Andreas H. Ewald

Towards a universal LC–MS screening procedure – can an LIT LC–MSn screening approach and reference library be used on a quadrupole‐LIT hybrid instrument?

In contrast to libraries with highly reproducible gas chromatography electron ionization mass spectra, current liquid chromatography (LC-MS) libraries are limited to specific instrument types. Therefore, the aim of the study was to prove whether a recently developed linear ion trap (LIT) LC-MS(n) screening approach and reference library can be transferred to an LC-MS/MS system with a quadrupole-LIT hybrid mass analyzer using SmileMS, a sophisticated search algorithm. The LIT reference library was built with MS² and MS³ wideband spectra recorded on a ThermoFisher LXQ LIT with electrospray ionization in positive mode and full-scan data-dependent acquisition (DDA). Collision parameter optimizations, including different scan types and energies, were performed on an Applied Biosystems QTRAP 4000 system using electrospray ionization in positive mode and full-scan DDA. Modified library sets were generated to improve the detection of a compound by the used search algorithm. Additionally, 100 authentic human urine samples were screened by both systems for proof of applicability. In the applicability study, 533 compounds were detected by the LXQ and 477 by the QTRAP system using enhanced product ion scan and a modified database. The presented data showed that the LIT screening approach and reference library could be used successfully on a QTRAP instrument with some limitations. These should be overcome by further optimizations regarding DDA settings for better sensitivity and further library modifications to reduce spectra mismatches.

Metabolism and toxicological detection of the designer drug 4-chloro-2,5-dimethoxyamphetamine in rat urine using gas chromatography-mass spectrometry

Studies are described on the metabolism and the toxicological analysis of the amphetamine-derived designer drug 4-chloro-2,5-dimethoxyamphetamine (DOC) in rat urine using gas chromatographic-mass spectrometric techniques. The metabolites identified indicated that DOC was metabolized by O-demethylation at position 2 or 5 of the phenyl ring partly followed by glucuronidation and/or sulfation. The authors’ systematic toxicological analysis procedure using full-scan gas chromatography-mass spectrometry after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of an intake of a dose of DOC in rat urine that corresponds to a common drug user’s dose. Assuming similar metabolism, the STA procedure described should be suitable as proof of an intake of DOC in human urine.

2,5-Dimethoxyamphetamine-derived designer drugs: studies on the identification of cytochrome P450 (CYP) isoenzymes involved in formation of their main metabolites and on their capability to inhibit CYP2D6.

The designer drugs 4-methyl-2,5-dimethoxy-amphetamine (DOM), 4-iodo-2,5-dimethoxy-amphetamine (DOI), 4-chloro-2,5-dimethoxy-amphetamine (DOC), 4-bromo-2,5-dimethoxy-amphetamine (DOB), 4-bromo-2,5-dimethoxy-methamphetamine (MDOB), and 2,4,5-trimethoxy-amphetamine (TMA-2) are potent serotonin 5HT(2) receptor agonists and have appeared on the illicit drug market. These drugs are mainly metabolized by O-demethylation or in case of DOM by hydroxylation of the methyl moiety. In an initial activity screening using microsomes of insect cells heterologously expressing human CYPs, CYP2D6 was found to be the only CYP isoenzyme involved in the above-mentioned main metabolic steps whereas the amounts of metabolites formed were very small. As inhibition of CYP2D6 by other amphetamines had been described, the inhibitory effects of the 2,5-dimethoxyamphetamine derivatives were studied using insect cell microsomes with heterologously expressed human CYP2D6 and pooled human liver microsomes (HLM) as enzyme sources and dextromethorphan O-demethylation as probe reaction. All studied drugs were observed to be non-mechanism-based competitive inhibitors of CYP2D6 with inhibition constants (K(i)) from 7.1 to 296microM using recombinant CYP2D6 and 2.7-19.9microM using HLM. For comparison, the K(i) values for quinidine and fluoxetine were 0.0092 and 8.2microM using recombinant CYP2D6 and 0.019 and 0.93microM using HLM. As the K(i) values of the drugs were much higher than that of quinidine and, with the exception of DOI, higher than that of fluoxetine, interactions with other CYP2D6 substrates are possible but rather unlikely.

Detection and quantification of benzodiazepines and Z-drugs in human whole blood, plasma, and serum samples as part of a comprehensive multi-analyte LC-MS/MS approach

For the first time, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) multi-analyte approach based on a simple liquid–liquid extraction was developed and validated for fast target screening and quantification of benzodiazepines and Z-drugs in case of driving ability and crime responsibility in the three most important biosamples whole blood, plasma, and serum. Whole blood, plasma, and serum (500xa0μL each) were extracted twice at pH 7.4 and at pH 10 with ether/ethyl acetate (1:1). Separation, detection, and quantification were performed using LC-MS/MS with electrospray ionization in positive mode. The method was validated with respect to selectivity, ion suppression/enhancement of co-eluting analytes, matrix effects, recovery, process efficiency, accuracy and precision, stabilities, and limits of detection and quantification. For accuracy and precision, full calibration was performed with ranges from subtherapeutic to toxic concentrations. The presented LC-MS/MS approach as part of a universal multi-analyte concept for over 100 drugs was applicable for selective detection as well as accurate and precise quantification in whole blood, plasma, and serum. The approach was selective, sensitive, accurate, and precise for 16 of the 19 tested drugs in whole blood, 18 in plasma, and 17 in serum. Only semiquantitative results could be obtained for zopiclone because of its instability in all tested biosamples.

A simple extraction and LC-MS/MS approach for the screening and identification of over 100 analytes in eight different matrices.

In the present study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) multi-analyte approach using one single work-up approach in whole blood, plasma, serum, post-mortem blood, liver tissue, gastric content, hair, and urine was developed for fast target screening and reliable identification of 130 analytes often requested in clinical and forensic toxicology. Samples (500 μL each) of whole blood, plasma, serum, post-mortem blood, tissue (homogenized 1u2009+u20094 with water), as well as 3 g of distilled gastric contents, 1 mL of urine, or 20 mg of pulverized hair were extracted at different pH values with an diethyl ether-ethyl acetate mixture (1:1). Separation and identification were performed using LC-QTRAP with electrospray ionization in positive mode. For identification 1 scheduled multi-reaction-mode (sMRM) method with 390 transitions was developed covering benzodiazepines, Z-drugs, antidepressants, neuroleptics, opioids, new synthetic drugs, and phosphodiesterase type 5 inhibitors. For positive sMRM transitions with intensities exceeding 5000 cps, dependent scans (EPI scan collision energy, 35 eV, collision energy spread, 15 eV) were performed for library search using our in-house library. The method was developed with respect to selectivity, matrix effects, recovery, process efficiency, limit of detection, and applicability. The simple work-up procedure was suitable for all biosamples with exception of urine in respect to low concentrated analytes, which showed median recovery values of 59%. The method was selective for 130 analytes in all 8 biosamples. For 106 analytes, the limit of detection in whole blood, plasma, and serum was lower than the lowest therapeutic concentration listed in blood level lists.

Quantification of 33 antidepressants by LC-MS/MS—comparative validation in whole blood, plasma, and serum

In the present study, a liquid chromatography–mass spectrometry (LC-MS/MS) multi-analyte approach based on a simple liquid–liquid extraction was developed for fast target screening and quantification of 33 antidepressants in whole blood, plasma, and serum. The method was validated with respect to selectivity, matrix effects, recovery, process efficiency, accuracy and precision, stabilities, and limits. In addition, cross-calibration between the three biosamples was done to assess the impact of the different matrices on the calibration. Whole blood, plasma, and serum (500xa0μL each) were extracted twice at pH 7.4 and at pH 10 with ether–ethyl acetate (1:1). Separation, detection, and quantification were performed using LC-MS/MS with electrospray ionization in positive mode. For accuracy and precision, full calibration was performed with ranges from subtherapeutic to toxic concentrations. The approach was sensitive and selective for 33 analytes in whole blood and 31 analytes in plasma and serum and accurate and precise for 30 of the 33 tested drugs in whole blood, 31 in plasma, and 28 in serum. Cross-calibration was successful only for 13 analytes in whole blood and 16 analytes in serum calculated over a calibration curve made in plasma, 12 analytes in whole blood and 15 analytes in plasma calculated over a calibration curve made in serum, and 10 analytes in plasma and 15 analytes in serum calculated over a calibration curve made in whole blood.

Pharmacokinetics of (synthetic) cannabinoids in pigs and their relevance for clinical and forensic toxicology

Synthetic cannabinoids (SCs) are gaining increasing importance in clinical and forensic toxicology. They are consumed without any preclinical safety studies. Thus, controlled human pharmacokinetic (PK) studies are not allowed, although being relevant for interpretation of analytical results in cases of misuse or poisoning. As alternative, in a controlled animal experiment, six pigs per drug received a single intravenous dose of 200μg/kg BW each of Δ(9)-tetrahydrocannabinol (THC), 4-ethylnaphthalen-1-yl-(1-pentylindol-3-yl)methanone (JWH-210), or 2-(4-methoxyphenyl)-1-(1-pentyl-indol-3-yl)methanone (RCS-4). In addition, six pigs received a combination of the three drugs with the identical dose each. The drugs were determined in serum using LC-MS/MS. A population (pop) PK analysis revealed that a three-compartment model described best the PK data of all three cannabinoids. Central volumes of distribution were estimated at 0.29L/kg, 0.20L/kg, and 0.67L/kg for THC, JWH-210, and RCS-4, respectively. Clearances were 0.042L/min/kg, 0.048L/min/kg, and 0.093L/min/kg for THC, JWH-210, and RCS-4, respectively. The popPK THC pig model was upscaled to humans using allometric techniques. Comparison with published human data revealed that the concentration-time profiles could successfully be predicted. These findings indicate that pigs in conjunction with PK modeling technique may serve as a tool for prediction of human PK of SCs.

Simultaneous LC-MS/MS determination of JWH-210, RCS-4, ∆9-tetrahydrocannabinol, and their main metabolites in pig and human serum, whole blood, and urine for comparing pharmacokinetic data

A series of new synthetic cannabinoids (SC) has been consumed without any toxicological testing. For example, pharmacokinetic data have to be collected from forensic toxicological case work and/or animal studies. To develop a corresponding model for assessing such data, samples of controlled pig studies with two selected SC (JWH-210, RCS-4) and, as reference, ∆9-tetrahydrocannabinol (THC) should be analyzed as well as those of human cases. Therefore, a method for determination of JWH-210, RCS-4, THC, and their main metabolites in pig and human serum, whole blood, and urine samples is presented. Specimens were analyzed by liquid-chromatography tandem mass spectrometry and multiple-reaction monitoring with three transitions per compound. Full validation was carried out for the pig specimens and cross-validation for the human specimens concerning precision and bias. For the pig studies, the limits of detection were between 0.05 and 0.50xa0ng/mL in serum and whole blood and between 0.05 and 1.0xa0ng/mL in urine, the lower limits of quantification between 0.25 and 1.0xa0ng/mL in serum and 0.50 and 2.0xa0ng/mL in whole blood and urine, and the intra- and interday precision values lower than 15xa0% and bias values within ±15xa0%. The applicability was tested with samples taken from a pharmacokinetic pilot study with pigs following intravenous administration of a mixture of 200xa0μg/kg body mass dose each of JWH-210, RCS-4, and THC. The cross-validation data for human serum, whole blood, and urine showed that this approach should also be suitable for human specimens, e.g., of clinical or forensic cases.

Development and validation of a multi-analyte LC-MS/MS approach for quantification of neuroleptics in whole blood, plasma, and serum.

Based on a similar approach for quantification of antidepressants, benzodiazepines, and z-drugs, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) multi-analyte approach with simple liquid-liquid extraction was extended for fast target screening and quantification of neuroleptics in whole blood, plasma, and serum. As this method is part of a multi-analyte procedure for over 100 analytes from different drug classes and as the extracts were additionally used in the authors laboratory for gas chromatography-mass spectrometry (GC-MS) analysis, one universal stable-isotope-labelled internal standard (SIL-IS) was used to save time and resource. The method was validated with respect to international guidelines. For accuracy and precision, full calibration was performed with ranges from subtherapeutic to toxic concentrations. Selectivity problems could not be observed, but matrix effects ranged from 68 to 211% in all samples. For the low quality control (QC), recovery ranged from 32 to 112%, process efficiency from 31 to 165% and for the high QC recovery from 42 to 141%, process efficiency from 29 to 154%. In addition statistical data evaluation of the variances of the recovery, matrix effects, and process efficiency data between whole blood vs. plasma, whole blood vs. serum, and plasma vs. serum were done. The presented LC-MS/MS approach was applicable for selective detection of 33 neuroleptics as well as accurate and precise quantification of 25 neuroleptics in whole blood, 19 in plasma, and 17 in serum. More significant matrix effects (ME) for neuropletic drugs overall in plasma and serum as compared with whole blood were detected. Copyright

Can JWH‐210 and JWH‐122 be detected in adipose tissue four weeks after single oral drug administration to rats?

Synthetic cannabinoids such as alkylindole derivatives entered the illicit drug market worldwide a few years ago. Only a few data are available concerning their pharmacokinetics, in particular their distribution and persistence in adipose tissue. For the present study, rats were administered a single 20 mg/kg oral dose of JWH-210 or JWH-122. After one month, they were dissected and adipose tissue was taken in order to study whether JWH-210 and JWH-122 persisted in this body sample. After extraction, the samples were analyzed by liquid chromatographic-mass spectrometry (LC-QTrap-MS). Validation of the analytical method for adipose tissue is also presented. The results of the matrix effects determination ranged between 30.6 and 43.8%. The limits of detection for JWH-210 and JWH-122 were 0.8 and 1.0 ng/g and lower limits of quantification were 3.7 and 2.1 ng/g, respectively. Calibration curves ranged from 10 to 75 ng/g for JWH-210 and from 5 to 50 ng/g for JWH-122. Intra- and interday precision values were lower than 15% and bias values within ±15%. Applying this method, in adipose tissue specimens obtained 4 weeks after single drug administration, JWH-210 and JWH-122 were detected in concentrations of 116 and 9 ng/g, respectively.