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.     

Mass spectrometric differentiation of the isomers of mono-methoxyethylamphetamines and mono-methoxydimethylamphetamines by GC–EI–MS–MS

Mass spectrometric differentiation of the six isomers of mono-methoxyethylamphetamines (MeO-EAs) and mono-methoxydimethylamphetamines (MeO-DMAs) by gas chromatography–electron ionization–tandem mass spectrometry (GC–EI–MS–MS) was investigated. Based on their EI-mass spectra, the fragment ions at m/z 121 and 72 were selected as precursor ions for their regioisomeric and structurally isomeric differentiation, respectively. Collision-induced dissociation provides intensity differences in product ions among the isomers, enabling mass spectrometric differentiation of the isomers. Furthermore, high reproducibility of the product ion spectra at the optimized collision energy was confirmed, demonstrating the reliability of the method. To our knowledge, this is the first report on mass spectrometric differentiation of the six isomers of MeO-EAs and MeO-DMAs by GC–EI–MS–MS. Isomeric differentiation by GC–EI–MS–MS has a high potential to discriminate isomers of newly encountered designer drugs, making GC–MS–MS a powerful tool in the forensic toxicology field.     

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.

     

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...     

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...     

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.

     

Strong evidence of drug-facilitated crimes by hair analysis using LC–MS/MS after micro-segmentation

Forensic Toxicology - Hair analysis can provide effective information to prove drug-facilitated crimes (DFCs). Herein, an analytical procedure for obtaining evidence of DFCs stronger than with...     

An LC–MS–MS method for quantitation of four new phenethylamines (BOX series) in plasma: in vivo application

The appearance of new “designer drugs” in the illicit market poses a serious health risk because they have unknown safety profiles, have a high potential for abuse, high potency, and can lead to devastating health consequences. For this reason, it is desirable to develop validated and reliable analytical screening tests that allow detection of amphetamines and related designer drugs in biological samples. We report a method for separation and quantitation of four new phenethylamines, 4-bromo-2,5-beta-trimethoxyphenethylamine (BOB), 4-methyl-2,5-beta-trimethoxyphenethylamine (BOD), 3,4-methylenedioxy-beta-methoxyphenethylamine (BOH), and 4-methyl-2,5-dimethoxy-beta-hydroxyphenethylamine (BOHD), in plasma. Quantitation was achieved via liquid chromatography–tandem mass spectrometry (LC–MS–MS) in the multiple reaction monitoring mode, using 2,3-dimethoxyphenethylamine-d ₃ as internal standard. The method was validated according to international guidelines. The parameters determined were selectivity, sensitivity, matrix effect, linearity, precision, recovery, and stability. All parameters were satisfactory. To remove matrix interference, solid-phase extraction was introduced in the method as clean-up step. The same method was applied in a pharmacokinetic study to monitor the target compounds in rat plasma after a single oral administration. The developed and validated LC–MS–MS method is the first available for quantitation of BOB, BOH, BOD, and BOHD in a biological matrix. This method is recommended for use in forensic and clinical toxicology, because of its sensitivity, selectivity, and simplicity. An important extension of this method could involve its application to other complex matrices.     

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.     

An LC–MS–MS method for quantitative analysis of six trimethoxyamphetamine designer drugs in rat plasma, and its application to a pharmacokinetic study

Trimethoxyamphetamines (TMAs) comprise a family of hallucinogenic drugs that includes various different positional isomers, which are important both for their hallucinogenic activity and their circulation in illicit drug markets. This report describes a method for identification and quantitation of six TMA isomers in rat plasma by solid-phase extraction and liquid chromatography–tandem mass spectrometry (LC–MS–MS) with electrospray ionization. Mescaline-d ₁ was used as internal standard. Multiple reaction monitoring on a triple quadrupole mass spectrometer operating in the positive ion mode was used for detection. The chromatographic system used a Varian Polaris C18-A column (2.0 × 100 mm i.d., 3 μm) and gradient elution with acetonitrile and 0.1 % formic acid in water. The calibration curves were linear over the concentration range from 10 to 200 ng/ml for all drugs with correlation coefficients that exceeded 0.998. The limits of detection and quantitation ranged from 1.1 to 2.3 ng/ml and from 6.9 to 10.2 ng/ml, respectively. The validation data, such as precision, accuracy, and recovery, showed good reproducibility and selectivity. This method was successfully applied to evaluating the pharmacokinetic profiles of TMAs in rats.     

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.     

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.     

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...     

Development for the measurement of serum thiosulfate using LC–MS/MS in forensic diagnosis of H2S poisoning

Thiosulfate measurement is crucial to diagnosis of hydrogen sulfide (H₂S) poisoning in forensic toxicology. Although GC–MS method is currently regarded as a standard thiosulfate measurement, it requires complicated sample preparation prior to analysis. This study presents a simple, rapid, and highly sensitive method for the quantitative analysis of serum thiosulfate by using liquid chromatography–tandem mass spectrometry (LC–MS/MS). This method is based on selected reaction monitoring and has high sensitivity with a lower quantification limit of 0.5 μM. Precision and accuracy of this method meet the basic requirements for quantitative analysis (intra- and inter-day tests have a relative standard deviation of ⩽10.4%; range of analytical recovery is 94.3–102.6%). On the measurements of serum thiosulfate by our developed method, a thiosulfate concentration as 57.5 μM was detected clearly in the H₂S poisoning case comparing to the non poisoning case in which only a trace amount of thiosulfate was observed.     

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.     

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.     

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.     

Preventing misidentification of 25I-NBOH as 2C-I on routine GC–MS analyses

25I-NBOH is a novel psychoactive substance (NPS) recently reported to have been found on blotter paper samples seized on the streets of Brazil, and used as a replacement for the NBOMes now scheduled in many countries. The presence of this NPS on the street market may go undetected, because the most widely and routinely utilised analytical technique for drug sample analyses is gas chromatography–mass spectrometry (GC–MS), which can misidentify 25I-NBOH (and indeed the other members of the NBOH series), because of its degradation into 2C-I (and corresponding 2C for the other members of the series) within the injector, unless a derivatization procedure is employed, which is often non-standard. While direct detection of 25I-NBOH under routine GC–MS conditions is still achieved, a slight adjustment in the standard GC–MS method, including shortening of the solvent delay window, was found to enable the detection of an additional peak due to 25I-NBOH degradation. Consequently, the presence of this secondary early chromatographic peak allowed for the distinction between 25I-NBOH and 2C-I using routine GC–MS without resorting to derivatization (or other analytical processes), thus preventing misidentification of 25I-NBOH as 2C-I.     

Development and validation of a sensitive LC–MS/MS method to analyze NBOMes in dried blood spots: evaluation of long-term stability

Purpose

We evaluated the use of dried blood spots (DBS) to determine seven NBOMes by liquid chromatography–tandem mass spectrometry (LC–MS/MS), and also evaluated the stability of these compounds in this dried matrix.

Methods

An LC–MS/MS method was developed and fully validated to quantify seven NBOMes (25C-, 25H-, 25I-, 25B-, 25G-, 25D- and 25E-NBOMe) in DBS samples. The extraction procedure was optimized using mixture design experiment. Stability study was performed in two different concentrations over 180 days at three different storage temperatures.

Results

Good linearity, and limits of detection and quantitation of 0.05 and 0.1 ng/mL, respectively, were obtained. The interday imprecision (n = 15) and bias (n = 15) were not higher than 11.4 and 10.3%, respectively, and no carryover was observed. All analytes remained stable in DBS at ? 20, 4 °C and even at room temperatures for 180 days, except 25B-NBOMe and 25I-NBOMe which experienced degradation (22 and 21%, respectively) of the initial concentration at room temperature after 180 days of study. The method was applied to a DBS of an authentic postmortem blood from an NBOMe user, and it was found to be reliable with good selectivity and specificity.

Conclusions

DBS has been found to allow reliable, sensitive, accurate and robust detection and quantification of seven NBOMes via LC–MS/MS. Also, DBS provided great stability to most of the compounds at room temperature, and no degradation was observed for DBS kept at 4 and ? 20 °C. This is the first trial to analyze NBOMes in DBS samples to our knowledge.