiac blood, femoral vein blood, urine, stomach contents, and seven solid tissues were collected and frozen until analysis to investigate the distribution of α-PVP and its metabolite 1-phenyl-2-(pyrrolidin-1-yl)pentan-1-ol (OH-α-PVP) in the body of the cadaver. After sample pretreatment, they were subjected to solid-phase extraction with Oasis HLB cartridges and analysis by liquid chromatography–tandem mass spectrometry. Because the distribution study dealt with different matrices, we used the standard addition method to overcome the matrix effects. The concentration of α-PVP in urine was much higher than in other specimens; the concentrations of α-PVP in solid tissues except for the spleen were about twofold those in blood specimens. Among the solid tissues, the highest α-PVP concentration was found in the pancreas, followed by the kidney. The extremely high concentration of the drug in urine and the relatively high concentration in the kidney suggested that α-PVP is rapidly excreted into urine via the kidney. The distribution profile of OH-α-PVP was generally similar to that of α-PVP in body fluids and solid tissues. The concentration of OH-α-PVP in urine was also much higher than those in other specimens. Among the solid tissues, the OH-α-PVP concentration was highest in the liver, followed by the kidney. The high concentration of OH-α-PVP in the liver was expected, because the metabolism of α-PVP was probably most active in the liver. The high levels of OH-α-PVP in urine and kidney also suggested that the metabolite was also rapidly excreted into urine via the kidney. To test whether conjugated metabolites were present in urine and solid tissues, urine and homogenates of the liver and spleen were incubated with β-glucuronidase/sulfatase at 37 °C for 5 h. Concentrations of OH-α-PVP were measured before and after the incubation, but differences in the concentrations before and after the incubation were within 10 % for the urine, liver, and spleen samples. To date, data on the distribution of α-PVP and OH-α-PVP in body fluids and solid tissues in a fatal α-PVP poisoning case have not been reported; thus, the findings of our study will be useful for assessment of future cases of α-PVP poisoning.     

Use of electrical impedance scanning in the differentiation of sonographically suspicious and highly suspicious lymph nodes of the head–neck region

Sonographic differentiation between inflammatory and malignant lymph nodes is difficult, due in part to almost unchanged morphology of small lymph node metastases; however, as cancer cells exhibit altered dielectric properties, measurement of local electrical field distortions may be useful as adjunct to ultrasound in detection of malignancy. In this study, we evaluated the ability of electrical impedance scanning (EIS) to differentiate cervically located sonographically suspicious or highly suspicious lymph nodes. Seventy patients with 106 sonographically suspicious lymph nodes (mean size 20 x 13 x 13 mm, mean depth 8 mm) were examined using TransScan TS2000 (Siemens, Erlangen, Germany; manufactured by TransScan Research and Development Co., Israel). Included in the study were cervical ( n=64), inframandibular/periparotideal ( n=32) and nuchal/supraclavicular ( n=10) nodes. The EIS results were compared with histopathological ( n=100) and serological ( n=6) findings. Sixty-two of 64 malignant lymph nodes were correctly detected using EIS; 19 of 42 inflammatory/benign lymph nodes were correctly identified as benign (true positive 96.9%, true negative 45.2%; accuracy 71.3%, negative predictive value 90.5%, positive predictive value 59.6%). The high tumour detection rate achieved in this study suggests that EIS may be of value as an adjunctive technique in differentiation of lymph nodes of the head-neck region. Software changes to reduce the high number of false-positive markers are, however, necessary to improve the value in the evaluation during a regular clinical routine.     

Determination of new psychoactive substances and other drugs in postmortem blood and urine by UHPLC–MS/MS: method validation and analysis of forensic samples

Forensic Toxicology - This study aimed to validate a modified QuEChERS method followed by ultra-high performance liquid chromatography–tandem mass spectrometry to determine 79 new...     

SPME–GC–MS analysis of α-pyrrolidinovaleorophenone in blood in a fatal poisoning case

We provided toxicological analytical support for a fatal case of abuse of α-pyrrolidinovaleorophenone (α-PVP). Solid-phase microextraction (SPME) and capillary gas chromatography coupled to mass spectrometry (GC–MS) was employed to quantify the drug in whole blood. The whole blood concentration of the drug in the heart was 486 ng/ml. This is the first report of α-PVP intoxication as ascertained by mass spectrometric identification of α-PVP in whole blood.