Dag Ekeberg

Determination of benzodiazepines in ante-mortem and post-mortem whole blood by solid-supported liquid-liquid extraction and UPLC-MS/MS

A solid-supported liquid-liquid extraction ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of benzodiazepines commonly found in Norway, for use in cases with suspected driving impairment and autopsy cases by analysis of human whole blood samples. The following compounds were included: alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, lorazepam, midazolam, nitrazepam, nordiazepam (metabolite of diazepam), oxazepam and phenazepam. Aliquots of 500 μL whole blood were added 500 μL of borate buffer pH 11 and extracted by solid-supported liquid-liquid extraction on ChemElut(®) columns using three times 2.5 mL of methyl tert-butyl ether. Deuterated analogues were used as internal standards (IS) for all analytes, except for midazolam, phenazepam and bromazepam which had no commercially available deuterated analogues at the time the method was developed, and therefore used diazepam-d(5), flunitrazepam-d(7) and nitrazepam-d(5), respectively. The analytes were separated using UPLC with a 2.1×100 mm BEH C(18)-column, 1.7 μm particle size, and quantified by MS/MS using multiple reaction monitoring (MRM) in positive mode. Two transitions were used for the analytes and one transition for the IS. The run time of the method was 8 min including equilibration time. The concentrations of the benzodiazepines in the method span a broad range varying from the lowest concentration of 0.005 μM for flunitrazepam to the highest of 20 μM for oxazepam. The calibration curves of extracted whole blood standards were fitted by second-order calibration curves weighted 1/x, with R(2) values ranging from 0.9981 to 0.9998. The intermediate precision had a CV (%) ranging between 2 and 19%. Recoveries of the analytes were from 71 to 96%. The LLOQs for the analytes varied from 0.0006 to 0.075 μM and the LODs from 0.005 to 3.0 nM. Matrix effects were studied by post extraction addition and found to be between 95 and 104% when calculated against an internal standard. A comparison with two other LC-MS methods was performed during method validation. Good correlation was seen for all analytes. The method has been running on a routine basis for several years, and has proven to be very robust and reliable with good results for external quality samples. The method also meets the requirements of the legislative limits for driving under the influence of non-alcohol drugs to be introduced in the Norwegian legislative system from 2012.

Negative-ion electrospray ionisation–mass spectrometry (ESI–MS) as a tool for analysing structural heterogeneity in kappa-carrageenan oligosaccharides

Oligosaccharides, enzymically produced from kappa-carrageenan, have been investigated by electrospray ionisation mass spectrometry (ESI-MS). The technique was used without prior derivatisation of the oligosaccharide originally obtained by size-exclusion chromatography (SEC). The structure of the oligosaccharides was mainly 4-sulphated neocarrabiose (A-G4S) with an increasing length ranging from di- to dodecasaccharides. However, in the larger oligosaccharides, structural motifs deviating from the perfect alternating A-G4S structure were detected, i.e. (A2S-G4S). Although resulting in reduced signal intensity, samples to which NaCl was added also gave rise to reliable mass spectra. Desulphation was induced at elevated cone voltages and in acidic or alkaline salt solutions.

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.

A GC – magnetic sector MS method for identification and quantification of fatty acids in ewe milk by different acquisition modes

A general method for qualitative and quantitative determination of fatty acids (FAs) using GC-MS was developed and tested on ewe milk. A total number of 38 poly unsaturated FAs, monounsaturated FAs and saturated FAs, from C6:0 to C24:1, were used in a comparative study of scan, reconstructed ion chromatogram and SIM. Fatty acid methyl esters in standard solutions as well as in milk from ewe were analyzed by these techniques, using a sector instrument. Instrument precision, linearity, LOD and LOQ, as well as calibration behavior and response factors were investigated for each approach. The quantitative results obtained by each technique were compared. All techniques had values for LOD and LOQ in the ng/mL region.

Base catalysed isomerisation of aldoses of the arabino and lyxo series in the presence of aluminate

Base-catalysed isomerisation of aldoses of the arabino and lyxo series in aluminate solution has been investigated. L-Arabinose and D-galactose give L-erythro-2-pentulose (L-ribulose) and D-lyxo-2-hexulose (D-tagatose), respectively, in good yields, whereas lower reactivity is observed for 6-deoxy-D-galactose (D-fucose). From D-lyxose, D-mannose and 6-deoxy-L-mannose (L-rhamnose) are obtained mixtures of ketoses and C-2 epimeric aldoses. Small amounts of the 3-epimers of the ketoses were also formed. 6-Deoxy-L-arabino-2-hexulose (6-deoxy-L-fructose) and 6-deoxy-L-glucose (L-quinovose) were formed in low yields from 6-deoxy-L-mannose and isolated as their O-isopropylidene derivatives. Explanations of the differences in reactivity and course of the reaction have been suggested on the basis of steric effects.

Determination of chlorinated pesticides by capillary supercritical fluid chromatography—mass spectrometry with positive- and negative-ion detection

Abstract An interface between a capillary supercritical fluid chromatograph and a double-focusing mass spectrometer was developed. Modification of the standard electron ionization (EI)-chemical ionization (CI) combination ion source was necessary to obtain useful mass spectra with negative-ion detection. A detection limit in the lower nanogram range of the chlorinated pesticides (DDT and dieldrin) was found irrespective of the mode of detection. Positive-ion methane CI resulted in a relatively abundant [M+ H]+ ion, whereas positive-ion isobutane and ammonia CI appeared not to be amenable to the detection of chlorinated pesticides. The EI-charge exchange mass spectra of the investigated pesticides generally did not match the library mass spectra. In the negative-ion mode, CO2 was an efficient moderating gas giving relatively large amounts of M−·, in addition to some fragment ions. More fragmentation was observed when N2O replaced CO2 as the mobile phase. No major effects on the mass spectra, obtained by using pure mobile phase, were observed on adding methane, isobutane or ammonia.

Determination of CH4, CO2 and N2O in air samples and soil atmosphere by gas chromatography mass spectrometry, GC-MS

A method for determination of the climate gases CH4, CO2 and N2O in air samples and soil atmosphere was developed using GC-MS. The method uses straightforward gas chromatography (separation of the gases) with a mass spectrometric detector in single ion mode (specific determination). The gases were determined with high sensitivity and high sample throughput (18 samples h(-1)). The LOD (3sigma) for the gases were 0.10 micro L L(-1) for CH4, 20 microL L(-1) for CO2 and 0.02 microL L(-1) for N2O. The linear range (R2 = 0.999) was up to 500 microL L(-1) for CH4, 4000 microL L(-1) for CO2 and 80 microL L(-1) for N2O. The samples were collected in 10 mL vials and a 5 microL aliquot was injected on column. The method was tested against certified gas references, the analytical data gave an accuracy within +/-5% and a precision of +/-3%. The presence of < or = 10% by volume of C2H2 (often used experimentally to prevent N2 formation from N2O) did not interfere with detection for the targeted trace gases.

Identification of Brominated Flame Retardants in Sediment and Soil by Cyclohexane Extraction and Gas Chromatography Mass Spectrometry

The accumulation of brominated fl ame retardants (BFRs) in the environment raises concern in light of observed detrimental effects on wildlife as well as on public health. We here present a recently modi fi ed method for the identi fi cation and quanti fi cation of the following selection of bromodiphenyl ether (BDE) fl ame retardants: BDE-17, -47, -66, -100, -153 and -183, in soil and sediments, using a new extraction procedure followed by gas chromatography mass spectrometry (GC-MS). Low- and high- resolution mass spectrometry (LRMS and HRMS, respectively) were compared and the latter was found to be superior with respect to both sensitivity and linear range. At LRMS mode the linear range was 3.8 – 19.2 ng/g dry weight (dw), while the use of HRMS more than doubled the linear range to 1.9 – 38.4 ng/g dry weight. Both methods were tested with regards to matrix associated effects on the limit of detection and quantitation. The use of HRMS yielded equal sensitivity for standards in solution and matrix. This was not the case when using LRMS. Here the limits of detection and quantitation were severely elevated by the matrix. Recoveries were comparable, but slightly higher at LRMS mode (77.0 – 121.9%) compared to HRMS (83.2 – 115.3%). The method described here is high throughput, low cost and will prove valuable in monitoring the levels of BFRs in the environment.

Ex vivo Digestion of Milk from Red Chittagong Cattle Focusing Proteolysis and Lipolysis

Ex vivo digestion of proteins and fat in Red Chittagong Cattle milk from Bangladesh was carried out using human gastrointestinal enzymes. This was done to investigate the protein digestion in this bovine breed’s milk with an especial focus on the degradation of the allergenic milk proteins; αs1-casein and β-lactoglobulin and also to record the generation of peptides. Lipolysis of the milk fat and release of fatty acids were also under consideration. After 40 min of gastric digestion, all the αs-caseins were digested completely while β-lactoglobulin remained intact. During 120 min of duodenal digestion β-lactoglobulin was reduced, however, still some intact β-lactoglobulin was observed. The highest number of peptides was identified from β-casein and almost all the peptides from κ-casein and β-lactoglobulin were identified from the gastric and duodenal samples, respectively. No lipolysis was observed in the gastric phase of digestion. After 120 min of duodenal digestion, milk fat showed 48% lipolysis. Medium (C10:0 to C16:0) and long (≥C17:0) chain fatty acids showed 6% to 19% less lipolysis than the short (C6:0 to C8:0) chain fatty acids. Among the unsaturated fatty acids C18:1∑others showed highest lipolysis (81%) which was more than three times of C18:2∑all and all other unsaturated fatty acids showed lipolysis ranging from 32% to 38%. The overall digestion of Bangladeshi Red Cattle milk was more or less similar to the digestion of Nordic bovine milk (Norwegian Red Cattle).

Phytol as a possible indicator of ozone stress by Picea abies

Abstract This work is based on a project where the effect of ozone ( 3 ) on hexane-soluble components of Picea abies needles was investigated ( Ogner (1993) Environ. Pollut. , 82 , 223-9 ). The trees representing six clones of Picea abies were fumigated in open-top chambers at two locations in Norway, Aas and Bergen, for one growth season. The gas chromatography-flame ionization detector (GC-FID) analysis showed that the concentrations of four of the compounds from the hexane extract changed with ozone dose in a way that made them promising as indicators. However, at that time no identification of the compounds was made. The same ozone treated plant material was utilized in this study and one of the compounds, analysed by gas chromatography-mass spectrometry (GC-MS), was identified as phytol. The remaining indicators were not identified due to either poorly separated peaks or too low a concentration.