Determination of 12 commonly found compounds in DUID cases in whole blood using fully automated supported liquid extraction and UHPLC-MS/MS.

A high-throughput UHPLC-MS/MS method for the most frequently found compounds; tetrahydrocannabinol (THC), amphetamine, methamphetamine, MDMA, clonazepam, diazepam, nordiazepam, oxazepam, alprazolam, nitrazepam, morphine, and codeine, in driving under the influence of drugs (DUID) cases in whole blood, is presented. Automated sample preparation by 96-well supported liquid extraction (SLE) plates with ethyl acetate + heptane (80 + 20, v/v) as organic solvent was carried out on a Freedom Evo 200 platform from Tecan. An aliquot of 100 µL whole blood was used. Sample preparation time for 96 samples was 1.5 h. Compounds were separated with gradient elution on a C18 column (50 × 2.1 mm, 1.7 µm) with a mobile phase consisting of 5 mM pH 10.2 ammonium formate and methanol. The run time was 4.5 min and 1 µL was injected on an Acquity UPLC I-Class system with a Xevo TQS tandem-quadrupole mass spectrometer in multiple-reaction monitoring mode (MRM) from Waters. Isotope labelled, 13C, internal standards (ISs) were used for all compounds except for alprazolam and morphine, which had deuterated analogs. Quantification was carried out with calibrators without whole blood matrix. Full validation was carried out according to international guidelines, and a new approach for evaluation of process efficiency (PE) has been presented. Linear or quadratic weighted (1/x) calibration curves were used with R2 ≥ 0.999. The method showed satisfactory deviations ±16% when compared to the existing methods, and satisfactory agreement with proficiency testing control samples (z-score -1.6 to 1.8, n = 16 samples). The precision, estimated as the relative standard deviation (RSD) of the concentration difference between results from two independent analyses of authentic whole blood samples, was ≤7.2% in antemortem and ≤9.3% in postmortem samples. Recovery was ≥85% for all the compounds, except morphine ≥62% and THC ≥ 50%. PE was satisfactory for all the compounds with low variation in IS response, RSD ≤ 16% (THC 27%) in antemortem samples and ≤34% (THC 66%) in postmortem samples. To the best of our knowledge, this is the first automated 96-well SLE UHPLC-MS/MS method developed for the simultaneous determination of these 12 compounds in whole blood covering the concentration ranges found in forensic samples. The method has been used in routine work during the last ten months, analysing about 9900 antemortem and 1000 postmortem whole blood samples, and has proven to be robust and reliable.

Quantitative determination of zopiclone and zolpidem in whole blood by liquid-liquid extraction and UHPLC-MS/MS.

An ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of zopiclone and zolpidem in whole blood, for use in cases with suspected driving under influence of drugs (DUID) and autopsy cases. Sample preparation was performed with liquid-liquid extraction (LLE) using ethyl acetate/n-heptane (80:20, v/v) and 0.1mL whole blood. Deuterated analogues were used as internal standards (IS) for both compounds. The compounds were separated using a reversed phase C18-column (2.1mm×100mm, 1.7µm), with a flow rate of 0.5mL/min, 1µL injected and gradient elution with 5mM ammonium formate pH 10.2 and acetonitrile. Quantification was done by MS/MS using multiple reaction monitoring (MRM) in positive mode. The run time of the method was 4.5min including equilibration time. The calibration curves of extracted whole blood standards were fitted by linear-order calibration curves weighted 1/x, with R(2) values above 0.999 for both compounds. Intermediate precision and accuracies (bias) were 2.4-12.9% RSD and from -5.9 to 6.8%, respectively. Recoveries of the compounds were ≥70%. The lower limit of quantification (LLOQ) for zopiclone was 0.50nmol/L (0.19ng/mL) or 0.05pg injected on column, and 3.5nmol/mL (1.10ng/mL) for zolpidem, or 0.27pg injected on column. The limit of detection (LOD) was 0.2nmol/L (0.08ng/mL) for zopiclone and 0.3nmol/L (0.09ng/mL) for zolpidem. Matrix effects (ME) were between 108 and 115% when calculated against IS. A comparison with former confirmation LC-MS method at the Norwegian Institute of Public Health, Division of Forensic Medicine (NIPH) was performed during method validation. Good correlation was seen for both compounds. The method has been running on a routine basis for two years, and has proven to be very robust and reliable with satisfactory long term precision and bias and with results for external quality samples corresponding well to consensus mean or median. Zopiclone and zolpidem concentrations in post mortem and ante mortem cases were reported. The method also meets the requirements of the legislative limits for driving under the influence of non-alcohol drugs introduced in the Norwegian Road Traffic Act Law from 2012.

Quantitative determination of fifteen basic pharmaceuticals in ante- and post-mortem whole blood by high pH mobile phase reversed phase ultra high performance liquid chromatography-tandem mass spectrometry.

An ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of fifteen basic pharmaceuticals, for analysis of post- and ante-mortem whole blood samples. The following compounds were included: amitriptyline and its metabolite nortriptyline, trimipramine, mianserin, mirtazapine, citalopram, paroxetine, sertraline, and venlafaxine (all antidepressants), levomepromazine and quetiapine (antipsychotics), ketobemidone and tramadol (analgesics), alimemazine (sedative antihistamine), and metoprolol (beta-blocker). The sample pretreatment consisted of liquid-liquid extraction (LLE) using ethylacetate:n-heptane (80:20, v/v). Six deuterated analogues were used as internal standards (IS). The compounds were separated using a reversed phase C18-column (2.1mm×100mm, 1.7µm), a flow rate of 0.5mL/min, and gradient elution with 5mM ammonium formate pH 10.2 and acetonitrile. Quantification was done by MS/MS using multiple reaction monitoring (MRM) in positive mode, using two transitions for the compounds and one transition for the IS. The run time of the method was 8min including equilibration time. The calibration curves had R(2) values above 0.995 for all the compounds. The intermediate precision had a relative standard deviation (RSD, %) ranging between 2.0 and 16%. Recoveries of the compounds were ≥81%. The lower limits of quantifications (LLOQs) for the compounds varied from 5.0nmol/L to 0.10µmol/L (1.3-26ng/mL) and the limits of detections (LODs) from 1.0 to 20nmol/L (0.24-5.3ng/mL). LLOQ corresponds to 0.28-5.5pg injected on column. Matrix effects (ME) were between 91 and 113% when calculated against an IS. A comparison with former confirmation LC-MS methods at the Norwegian Institute of Public Health, Division of Forensic Medicine and Drug Abuse Research (NIPH) was performed during method validation. Good correlation was seen for all compounds except sertraline, where the old LC-MS method was showing 33% higher results. The method has been running on a routine basis for more than a year, and has proven to be very robust and reliable with results for external quality samples, including sertaline, corresponding well to consensus mean or median.

Drug screening of hair by liquid chromatography-tandem mass spectrometry.

Hair has become an important matrix for drug analysis, complementary to blood and urine as a matrix. A prolonged detection window makes hair analysis suitable for the detection of exposure to illegal and medicinal drugs for periods up to 12 months. In the present study, a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for drug screening in hair was developed and validated. To 20 mg of hair, 0.45 mL of acetonitrile/25 mM formic acid (5:95 v/v) and 50 microL of deuterated internal standards were added, and the sample was incubated in a water bath at 37 degrees C for 18 h. LC separation was achieved with a Zorbax SB-Phenyl column (2.1 x 100 mm, 3.5-microm particle). Mass detection was performed by positive ion mode electrospray LC-MS-MS and included the following drugs/metabolites: nicotine, cotinine, morphine, 6-monoacetylmorphine, codeine, amphetamine, methamphetamine, 3,4-methylenedioxymeth-amphetamine, cocaine, benzoylecgonine, 7-aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, oxazepam, diazepam, alprazolam, zopiclone, zolpidem, carisoprodol, meprobamate, buprenorphine, and methadone. Within- and between-assay relative standard deviations varied from 2.0% to 12% and 2.7% to 15%, respectively. The accuracies were in the range of -24% to 16%, and recoveries ranged from 25% to 100%. The LC-MS-MS method proved to be simple and robust for the determination of drugs in hair. It has been used for authentic samples in our laboratory in the past year.

Relative validity of a food frequency questionnaire used in the Inter99 study.

OBJECTIVE: To evaluate the validity of the Inter99 food frequency questionnaire (FFQ) compared with a 28-days' diet history and biomarkers. SUBJECTS: A random sample of 13 016 individuals were drawn from a general population and invited for a health screening programme. Participation rate was 52.5%. All high-risk individuals were re-invited for assessment after 1 and 3 years and completed a 198-item FFQ at all three occasions. Participants attending for 3 years follow-up were invited to participate in the validation study, including a 28-days' diet history, a 24-h urine collection and a fasting blood sample. Overall, 264 subjects participated. RESULTS: Spearman's rank correlation coefficients between the two dietary methods ranged from 0.31(beta-carotene) to 0.64 (fruits) in men and from 0.31 (polyunsaturated fat and sodium) to 0.64 (fruits) for women. The proportion of individuals classified in the same or adjacent quintiles were, on average, 72% for men and 69% for women. Gross misclassification was found on average in 2%. The correlation coefficients of the residuals ranged from 0.27 (sodium) to 0.61 (fruits) for men and from 0.21 (sodium) to 0.62 (B12-vitamin) for women. Correlation coefficients between fruit and vegetable intake and carotenoids ranged from -0.08 (lycopene) to 0.44 (alpha-carotene). For the residuals the correlation coefficients ranged from -0.004 (lycopene) to 0.47 (alpha-carotene). CONCLUSION: The Inter99 FFQ and the residuals of the intake provide acceptable classification of individuals according to their dietary intakes and the FFQ gives a good quantitative measurement of key dietary components.

The Dietary Quality Score: validation and association with cardiovascular risk factors: the Inter99 study.

OBJECTIVE: To develop and assess the validity of the Dietary Quality Score (DQS) and investigate the association with cardiovascular risk factors. DESIGN: Cross-sectional population-based study. SETTING: Copenhagen County, Glostrup, Denmark. SUBJECTS: A random sample of 12,934 men and women aged 30-60 years were invited to a health examination. A total of 6542 participants were included in the statistical analysis. METHODS: The DQS was developed using eight questions from a 48-item food frequency questionnaire (FFQ) and validated using a 198-item FFQ. Associations between the DQS and fasting serum lipids, homocysteine, blood pressure and the absolute risk of ischaemic heart disease (IHD) were explored. RESULTS: A higher DQS was shown to be associated with higher dietary quality in general, including a low intake of fat, especially saturated fat; a high intake of fibre; various vitamins and minerals; and fruit, fish, vegetables and whole-grain products. A higher score according to the DQS was significantly negatively associated with total cholesterol (P=0.0031), triglyceride (P=0.0406), low-density lipoprotein-cholesterol (P=0.0071), homocysteine (P<0.0001) and the absolute risk of IHD (P<0.0001), adjusted for sex, age, smoking habits and physical activity level. CONCLUSIONS: The DQS is a simple, valid and quick tool to make a rough classification of individuals into groups with high, average and low dietary quality. The DQS is negatively associated with serum lipids, homocysteine and the absolute risk of IHD. SPONSORSHIP: The Inter99 study is supported economically by The Danish Medical Research Council, The Danish Centre for Evaluation and Health Technology Assessment, Novo Nordisk, Copenhagen County, The Danish Heart Foundation, The Danish Pharmaceutical Association, Augustinus Foundation, Ib Henriksen Foundation and Becket Foundation, Copenhagen County.

Molecular population divergence and sexual selection on morphology in the banded demoiselle (Calopteryx splendens).

The importance of sexual selection in population divergence is of much interest, mainly because it is thought to cause reproductive isolation and hence could lead to speciation. Sexually selected traits have been hypothesized to diverge faster between populations than other traits, presumably because of differences in the strength, mechanism or dynamics of selection. We investigated this by quantifying population divergence in eight morphological characters in 12 south Swedish populations of a sexually dimorphic damselfly, the banded demoiselle (Calopteryx splendens). The morphological characters included a secondary sexual character, the male melanized wing spot, which has an important function in both inter- and intrasexual selection. In addition, we investigated molecular population divergence, revealed by amplified fragment length polymorphism (AFLP) analysis. Molecular population divergence was highly significant among these Northern European populations (overall F(st)=0.054; pairwise population F(st)'s ranged from approximately 0 to 0.13). We found evidence for isolation-by-distance (r=0.70) for the molecular markers and a significant correlation between molecular and phenotypic population divergence (r=0.39). One interpretation is that population divergence for the AFLP loci are affected by genetic drift, but is also indirectly influenced by selection, due to linkage with loci for the phenotypic traits. Field estimates of sexual and natural selection from two of the populations revealed fairly strong sexual selection on wing spot length, indicating that this trait has the potential to rapidly diverge, provided that variation is heritable and the observed selection is chronic.

Simultaneous determination of citalopram, fluoxetine, paroxetine and their metabolites in plasma by temperature-programmed packed capillary liquid chromatography with on-column focusing of large injection volumes.

A miniaturized temperature-programmed packed capillary liquid chromatographic method with on-column large volume injection and UV detection for the simultaneous determination of the three selective serotonin reuptake inhibitors citalopram, fluoxetine, paroxetine and their metabolites in plasma is presented. An established reversed-phase C8 solid-phase extraction method was employed, and the separation was carried out on a 3.5-microm Kromasil C18 0.32x300 mm column with temperature-programming from 35 (3 min) to 100 degrees C (10 min) at 1.3 degrees C/min. The mobile phase consisted of acetonitrile-45 mM ammonium formate (pH 4.00) (25:75, v/v). The non-eluting sample focusing solvent composition acetonitrile-45 mM ammonium formate (pH 4.00) (3:97, v/v) allowed injection of 10 microl or more of the plasma extracts. The method was validated for the concentration range 0.05-5.0 microM, and the calibration curves were linear with coefficients of correlation >0.993. The limits of quantification for the antidepressants and their metabolites ranged from 0.05 to 0.26 microM. The within and between assay precision of relative peak height were in the range 2-22 and 2-15% relative standard deviation, respectively. The within and between assay recoveries were in the 61-99 and 54-92% range for the antidepressants, respectively, and between 52-102 and 51-102% for the metabolites.

Simultaneous determination of citalopram, fluoxetine, paroxetine and their metabolites in plasma and whole blood by high-performance liquid chromatography with ultraviolet and fluorescence detection.

A method for the simultaneous determination of the three selective serotonin reuptake inhibitors (SSRIs) citalopram, fluoxetine, paroxetine and their metabolites in whole blood and plasma was developed. Sample clean-up and separation were achieved using a solid-phase extraction method with C8 non-endcapped columns followed by reversed-phase high-performance liquid chromatography with fluorescence and ultraviolet detection. The robustness of the solid-phase extraction method was tested for citalopram, fluoxetine, paroxetine, Cl-citalopram and the internal standard, protriptyline, using a fractional factorial design with nine factors at two levels. The fractional factorial design showed two significant effects for paroxetine in whole blood. The robustness testing for citalopram, fluoxetine, Cl-citalopram and the internal standard revealed no significant main effects in whole blood and plasma. The optimization and the robustness of the high-performance liquid chromatographic separation were investigated with regard to pH and relative amount of acetonitrile in the mobile phase by a central composite design circumscribed. No alteration in the elution order and no significant change in resolution for a deviation of +/-1% acetonitrile and +/-0.3 pH units from the specified conditions were observed. The method was validated for the concentration range 0.050-5.0 micromol/l with fluorescence detection and 0.12-5.0 micromol/l with ultraviolet detection. The limits of quantitation were 0.025 micromol/l for citalopram and paroxetine, 0.050 micromol/l for desmethyl citalopram, di-desmethyl citalopram and citalopram-N-oxide, 0.12 micromol/l for the paroxetine metabolites by fluorescence detection, and 0.10 micromol/l for fluoxetine and norfluoxetine by ultraviolet detection. Relative standard deviations for the within-day and between-day precision were in the ranges 1.4-10.6% and 3.1-20.3%, respectively. Recoveries were in the 63-114% range for citalopram, fluoxetine and paroxetine, and in the 38-95% range for the metabolites. The method has been used for the analysis of whole blood and plasma samples from SSRI-exposed patients and forensic cases.