Occurrence and fate of CECs (OMPs, ARGs and pathogens) during decentralised treatment of black water and grey water.

Decentralised wastewater treatment is becoming a suitable strategy to reduce cost and environmental impact. In this research, the performance of two technologies treating black water (BW) and grey water (GW) fractions of urban sewage is carried out in a decentralised treatment of the wastewater produced in three office buildings. An Anaerobic Membrane Bioreactor (AnMBR) treating BW and a Hybrid preanoxic Membrane Bioreactor (H-MBR) containing small plastic carrier elements, treating GW were operated at pilot scale. Their potential on reducing the release of contaminants of emerging concern (CECs) such as Organic Micropollutants (OMPs), Antibiotic Resistance Genes (ARGs) and pathogens was studied. After 226 d of operation, a stable operation was achieved in both systems: the AnMBR removed 92.4 ± 2.5 % of influent COD, and H-MBR removed 89.7 ± 3.5 %. Regarding OMPs, the profile of compounds differed between BW and GW, being BW the matrix with more compounds detected at higher concentrations (up to µg L-1). For example, in the case of ibuprofen the concentrations in BW were 23.63 ± 3.97 µg L-1, 3 orders of magnitude higher than those detected in GW. The most abundant ARGs were sulfonamide resistant genes (sul1) and integron class 1 (intl1) in both BW and GW. Pathogenic bacteria counts were reduced between 1 and 3 log units in the AnMBR. Bacterial loads in GW were much lower than in BW, being no bacterial re-growth observed for the GW effluents after treatment in the H-MBR. None of the selected enteric viruses was detected in GW treatment line.

LC-MS-MS Method for the Determination of Antidepressants and Benzodiazepines in Meconium.

An LC-MS-MS method for the determination of 14 benzodiazepines (BZDs) (alprazolam, α-hydroxyalprazolam, clonazepam, bromazepam, diazepam, nordiazepam, lorazepam, lormetazepam, oxazepam, flunitrazepam, 7-aminoflunitrazepam, triazolam, midazolam and zolpidem) and 15 antidepressants (ADs) (amitriptyline, nortriptyline, imipramine, desipramine, clomipramine, norclomipramine, fluoxetine, norfluoxetine, sertraline, norsertraline, paroxetine, venlafaxine, desmethylvenlafaxine, citalopram and desmethylcitalopram) in meconium was developed and validated. Meconium samples (0.25 ± 0.02 g) were homogenized in methanol and subjected to mixed-mode cation exchange solid-phase extraction. Chromatographic separation was performed in reversed phase, with a gradient of 0.1% formic acid in 2 mM ammonium formate and acetonitrile. Two different chromatographic gradient methods were employed, one for the separation of ADs and another for BZDs. Analytes were monitored by tandem mass spectrometry employing electrospray positive mode in MRM mode (2 transitions per compound). Method validation included: linearity [n = 5, limit of quantification (LOQ) to 400 ng/g], limits of detection (n = 6, 1-20 ng/g), LOQ (n = 9, 5-20 ng/g), selectivity (no endogenous or exogenous interferences), accuracy (n = 15, 90.6-111.5%), imprecision (n = 15, 0-14.6%), matrix effect (n = 10, -73 to 194.9%), extraction efficiency (n = 6, 35.9-91.2%), process efficiency (n = 6, 20.1-188.2%), stability 72 h in the autosampler (n = 3, -8.5 to 9%) and freeze/thaw stability (n = 3, -1.2 to -47%). The method was applied to four meconium specimens, which were analyzed with and without hydrolysis (enzymatic and alkaline). The authentic meconium samples tested positive for alprazolam, α-hydroxyalprazolam, clonazepam, diazepam, nordiazepam, fluoxetine, norfluoxetine, clomipramine and norclomipramine. Therefore, the present LC-MS-MS method allows a high throughput determination of the most common BZDs and ADs in meconium, which could be useful in clinical and forensic settings.

Intoxicaciones inadvertidas por monóxido de carbono: una epidemia oculta / Intoxications inadvertent carbon monoxide: an epidemic hidden

Diferentes autores afirman que existe un elevado volumen de intoxicaciones por monóxido de carbono que pasan inadvertidas para el personal sanitario, lo que representa un importante problema de salud. Para confirmar esta hipótesis en un área de salud determinada, sobre la que se dispone de reciente información en relación al volumen de intoxicaciones que sí son detectadas, se realizó este trabajo. Para ello, se seleccionó como población de estudio al conjunto de pacientes que acudieron al Servicio de Urgencias del Hospital do Salnes, por cualquier motivo, durante el mes de febrero de 2013. Se recogió una amplia muestra representativa de esta población por un sistema aleatorio sistemático, quedando constituida por 1501 pacientes. A todos ellos se les realizó una medición no invasiva de carboxihemoglobina mediante pulsicooximetría con el objetivo de detectar todas las intoxicaciones, tanto las sospechadas por el personal sanitario como las que pasarían inadvertidas. El número de intoxicaciones detectadas en la muestra fue de 10, lo que representa el 0,7% (± 0.34% p< 0.05) de las urgencias atendidas, siendo 18 (8,8-27,1) el número de casos esperados para la población de estudio. Esta cifra fue muy superior a la de intoxicados que se detectaron en el trabajo ordinario del personal sanitario durante los diez años previos, en el mismo mes de febrero y en el mismo centro sanitario, con una media anual de 1.3 casos (DS: 2.8), por lo que se concluye confirmando la hipótesis de que el número de intoxicaciones inadvertidas por monóxido de carbono es muy elevado (AU)

Different authors state that there is a high volume of carbon monoxide poisoning that go unnoticed for health workers, representing a major health problem. To confirm this hypothesis in a particular area of ​​health, on which recent information regarding the volume of poisonings that are detected itself, this work was performed. To do this, it was selected as study population to all patients presenting to the emergency department of Salnés Hospital, for any reason, during the month of February 2013. A broad cross-section of the population was collected by a systematic random system , it is composed of 1501 patients. All subjects underwent a noninvasive measurement of carboxyhemoglobin by pulsicooximetría in order to detect all poisonings, both suspected by medical personnel as they go unnoticed. The number of poisonings detected in the sample was 10, representing 0.7% (± 0.34% p <0.05) of emergencies attended, with 18 (8.8 to 27.1) for the number of expected cases the study population. This figure was much higher than that of intoxicated that were detected in the ordinary work of health workers during the previous ten years, in the month of February and at the same health center with an annual average of 1.3 cases (DS: 2.8) , so it is concluded confirming the hypothesis that the number of intoxications inadvertent carbon monoxide is very high

Molecularly imprinted polymer for selective determination of Δ9-tetrahydrocannabinol and 11-nor-Δ9-tetrahydrocannabinol carboxylic acid using LC-MS/MS in urine and oral fluid.

The use of molecularly imprinted polymers (MIPs) for solid phase extraction (MISPE) allows a rapid and selective extraction compared with traditional methods. Determination of Δ(9)-tetrahydrocannabinol (THC) and 11-nor-Δ(9)-tetrahydrocannabinol carboxylic acid (THC-COOH) in oral fluid (OF) and urine was performed using homemade MISPEs for sample clean-up and liquid chromatography tandem mass spectrometry (LC-MS/MS). Cylindrical MISPE shaped pills were synthesized using catechin as a mimic template. MISPEs were added to 0.5 mL OF or urine sample and sonicated 30 min for adsorption of analytes. For desorption, the MISPE was transfered to a clean tube, and sonicated for 15 min with 2 mL acetone:acetonitrile (3:1, v/v). The elution solvent was evaporated and reconstituted in mobile phase. Chromatographic separation was performed using a SunFire C18 (2.5 µm; 2.1 × 20 mm) column, and formic acid 0.1% and acetonitrile as mobile phase, with a total run time of 5 min. The method was fully validated including selectivity (no endogenous or exogenous interferences), linearity (1-500 ng/mL in OF, and 2.5-500 ng/mL in urine), limit of detection (0.75 and 1 ng/mL in OF and urine, respectively), imprecision (%CV <12.3%), accuracy (98.2-107.0% of target), extraction recovery (15.9-53.5%), process efficiency (10.1-46.2%), and matrix effect (<-55%). Analytes were stable for 72 h in the autosampler. Dilution 1:10 was assured in OF, and Quantisal™ matrix effect showed ion suppression (<-80.4%). The method was applied to the analysis of 20 OF and 11 urine specimens. This is the first method for determination of THC and THC-COOH in OF using MISPE technology.

Bioanalytical procedures and recent developments in the determination of opiates/opioids in human biological samples.

The use and abuse of illegal drugs affects all modern societies, and therefore the assessment of drug exposure is an important task that needs to be accomplished. For this reason, the reliable determination of these drugs and their metabolites in biological specimens is an issue of utmost relevance for both clinical and forensic toxicology laboratories in their fields of expertise, including in utero drug exposure, driving under the influence of drugs and drug use in workplace scenarios. Most of the confirmatory analyses for abused drugs in biological samples are performed by gas chromatographic-mass spectrometric methods, but use of the more recent and sensitive liquid chromatography-(tandem) mass spectrometry technology is increasing dramatically. This article reviews recently published articles that describe procedures for the detection of opiates in the most commonly used human biological matrices, blood and urine, and also in unconventional ones, e.g. oral fluid, hair, and meconium. Special attention will be paid to sample preparation and chromatographic analysis.

Ginecomastia y cáncer de mama en el varón. Estudio de 49 casos / No disponible

Objetivo: Estudiar la posible relación entre la ginecomastia y el cáncer de mama en el varón. Material y métodos: Estudio descriptivo retrospectivo con revisión de los pacientes varones, mayores de 14 años, con ginecomastia intervenidos quirúrgicamente en nuestro hospital desde enero del año 1996 hasta diciembre de 2009. Para el análisis de los datos, los casos se dividieron en 2 grupos, atendiendo a la benignidad o malignidad en el estudio anatomopatológico. Resultados: El número total de casos analizados fue de 49, de los que 10 presentaron un diagnóstico anatomopatológico de cáncer de mama y 39 de patología benigna. Se ha encontrado una relación estadísticamente significativa entre la edad de presentación de la ginecomastia y el riesgo relativo de padecer cáncer de mama (p = 0,002). Conclusiones: Aunque no se ha podido demostrar que la ginecomastia sea un factor de riesgo para el cáncer de mama, este debe ser un diagnóstico diferencial a tener en cuenta en pacientes con ginecomastia. Según nuestros resultados, esta sospecha debería ser mayor cuanto mayor sea la edad del paciente(AU)

Objetive: To study the possible association between gynecomastia and breast cancer in men. Material and methods: A retrospective case-control study was designed. Male patients older than 14 years old with breast disorder undergoing surgery in our hospital between January 1996 and December 2009 were included. Two groups of patients were made, those with malignant disease and those with benign lesions. Results: The total number of cases analyzed was 49, 10 with pathological diagnosis of breast cancer and 39 of benign disease. A significant difference was found only for age at onset of gynecomastia and relative risk of breast cancer (p = 0.002). Conclusions: Although it was not possible to demonstrate that gynecomastia is a risk factor for breast cancer, it must be a differential diagnosis to be considered in patients with gynecomastia. According to our results, this suspicion should be higher with increasing patient age(AU)

Mixed-mode solid-phase extraction for sample cleanup in hair analysis for methadone and its main metabolite.

A simple and rapid method for the determination of methadone and its main metabolite EDDP in hair has been developed and validated. The analytes were completely extracted from the matrix after a short alkaline incubation, and the extracts were further cleaned up by solid-phase extraction using mixed-mode cartridges. Linearity was obtained from 0.1 (lower limit of quantitation, LLOQ) to 30 ng/mg for both compounds, with correlation coefficients higher than 0.99. Intra- and interday precision and accuracy were in conformity with internationally accepted guidelines for bioanalytical method validation, and the cleanup procedure presented mean extraction efficiencies higher than 90% for both analytes. This high efficiency greatly contributed to the low limits of quantitation achieved, and therefore this method can be successfully applied in the determination of methadone and EDDP in hair samples in clinical and forensic scenarios where these compounds are involved.

Analysis of phenylpiperazine-like stimulants in human hair as trimethylsilyl derivatives by gas chromatography-mass spectrometry.

A simple and sensitive procedure, using p-tolylpiperazine (pTP) as internal standard (IS), has been developed and validated for the qualitative and quantitative analysis of 1-(3-trifuoromethylphenyl)piperazine (TFMPP), 1-(3-chlorophenyl)piperazine (mCPP) and 1-(4-methoxyphenyl)piperazine (MeOPP) in hair. Drug extraction was performed by incubation with 1 M sodium hydroxide at 50°C for 40 min, and the extracts were cleaned up using mixed-mode solid-phase extraction. The analytes were derivatized with N-methyl-N-(trimethylsilyl) trifluoroacetamide with 5% trimethylchlorosilane and analysed by gas chromatography-mass spectrometry in the selected ion monitoring mode. The method was linear from 0.05 (lower limit of quantitation) to 4 ng mg(-1), with correlation coefficients higher than 0.99 for all the compounds. Intra- and interday precision and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation, and the sample cleanup step presented a mean efficiency higher than 90% for all the analytes. Due to its simplicity and speed, this method can be successfully applied in the screening and quantitation of these compounds in hair samples, and is suitable for application in forensic toxicology routine analysis.

Simultaneous quantitation of morphine, 6-acetylmorphine, codeine, 6-acetylcodeine and tramadol in hair using mixed-mode solid-phase extraction and gas chromatography-mass spectrometry.

A simple procedure has been developed and validated for the qualitative and quantitative analysis of several opiates (morphine, 6-acetylmorphine, codeine, 6-acetylcodeine) and tramadol in hair. The analytes were extracted from within the matrix via an overnight incubation with methanol at 65 degrees C, and afterwards the samples were cleaned up by mixed-mode solid-phase extraction. The extracts were derivatized with N-methyl-N-(trimethylsilyl) trifluoroacetamide with 5% trimethylchlorosilane and analyzed by gas chromatography-mass spectrometry in the selected ion monitoring mode. The method was linear from 0.05 (lower limit of quantitation) to 50 ng/mg (40 ng/mg for tramadol), with correlation coefficients higher than 0.99 for all compounds, accomplishing the cut-off values proposed by the Society of Hair Testing for the detection of these substances in hair (0.2 ng/mg). Intra- and interday precision and trueness were in conformity with the criteria normally accepted in bioanalytical method validation, and the sample cleanup step presented a mean efficiency higher than 90% for all analytes. Furthermore, using these incubation conditions, 6-acetylmorphine did not significantly hydrolyze to morphine. For these reasons, and because of its simplicity, the proposed method can be successfully applied in the determination of these compounds in hair samples, and is suitable for application in routine analysis with forensic purposes.

Development and validation of an analytical method for the simultaneous determination of cocaine and its main metabolite, benzoylecgonine, in human hair by gas chromatography/mass spectrometry.

A new, simple and rapid procedure has been developed and validated for the determination of cocaine and its main metabolite, benzoylecgonine, in human hair samples. After extraction from within the hair matrix by a mixture of methanol/hydrochloric acid (2:1) at 65 degrees C for 3 h, and sample cleanup by mixed-mode solid-phase extraction (SPE), the extracts were analyzed by gas chromatography/mass spectrometry (GC/MS), after derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide with 5% chlorotrimethylsilane. Using a sample size of only 20 mg of hair, limits of detection (LODs) and quantitation (LOQs) were, respectively, 20 and 50 pg/mg for cocaine, and 15 and 50 pg/mg for benzoylecgonine, achieving the cut-off values proposed by the Society of Hair Testing for the analysis of these compounds in hair. The method was found to be linear (weighing factor of 1/x) between the LOQ and 20 ng/mg for both compounds, with correlation coefficients ranging from 0.9974 to 0.9996 for cocaine; and from 0.9981 to 0.9994 for benzoylecgonine. Intra- and interday precision and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The sample cleanup step presented a mean absolute recovery greater than 90% for both compounds. The developed method may be useful in forensic toxicology laboratories for the analysis of cocaine and benzoylecgonine in hair samples, taking into account its speed (only 3 h are required for the extraction of the analytes from within the matrix, whereas 5 h or even overnight extractions have been reported) and the low limits achieved (using a single quadrupole mass spectrometer, which is available in most laboratories).

LC-MS/MS method for the determination of nine antidepressants and some of their main metabolites in oral fluid and plasma. Study of correlation between venlafaxine concentrations in both matrices.

In this paper, a fast, sensitive and selective LC-MS/MS method is described for the simultaneous determination of amitriptyline, imipramine, clomipramine, fluoxetine, paroxetine, sertraline, fluvoxamine, citalopram and venlafaxine, as well as some of their main metabolites (nortriptyline, desipramine, norclomipramine and norfluoxetine), in oral fluid and plasma. The sample (0.2 mL) was extracted with an automated solid-phase extraction system (ASPEC XL), using mixed mode OASIS MCX cartridges. Chromatographic separation was performed in a Sunfire C18 IS column (20 mmx2.1 mm, 3.5 microm), using a gradient of acetonitrile and ammonium formate (pH 3; 2 mM) as mobile phase, which allowed the elution of all the compounds in less than 5 min. The method has been fully validated in both specimens. This method was initially applied to the analysis of oral fluid and plasma samples from patients on antidepressant treatment in order to assess for which compounds it was likely to find a good correlation between both matrices. The best results were obtained for venlafaxine, so the study was extended for this compound, comparing the ratio between oral fluid and plasma concentrations (ROF/PL) in five patients on venlafaxine treatment when both samples were collected simultaneously on four different occasions. An important inter and intraindividual variability was found in oral fluid concentrations for 150 mg dose (mean=287.5 ng/m, range 58.8-531.2 ng/mL) and for 75 mg dose (mean=186.3 ng/mL, range=82.1-289.2 ng/mL). R(OF/PL) was calculated for each patient on the four different occasions, showing also a high variability (CV=24.2-69.6%).

Determination of parathion in biological fluids by means of direct solid-phase microextraction.

A new and simple procedure for the determination of parathion in human whole blood and urine using direct immersion (DI) solid-phase microextraction (SPME) and gas chromatography/mass spectrometry (GC/MS) is presented. This technique was developed using only 100 microL of sample, and ethion was used as internal standard (IS). A 65-microm Carbowax/divinylbenzene (CW/DVB) SPME fibre was selected for sampling, and the main parameters affecting the SPME process such as extraction temperature, adsorption and desorption time, salt addition, agitation and pH effect were optimized to enhance the sensitivity of the method. This optimization was also performed to allow the qualitative determination of parathion's main metabolite, paraoxon, in blood. The limits of detection and quantitation for parathion were 3 and 10 ng/mL for urine and 25 and 50 ng/mL for blood, respectively. For paraoxon, the limit of detection was 50 ng/mL in blood. The method showed linearity between the LOQ and 50 microg/mL for both matrices, with correlation coefficients ranging from 0.9954 to 0.9999. Precision and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The mean absolute recoveries were 35.1% for urine and 6.7% for blood. Other parameters such as dilution of sample and stability were also validated. Its simplicity and the fact that only 100 microL of sample is required to accomplish the analysis make this method useful in forensic toxicology laboratories to determine this compound in intoxications, and it can be considered an alternative to other methods normally used for the determination of this compound in biological media.

A high-performance liquid chromatography method for hypoxanthine determination in vitreous humour: application to estimation of post mortem interval.

We have developed a new technique to determine the concentration of hypoxanthine [Hx] in a reverse phase column using a modified high-performance liquid chromatography (HPLC) method that is faster and more reliable than those previously described. In this paper we present a formula for estimating the post mortem interval (PMI) based on this HPLC method by applying the inverse prediction method. The regression line obtained by changing the variables gives PMI = 0.183 [Hx] + 0.599 (PMI in hours, [Hx] in micromol/L, R2 = 0.531, P < 0.05).

Determination of methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine and alprazolam in human plasma by liquid chromatography-electrospray ionization mass spectrometry.

A fast liquid chromatographic assay with mass spectrometric detection (LC/MS) has been developed and validated for the simultaneous determination of methadone (MT), its primary metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and alprazolam, in human plasma. The extraction procedure was performed with automatic solid phase extraction, and the compounds were separated with a Sunfire column using a gradient mode. Deuterated analogues for all of the analytes of interest were used for quantitation. Limits of detection (LOD) were established between 0.5 and 1 ng/ml. Linearity was obtained over a range of 2-2,000 ng/ml with an average correlation coefficient (R(2)) of >0.99. Intra- and inter-batch coefficients of variation and relative mean errors were less than 10% for all analytes and concentrations. The recoveries were higher than 50.0% in all cases. The method proved to be suitable for evaluation of plasma obtained from patients enrolled in a MT-maintenance program who are frequently treated with alprazolam as a sedative.

Mutant p53 protein in serum could be used as a molecular marker in human breast cancer.

p53 wild-type is a tumor suppressor gene involved in DNA gene transcription or DNA repair mechanisms. When damage to DNA is unrepairable, p53 induces programmed cell death (apoptosis). The mutant p53 gene is the most frequent molecular alteration in human cancer, including breast cancer. Here, we analyzed the genetic alterations in p53 oncogene expression in 55 patients with breast cancer at different stages and in 8 normal women. We measured by ELISA assay the serum levels of p53 mutant protein and p53 antibodies. Immunohistochemistry and RT-PCR using specific p53 primers as well as mutation detection by DNA sequencing were also evaluated in breast tumor tissue. Serological p53 antibody analysis detected 0/8 (0%), 0/4 (0%) and 9/55 (16.36%) positive cases in normal women, in patients with benign breast disease and in breast carcinoma, respectively. We found positive p53 mutant in the sera of 0/8 (0.0%) normal women, 0/4 (0%) with benign breast disease and 29/55 (52.72%) with breast carcinoma. Immunohistochemistry evaluation was positive in 29/55 (52.73%) with mammary carcinoma and 0/4 (0%) with benign breast disease. A very good correlation between p53 mutant protein detected in serum and p53 accumulation by immunohistochemistry (83.3% positive in both assays) was found in this study. These data suggest that detection of mutated p53 could be a useful serological marker for diagnostic purposes.

Solid-phase microextraction for gas chromatographic/mass spectrometric analysis of dimethoate in human biological samples.

A new, simple and rapid procedure for the determination of dimethoate in urine and blood samples was developed using direct immersion solid-phase microextraction and gas chromatography/mass spectrometry. This technique required only 0.1 mL of sample, and ethion was used as internal standard. Two types of coated fibre were compared (100 microm polydimethylsiloxane, and 65 microm Carbowax/divinylbenzene). Other parameters, such as extraction temperature, adsorption and desorption time, salt addition, agitation and pH, were optimized to enhance the sensitivity of the method. Limits of detection (LODs) and quantitation (LOQs) were 50 and 100 ng/mL for urine and 200 and 500 ng/mL for blood, respectively. The method was found to be linear between the LOQ and 40 microg/mL for urine, and between the LOQ and 50 microg/mL for blood, with correlation coefficients ranging from 0.9923-0.9996. Precision (intra- and interday) and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The mean absolute recoveries of dimethoate were 1.24 and 0.50% for urine and blood, respectively. Because of its simplicity and the fact that small volumes of sample are used, the described method can be successfully used in the diagnosis of poisoning by this pesticide, namely in those situations where the sample volume is limited, as frequently occurs in forensic toxicology.

Determination of quinalphos in blood and urine by direct solid-phase microextraction combined with gas chromatography-mass spectrometry.

A new method based on direct solid-phase microextraction (DI-SPME) followed by gas chromatography-mass spectrometry was developed for the purpose of determining quinalphos in blood and urine. Two types of coated fibre have been assayed and compared: carbowax/divinylbenzene (CW/DVB 65 microm) and polydimethylsiloxane (PDMS 100 microm). The main parameters affecting the SPME process such as temperature, salt addition, pH, stirring and adsorption/desorption time profiles were optimized to enhance the sensitivity of the procedure. The method was developed using only 100 microL of blood and urine. Limits of detection of the method for blood and urine matrices were, respectively, 10 and 2 ng/mL. Linearity was established over concentration ranges from 0.05 to 50 microg/mL for blood, and 0.01 to 50 microg/mL for urine, with regression coefficients ranging between 0.9991 and 0.9999. Intra- and interday precision values were less than 13%, and accuracy was within +/-15% of the nominal concentration for all studied levels in both matrices. Absolute recoveries were 14 and 26% for blood and urine, respectively.

Liquid chromatography-electrospray ionisation mass spectrometry for the determination of nine selected benzodiazepines in human plasma and oral fluid.

A new simple and rapid liquid chromatographic-mass spectrometric technique was designed for the determination of nine benzodiazepines in plasma and oral fluid. Benzodiazepines were extracted from alkalinised spiked and clinical plasma and oral fluid samples using a single step, liquid-liquid extraction procedure with diethyl ether. The chromatographic separation was performed with a Xterra RP18, 5 microm (150 x 2.1 mm i.d.) reversed-phase column using deuterated analogues of the analytes as internal standard. The recovery ranged from 70.3 to 86.9% for plasma and 63.9 to 77.2% for oral fluid. The limits of detection ranged from 0.5 to 1 ng/ml in plasma and 0.1 to 0.2 ng/ml for oral fluid. The method was validated for all the compounds, including linearity and the main precision parameters. The procedure, showed to be sensitive and specific, was applied to real plasma and oral fluid samples. The method is especially useful to analyse saliva samples from drivers undergoing roadside drug controls.