Lambert K. Sørensen

Determination of cathinones and related ephedrines in forensic whole-blood samples by liquid-chromatography–electrospray tandem mass spectrometry

A liquid-chromatography-tandem-mass-spectrometry method using pneumatically assisted electrospray ionisation (LC-ESI-MS/MS) was developed for the simultaneous determination of cathinone, methcathinone, ethcathinone, amfepramone, mephedrone, flephedrone, methedrone, methylone, butylone, cathine, norephedrine, ephedrine, pseudoephedrine, methylephedrine and methylpseudoephedrine in human live and post-mortem whole blood. The blood proteins were precipitated by the addition of methanol, and the extract was purified by ultrafiltration. The separation of diastereomeric ephedrines was achieved on an ethyl-linked phenyl column. Matrix-matched calibrants combined with the isotope dilution of selected substances were used for quantitative analysis. The relative intra-laboratory reproducibility standard deviations were generally better than 7% at concentrations of 20 μg/L, and the mean true recoveries were 87-106% in the concentration range of 10-250 μg/L. The detection limits were in the range of 0.5-3 μg/L. The cathinones were unstable in whole blood and sample extracts under neutral conditions, but the stability could be improved by the acidification of the sample matrix.

Determination of acidic and neutral therapeutic drugs in human blood by liquid chromatography–electrospray tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry method using pneumatically assisted electrospray ionisation (LC-ESI-MS/MS) was developed for the simultaneous determination of paracetamol, naproxen, ibuprofen, etodolac, diclofenac, salicylic acid, lamotrigine, carbamazepine, 10-OH-carbazepine, phenytoin, phenobarbital, barbital, cyclobarbital, pentobarbital, amobarbital and caffeine in human live and post-mortem whole blood. The blood proteins were precipitated in a mixture of methanol and acetonitrile, and the extract was purified by ultrafiltration. The separation was performed on an ether-linked phenyl column with polar endcapping, and both negative and positive ESI were applied. Matrix-matched standards were used for calibration. The relative intra-laboratory reproducibility standard deviations were, in general, better than 8% at concentrations in the therapeutic range. The mean true recoveries were in the range 92-101% for the live blood and 84-101% for the post-mortem blood.

A hydrophilic interaction liquid chromatography electrospray tandem mass spectrometry method for the simultaneous determination of γ-hydroxybutyrate and its precursors in forensic whole blood.

A liquid-chromatography-tandem-mass-spectrometry method using pneumatically assisted electrospray ionisation (LC-ESI-MS/MS) was developed for the simultaneous determination of γ-hydroxybutyric acid (GHB), γ-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) in human ante-mortem and post-mortem whole blood. The blood proteins were precipitated using a mixture of methanol and acetonitrile, and the extract was cleaned-up by passage through a polymeric strong cation exchange sorbent. Separation of the analytes and their structural isomers was obtained using a column with a zwitterionic stationary phase. Matrix-matched calibrants, combined with isotope dilution, were used for quantitative analysis. GHB was determined in both positive and negative ion modes. The relative intra-laboratory reproducibility standard deviations were better than 10% and 6% for blood samples at concentrations of 2 mg/L and 20-150 mg/L, respectively. The mean true extraction recoveries were 80% for GHB and greater than 90% for GBL and 1,4-BD at concentration levels of 20-50 mg/L. The limits of detection were approximately 0.5 mg/L for GHB and GBL, and 0.02 mg/L for 1,4-BD in ante-mortem blood. The corresponding lower limits of quantification were less than 1 mg/L for GHB and GBL, and less than 0.1 mg/L for 1,4-BD. GBL was unstable in whole blood freshly preserved with a sodium fluoride oxalate mixture, but the stability could be improved significantly by preservation with a sodium fluoride citrate EDTA mixture.

Determination of metformin and other biguanides in forensic whole blood samples by hydrophilic interaction liquid chromatography–electrospray tandem mass spectrometry

Metformin is an anti-diabetic drug in the biguanide class which also includes phenformin and buformin. Because of the potential adverse effects of the biguanides, a reliable liquid chromatography-tandem mass spectrometry method using pneumatically assisted electrospray ionization was developed for the quantification of the drugs in both live and post-mortem human whole blood. The blood proteins were precipitated by the addition of a mixture of methanol and acetonitrile, and the extract was cleaned up by cation-exchange solid-phase extraction to eliminate ion suppression effects. The separation was performed by hydrophilic interaction liquid chromatography. Matrix-matched calibrants combined with isotope dilution of metformin were used for calibration. The detection limits were 0.01 mg/L for metformin and phenformin and the relative intra-laboratory reproducibility standard deviations were less than 6% at concentrations of 1-10 mg/L. The mean true recoveries were greater than 86%.

Simultaneous determination of β-hydroxybutyrate and β-hydroxy-β-methylbutyrate in human whole blood using hydrophilic interaction liquid chromatography electrospray tandem mass spectrometry.

OBJECTIVES For the quantification of β-hydroxybutyrate (BHB) and β-hydroxy-β-methylbutyrate (HMB) in human whole blood, a method using hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) was developed, which does not require chemical modification of the analytes. DESIGN AND METHODS Samples were deproteinised by a mixture of methanol and acetonitrile, and the extracts were cleaned-up using both polymeric strong cation exchange and strong anion exchange sorbents. The analytes and their structural isomers were separated using a column with a zwitterionic stationary phase. Isotope dilution of both analytes was used for quantitative analysis. RESULTS Separation of BHB from isobaric interferences was achieved through chromatography. The relative intra-laboratory reproducibility standard deviations were better than 10% for blood samples at concentration levels of 10-20μM BHB and 1μM HMB and better than 5% at concentration levels 10 times higher. The mean true extraction recoveries were close to 100%. The trueness expressed as the relative bias of test results was within ±5% at concentration levels of 10-1000μM BHB and 1-20μM HMB. The lower limits of quantification were estimated to be 3μM for BHB and 0.4μM for HMB. CONCLUSIONS A simple and highly sensitive and selective HILIC-MS/MS method was developed that is suitable for the quantification of BHB and HMB in whole blood.

A high-throughput multi-class liquid chromatography tandem mass spectrometry method for quantitative determination of licit and illicit drugs in whole blood

An ultra-high-performance liquid chromatography tandem mass spectrometry confirmatory method using pneumatically assisted electrospray ionisation (UPLC-ESI-MS/MS) was developed for the simultaneous quantification of licit and illicit drugs in whole blood across multiple drug classes. The method was validated on 51 frequently detected drugs and metabolites in human ante-mortem and post-mortem whole blood. The generic sample treatment procedure was based on deproteinisation by a mixture of methanol and acetonitrile followed by ultrafiltration of the extract. Matrix-matched calibrants combined with isotope dilution of almost all substances were used for quantitative analysis. A high sample throughput was obtained by using a 96-well format. The total instrumental run-time between injections was 16 min. The relative intra-laboratory reproducibility standard deviations were generally below 8% and 15% at concentrations of 250 μg L−1 and 5 μg L−1, respectively. The mean true recoveries were in most cases greater than 90% for ante-mortem samples and greater than 80% for post-mortem samples. For most substances, the limits of detection and quantification were less than 1 μg L−1 and 3 μg L−1, respectively.

Simultaneous determination of propofol and its glucuronide in whole blood by liquid chromatography-electrospray tandem mass spectrometry and the influence of sample storage conditions on the reliability of the test results.

Propofol (2,6-diisopropylphenol) is commonly used as an anaesthetic agent but is also abused for recreational purposes. Several cases of fatalities involving self-administered propofol have been reported. For rapid quantification of propofol and propofol β-d-glucuronide (propofol G) in clinical and forensic cases, an ultra-performance liquid chromatography-tandem mass spectrometry method using pneumatically assisted electrospray ionisation has been developed. The technique has been validated on both ante-mortem and post-mortem human whole blood. The proteins in the blood samples were removed by the addition of a mixture of methanol and acetonitrile, and the extract was cleaned up by solid phase extraction. The extract was concentrated in dimethyl sulphoxide. The system was calibrated using matrix-matched calibrants combined with isotope dilution. The lower limits of quantification were 0.01 and 0.02mg/L for propofol and 0.02 and 0.04mg/L for propofol G in ante-mortem and post-mortem whole blood, respectively. The relative intra-laboratory reproducibility standard deviation was less than 10% at concentrations of 0.2mg/L or higher. The mean true extraction recovery was 85% for propofol and 81% for propofol G. The trueness of the propofol determination expressed as the relative bias of the test results was within ±6% at concentration levels of 0.01-8.5mg/L. Propofol was less stable in blood stabilised with a citrate-EDTA-fluoride mixture than in blood stabilised with an oxalate-fluoride mixture. The stability was lower at -20°C than at 5°C and -80°C. Propofol G did not show instability under the storage conditions tested.

Determination of therapeutic γ-aminobutyric acid analogs in forensic whole blood by hydrophilic interaction liquid chromatography-electrospray tandem mass spectrometry.

Vigabatrin, pregabalin, gabapentin and baclofen are γ-aminobutyric acid analogs that are used in the treatment of epileptic seizures (vigabatrin, pregabalin and gabapentin) and spasticity (baclofen). The intake of these drugs may induce adverse reactions and impair the ability of an individual to drive a vehicle. There have also been reports of cases of intoxication and fatalities from overdoses. For rapid and accurate quantification of these drugs in forensic cases, an ultraperformance liquid chromatography tandem mass spectrometry method using pneumatically assisted electrospray ionization has been developed. The technique has been validated on both ante- and postmortem human whole blood. The protein in the blood samples was removed by the addition of a mixture of methanol and acetonitrile, and the extract was ultrafiltered and diluted with acetonitrile. The separation was performed by hydrophilic interaction liquid chromatography. Calibration of the system was achieved through use of matrix-matched calibrants combined with isotope dilution. The lower limits of quantification were 0.02-0.04 mg/L, and the relative intra-laboratory reproducibility standard deviations were <4 and 8% at concentrations of 10 and 1 mg/L, respectively. The mean true recoveries were >89%. The trueness expressed as the relative bias of the test results was within ±7% at concentrations of 1-40 mg/L for vigabatrin, pregabalin and gabapentin and of 0.1-4 mg/L for baclofen.

A Liquid Chromatography -Electrospray Tandem Mass Spectrometry Method for the Determination of Antiarrhythmic Drugs and Their Metabolites in Forensic Whole Blood Samples

A liquid chromatography-tandem mass spectrometry method, using pneumatically assisted electrospray ionization was developed for the determination of amiodarone, desethylamiodarone, propafenone, N-depropylpropafenone, 5-OH-propafenone, flecainide, and sotalol in human antemortem and postmortem whole blood. A mixture of methanol and acetonitrile was used to extract the samples, and the clear extracts obtained from the protein precipitation were injected directly onto an ethyl-linked phenyl LC column. The isotope dilution technique was applied for quantitative analysis. The relative intralaboratory reproducibility standard deviations were 5-9% at a concentration range of 1-5 mg/L and 9-12% at a concentration of 0.1 mg/L. The mean true recoveries were greater than 91% in the concentration range of 0.05-5 mg/L. The detection limits were in the range of 8-18 µg/L.

Sensitive Determination of Cannabinoids in Whole Blood by LC–MS-MS After Rapid Removal of Phospholipids by Filtration

Direct analysis of Δ9-tetrahydrocannabinol (THC) and other cannabinoids in crude acetonitrile extracts of whole blood by liquid chromatography-tandem mass spectrometry using pneumatically assisted electrospray ionization (LC-ESI-MS-MS) was subjected to pronounced ion suppression from co-eluting phospholipids (PLs). The interferences were mainly caused by the lysophosphatidylcholine and lysophosphatidylethanolamine classes of PLs. The PLs were easily removed from crude extracts by filtration through a sorbent with Lewis acid properties, which typically increased the THC and cannabinol (CBN) signal intensities by a factor of 5. Based on this technique, a simple high-throughput LC-MS-MS method was developed for the determination of cannabinoids in 100 μL samples of whole blood. The lower limits of quantification were 0.2 μg/L for THC, CBN, cannabidiol (CBD) and Δ9-tetrahydrocannabinolic acid A (THCA-A) and 0.5 μg/L for 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH). The mean ion suppression levels after clean-up were 10% (THC), 9% (CBN), 17% (CBD), 0% (THC-OH), 2% (THC-COOH) and 9% (THCA-A) at blood concentration levels of 1-10 μg/L. The mean true extraction recoveries were 97% (THC), 101% (CBN), 101% (CBD), 98% (THC-OH), 95% (THC-COOH) and 90% (THCA-A) at the same concentration levels. The relative intra-laboratory reproducibility standard deviations were <9% at concentrations of 1 μg/L or higher. The trueness expressed as the relative bias of the test results was within ±4% at concentrations of 1 μg/L or higher.