Journal of Chromatography B (v.769, #2)

News Section (N1-N3).

Biological monitoring of pesticide exposure: a review of analytical methods by Cristina Aprea; Claudio Colosio; Teresa Mammone; Claudio Minoia; Marco Maroni (191-219).
A wide range of studies concerned with analytical methods for biological monitoring of exposure to pesticides is reviewed. All phases of analytical procedures are assessed, including sampling and storage, sample preparation and analysis, and validation of methods. Most of the studies aimed at measuring metabolites or unchanged compounds in urine and/or blood as biological indicators of exposure or dose. Biological indicators of effect, such as cholinesterase, are also evaluated. The principal groups of pesticides are considered: organophosphorus pesticides, carbamate pesticides, organochlorine pesticides, pyrethroid pesticides, herbicides, fungicides and other compounds. Choice of the method for biological monitoring of exposure depends on the study population: a detection limit of 1 μg/l or less is required for the general population; higher values are adequate for occupationally exposed subjects. Interpretation of results is also discussed. Since biological indices of exposure are only available for a few compounds, biological reference values, established for the general population, may be used for comparison with levels of professionally exposed subjects.
Keywords: Pesticides;

Capillary gas chromatographic determination of 1,4-butanediol and γ-hydroxybutyrate in human plasma and urine by Benoı̂t Blanchet; Karine Morand; Anne Hulin; Alain Astier (221-226).
This article describes two methods for the determination of 1,4-butanediol and γ-hydroxybutyrate in human plasma and urine using capillary gas chromatography. For 1,4-butanediol, plasma or urine samples (500 μl) were extracted by protein precipitation whereas for γ-hydroxybutyrate, plasma or urine samples (500 μl) were extracted and derivatised with BF3–butanol. The compounds were separated on a Supelcowax-10 column and detection was achieved using a flame ionization detector. The methods are linear over the specific ranges investigated, accurate (with a percentage of the nominal concentration <109.8%) and showed intra-day and inter-day precision within the ranges of 5.0–12.0 and 7.0–10.1%, respectively. No interferences were observed in plasma and urine from hospitalized patients.
Keywords: 1,4-Butanediol; γ-Hydroxybutyrate;

Sufentanil is the most potent synthetic opioid currently in use. Extremely low serum concentrations have to be determined for therapeutic drug monitoring and to support brain death diagnosis by excluding opioid induced coma. The described method utilizes a HPLC–MS–MS system with an electrospray ion source and an ion-trap mass spectrometer. The serum samples were extracted under basic conditions with toluene–2-propanol (10:1, v/v). Chromatographic separation was achieved on a RP-18 70×2 mm column with a 0.02% trifluoroacetic acid in a water–acetonitrile gradient as mobile phase. The limits of detection and quantification are 3 and 10 pg/ml, respectively. At the limit of quantification, the intra-day relative standard deviation of the assay is 12.6% and the inter-day relative standard deviation is 14%.
Keywords: Sufentanil;

A rapid and simple capillary electrophoresis (CE) assay for measuring the stability of luteinizing hormone-releasing hormone (LHRH) analogues in the presence of intestinal enzymes has been developed and validated. Buffer pH and sample stacking were important factors in controlling resolution and reproducibility. The CE assay for human (h) and salmon LHRH analogues between 0.05 and 0.25 mM was linear for peak height versus concentration (r 2>0.99). Analysis of hLHRH at 0.1 mM had an intra-day relative standard deviation of 1.25% and an inter-day relative standard deviation of 5.0%. The method was applied to the stability of LHRH analogues in salmon intestinal digests.
Keywords: Luteinizing hormone-releasing hormones;

The need for analytical screening tests more reliable and valid to detect amphetamine and related “designer drugs” in biological samples is becoming critical, due to the increasing diffusion of these drugs on the European illegal market. The most common screening procedures based on immunoassays suffer a number of limitations, including low sensitivity, lack of specificity and limited number of detectable substances. This paper describes a screening method based on gas-chromatography-mass–spectrometry (GC/MS) using positive chemical ionisation (PCI) detection. Methanol was used as reactant gas in the ionisation chamber. Molecular ions of different compounds were monitored, allowing a sensitivity of 5–10 ng/ml with high selectivity. The sensitivity of the method gives positive results in samples taken 48–72 h after intake of one dose of 50–100 mg. The method is simple and rapid. Sample preparation was limited to one liquid–liquid extraction, without any hydrolysis and derivatisation. Hydrolysis is critical to identify metabolites excreted as conjugates. Blank urine samples spiked with known amounts of amphetamine (AM), methylamphetamine (MA), methylenedioxyamphetamine (MDA), methylenedioxymethylamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) and methylenedioxyphenyl-N-methyl-2-butanamine (MBDB) were analysed. The method was successfully tested on real samples of urine from people, whose use of amphetamine was suspected, and results were compared with results obtained with immunoassays.
Keywords: Ecstasy; Amphetamines;

A new method for simultaneous quantification of trimethoprim, sulfadiazine and N 4-acetylsulfadiazine in plasma of broilers at levels down to 13–16 ng/ml has been developed. Samples were deproteinized with acetonitrile, defatted with hexane, and extracted with dichloromethane. Chromatographic analysis was carried out on a C18 column in the presence of tetrabutylammonium hydrogen sulfate, a competing base, while detection was performed at 240 nm for trimethoprim, and 270 nm for both sulfadiazine and N 4-acetylsulfadiazine. Accuracy and precision data showed recoveries and relative standard deviation values better than 87.3% and 3.1%, respectively, for all three analytes. The good analytical characteristics of the method could allow limits of detection in the low ng/ml range to be realised. The method was successfully applied to determine drug concentrations in plasma samples from broilers administered a combination of sulfadiazine and trimethoprim.
Keywords: Trimethoprim; Sulfadiazine; N 4-Acetylsulfadiazine;

A micellar electrokinetic capillary chromatography (MEKC) for determining fluoxetine and its metabolite (norfluoxetine) is proposed. Optimal conditions for the quantitative separation were investigated. A background electrolyte solution consisting of 5 mM phosphate buffer adjusted to pH 12.3 and 40 mM of 1-decanesulfonic acid sodium salt (DSS), hydrodynamic injection and 25 kV of separation voltage were used. Good linearity and precision were obtained for both compounds. Detection limits of 0.2 mg/l for fluoxetine and norfluoxetine were obtained. The developed method is rapid and it has been applied to determine fluoxetine and its metabolite in human serum and urine. The samples were purified and enriched by means of extraction–preconcentration step with a preconditioned C18 cartridge and eluting the compounds with methanol.
Keywords: Fluoxetine; Norfluoxetine;

An isocratic reversed-phase HPLC method for the simultaneous quantitation of α-lipoic acid and five of its metabolites in human plasma as well as in human urine employing solid-phase extraction and pulsed amperometric detection was developed and validated. The method was found to be sufficiently precise and accurate for the measurement of α-lipoic acid and its metabolites 6,8-bis(methylthio)octanoic acid, 4,6-bis(methylthio)hexanoic acid and 2,4-bis(methylthio)butanoic acid in plasma and urine samples, obtained from patients suffering from diabetic neuropathy as well as from healthy volunteers following daily oral administration of 600 mg α-lipoic acid. The quantitation of the metabolite bisnorlipoic acid was often impaired by interferences caused by an unidentified metabolite. However, bisnorlipoic acid was detected in few test samples and the concentrations were consistently low. Despite the poor recovery of the metabolite tetranorlipoic acid from plasma, reproducibility and accuracy were found to be from acceptable magnitude to assess concentration time courses. Thus, the obtained analytical results are considered as reliable and well suited for pharmacokinetic studies of α-lipoic acid and its metabolites.
Keywords: α-Lipoic acid;

In this study, nucleotide adsorption–desorption behaviour of boronic acid-carrying uniform, porous particles was investigated. The particles were produced by a “multi-step microsuspension polymerization” in the form of poly(styrene–vinylphenyl boronic acid–divinylbenzene) terpolymer. In the first step of the production method, uniform polystyrene latex particles (6.2 μm in size) were obtained by dispersion polymerization. These particles were first swollen by a low molecular mass organic agent (i.e. dibutylphthalate, DBP) and then by a monomer mixture including styrene (S), 4-vinylphenyl boronic acid (VPBA) and divinylbenzene (DVB). The particle uniformity was protected in both swelling stages by adjusting DBP/polystyrene latex and monomer mixture/polystyrene latex ratios. Polymerization of the monomer mixture in the swollen seed particles provided boronic acid-carrying uniform, porous particles 11–12 μm in size. To have uniform particles with different porosities and boronic acid contents, the feed concentration of boronic acid-carrying monomer and the monomer/seed latex ratio were changed. The particles were tried as sorbent for the adsorption of a model nucleotide (i.e., β-nicotinamide adenine dinucleotide, β-NAD). In the β-NAD adsorption experiments, the maximum equilibrium adsorption was obtained at pH 8.5 which was very close to pK a of boronic acid. The incorporation of boronic acid functionality provided a significant increase in the β-NAD adsorption. In contrast to plain poly(styrene-co-divinylbenzene) particles, four-fold higher β-NAD adsorption was obtained with the boronic acid functionalized particles. β-NAD was desorbed from the particles with the yields higher than 90% by weight.
Keywords: Nucleotides; Boronic acid;

Delavirdine is a newly developed anti-HIV-1 drug for AIDS therapy. This study describes a sensitive high-performance liquid chromatographic method for the determination of delavirdine in 50 μl of plasma. Samples were deproteinized with 150 μl of a solution of internal standard (cisapride 10 μg/ml) in acetonitrile. An aliquot of the supernatant was injected onto the column. HPLC separation was achieved on a C18 column with the mobile phase of acetonitrile-50 mM sodium dihydrogen phosphate (60:40, v/v) at a flow-rate of 1 ml/min. The eluants were measured by fluorescence detection with excitation at 295 nm and emission filtration at 425 nm. The retention time was about 5.3 min for delavirdine and 6.5 min for cisapride. The specificity was demonstrated, as there were no interferences from plasma samples of different batches in the regions of peak interest. Calibration curves were linear from 25 to 25000 ng/ml. The limit of quantitation was 25 ng/ml. The within- and between-day precision (C.V.) was 9.3%, or less, and the accuracy was within 9.2% of the nominal concentration. The small sample volume needed is especially advantageous for the application both in pharmacokinetic studies in HIV-infected adults and pediatric patients, and in small animals, where limited samples are available.
Keywords: Delavirdine;

An gas chromatography–electron-capture detection method has been developed for simultaneous determination of fluoxetine and p-trifluoromethylphenol (TFMP), an O-dealkylated metabolite of fluoxetine in human liver microsomes. Prior to the analysis, aliquots of alkalinized microsomal mixture were extracted with ethyl acetate solvent containing acetonitrile (10%, v/v) and the derivatizing reagent, pentafluorobenzenesulfonyl chloride (0.1%, v/v). The organ phase was retained and taken to dryness, the residue was reconstituted in methanol, and the aliquot of extracts was injected directly into a gas chromatograph equipped with an electron-capture detector. 2,4-Dichlorophenol was added to the initial incubation mixture and carried through the procedure as the internal standard. The method provided the mean recoveries of up to 103% for fluoxetine and 104% for TFMP. Acceptable relative standard deviations were found for both within-run and day-to-day assays. The practical limit of detection (signal-to-noise ratio=3) was 1.62 ng/ml for TFMP and 6.92 ng/ml for fluoxetine in human liver microsomes, and the limit of quantitation was 8.1 pg for TFMP and 34.6 pg for fluoxetine. The assay is rapid and sensitive and has been applied successfully to simultaneous quantification of fluoxetine and TFMP in human liver microsomes with different CYP2C19 genotypes.
Keywords: Fluoxetine; p-Trifluoromethylphenol;

High-performance liquid chromatography with electrochemical detection applied to the analysis of 3,4-dihydroxymethamphetamine in human plasma and urine by M Segura; J Ortuño; J.A McLure; M Pujadas; N Pizarro; M Farré; A Llebaria; J Joglar; J Segura; R de la Torre (313-321).
Metabolic activation in the disposition of 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”) has been implicated in some of its pharmacological and toxicological effects, with the major metabolite 3,4-dihydroxymethamphetamine (HHMA) as a putative toxicant through the formation of thioether adducts. We describe the first validated method for HHMA determination based on acid hydrolysis of plasma and urine samples, further extraction by a solid-phase strong cation-exchange resin (SCX, benzenesulfonic acid), and analysis of extracts by high-performance liquid chromatography with electrochemical detection. The chromatographic separation was performed in an n-butyl-silane (C4) column and the mobile phase was a mixture of 0.1 M sodium acetate containing 0.1 M 1-octanesulphonic acid and 4 mM EDTA (pH 3.1) and acetonitrile (82:18, v/v). Compounds were monitored with an electrochemical cell (working potentials 1 and 2, +0.05 and +0.35 V, respectively, gain 60 μA). A mobile phase conditioning cell with a potential set at +0.40 V was connected between the pumping system and the injector. Calibration curves were linear within the working concentration ranges of 50–1000 μg/L for urine and plasma. Limits of detection and quantification were 10.5 and 31.8 μg/L for urine and 9.2 and 28.2 μg/L for plasma. Recoveries for HHMA and DHBA (3,4-dihydroxybenzylamine, internal standard) were close to 50% for both biological matrices. Intermediate precision and inter-day accuracy were within 3.9–6.5% and 7.4–15.3% for urine and 5.0–10.8% and 9.2–13.4% for plasma.
Keywords: 3,4-Dihydroxymethamphetamine;

Proteomic analysis of rat soleus and tibialis anterior muscle following immobilization by Robert J Isfort; Feng Wang; Kenneth D Greis; Yiping Sun; Thomas W Keough; Sue C Bodine; N.Leigh Anderson (323-332).
A proteomic analysis was performed comparing normal slow twitch type fiber rat soleus muscle and normal fast twitch type fiber tibialis anterior muscle to immobilized soleus and tibialis anterior muscles at 0.5, 1, 2, 4, 6, 8 and 10 days post immobilization. Muscle mass measurements demonstrate mass changes throughout the period of immobilization. Proteomic analysis of normal and atrophied soleus muscle demonstrated statistically significant changes in the relative levels of 17 proteins. Proteomic analysis of normal and atrophied tibialis anterior muscle demonstrated statistically significant changes in the relative levels of 45 proteins. Protein identification using mass spectrometry was attempted for all differentially regulated proteins from both soleus and tibialis anterior muscles. Four differentially regulated soleus proteins and six differentially regulated tibialis anterior proteins were identified. The identified proteins can be grouped according to function as metabolic proteins, chaperone proteins, and contractile apparatus proteins. Together these data demonstrate that coordinated temporally regulated changes in the proteome occur during immobilization-induced atrophy in both slow twitch and fast twitch fiber type skeletal muscle.
Keywords: Proteins;

Rapid and sensitive determination of sertraline in human plasma using gas chromatography–mass spectrometry by Kyung Mee Kim; Byung Hwa Jung; Man Ho Choi; Jong Soo Woo; Ki-Jung Paeng; Bong Chul Chung (333-339).
A method for the determination of sertraline in human plasma using gas chromatography–mass spectrometry (GC–MS), with the selected ion-monitoring (SIM) mode, was described. The following was used in this study: (1) single liquid–liquid extraction at alkaline pH after deproteinization of plasma protein and (2) perfluoroacylation with HFBA, which has higher sensitivity (about 10-fold) compared with previous reported derivatization. The detection limit for the SIM of sertraline as an N-HFB derivative was 0.1 ng/ml, and its recovery was 80–85%. The linear response was obtained in the range of 0.2–10.0 ng/ml with a correlation coefficient of 0.999. The coefficient of variation (C.V.%) was less than 12.1% in the 1–30 ng/ml, and less than 18.2% at 0.2 ng/ml, and the accuracy was less than 10% at all of the concentration range. These findings indicate that this assay method has adequate precision and accuracy to determine the amount of sertraline in human plasma. After pharmacokinetics was performed with this assay method following oral administration of sertraline hydrochloride in man, moment analysis revealed that pharmacokinetic parameters for sertraline (C max, 10.3 ng/ml; T max, 8.0 h; T 1/2, 28.6 h) were similar to previously reported results. These results indicate that this simple and sensitive assay method is readily applicable to the pharmacokinetic studies of sertraline.
Keywords: Sertraline;

The clinical utility of a one-step extraction procedure based on the retention of a diphenyl boronate–catecholamine complex on a C18 solid-phase extraction sorbent was investigated for the measurement of urinary catecholamines. Although recoveries with the extraction procedure were optimal over a relatively broad pH range (7.5–9.5), analytical factors such as sample loading and elution flow-rates, wash step and elution conditions, the concentration of catecholamines in urine to be extracted and the type of C18 sorbent used for extraction were found to influence the efficiency of this procedure and would therefore need to be controlled for optimal recoveries. Under optimal conditions the recovery of noradrenaline, adrenaline and dopamine from spiked urine was high and reproducible (mean recoveries were >85% for all catecholamines). The effectiveness of sample clean-up step was demonstrated by reverse phase, ion pair high-performance liquid chromatography with electrochemical detection. The method described was found to be suitable for the routine measurement of catecholamines in urine in clinical biochemistry laboratories. It has a high sample extraction throughput (40/h) and has adequate precision (between batch C.V.<8%) and sensitivity (LOD<30 nmol/l; LOQ<65 nmol/l) for all the catecholamines measured. The method has acceptable accuracy, showing a mean bias of 6.6% for noradrenaline, 7.3% for adrenaline and 6.8% for dopamine from the mean value of laboratories (N=69) participating in an External Quality Assurance scheme for greater than 12 months.
Keywords: Catecholamines; Diphenyl boronate;

Diclofenac is a potent inhibitor of prostaglandin synthesis, as well as an established antipyretic and analgesic agent. To determine diclofenac in rat bile and investigate its hepatobiliary excretion, a procedure using rapid and sensitive high-performance liquid chromatography coupled to microdialysis sampling system was developed. A shunt linear microdialysis probe was inserted into the common bile duct between the liver and the duodenum for continuous sampling of the drug from bile fluids following intravenous administration of diclofenac (1 mg/kg). Separation and quantitation of diclofenac in the bile dialysates were achieved using a microbore reversed-phase C18 column (150×1.0 mm I.D.; particle size 5 μm) maintained at ambient temperature. Samples were eluted with a mobile phase containing 100 mM sodium dihydrogenphosphate (pH 3.1)–acetonitrile (30:70, v/v), and the flow-rate of the mobile phase was 0.05 ml/min. The UV detector wavelength was set at 280 nm. The concentration–response relationship from the present method indicated linearity (r 2>0.995) over a concentration range of 5–5000 ng/ml for diclofenac. Intra-assay and inter-assay precision and accuracy of diclofenac fell well within the predefined limits of acceptability (≤15%). The diclofenac in rat bile appeared to have a slow elimination phase, with a peak concentration at 20 min following diclofenac administration. The results demonstrated that diclofenac might be secreted into bile in unconjugated form by a canalicular bile acid transporter, and then go through hepatobiliary excretion. These results may provide good clinical evidence showing the value of diclofenac for the treatment of biliary colic. The elimination half-life of diclofenac in the biliary elimination was prolonged by treatment with cyclosporin A, indicating that the drug–drug interaction might affect the hepatobiliary excretion of diclofenac.
Keywords: Diclofenac; Cyclosporin A;

The application of coupled-column liquid chromatographic analysis to pharmacokinetic studies eliminates the need for sample clean-up from plasma. Considering lipophilic antineoplastic agents, we tested this approach to analyze paclitaxel under unfavourable circumstances (i.e., weekly low-dose regimen, plasma protein binding >90%, UV detection at 229 nm). The excellent quality control data (recovery: 95.6–100.7%, inter-assay relative standard deviation on 5 days: 1.3–3.2%, accuracy: 0.9–2.7%) and the detection limit of 19 nM indicates the usefulness of this method for the analysis of paclitaxel in plasma using on-line solid-phase extraction.
Keywords: Paclitaxel;

A new micro-technique is developed for purification of amyloid β peptides (Aβ) extracted from brain tissues of patients with Alzheimer’s disease (AD). It includes SDS–polyacrylamide gel electrophoresis of the extracted brain tissue material, electroblotting onto supporting membranes, and reversed-phase HPLC of the proteins eluted from membranes. By this technique, the extracted Aβ are first separated electrophoretically from the higher and lower molecular mass tissue components, and then purified by reversed-phase HPLC from the contaminants having similar molecular masses, but different retention times on the column. In contrast to the common large-scale isolation procedures employing density gradient centrifugation, enzymatic digestions and size-exclusion chromatography, the developed micro-technique might be applied for biochemical analysis of Aβ contained in small AD brain tissue specimens.
Keywords: Amyloid β peptides; Peptides;

Author Index 769 (371-373).

Compound Index 769 (375-377).