Development and validation of a magnetic solid‐phase extraction with high‐performance liquid chromatography method for the simultaneous determination of amphetamine and methadone in urine

The simultaneous determination of amphetamine and methadone was carried out by magnetic graphene oxide nanoparticles, a magnetic solid‐phase extraction adsorbent, as a new sample treatment technique. The main factors (the amounts of sample volume, amount of adsorbent, type and amount of extraction organic solvent, time of extraction and desorption, pH, the ionic strength of extraction medium, and agitation rate) influencing the extraction efficiency were investigated and optimized. Under the optimized conditions, good linearity was observed in the range of 100–1500 ng/mL for amphetamine and 100–1000 ng/mL for methadone. The method was evaluated for determination of AM and methadone in positive urine samples, satisfactory results were obtained, therefore magnetic solid‐phase extraction can be applied as a novel method for the determination of drugs of abuse in forensic laboratories.     

Stereoselective screening for and confirmation of urinary enantiomers of amphetamine, methamphetamine, designer drugs, methadone and selected metabolites by capillary electrophoresis.

Data presented in this paper demonstrate that a competitive binding, electrokinetic capillary-based immunoassay previously used for screening of urinary amphetamine and analogs cannot be employed to distinguish between the enantiomers of amphetamine and methamphetamine. However, capillary zone electrophoresis with a pH 2.5 buffer containing (2-hydroxypropyl)-beta-cyclodextrin as chiral selector is shown to permit the enantioselective analysis of urinary extracts containing methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (Ecstasy) and other designer drugs, and methadone together with its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine. In that approach, enantiomer identification is based upon comparison of extracted polychrome UV absorption data and electropherograms obtained by rerunning of spiked extracts with spectra and electropherograms monitored after extraction of fortified blank urine. The suitability of the described chiral electrokinetic capillary method for drug screening and confirmation is demonstrated via analysis of unhydrolyzed quality control urines containing a variety of drugs of abuse. Furthermore, in a urine of a patient under selegiline pharmacotherapy, the presence of the R-(-)-enantiomers of methamphetamine and amphetamine could be unambiguously identified. Direct intake of an R-enantiomer or ingestion of drugs that metabolize to the R-enantiomers can be distinguished from the intake of S-(+)-enantiomers (drug abuse) or prescribed drugs that metabolize to the S-enantiomers of methamphetamine and amphetamine. The described approach is simple, reproducible, inexpensive and reliable (free of interferences of other major basic drugs that are frequently found in toxicological urines) and could thus be used for screening for and confirmation of urinary enantiomers in a routine laboratory.     

液-液-液微萃取-高效液相色谱法测定人尿液中的美沙酮

建立了液-液-液微萃取与高效液相色谱联用技术快速分析尿样中美沙酮的方法.对有机溶剂种类、体积、样品溶液的pH值、萃取时间、搅拌速度进行了优化.方法的线性范围为0.05～10 mg/L,检出限为0.025 mg/L,相对标准偏差小于5%.     

Fluorometric determination of propranolol and its metabolite n-desisopropylpropranolol in plasma and urine by direct meausrement of thin-layer chromatographic plates (author's transl

The quantitative analysis of propranolol and its metabolite N-desisopropylpropranolol in plasma and urine is described. The drugs are extracted into 2-pentanol-heptane, and the solvent is concentrated. The whole residue is chromatographed on silica gel plates. The compounds are determined directly on the thin-layer plates without derivatization. The recovery of propranolol from plasma is 70%, with a standard deviation of +/- 4%.     

Determination of amphetamine and its metabolite p-hydroxyamphetamine in rat urine by reversed-phase high-performance liquid chromatography after dabsyl derivatization.

The consumption of amphetamine is illicit and controlled due to both the elicited behavioural deviations and the toxicity effects reported in abusers. Thus, amphetamine levels in biological samples must be monitored in several clinical and forensic circumstances. In spite of the interspecies differences in the preferred route of biotransformation, benzylmethylketone, benzoic acid and 4-hydroxyamphetamine are the principal metabolites of amphetamine. However, the clinical and forensic studies are focused in the parent compound and in 4-hydroxyamphetamine since benzylmethylketone is a minor metabolite in human and benzoic acid is also an endogenous compound. In the present study amphetamine and its metabolite, 4-hydroxyamphetamine, are quantified in urine by HPLC after derivatization with 4-dimethylaminoazobenzene-4'-sulfonyl chloride (dabsyl chloride). This derivatization procedure transforms amphetamine and its hydroxylated metabolite in compounds with similar lipofilicity, enabling their quantitative and simultaneous extraction with an organic solvent. The precision of the HPLC technique was 7.3 and 10.0% for amphetamine and 4-hydroxyamphetamine derivatives, respectively. For the overall procedure, including enzymatic hydrolysis, derivatization and extraction of the derivatives, the obtained values were 9.3 and 6.2%. Recoveries obtained from spiked urines for amphetamine and 4-hydroxyamphetamine were better than 97% and 94% (mean value), respectively. The detection limits of the method was 10 ng for both compounds. The principal advantages of the present proposed method are the stability of the dabsyl derivatives at room temperature and the detection carried out in the visible region, reducing the interferences detected.     

Simultaneous detection of a wide variety of commonly abused drugs in a urine screening program using thin-layer identification techniques.

A single-step extraction method and thin-layer identification techniques capable of testing a wide variety of drugs of abuse are presented. These techniques are well suited for large and/or small drug programs involved in urine testing because they provide substantial economic benefits and improve clinical functioning. The drugs are absorbed on a 6 X 6 cm piece of paper loaded with cation-exchange resin and then eluted from the paper at pH 10.1 using ammonium chloride-ammonia buffer. The simultaneous thin-layer detection of sedatives, hypnotics, narcotic analgesics, central nervous system stimulants and miscellaneous drugs is accomplished by spotting the solution of extracted residue on a 20 X 20 cm Gelman pre-coated silica gel glass microfiber sheet (ITLC Type SA). A two-stage solvent system is used in order to obtain a chromatogram with optimum separation of a wide range of drugs. This system can separate methadone and/or cocaine from propoxyphene, methaqualone, methylphenidate, pentazocine, pipradrol, Doxepin, chlorpromazine, phenazocine, naloxone, naltrexone, imipramine and trimeprazine; amphetamine from phenylpropanolamine and dimethyltryptamine; codeine from dextromethorphan; methamphetamine from dimethyltryptamine, etc. Different detection reagents are then applied in succession to different marked areas of the developed chromatogram. This elegant method of extraction and spraying has enabled us to detect morphine base at a sensitivity level of 0.15 mug/ml, amphetamine sulfate at 1.0 mug/ml, methamphetamine hydrochloride at 0.5 mug/ml, phenmetrazine hydrochloride at 0.5 mug/ml, codeine phosphate at 0.5 mug/ml, methadone hydrochloride at 1.0 mug/ml, secobarbital at 0.36 mug/ml and phenobarbital at 0.5 mug/ml in urine. The minimum volume of urine needed to achieve these sensitivities is 20 ml. The cost of analysis per urine specimen using these techniques for concomitant screening of these drugs is less than US$ 1.     

Sensitive determination of methadone in human serum and urine by dispersive liquid–liquid microextraction based on the solidification of a floating organic droplet followed by HPLC–UV

Dispersive liquid–liquid microextraction based on solidification of floating organic droplet was developed for the extraction of methadone and determination by high‐performance liquid chromatography with UV detection. In this method, no microsyringe or fiber is required to support the organic microdrop due to the usage of an organic solvent with a low density and appropriate melting point. Furthermore, the extractant droplet can be collected easily by solidifying it at low temperature. 1‐Undecanol and methanol were chosen as extraction and disperser solvents, respectively. Parameters that influence extraction efficiency, i.e. volumes of extracting and dispersing solvents, pH, and salt effect, were optimized by using response surface methodology. Under optimal conditions, enrichment factor for methadone was 134 and 160 in serum and urine samples, respectively. The limit of detection was 3.34 ng/mmL in serum and 1.67 ng/mL in urine samples. Compared with the traditional dispersive liquid–liquid microextraction, the proposed method obtained lower limit of detection. Moreover, the solidification of floating organic solvent facilitated the phase transfer. And most importantly, it avoided using high‐density and toxic solvents of traditional dispersive liquid–liquid microextraction method. The proposed method was successfully applied to the determination of methadone in serum and urine samples of an addicted individual under methadone therapy.     

Single drop liquid-liquid-liquid microextraction of methamphetamine and amphetamine in urine

Single drop liquid-liquid-liquid microextraction (LLLME) combined with high performance liquid chromatography (HPLC)-UV detection was investigated for the determination of a popular drug of abuse, methamphetamine (MAP), and its major metabolite, amphetamine (AP), in urine samples. The target compounds were extracted from NaOH modified sample solution to a thin layer of organic solvent membrane, and back-extracted to an acidic acceptor drop suspended on the tip of a 50-microL HPLC syringe in the aforementioned organic layer. This syringe was also used for direct injection after extraction. Factors affecting extraction efficiency were studied. At optimal conditions, the overall enrichment factor (EF) was 500-fold for AP and 730-fold for MAP, respectively. The method exhibited a wide linear range (1.0-1500 microg/L), low detection limit (0.5 microg/L), and good repeatability (RSD<5.0%) for both analytes. The feasibility of the method was demonstrated by the analysis of human urine samples.     

Fast gas chromatography/mass spectrometric assay for the validated quantitative determination of methadone and the primary metabolite EDDP in whole blood

A toxicological analysis was developed and validated for simultaneous screening and quantification of methadone (METH) and its primary metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). The method employs microscale liquid-liquid extraction (microLLE) and direct injection of a separated aliquot of the organic layer into a gas chromatography/mass spectrometric (GC/MS) system without any other pre-treatment stages. A fast GC/MS runtime (total 5.8 min; METH, Rt = 3.55 min; EDDP, Rt = 3.40 min) combined with rapid sample preparation allowed cost-efficient and routinely applicable performance with a low amount of manual work. The validated parameters included: linearity (25-1000 ng mL(-1) both; R(METH)2 = 0.998 and R(EDDP)2 = 0.997), accuracy (Bias(METH): from -0.05 to 11.3%, Bias(EDDP): from 1.11 to 4.37%); intra and inter-assay precision (RSD(METH): from 2.4 to 3.9%, from 4.89 to 10.3%; RSD(EDDP): from 4.50 to 6.20%, from 4.57 to 15.2%), extraction efficiency (METH = 95.5%; EDDP = 90.6%), LOQ(Meth,EDDP) = 25 ng mL(-1). Samples were stable for at least 25 h and no selectivity problems or baseline interference were observed. The method should be applicable for identifying and quantitative confirmation of possible misuse and/or illegal use of METH in toxicological cases.     

Automated headspace solid-phase microextraction and in-matrix derivatization for the determination of amphetamine-related drugs in human urine by gas chromatography-mass spectrometry

An automated extraction and determination method for the gas chromatography (GC)-mass spectrometry (MS) analysis of amphetamine-related drugs in human urine is developed using headspace solid-phase microextraction (SPME) and in-matrix derivatization. A urine sample (0.5 mL, potassium carbonate (5 M, 1.0 mL), sodium chloride (0.5 g), and ethylchloroformate (20 microL) are put in a sample vial. Amphetamine-related drugs are converted to ethylformate derivatives (carbamates) in the vial because amphetamine-related drugs in urine are quickly reacted with ethylchloroformate. An SPME fiber is then exposed at 80 degrees C for 15 min in the headspace of the vial. The extracted derivatives to the fiber are desorbed by exposing the fiber in the injection port of a GC-MS. The calibration curves show linearity in the range of 1.0 to 1000 ng/mL for methamphetamine, fenfluramine, and methylenedioxymethamphetamine; 2.0 to 1000 ng/mL for amphetamine and phentermine; 5.0 to 1000 ng/mL for methylenedioxyamphetamine; 10 to 1000 ng/mL for phenethylamine; and 50 to 1000 ng/mL for 4-bromo-2,5-dimethoxyphenethylamine in urine. No interferences are found, and the time for analysis is 30 min for one sample. Furthermore, this proposed method is applied to some clinical and medico-legal cases by taking methamphetamine. Methamphetamine and its metabolite amphetamine are detected in the urine samples collected from the patients involved in the clinical cases. Methamphetamine, amphetamine, and phenethylamine are detected in the urine sample collected from the victim of a medico-legal case.     

Ion chromatographic analysis of anions in ammonium hydroxide,hydrofluoric acid,and slurries,used in semiconductor processing

In this study, ion chromatography (IC) with suppressed conductivity detection was used for the determination of trace anions in 29% (w/w) ammonium hydroxide, 49% (w/w) hydrofluoric acid and slurries. For these samples, various sample pretreatment methods were applied to eliminate matrix interferences. For concentrated ammonium hydroxide, an on-line electrochemical neutralizer (SP10 AutoNeutralization module) was used to neutralize the base prior to the IC analysis. For concentrated hydrofluoric acid, a heart cutting technique with an ion-exclusion column was used to separate the anions of interest prior to an IC separation. A method was also developed to analyze chloride in silica slurries by IC.     

Properties of low-capacity macroporous anion-exchangers and their use with high-pH eluents for the determination of weak acids in urine

Low-capacity anion-exchangers have been synthesized from the macroporous styrene-divinylbenzene co-polymer Hamilton PRP-1. These anion-exchangers have non-polar adsorbent properties for neutral sample molecules. The dependence of the capacity factor for a polar weak-acid metabolite of the drug polythiazide on the type and concentration of electrolyte, the organic solvent concentration, and pH of aqueous eluents has been studied to characterize the exchangers. As an application, a method has been developed to determine the metabolite in urine at levels as low as 10 ng ml . The method employs the exchangers in both a precolumn, which is used to preconcentrate and clean-up the metabolite from urine, and an analytical column. Eluents containing 0.001-0.01M sodium hydroxide are used to ionize the weak-acid metabolite.     

Solid-phase microextraction for the determination of pethidine and methadone in human urine using gas chromatography with nitrogen-phosphorus detection

A simple and rapid analytical method is presented for the determination of pethidine (meperidine) and methadone in human urine using solid-phase microextraction (SPME) and gas chromatography with nitrogen-phosphorus detection (GC-NPD). After the analytes had been partitioned between an extracting phase and the aqueous sample matrix, the needle of the coating fiber assembly was injected directly into the GC injector. The analytes were thermally desorbed in the heated injector (240 degrees C) and subsequently separated and detected by the GC-NPD system. The factors influencing the SPME method, such as the salt (NaCl) effect (15%), pH (pH 11), and equilibration time (30 min), were optimized. The calibration graphs for urine samples showed a good linearity. The detection limit was below 1 ng ml-1 for both drugs.     

Confirmation testing of amphetamines and designer drugs in human urine by capillary electrophoresis-ion trap mass spectrometry

Monitoring of amphetamines and designer drugs in human urine is a timely topic in clinical toxicology, surveillance of drug substitution, forensic science, drug testing at the workplace, and doping control. Confirmation testing of urinary amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) and 3,4-methylenedioxyamphetamine (MDA) by capillary electrophoresis (CE) combined with atmospheric pressure electrospray ionization and ion trap mass spectrometry (MS) is described. Using an aqueous pH 4.6 buffer composed of ammonium acetate/acetic acid, CE-MS and CE-MS2 provided data that permitted the unambiguous confirmation of these drugs in external quality control urines. Furthermore, other drugs of abuse present in alkaline urinary extracts, including methadone and morphine, could also be monitored. The data presented illustrate that the sensitivity achieved with the benchtop MS is comparable to that observed by CE with UV absorption detection. CE-MS2 is further shown to be capable of identifying comigrating compounds, including the comigration of amphetamine with nicotine.     

Comparison of microemulsion electrokinetic chromatography and solvent modified micellar electrokinetic chromatography on rapid separation of heroin, amphetamine and their basic impurities

Microemulsion electrokinetic chromatography (MEEKC) and solvent modified micellar electrokinetic chromatography (MEKC) were investigated with the goal of the rapid separation of complex heroin and amphetamine samples. The rapid simultaneous separation of 17 species of heroin, amphetamine and their basic impurities and adulterants was performed within about 10 min using MEEKC for the first time, whereas solvent modified MEKCs were unable to resolve all the components. The comparisons between MEEKC and solvent modified MEKC proved internal lipophilic organic phase in microemulsions played an important role in improving the separation performance with respect to efficiency. However, the role of internal lipophilic organic phase in MEEKC was disgusted at high concentrations of cosurfactant, and the separations of MEEKC and 1-butanol modified MEKC became similar at high concentrations of 1-butanol. The evaluation of reproducibility, linearity and detection limit of optimized MEEKC method provided good results for all the analytes investigated, thus allowing its application to real controlled drug preparation analysis.     

Enantiomer-specific high-performance liquid chromatography with fluorescence detection of methamphetamines in abusers' hair and urine

Enantiomer-specific high-performance liquid chromatography with fluorescence detection using 4-(4,5-diphenyl-1H-imidazol-2-yl)-benzoyl chloride as a fluorescence labeling reagent was applied to determine methamphetamine and its metabolites in abusers' hair and urine. Hair samples were segmentally analyzed based on 1 cm long segments. In four hair samples, only the S(+)-enantiomers of methamphetamine and its N-demethylated metabolite, S(+)-amphetamine were detected. Satisfactory correlation (r = 0.901) between the results of high-performance liquid chromatography-fluorescence and those of gas chromatography-nitrogen phosphorous detection was obtained (n = 19). In an abuser's urine sample, the S(+)- and R(-)-enantiomers of methamphetamine, amphetamine and para-hydroxymethamphetamine were detected. The degree of N-demethylation of S(+)-methamphetamine into the corresponding metabolite of amphetamine was significantly higher than that of the R(-)-enantiomer.     

Micelle to solvent stacking of organic cations in micellar electrokinetic chromatography with sodium dodecyl sulfate

The on-line sample concentration technique, micelle to solvent stacking (MSS), was studied for small organic cations (quaternary ammonium herbicides, β-blocker drugs, and tricyclic antidepressant drugs) in reversed migration micellar electrokinetic chromatography. Electrokinetic chromatography was carried out in fused silica capillaries with a background solution of sodium dodecyl sulfate (SDS) in a low pH phosphate buffer. MSS was performed using anionic SDS micelles in the sample solution for analyte transport and methanol or acetonitrile as organic solvent in the background solution for analyte effective electrophoretic mobility reversal. The solvent also allowed for the separation of the analyte test mixtures. A model for focusing and separation was developed and the mobility reversal that involved micelle collapse was experimentally verified. The effect of analyte retention factor was observed by changing the % organic solvent in the background solution or the concentration of SDS in the sample matrix. With an injection length of 31.9 cm (77% of effective capillary length) for the 7 test drugs, the LODs (S/N=3) of 5-14 ng/mL were 101-346-fold better when compared to typical injection. The linearity (R(2), range=0.025-0.8 μg/mL), intraday and interday repeatability (%RSD, n=10) were ≥0.988, <6.0% and <8.5%, respectively. In addition, analysis of spiked urine samples after 10-fold dilution with the sample matrix yielded LODs=0.02-0.10 μg/mL. These LODs are comparable to published electrophoretic methods that required off-line sample concentration. However, the practicality of the technique for more complex samples will rely on dedicated sample preparation schemes.     

Liquid-phase microextraction of protein-bound drugs under non-equilibrium conditions

Recently, we introduced an inexpensive and disposable hollow fiber-based device for liquid-phase microextraction (LPME) where ionic analytes typically were extracted and preconcentrated from 1-4 mL aqueous samples (such as plasma and urine) through an organic solvent immobilized in the pores of a polypropylene hollow fiber and into a 10-25 microL volume of acceptor phase present inside the lumen of the hollow fiber. Subsequently, the acceptor phase was directly subjected to the final analysis by a chromatographic or electrophoretic method. In the present work, attention was focused on LPME of the basic drugs amphetamine, pethidine, promethazine, methadone and haloperidol characterized by substantial differences in the degree of protein binding. Drug-protein interactions in plasma resulted in reduced recoveries and substantially increased extraction times compared with extraction of the drugs from a pure water matrix. However, by addition of 5-50% methanol to the plasma samples, recoveries were comparable with LPME from water samples and ranged between 75 and 100%. The addition of methanol was found not to speed up the LPME process and extractions from plasma were performed in 45 min to reach equilibrium. Because approximately 55-70% of the final analyte concentrations were achieved within the initial 10 min of the LPME process, validation was accomplished after 10 and 45 min of LPME. In general, the results with 10 and 45 min were almost comparable, with precision data in the range 1.2-11.1% (RSD) and with linearity in the concentration range 20-1000 ng mL(-1) (r = 0.999). In conclusion, excellent LPME results may be achieved in a short time under non-equilibrium conditions with a minor loss of sensitivity. In cases of drug-protein interactions, methanol may be added to ensure a high extraction recovery.     

Fast GC-MS method for the simultaneous screening of THC-COOH, cocaine, opiates and analogues including buprenorphine and fentanyl, and their metabolites in urine

A fast gas chromatography (GC)-MS method has been developed and validated for the simultaneous screening of different classes of drugs of abuse in urine. Tetrahydrocannabinol metabolite, cocaine, opiates such as morphine, O-6-monoacetylmorphine (O-6-MAM), codeine, opioids such as buprenorphine, methadone, pentazocine, fentanyl and analogues and their main metabolites can be detected and quantified after a simple liquid-liquid extraction in alkaline conditions and derivatisation to obtain the corresponding trimethylsilyl derivatives. The chromatographic separation is performed in a total time of 6 min, using a short GC column (5% phenyl methyl silicone, 10-m length × 0.18-mm internal diameter). The Limits of Detection are satisfactory for forensic purposes for all the substances; the repeatability of concentrations (percent coefficients of variation) are always lower than 15% at high and low concentration levels, and accuracy, intended as % error on the true value, is always lower than 15% for all the analytes. The method can successfully be applied for screening analyses in many fields of forensic toxicology.     

Determination of metoprolol and two major metabolites in plasma and urine by column liquid chromatography and fluorometric detection

Metoprolol and its alpha-hydroxy metabolite were determined in plasma down to 2 nmol/l (S.D. 10-15%) after solvent extraction and bonded-phase liquid chromatography with fluorometric detection. The major metabolite with a carboxylic function was also measured in plasma when liquid-solid extraction on a column activated with dodecyl sulphate was applied. In urine the three components were assayed by direct injection of a diluted sample.