Maria Elisa Pereira Bastos de Siqueira

Simultaneous determination of multibenzodiazepines by HPLC/UV: Investigation of liquid–liquid and solid-phase extractions in human plasma

A method for simultaneous determination of seven benzodiazepines (BZPs) (flunitrazepam, clonazepam, oxazepam, lorazepam, chlordiazepoxide, nordiazepam and diazepam using N-desalkylflurazepam as internal standard) in human plasma using liquid-liquid and solid-phase extractions followed by high-performance liquid chromatography (HPLC) is described. The analytes were separated employing a LC-18 DB column (250 mm x 4.6 mm, 5 microm) at 35 degrees C under isocratic conditions using 5mM KH(2)PO(4) buffer solution pH 6.0:methanol:diethyl ether (55:40:5, v/v/v) as mobile phase at a flow rate of 0.8 mL min(-1). UV detection was carried out at 245 nm. Employing LLE, the best conditions were achieved with double extraction of 0.5 mL plasma using ethyl acetate and Na(2)HPO(4) pH 9.5 for pH adjusting. Employing SPE, the best conditions were achieved with 0.5 mL plasma plus 3 mL 0.1M borate buffer pH 9.5, which were then passed through a C18 cartridge previously conditioned, washed for 3 times with these solvents: 3 mL 0.1M borate buffer pH 9.5, 4 mL Milli-Q water and 1 mL acetonitrile 5%, finally the BZPs elution was carried with diethyl ether:n-hexane:methanol (50:30:20). In both methods the solvent was evaporated at 40 degrees C under nitrogen flow. The validation parameters obtained in LLE were linearity range of 50-1200 ng mL(-1) plasma (r>or=0.9927), limits of quantification of 50 ng mL(-1) plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15%, and recovery above 65% for all BZPs. In SPE, the parameter obtained were linearity range of 30-1200 ng mL(-1) plasma (r>or=0.9900), limits of quantification of 30 ng mL(-1) plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15% and recovery above 55% for all BZPs. These extracting procedures followed by HPLC analysis showed their suitable applicability in order to examine one or more BZPs in human plasma. Moreover, it could be suggested that these procedures might be employed in various analytical applications, in special for toxicological/forensic analysis.

On-line molecularly imprinted solid-phase extraction for the selective spectrophotometric determination of nicotine in the urine of smokers

This work describes an on-line molecularly imprinted solid-phase extraction (MISPE) method for spectrophotometric determination of nicotine in urine samples of smokers. This method is based on manganese (VII) to manganese (VI) reduction in an alkaline medium, promoted by nicotine. Two wash solutions (1:4 (v/v) acetonitrile:sodium hydroxide--pH 11.4, and nitric acid--pH 2.5) were employed to circumvent interferences. Aqueous solutions containing nicotine plus different possible concomitants (cotinine, anabasine, norcotinine and caffeine) were tested individually. The analytical calibration curve was prepared in urine samples collected from non-smokers and spiked with nicotine standard from 1.1 to 60 micromol L(-1) (r(2)>0.998). The limit of quantification and the analytical frequency were 1.1 micromol L(-1) and 11 h(-1), respectively. The precision, evaluated using 3, 10 and 30 micromol L(-1) nicotine in urine, was 10, 10 and 4% (intra-day precision) and 12, 13 and 5% (inter-day precision), respectively. Accuracy was checked through high performance liquid chromatography and the results did not present significant differences at the 95% confidence level according to the Students t-test.

Three-phase, liquid-phase microextraction combined with high performance liquid chromatography-fluorescence detection for the simultaneous determination of fluoxetine and norfluoxetine in human plasma

A three-phase, liquid-phase microextraction using a hollow fibre (HF-LPME) combined with high performance liquid chromatography-fluorescence detection (HPLC-FL) was developed for the analysis of fluoxetine (FLX) and its active metabolite, norfluoxetine (NFLX), in human plasma. An HF-LPME system using a disposable 7-cm polypropylene porous hollow fibre, 5 mL of alkaline plasma solution (donor phase), n-hexyl ether (extraction solvent) and 20 mM hydrochloric acid (acceptor phase) was used in the extraction. The method was validated after optimisation of several parameters that influence LPME efficiency. A reverse-phase LiChrospher 60 RP-Select B column (125 mm x 4 mm, 5 microm particle size) was used with 0.005 M sodium acetate buffer (pH 4.5) and acetonitrile at a 50:50 (v/v) as the mobile phase at a flow rate of 0.6 mL min(-1). In these conditions satisfactory chromatographic resolution and efficiency for the analytes were obtained. Fluorescence detection at 230 nm excitation wavelength and 290 nm emission wavelength was performed. Linearity over a range of 5-500 ng mL(-1), with determination coefficients (R(2)) of 0.9999 and 0.9962 for FLX and NFLX, respectively, was established. Venlafaxine was used as the internal standard for both analytes. Extraction recoveries from plasma samples were 70.9% for FLX and 59.7% for NFLX. The intra-day coefficients of variation (CVs) were below 5.4%, and inter-day CVs were below 13.0%, for both analytes at concentrations of 20, 80 and 160 ng mL(-1). HF-LPME extraction followed by HPLC-FL detection for FLX and NFLX analyses demonstrated excellent sample clean-up and selectivity. This method was simple, cheap, and easy to perform, yielding substantial analytes enrichment. The method was applied to the analysis of samples from 12 patients under fluoxetine treatment and proved suitable for routine therapeutic drug monitoring for this antidepressant.

Analysis of ortho-cresol in urine by solid phase microextraction-capillary gas chromatography

Neste trabalho foi desenvolvido um metodo usando a microextracao em fase solida (SPME) para analise cromatografica em fase gasosa capilar com detector de ionizacao por chama (CG/IC) de orto-cresol urinario, metabolito do tolueno. Apos otimizacao das variaveis da SPME e da validacao do metodo, foram analisadas amostras de urina de 27 trabalhadores expostos a solventes em oficinas de reparo automotivo. As melhores condicoes de extracao foram obtidas com a hidrolise acida da urina, extracao em fibra de carbowax/divinilbenzeno (CW/DVB) 70 µm por 20 min em pH neutro, com adicao de 3 g de Na2SO4, sob agitacao. O metodo mostrou linearidade entre 0,1 e 1,5 mg L-1 de o-cresol em urina, LOQ de 0,1 mg L-1, repetibilidade entre 6,8 e 7,8%. O valor medio de o-cresol em urina dos trabalhadores foi de 0,35 ± 0,23 mg L-1. O metodo SPME/CG/DIC mostrou ser rapido, simples e aplicavel para fins de biomonitorizacao de trabalhadores expostos.

Determination of benzene, toluene and N-hexane in urine and blood by headspace solid-phase microextration/gas-chromatography for the biomonitoring of occupational exposure

Analysis of unchanged toluene, benzene and n-hexane in human blood and urine can be useful to evaluate occupational exposure to these solvents. A simple method was developed using headspace-solid phase microextraction (HS-SPME) for simultaneous gas-chromatography analysis of toluene, benzene and n-hexane in urine and blood; with purpose of biological monitoring of occupational exposure. Carboxen/polidimethylsiloxane fiber (CAR-PDMS) and PDMS fiber coating were employed in this analysis. Blood and urine samples were collected from handling glue workers. The toluene was detected in all blood samples collected from workers handling glue in shoe repair shops, when using CAR-PDMS fibers (16.0-55.2 μg L-1, n=7). n-Hexane was only detected in two blood samples (33.0 and 41.3 μg L-1) and benzene was not detected in anyone. No solvent could be quantified in urine samples.

Influência do hábito de fumar na excreção urinária do ácido trans, trans-mucônico

Tobacco smoking is the major individual benzene source for no occupationally exposed subjects. This solvent is a volatile carcinogen and can induce serious health problems. Several biomarkers for benzene have been proposed, the most used is its metabolite trans,trans-muconic acid (ttMA) in urine, a suitable indicator since it reflects the internal dose of low benzene exposure. The present study aimed to evaluate the influence of smoking on urinary ttMA excretion. The analysis was performed by High Pressure Liquid Chromatography in a reversed-phase column and UV detection, after extraction of ttAM from urine using SAX cartridges. The method was validated, showing linearity between 0.006 to 10 µg mL-1 (r= 0.996), detection and quantification limits of 1.41 x 10-3 µg mL-1 and 6.0 x 10-3 µg mL-1 respectively, CVs from 1.79 to 2.91% (intra assay precision) and 3.53 to 18.37% (interassay precision). A significant positive correlation was observed between ttMA excretion and smoking (p= 0.005388). However, the cigarette number smoked by day seems have no influence on the urinary ttMA excretion.

Headspace solid-phase microextraction procedure for gas-chromatography analysis of toluene in urine

Toluene is widely used in industrial and laboratory applications and in many countries is related with social problems of abuse. Unaltered urinary toluene was introduced as a bioindicator of occupational exposure to the solvent, but its analysis presents difficulties due to the low levels of the compound excreted in urine. A gas-chromatography/flame ionization method for toluene in urine is described using headspace solid-phase microextraction and establishing the better conditions for two different extracting phases: polydimethylsiloxane (PDMS) and carboxen-PDMS. The carboxen-PDMS fiber showed lower quantifying limit (12.5 ng mL-1), better extraction efficiency (up to 28.1%) and repeatability (CV < 4.9%) than PDMS coating. The method was applied to analysis of toluene in urine of workers from car repair shops exposed to low solvent levels.

Single gas chromatography method with nitrogen phosphorus detector for urinary cotinine determination in passive and active smokers

Nicotine is a major addictive compound in cigarettes and is rapidly and extensively metabolized to several metabolites in humans, including urinary cotinine, considered a biomarker due to its high concentration compared to other metabolites. The aim of this study was to develop a single method for determination of urinary cotinine, in active and passive smokers, by gas chromatography with a nitrogen phosphorus detector (GC-NPD). Urine (5.0 mL) was extracted with 1.0 mL of sodium hydroxide 5 mol L-1, 5.0 mL of chloroform, and lidocaine used as the internal standard. Injection volume was 1 μL in GC-NPD. Limit of quantification was 10 ng mL-1. Linearity was evaluated in the ranges 10-1000 ng mL-1 and 500-6000 ng mL-1, with determination coefficients of 0.9986 and 0.9952, respectively. Intra- and inter-assay standard relative deviations were lower than 14.2 %, while inaccuracy (bias) was less than +11.9%. The efficiency of extraction was greater than 88.5%. Ruggedness was verified, according to Youdens test. Means of cotinine concentrations observed were 2,980 ng mL-1 for active smokers and 132 ng mL-1, for passive smokers. The results revealed that satisfactory chromatographic separation between the analyte and interferents was obtained with a ZB-1 column. This method is reliable, precise, linear and presented ruggedness in the range evaluated. The results suggest that it can be applied in routine analysis for passive and active smokers, since it is able to quantify a wide range of cotinine concentrations in urine.

Determination of Diazepam and Nordiazepam in the Plasma of Psychiatric Patients in Long-Term Treatment Employing Liquid-Liquid and Solid Phase Extraction by Gas Chromatography with Electron Capture Detection

A sensitive, rapid, and reproducible gas chromatography-electron capture detection (GC-ECD) method was developed for the determination of diazepam (DZP) and N-desmethyldiazepam (nordiazepam, NDZP) in plasma of psychiatric patients in long-term treatment. Liquid-liquid (LLE) and solid phase (SPE) extractions were employed as techniques of sample preparation. For both techniques, the extraction solvent was hexane: methanol (9: 1) at pH 9.0. The recoveries ranged from 89 to 99% for DZP and from 53 to 69% for NDZ. Some validation parameters were found: linearity in spiked human plasma from 5 to 1200 ng mL−1 for DZP and 10 to 1200 ng mL−1 for NDZP using SPE and LLE techniques. The values for within- and between-day precision (% coefficient of variation) and accuracy (% relative error) were lower than 15%. The methods were applied to the analysis of samples from 54 patients under DZP treatment and proved to be suitable for routine therapeutic drug monitoring. Both studied techniques showed similar results with correlation coefficients higher than 0.98 for the determination of DZP and NDZP in sample plasma of psychiatric patients in continuous use of DZP. The increase in DZP plasma levels was not linear with the increase in administered drug dose, denoting the influence of individual factors, and the magnitude of the therapeutical monitoring to verify the clinical response.