Elsa Lundanes

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.

Determination of opiates and cocaine in urine by high pH mobile phase reversed phase UPLC-MS/MS

A fast and selective ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of opiates (morphine, codeine, 6-monoacetylmorphine (6-MAM), pholcodine, oxycodone, ethylmorphine), cocaine and benzoylecgonine in urine has been developed and validated. Sample preparation was performed by solid phase extraction (SPE) on a mixed mode cation exchange (MCX) cartridge. For optimized chromatographic performance with repeatable retention times, narrow and symmetrical peaks, and focusing of all analytes at the column inlet at gradient start, a basic mobile phase consisting of 5mM ammonium bicarbonate, pH 10.2, and methanol (MeOH) was chosen. Positive electrospray ionization (ESI(+)) MS/MS detection was performed with a minimum of two multiple reaction monitoring (MRM) transitions for each analyte. Deuterium labelled-internal standards were used for six of the analytes. Between-assay retention time repeatabilities (n=10 series, 225 injections in total) had relative standard deviation (RSD) values within 0.1-0.6%. Limit of detection (LOD) and limit of quantification (LOQ) values were in the range 0.003-0.05 microM (0.001-0.02 microg/mL) and 0.01-0.16 microM (0.003-0.06 microg/mL), respectively. The RSD values of the between-assay repeatabilities of concentrations were <or=10% at five concentration levels for all analytes, except for pholcodine. Specificity was investigated by determination of the retention times of 96 drugs and internal standards in total. Co-eluting compounds were in all cases separated by the MS/MS detection. No or only minor matrix effects were observed. Total run time, including injection and equilibration time was 5.7 min. The method has been routinely used at the Norwegian Institute of Public Health (NIPH) since August 2007 for qualitative detection of opiates, cocaine and benzoylecgonine in more than 2000 urine samples with two replicates of each sample.

Determination of chlorinated phenols by high-performance liquid chromatography

The separation of mono-, di-, tri- and tetrachlorinated phenols and pentachlorophenol has been investigated by high-performance liquid chromatography using three different systems, adsorption chromatography on silica and reversedphase chromatography on a polar bonded phase (aminoalkyl) and a non-polar bonded phase (octadecyl). A satisfactory group separation as well as a satisfactory separation of isomers could not be obtained by adsorption chromatography on silica. A reasonable separation of isomers was obtained with amino columns, but the group separation was not satisfactory. The octadecyl columns were superior with reference both to separation of groups and of isomers within these groups as well as to stability and speed of analysis. A mixture of nineteen different phenols was resolved on a C18 column with a 30-min linear gradient of 56–80% methanol and 44-20% of 0.02 M KH2PO4 (pH 4.0).

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.

Reversed-phase chromatography of peptides

Summary The chromatographic behaviour of underivatized peptides on reversed-phase packings has been examined. To study the effects of different amino acids in peptides, simple model substances, such as amino acids and dipeptides, were chosen. The capacity factors of amino acids, l,l -dipeptides and diastereoisomeric dipeptides were measured, and side-effects such as loss by adsorption, double-peak formation and load-retention dependence were studied. Of the four different reversed phases examined, the ODS packings appeared to give both the best selectivity and the best efficiency in the separation of most peptides and diastereoisomers. A marked difference in the retention of basic functions was obtained on two ODS packings of different manufacture.

Recent advances in on-line multidimensional liquid chromatography

Multidimensional liquid chromatography (MD LC) has, due to enhanced peak capacity compared to one-dimensional LC, become an important analytical tool for separating components in complex samples, e.g. in proteomics. MD LC can be performed both on-line and off-line. Because of the advantages like possible automation and minimal sample loss, on-line MD LC has appeared to be very attractive also for high throughput analysis. This review includes only on-line coupled MD LC. Different aspects related to the compatibility of separation principles and column dimensions are highlighted and recent applications using on-line mainly two-dimensional (2D) LC have been included.

Comparing electron ionization high-resolution and electron capture low-resolution mass spectrometric determination of polybrominated diphenyl ethers in plasma, serum and milk.

Gas chromatography coupled to low-resolution mass spectrometry with electron capture negative ionization as detection mode (GC-LRMS (ECNI)) has been compared to gas chromatography coupled to high-resolution mass spectrometry using electron ionization as detection mode (GC-HRMS (EI)) for determination of polybrominated diphenyl ethers (PBDEs) in biological samples. Extracts of 5.0 g plasma, serum and milk samples were analyzed using both methods. The GC-LRMS (ECNI) and GC-HRMS (EI) systems were found to be equally well suited for determination of PBDEs in the biological samples, as well as in standard solutions, with respect to response, detection limits and repeatability at the pg-level. The estimated limits of detection (LOD) in milk extracts ranged from 0.3-0.6 pg PBDE/g milk and 0.4-0.7 pg PBDE/g milk, for the GC-LRMS (ECNI) and GC-HRMS (EI) systems, respectively. The method repeatability including sample preparation was in the range 4.7-8.4% and 0.6-10% relative standard deviation (RSD) for the GC-LRMS (ECNI) and GC-HRMS (EI) systems, respectively.

Determination of long-chained fatty acids using non-aqueous capillary electrophoresis and indirect UV detection

Abstract A method for separation and determination of free saturated long-chained fatty acids ( n -C 14 −n-C 26 ) has been developed using non-aqueous capillary electrophoresis with indirect UV detection at 264 nm. The separation medium consisted of 2.5m M anthraquinone-2-car☐ylic acid and 40m M Tris in N-methylformamide-dioxane (3:1, v/v). The electroosmotic mobility was about 3·10 −4 cm 2 /V s resulting in a separation time of about 15 min. Injection was done at the anode. Sorbic acid and the low mobility anthraquinone-2-sulphonic acid, 2,6-dihydroxy anthraquinone and all- trans -retinoic acid were less suitable as background absorbent. Maximum efficiency (up to 3.2·10 5 plates) were obtained in a 54 cm effective length×50 μm I.D. capillary at 300 V/cm. However, due to better sensitivity, 75 μm I.D. capillaries were preferred. Plate numbers of 1.4·10 5 were achieved in 75 μm I.D. capillaries of 46 cm effective length at 300 V/cm. Linear calibration curves were established for the fatty acids n -C 16 −n-C 20 (0.025−1.0mM), n-C 22 (0.025−0.6mM) and n -C 24 − n-C 26 (0.025−0.3mM) with correlation coefficients better than 0.985, using corrected peak area ratio and n -C 14 fatty acid as internal standard. The concentration limits of detection were about 0.025m M . The method has been applied for separation of the components in a hydrogenated fish oil, and for separation of dimeric and trimeric acids as well.

A simplified method for determination of tetrabromobisphenol A and polybrominated diphenyl ethers in human plasma and serum

A method utilizing solid-phase extraction (SPE) and gas chromatography with electron capture mass spectrometric detection (GC-ECMS) has been developed for simultaneous determination of seven polybrominated diphenyl ethers (PBDEs) and tetrabromobisphenol A (TBBP-A) in plasma and serum. Plasma and serum samples of 5.0 g were diluted and applied to a polystyrene-divinylbenzene SPE column. The lipids were decomposed by treatment with concentrated sulfuric acid directly on the SPE column, prior to elution of the brominated flame retardants by dichloromethane-methanol (7 + 3, v/v). Following diazomethane derivatization, the samples were analyzed by GC-ECMS. The method has been validated in the concentration range of 1.8-90 pg brominated flame retardants/g plasma or serum. The average absolute recoveries were in the range of 56-111% and 40-90% for plasma and serum, respectively. The average recoveries of the analytes relative to the internal standards were 75-108% and 88-111% for plasma and serum, respectively. The repeatability of the method was in the range of 0.6-27% RSD, and the estimated detection limits were in the range of 0.4-9.8 pg/g plasma or serum. The human plasma used for validation contained all the investigated analytes, in concentrations below 4 ng/g lipid weight.

Group separation of oil residues by supercritical fluid chromatography

Abstract The group separation by supercritical fluid chromatography of high-boiling residues (b.p. higher than 350°C) or North Sea oil is described. The chromatographic separation used a three-column system (cyano and silica and silver-impregnated silica) with column-switching and back-flushing with supercritical carbon dioxide as the mobile phase. The flame ionization detector could be used directly by placing the restrictor in the jet. The quantitative aspects are discussed in relation to the results obtained by liquid chromatographic methods.