Simultaneous analysis of sildenafil, vardenafil, tadalafil, and their desalkyl metabolites in human whole blood and urine by isotope dilution LC–MS–MS

Given that there are many autopsy cases in which erectile dysfunction (ED) treatment drugs can be detected from elderly men who are diagnosed to have died of cardiovascular disorders, the degree of cardiovascular risk posed by ED treatment drugs is still controversial. Moreover, counterfeit ED drugs or illegal dietary supplements containing ED drugs are also threats to public health. In this study, we established a detailed procedure for simultaneous analysis of typical ED drugs (sildenafil, vardenafil, tadalafil) and their metabolites in human blood and urine by isotope dilution liquid chromatography-tandem mass spectrometry (LC–MS–MS). Each sample of whole blood and urine containing the three ED treatment drugs, their metabolites, and deuterated internal standards (ISs) was diluted with alkalinized water, loaded onto an Oasis HLB cartridge, washed with dilute ammonium hydroxide solution, and eluted with chloroform. The eluate was acidified with methanol and concentrated HCl and evaporated to dryness. The resulting residue was reconstituted with methanol and mobile phase solution, and 5 μl of the solution was injected into an LC–MS–MS instrument. The determinations were made by multiple reaction monitoring using each product ion. The recovery rates of the drugs, metabolites, and ISs from whole blood and urine ranged from 80.7 to 127%. Good linearity was obtained for all drugs and their metabolites in the range of 1–100 ng/ml in whole blood and urine with correlation coefficients greater than 0.99. The detection limits (signal-to-noise ratio = 3) for all compounds were not higher than 0.05 ng/ml. Intraday and interday accuracy and precision data were also satisfactory for all compounds in whole blood and urine. Actual measurements of sildenafil and N-desmethyl sildenafil were also demonstrated by analysis of whole blood and urine specimens from two male volunteers after ingestion of a 25-mg tablet of sildenafil.     

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.

     

Sensitive quantification of 5F-NNEI and characterization of its several metabolites in authentic urine and/or serum specimens obtained from three individuals by LC–QTRAP-MS/MS and high-resolution LC–Orbitrap-MS/MS

Forensic Toxicology - A synthetic cannabinoid 5F-NNEI and its metabolites in authentic human specimens have not been reported yet. The aim of this study is, firstly, to establish a sensitive...     

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.     

LC–MS assay for quantitative determination of cardio glycoside in human blood samples

A method is described for liquid chromatography-mass spectrometry analysis of the cardio glycosides digoxin and digitoxin in biological samples. The method was optimized for use in the forensic field and, therefore, comprises the determination from whole blood and tissue samples. Sample cleanup by solid phase extraction (SPE) on a functionalized polymeric phase was sufficient to limit matrix suppression to <10% for all analytes. Chromatographic separation was achieved using an RP-8 column. Detection of the cardio glycosides was performed with electrospray ionization in the positive mode. The system was run in single ion monitoring mode, measuring the sodium adducts (M + Na)+ of the analyte and of the internal standard, respectively. The method was fully validated for the analysis of blood samples and was also successfully applied in forensic cases. The method was accurate and precise over a linear concentration range up to 50 ng/g blood. Lower limit of quantitation was 0.2 ng/g for digoxin and 2 ng/g for digitoxin, respectively. As deuterated analyte was used as internal standard, we also present a new microwave-enhanced method for the fast preparation of the labelled analyte within 20 min.     

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.

     

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...     

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...     

Sensitive quantification of BB-22 and its metabolite BB-22 3-carboxyindole,and characterization of new metabolites in authentic urine and/or serum specimens obtained from three individuals by LC–QTRAP-MS/MS and high-resolution LC–Orbitrap-MS/MS

Forensic Toxicology - A synthetic cannabinoid BB-22 and its metabolite BB-22 3-carboxyindole have not yet been quantified&nbsp;in human urine. The aim of this study is to establish a sensitive...     

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...     

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.     

Detection of metabolites of two synthetic cannabimimetics,MDMB-FUBINACA and ADB-FUBINACA,in authentic human urine specimens by accurate mass LC–MS: a comparison of intersecting metabolic patterns

The structures of two indazole-derived synthetic cannabimimetics, a methyl ester, MDMB-FUBINACA, and an amide, ADB-FUBINACA, differ only in the terminal groups on the side chains. Based upon liquid chromatography–quadrupole time-of-flight-mass spectrometry analysis of urine and blood samples collected from patients who were admitted to hospital with suspected drug intoxications and from postmortem forensic investigations, 38 metabolites were tentatively identified. Hydrolysis of the terminal groups (methyl ester and amide for MDMB-FUBINACA and ADB-FUBINACA, respectively) was found to be a common metabolic pathway for both compounds, leading to the formation of other common metabolites. Hydrolysed metabolites undergo subsequent monohydroxylation, dihydrodiol formation, fluorobenzyl loss and dehydrogenation. Most of the metabolites of MDMB-FUBINACA were products of ester hydrolysis. Metabolites formed by hydrolysis, additional monohydroxylation, dihydrodiol formation and fluorobenzyl loss were also detected in forms of glucuronides. Two unhydrolysed metabolites were identified as products of hydroxylation and fluorobenzyl loss with subsequent glucuronidation. In the case of ADB-FUBINACA, products of mono- and dihydroxylation, dihydrodiol formation, dihydrodiol formation combined with monohydroxylation, monohydroxylation combined with dehydrogenation and fluorobenzyl loss with monohydroxylation were identified. Monohydroxylated and dihydrodiol metabolites were also detected in the form of glucuronides. The most abundant metabolites were products of ester hydrolysis in free and glucuronidated forms (for MDMB-FUBINACA) and of dihydrodiol formation (for ADB-FUBINACA). These compounds are recommended for toxicological screening. To our knowledge, there are no reports dealing with the detection of metabolites of MBDB-FUBINACA and ADB-FUBINACA in authentic human urine specimens.     

In vitro and in vivo human metabolism of a synthetic cannabinoid EAM-2201 detected by LC–quadrupole-ion trap-MS/MS and high-resolution LC–Orbitrap-MS/MS

Forensic Toxicology - The aim of this study is to characterize the metabolites of EAM-2201 in human hepatocytes obtained in vitro and those in liver and urine specimens obtained in vivo from the...     

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.

     

Quantification of seven novel synthetic opioids in blood using LC–MS/MS

Forensic Toxicology - The objective of this study was to develop, optimize, and validate a method for the simultaneous quantification of U-47700, AH-7921, U-49900, U-50488, MT-45, W-18, and W-15 in...     

A fatal case involved in pyrethroid insecticide ingestion: quantification of tetramethrin and resmethrin in body fluids of a deceased by LC–MS/MS

Forensic Toxicology - The quantification of parent molecules of pyrethroids tetramethrin and resmethrin in human specimens by a mass spectrometry (MS) technique has not been reported yet. A woman...     

Simultaneous quantification by LC/ESI–MS/MS of chlorinated tyrosine derivatives in the autopsy sample of a victim of chlorine exposure

Direct detection and accurate quantification of chlorine in autopsy samples are difficult because of the volatility and rapid metabolism of chlorine. Here, we developed and validated a method for quantitative analysis of 3-chloro-l-tyrosine (Cl-Tyr) and 3,5-dichloro-l-tyrosine (DiCl-Tyr) as stable markers of chlorine exposure. Chemical derivatization followed by liquid chromatography coupled with electrospray ionization–tandem mass spectrometry (LC/ESI–MS/MS) enabled us to simultaneously analyze both Cl-Tyr and DiCl-Tyr in an autopsy sample from the victim of chlorine exposure. Cl-Tyr was detected in the heart blood (53.6 ng/mL), urine (9.5 ng/mL), and lung tissue (211.1 ng/g); however, DiCl-Tyr was detected only in the lung tissue (10.3 ng/g). In contrast, in autopsy samples obtained from cases without exposure to chlorine, DiCl-Tyr was not detected in any matrixes. Our result suggested that the simultaneous detection of Cl-Tyr and DiCl-Ty may provide a better appreciation of chlorine exposure. To our knowledge, this is the first time Cl-Tyr and DiCl-Tyr have been determined simultaneously in a real human autopsy sample from a victim of chlorine exposure.     

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.     

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.