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.

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.

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.

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.

Determination of strychnine in human blood using solid-phase extraction and GC-EI-MS.

A rapid, simple, and sensitive method has been developed for the identification and quantitation of strychnine in human blood. The sample cleanup procedure involved solid-phase extraction with Oasis(R) HLB cartridges. The extracts were analyzed by gas chromatography-electron impact ionization-mass spectrometry. Limits of detection (LOD) and quantitation (LOQ) were 0.03 and 0.10 microg/mL, respectively, and the method was found to be linear between the LOQ and 2.5 microg/mL, with a correlation coefficient of 0.9994. Intra- and interday precision and accuracy were determined at both low and high concentrations (0.50 and 2.00 microg/mL). The CVs ranged from 5.63 to 8.50% and bias was within +/- 10% of the true value. Mean recovery of strychnine was 90.7%. Because of its simplicity and speed, the described method can be applied in forensic toxicology laboratories to determine this alkaloid in whole blood samples. Also, the fact that only 0.5 mL of blood is required to accomplish the analysis make this procedure useful in situations where several exams are needed and the sample volume is limited.

Application of solid phase microextraction to the determination of strychnine in blood.

A simple and rapid method based on solid phase microextraction (SPME) via direct immersion followed by gas chromatography coupled with electron impact ionization/mass spectrometry (GC/EI-MS) was developed for the determination of strychnine in blood. Papaverine was used as internal standard (I.S.). Two types of fibre coating were tested, 100 microm polydimethylsiloxane and 65 microm Carbowax/Divinylbenzene, the latter giving higher recoveries of the compound. The main factors affecting the SPME process, such as sample dilution (1:10), adsorption and desorption times (20 and 10 min, respectively), carry-over effect (not observed), pH and salt addition (no modifications on pH or salt concentration) were optimized. The procedure was validated in terms of linearity (r(2)=0.9992 for concentrations ranging from 0.10 to 5.00 microg/mL), intra and interday precision (0.93 and 4.62%, respectively at 0.50 microg/mL; 3.33 and 8.06%, respectively at 2.50 microg/mL), sensitivity (6.83 and 8.91 ng/mL for LOD and LOQ, respectively) and extraction recovery (0.54 and 0.39% at 0.50 and 2.50 microg/mL, respectively). The developed procedure was found suitable for forensic investigations and was considered a good alternative to the liquid-liquid extraction methods normally used for the determination of this compound in biological media.

Development and validation of a method for the quantitation of Delta9tetrahydrocannabinol in oral fluid by liquid chromatography electrospray-mass-spectrometry.

Analysis of Delta(9)tetrahydrocannabinol (Delta(9)THC) and its metabolites in biological samples is of great relevance for forensic purposes. In the case of oral fluid (OF), the analysis should determine Delta(9)THC, whereas in urine, it detects the inactive metabolite tetrahydrocannabinol carboxylic acid (THC-COOH). Most laboratories analyze Delta(9)THC in such samples using GC-MS methods, but these procedures are time-consuming and involve unavoidable previous extraction and derivatization. No data is yet available on the application of liquid chromatography-mass-spectrometry to detect Delta(9)THC in oral fluid. We report a validation method in which the Delta(9)THC is isolated from oral fluid by a simple liquid-liquid extraction with hexane and subsequently analyzed by liquid chromatography-mass-spectrometry. The method here reported for the determination of Delta(9)THC in oral fluid only requires 200 microl of sample and achieves limits of detection of 2 ng/ml, and has been used to analyze oral fluid samples collected from current drug users.

Vitreous humour as a complementary sample to blood for the detection/confirmation of diazepam: ante-mortem and post-mortem studies in an animal model.

The quantification of medical or toxic substances in vitreous humour (VH) could be very useful in forensic toxicology when blood sample determinations are impossible due to absence or deterioration. However, few studies have been made in this area and even fewer have tried to find a relationship between drug levels in both samples. To determine a correlation ratio between blood and VH diazepam (DZ) levels, we performed an experimental study using rabbits administered with a sub-toxic dose of DZ under known and controlled conditions. Blood and VH samples were collected 0.5, 1, 2, 3 and 6 hours after the drug administration in order to determine DZ and its main active metabolite, desmethyldiazepam (DMD). In addition, we have studied an animal group sacrificed 2 hours after intramuscular (i.m.) drug administration with blood and VH collection 24 hours later, to evaluate the existence of possible post-mortem changes. After DZ administration, a fast absorption phase was observed with a plasma Cmax value 1 hour after, followed by a rapid concentration decrease, with a half-life of 1 hour, indicating that, besides elimination, a fast distribution to other organs and tissues and/or hepatic metabolism occurred. Diazepam Cmax value in VH was achieved between 1 and 2 hours, when plasma concentrations had already decreased to half the value. The plasma/VH DZ ratio calculated at this time was 10. In the post-mortem study, while plasma DZ concentration at 24 hours was smaller, DMD levels were higher than those at the time of death. In the VH, both DZ and DMD concentrations at 24 hours were higher than those obtained at the time of death. That is, in both fluids DZ and DMD concentrations were different from those at the time of death and post-mortem distribution and redistribution phenomena occurred. The combination of ante-mortem and post-mortem studies has allowed the determination of a correlation ratio for DZ in the rabbit of 6 x, comparing the concentrations in VH collected 24 hours after death with the concentrations detected in plasma at the time of death. This study opens new perspectives for the use of VH as a complementary sample to blood for DZ detection and confirmation. The putative relevance of the correlation ratio obtained, for forensic toxicology practice with medical substances, namely benzodiazepines, recommends further studies in humans.

Determination of methemoglobin and total hemoglobin in toxicological studies by derivative spectrophotometry.

A method for the determination of methemoglobin in the presence of other hemoglobin subforms (i.e., oxy-, deoxy-, and carboxyhemoglobin) by use of derivative spectrophotometry is proposed. The method, which uses the first-derivative of the spectrum at 645 nm, is straightforward and expeditious, so it is of special interest to forensic toxicology laboratories as it allows the simultaneous determination of the methemoglobin saturation percentage and the hemoglobin concentration. This facilitates interpretation of the results and provides a better understanding of the significance of methemoglobin saturation in specific cases. Based on an analysis of interferences, the presence of other hemoglobin subforms or of endogenous components of plasma does not detract in any way from the performance of the method.