P. Fernández

Use of solid-phase microextraction (SPME) for the determination of methadone and EDDP in human hair by GC–MS

Solid-phase microextraction (SPME) is a new extraction technique with many advantages: small sample volume, simplicity, quickness and solvent-free. It is mainly applied to environmental analysis, but is also useful for the extraction of drugs from biological samples. In this paper the use of SPME is proposed for the determination of methadone and its main metabolite EDDP in hair by GC-MS. The hair samples were washed, cut into 1-mm segments, and incubated with Pronase E for 12 h. A 100-micron polydimethylsiloxane (PDMS) film fibre was submerged for 30 min in a diluted solution of the hydrolysis liquid (1:4 with borax buffer) containing methadone-d3 and EDDP-d3 as internal standards. Once the microextraction was concluded the fibre was directly inserted into the CG injection port. Linearity was found for methadone and EDDP in the range studied, 1.0-50 ng/mg hair, with correlation coefficients higher than 0.99. Interassay relative standard deviation (R.S.D) was determined to be less than 13.30% for methadone and less than 8.94% for EDDP, at 3.0 and 30.0 ng/mg. Analytical recoveries were close to 100% for both compounds on spiked samples. The method was applied to the analysis of real hair samples from eight patients of a methadone maintenance programme. The concentration of methadone in hair ranged from 2.45 to 78.10 ng/mg, and for EDDP from 0.98 to 7.76 ng/mg of hair.

Optimization of a rapid microwave-assisted extraction method for the simultaneous determination of opiates, cocaine and their metabolites in human hair

A rapid and cleanup-free microwave-assisted extraction (MAE) method is proposed for the simultaneous extraction of six illegal drugs of abuse - cocaine, benzoylecgonine (BZE), cocaethylene (CCE), morphine, 6-monoacethylmorphine (6AM) and codeine - from human hair samples. The analytes were determined using high performance liquid chromatography (HPLC) with photodiode array UV detection. The influence of several variables on the efficiency of the MAE procedure was investigated in detail by a multi-objective optimization approach based on a hybrid experimental design (17 experiments) and desirability functions. Six drugs were successfully extracted from human hair with recoveries close to 100% and good reproducibility (<3.6% RSD) under the optimal MAE conditions: 11 mL dichloromethane (DCM) extraction solvent, 60 degrees C extraction temperature, 9 min extraction time and 0.5 mL of methanol (MeOH) added to 50mg of the hair sample in the extraction vessels. Limits of quantification of 0.2 ng mg(-1) were found for the studied compounds. A comparison of sample preparation procedures, including MAE, enzymatic digestion and digestion by aqueous acids, was also conducted. The results indicated that the global behaviour of sample procedures provided similar satisfactory recoveries ranging from 86 to 100%. Indeed, the MAE procedure resulted in a reduction of extraction time by 100-fold and the elimination of cleanup steps. Slightly higher recoveries of morphine, 6AM, BZE and CCE, at 1 ng mg(-1) concentration level and cocaine at 40 ng mg(-1) concentration level, were achieved using MAE. Lastly, the proposed MAE method was applied to several human hair samples from multidrug abusers.

Microwave-assisted extraction: a simpler and faster method for the determination of ethyl glucuronide in hair by gas chromatography–mass spectrometry

Alcohol is the most frequently abused “addictive substance” that causes serious social problems throughout the world; thus, alcoholism is of particular interest in clinical and forensic medicine. Alcohol biomarkers are physiological indicators of alcohol exposure or ingestion and may reflect the presence of an alcohol use disorder. The glucuronide conjugation is a minor pathway of ethanol metabolism. Ethyl glucuronide (EtG) is a marker of recent alcohol consumption that detects alcohol use reliably over a definite time period. The present paper describes a new method for the determination of EtG in hair. It is based both in the microwave-assisted extraction (MAE), to extract the analyte from hair samples, and gas chromatography–mass spectrometry (GC-MS), to identify and quantify the EtG in selected ion monitoring (SIM) mode. The method was applied to 15 hair samples from occasional alcohol users, obtaining positive results in all cases. It was fully validated, including a linear range (0.3–10xa0ng/mg) and the main precision parameters. In summary, the use of microwave-assisted extraction turned out to be a substantially simpler, faster, and a more sensitive procedure than any other conventional sample preparations.

Application of dispersive liquid-liquid microextraction for the determination of phosphatidylethanol in blood by liquid chromatography tandem mass spectrometry.

Phosphatidylethanol (PEth) is a phospholipid which requires for its metabolic formation the presence of relatively high ethanol levels. PEth is thus a promising marker to quentify ethanol abuse. Dispersive liquid-liquid microextraction has become a popular technique because it is fast, inexpensive, easy to operate and consumes low volume of organic solvent. In this method, the appropriate mixture of extraction solvent (230 μL dichloromethane) and disperser solvent (630 μL acetone) are injected into the sample by syringe, rapidly. The liquid chromatography method using a reversed phase-C8 column and a negative ion mode electrospray ionization tandem mass spectrometry detection instrument was developed for the determination of small amounts of PEth that might be present in blood samples, using phosphatidylbutanol (PBut) as an internal standard. The sensitivity of detection obtained with tandem MS was better than that of previous methods. Good linearity was obtained for a range of LOQ-10 μg/mL for PEth, whereas all of the deviations in precision and accuracy were less than 15% except for the LLOQ, where it should not exceed 20%. A set of 50 blood samples were analyzed by such method and whole blood concentrations of PEth 16:0/18:1 ranged from LLOQ to 1.71 μg/mL.

Optimization of ultrasound assisted dispersive liquid-liquid microextraction of six antidepressants in human plasma using experimental design

A simple Ultrasounds Assisted-Dispersive Liquid Liquid Microextraction (UA-DLLME) method is presented for the simultaneous determination of six second-generation antidepressants in plasma by Ultra Performance Liquid Chromatography with Photodiode Array Detector (UPLC-PDA). The main factors that potentially affect to DLLME were optimized by a screening design followed by a response surface design and desirability functions. The optimal conditions were 2.5 mL of acetonitrile as dispersant solvent, 0.2 mL of chloroform as extractant solvent, 3 min of ultrasounds stirring and extraction pH 9.8.Under optimized conditions, the UPLC-PDA method showed good separation of antidepressants in 2.5 min and good linearity in the range of 0.02-4 μg mL(-1), with determination coefficients higher than 0.998. The limits of detection were in the range 4-5 ng mL(-1). The method precision (n=5) was evaluated showing relative standard deviations (RSD) lower than 8.1% for all compounds. The average recoveries ranged from 92.5% for fluoxetine to 110% for mirtazapine. The applicability of DLLME/UPLC-PDA was successfully tested in twenty nine plasma samples from antidepressant consumers. Real samples were analyzed by the proposed method and the results were successfully submitted to comparison with those obtained by a Liquid Liquid Extraction-Gas Chromatography - Mass Spectrometry (LLE-GC-MS) method. The results confirmed the presence of venlafaxine in most cases (19 cases), followed by sertraline (3 cases) and fluoxetine (3 cases) at concentrations below toxic levels.

Determination of Cocaine and Heroin with Their Respective Metabolites in Human Hair using Gas Chromatography‐Mass Spectrometry

Abstract This paper describes a gas chromatography‐mass spectrometry method for the simultaneous detection of opiates, cocaine, and benzoylecgonine from human hair samples. Conditioning samples were extracted with Waters Oasis HLB (hydrophilic‐lipophilic balance) cartridges. The detector response was linear for the drugs studied over the range 0.5–20 ng/mg. The limits of quantitation and detection were found to be acceptable. Intra‐ and interbatch coefficients of variation oscillated between 0.2 and 17.9%, and mean relative errors were in the range of 0.04–18.2%. The recoveries were higher than 66.7% in all cases. Finally, the method was applied to 20 hair samples from drug users, obtaining positive results in all cases.

Determination of colchicine in biological fluids by reverse-phase HPLC. Variation of colchicine levels in rats☆

A reverse-phase HPLC method for the determination of colchicine in biological fluids is proposed. Blood, liver and kidney colchicine concentrations were determined in rats at different times following intraperitoneal (i.p.) injection of 10 mg/kg of the drug. Colchicine was extracted from the samples studied using dichloromethane at pH 8. The dry extract was redissolved in the mobile phase and analyzed with simultaneous detection at 254 and 350 nm.

A sequential spectrophotometric method for the determination of paraquat and diquat in plasma

Abstract A sequential spectrophotometric method for the simultaneous determination of paraquat and diquat in plasma using second-derivative spectroscopy after ion-pair liquid-liquid extraction was developed. The paraquat and diquat in the last extract are reduced with alkaline sodium dithionite to coloured complexes whose absorption peaks appear at a different wavelength. The two herbicides can thus be determined sequentially over the wavelength ranges 454.3—446.4 nm (diquat) and 460—396 nm (paraquat), respectively.

Chromatographic determination of drugs of abuse in vitreous humor using solid-phase extraction

A simple method is presented for the simultaneous determination of morphine, 6‐acetylmorphine, codeine, cocaine, benzoylecgonine, cocaethylene, methadone and 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP) in vitreous humor by high‐performance liquid chromatography with photodiode array detector after solid‐phase extraction with Oasis® HLB cartridges and dichloromethane as eluent. The chromatographic process was carried out using an XTerra® RP8 column (250u2009×u20094.6u2009mm i.d., 5u2009µm particle size) and a mobile phase composed of acetonitrile and pH 6.5 phosphate buffer in gradient mode. A linear response from the detector was obtained within the concentration range of 0.1–4u2009µg ml−1, with correlation coefficients higher than 0.99. The limits of detection were lower than 30u2009ng ml−1 for all the drugs studied, the coefficients of variation fluctuated between 0.1 and 12.4%, and the average recoveries were higher than 78% for all the drugs except for EDDP, with a value of 66.4%. Finally, the proposed method was applied to 15 vitreous humor samples coming from individuals who had died from opiate and/or cocaine overdose, showing consumption of cocaine in 14 cases, methadone in five cases and heroin in three cases. Average concentrations of 0.30u2009µg ml−1 for morphine, 0.24u2009µg ml−1 for 6‐acetylmorphine, 0.10u2009µg ml−1 for codeine, 0.81u2009µg ml−1 for cocaine, 1.26u2009µg ml−1 for benzoylecgonine, 0.15u2009µg ml−1 for cocaethylene, 0.11u2009µg ml−1 for methadone and 0.68u2009µg ml−1 for EDDP were obtained. Copyright

Simultaneous Determination of Diazepam and its metabolites in Plasma by High-Performance Liquid Chromatography

Abstract A reversed phase high-performance liquid chromatographic method (HPLC) for the simultaneous determination of diazepam and its three active metabolites, nordazepam, oxazepam and temazepam, in plasma was proposed. The compounds were isolated by solid-phase extraction. The chromatographic mobile phase was metanol-water (55:45, v/v) at a flow rate of 1 mL/min. UV detection was performed concurrently at 240 and 254 nm.