Drugs In Human Urine Research Articles

Direct capillary electrophoretic determination of three chemotherapeutic drugs in human urine

Capillary zone electrophoresis (CZE) has been used for direct determination of 6-thioguanine, methotrexate, and 5-fluorouracil in human urine, by use of a fused-silica capillary (60.2 cm×75 μm i.d.). Separation was performed after hydrodynamic injection for 7 s; the separation potential and capillary temperature were 25 kV and 35°C, respectively. A 45mm borate buffer solution (pH 9.2) was used as separation electrolyte. Under these conditions the analysis takes approximately 10 min and interday precision of migration times and corrected peak areas is satisfactory. A linear response over the concentration range 3.0–20.0 mg L1 was observed for the three chemotherapeutic drugs in diluted human urine. Detection limits (s/n=3) for 6-thioguanine and methotrexate were approximately 1.60 mg L1 in diluted human urine; that for fluorouracil was 2.60 mg L1. A 2-ml volume of human urine was diluted with 2-mL of water and introduced directly into the electrophoresis system. CZE was shown to be a good method with regard to simplicity, satisfactory precision, and sensitivity. This method resulted in especially excellent recoveries for determination of methotrexate in all the different urine samples analysed (n=10).

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.

Sample preparation with fiber-in-tube solid-phase microextraction for capillary electrophoretic separation of tricyclic antidepressant drugs in human urine.

Solid-phase microextraction (SPME) is a solvent-free sample preparation technique using a thin coating attached to the surface of a fused silica-fiber as the extraction medium, which has been successfully applied to the analysis of a wide variety of compounds by coupling to gas chromatography (GC). In recent years, in-tube SPME using GC capillary column as the extraction medium has also been developed and coupled with liquid chromatography (LC) for the preconcentration of nonvolatile compounds. In this study, an on-line interface between the fiber-in-tube SPME and capillary electrophoresis (CE) has been developed, and the preconcentration and separation of four tricyclic antidepressant (TCA) drugs, amitriptyline, imipramine, nortriptyline, and desipramine, were performed with the hyphenated system. Under the optimized condition, a better extraction performance than conventional in-tube SPME was obtained, even the length of the extraction medium was much shorter. The results clearly indicated that the fiber was working effectively as an extraction medium. For the separation of these four TCAs, capillary electrophoretic separation with beta-cyclodextrin as the buffer additive has been employed and the application of the developed system to the analysis of complex sample mixtures in a biological matrix is also demonstrated.

Analysis of codeine, dihydrocodeine and their glucuronides in human urine by electrokinetic capillary immunoassays and capillary electrophoresis–ion trap mass spectrometry

Screening for and confirmation of illicit, abused and banned drugs in human urine is a timely topic in which capillary separation techniques play a key role. Capillary electrophoresis (CE) represents the newest technology employed in this field of analysis. Two rapid competitive binding, electrokinetic capillary-based immunoassays are shown to be capable of recognizing the presence, but not the identity, of urinary opioids, namely codeine (COD), codeine-6-glucuronide, dihydrocodeine (DHC), dihydrocodeine-6-glucuronide, morphine (MOR), morphine-3-glucuronide and ethylmorphine (EMOR). In these approaches, aliquots of urine and immunoreagents of a commercial, broadly cross-reacting fluorescence polarization immunoassay for opiates were combined and analyzed by capillary zone electrophoresis or micellar electrokinetic capillary chromatography with laser induced fluorescence detection. With the fluorescent tracer solution employed, the former method is shown to provide simple electropherograms which are characterized by an opioid concentration dependent magnitude of the free tracer peak. In presence of dodecyl sulfate micelles, however, two tracer peaks with equal opioid concentration sensitivity are monitored. These data suggest the presence of two fluorescent tracers which react competitively with the urinary opioids for the binding sites of the antibody. Assay sensitivities for COD and MOR are comparable (10 ng/ml), whereas those for DHC and EMOR are about four-fold lower. Furthermore, glucuronides are shown to react like the corresponding free opioids. Analysis of urines that were collected after administration of 7 mg COD and 25 mg DHC tested positively in both assay formats. The presence of the free and conjugated codeinoids in these urines and their identification was accomplished by capillary electrophoresis–ion trap mass spectrometry (CE–MS). This confirmatory assay is based upon solid-phase extraction using a mixed-mode polymer cartridge followed by CE hyphenated to the LCQ mass spectrometer with electrospray ionization in the positive ion mode. With this technology, MS 2 is employed for proper identification of COD ( m/z 300.4) and DHC ( m/z 302.4) whereas MS 3 provides unambiguous identification of the glucuronides of COD ( m/z 476.5) and DHC ( m/z 478.5) via their fragmentation to COD and DHC, respectively. MS n ( n≥2) is shown to be capable of properly identifying the urinary codeinoids on the 100–200 ng/ml concentration level.

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.

Determination of Methylamphetamine in Urine by Differential Pulse Polarography

A new electroanalytical method for detecting methylamphetamine as a stimulant drug in human urine has been proposed for forensic purposes. Although methylamphetamine itself has no remarkable voltammetric waves at any electrode in various electrolytes, we have found that the copper complex of methylamphetamine obtained by the method of Feigl showed a well-defined peak at -0.56 V (vs. SCE) at a mercury electrode by differential pulse polarography (DPP) in an aqueous ethanol solution containing 0.25 M ammonium acetate. The peak height was linear as a function of the methyl-amphetamine concentration in the original test solutions in the region of at least 0 - 50 µg/ml. The concentrations of methylamphetamine determined by the present method for eight abusers’ urine samples were in excellent agreement with those obtained by the conventional methods (GC-MS analysis) so far used. The proposed method is rather simple in procedure, rapid and selective with a satisfactory detection limit (<0.125 µg/ml) for analysis of methylamphetamine, and would also be applicable for field use.

Screening procedure for beta-adrenergic drugs in sports drug testing by immunological methods.

A systematic study of the cross-reactivities of beta-adrenergic agonists and antagonists and their metabolites with an ELISA test designed to detect terbutaline and other beta 2-agonists has been performed in order to evaluate the applicability of this immunological test as screening method in routine human sports drug testing. Concentration response curves were calculated. IC50 values from 1.7 to 21.5 ng/mL were obtained for beta 2-agonists, and values from 3.1 to 520.8 ng/mL were obtained for most of the beta-blockers. The highest cross-reactivity was mainly depending on the amino substituent of the molecules although the structure of the aromatic ring was also important. Minor changes in the aromatic ring, such as 4-hydroxylation for propranolol, did not largely influence the IC50 value. Distribution of control activities (percentage of optical density as compared with a blank sample) of urines obtained in controlled excretion studies (70 samples) and urines collected after competition in sports in which beta-blockers are recommended to be tested (147 samples) showed a very small overlapping between the subpopulations of positive and negative samples. Cutoff values of 45 or 60% control activity were proposed for routine analysis. Sensitivity and specificity values of 92.8 and 98.8%, respectively (cutoff 45%), or 98.2 and 95.0%, respectively (cutoff 60%), were obtained. Because the known prevalence of positive results of beta-blockers and beta 2-agonists in the target population was very low, the predicted percentage of presumptive positive cases that would need further confirmation by gas chromatography-mass spectrometry was also low, accounting for a 1.48 and 5.28% (for the cutoffs of 45 and 60%, respectively) of the total number of samples to be screened by the presented ELISA test. Thus, in terms of time and cost savings, the ELISA test is a powerful tool for the purpose of screening for beta-adrenergic drugs in human urine.

Use of liquid chromatography–tandem mass spectrometry for the quantitative and qualitative analysis of an antipsychotic agent and its metabolites in human plasma and urine

The use of liquid chromatography–tandem mass spectrometry (LC–MS–MS) for the quantitative analysis of an `atypical' antipsychotic agent in human plasma is described. The method uses atmospheric pressure chemical ionisation, and offers increased sensitivity, selectivity and speed of analysis over an existing high-performance liquid chromatography method using fluorescence detection. This method enabled same day turn around of results for in excess of 100 samples, including sample preparation, data acquisition and processing. LC–MS–MS was also used to detect and characterise known and unknown in vivo metabolites of the drug in human urine and plasma, using electrospray ionisation.

Simultaneous analysis of several non-steroidal anti-inflammatory drugs in human urine by high-performance liquid chromatography with normal solid-phase extraction.

A practical and reproducible high-performance liquid chromatographic method using normal solid-phase extraction has been developed for the simultaneous analysis of twelve non-steroidal anti-inflammatory drugs (NSAIDs) in human urine. A urine specimen mixed with acetate buffer pH 5.0 was purified by solid-phase extraction on a Sep-Pak Silica cartridge. The analyte was chromatographed by a reversed-phase Inertsil ODS-2 column using a phosphate buffer-acetonitrile at pH 5.0 as the mobile phase, and the effluent from the column was monitored at 230 or 320 nm. Absolute recoveries were greater than 73% for all of the twelve NSAIDs. The present method enabled simple manipulation and isocratic HPLC with UV analysis as well as high sensitivity of 0.005 microg/ml for naproxen, and 0.05 microg/ml for sulindac, piroxicam, loxoprofen, ketoprofen, felbinac, fenbufen, flurbiprofen, diclofenac, ibuprofen and mefenamic acid as the quantitation limit in human urine using indomethacin as an internal standard.

Rapid Solid-phase Extraction/Derivatization System for Sample Preparation and Gas Chromatographic/Mass Spectrometric Determination of Drugs in Human Urine

Rapid Solid-phase Extraction/Derivatization System for Sample Preparation and Gas Chromatographic/Mass Spectrometric Determination of Drugs in Human Urine

Rapid solid-phase extraction/derivatization system for sample preparation and gas chromatographic/mass spectrometric determination of drugs in human urine.

A novel automatic system has been developed for the rapid solid-extraction and derivatization of different drugs and their metabolites from urine, prior to further analysis. Manual injection of underivatized/derivatized drugs in a GC/MS system, in which the quadrupole mass filter was operated conventionally, provided high sensitivity and selectivity at the selected m/z value. The precision (2.1-6.8%) is acceptable and the overall recovery from urine was determined to be ca. 95%. The effectiveness of this approach is demonstrated with data obtained from different urine samples which had tested positively.

Simultaneous Determination of Illicit Drugs in Human Urine by Liquid Chromatography-Mass Spectrometry

The method for simultaneous determination and confirmation of illicit drugs (e.g., methamphetamine, amphetamine, ephedrine, methylephedrine, morphine, morphine-3-glucuronide, morphine-6-glucuronide, 6-acetylmorphine, cocaine, and benzoylecgonine) in human urine by thermospray liquid chromatography-mass spectrometry (LC-MS) was studied. The LC-MS separation was performed on a reversed phase column (L-column ODS; 150 mm x 4.6-mm i.d.) using a gradient mobile phase system of 100mM ammonium acetate for 1 min then linear ramps to 100mM ammonium acetate including 40% acetonitrile at 20 min. Extraction was conducted by solid-phase extraction using a Sep-pak C18 cartridge. The drugs were eluted with 2 mliters of 40% acetonitrile in 100 mM ammonium acetate, pH3 (adjusted with acetic acid), from the cartridge. A 50-microL volume of the eluate was injected into the LC-MS. The recoveries by this extraction were 88 to 99%. The mass spectra of methamphetamine, amphetamine, ephedrine, methylephedrine, morphine, 6-acetylmorphine, cocaine, and benzoylecgonine showed the quasi-molecular [M + H]+ ion as the base peak, whereas morphine-3-glucuronide and morphine-6-glucuronide showed [MH-glucuronide]+ ion as the base peak. The calibration graphs were linear and reproducible. Detection limits of these drugs ranged from 2 to 40 ng/mliters by selected ion monitoring (SIM) mode and from 50 to 400 ng/mliters by scan mode. The coefficients of variation for the analysis of these drugs ranged from 4.5 to 9.5% at a concentration of 0.4 micrograms/mliters (n = 10).

Analysis of Abused Drugs in Human Urine with Commercial Enzyme Immunoassay Reagents.

A simple, rapid and sensitive screening method and semiquantitative analysis for abused drugs, methamphetamine (MA), morphine (MO), cocaine (COC) and cannabinoid (CN), in the urine sample were investigated usine the Syva Emit stTM urine assay reagent with Shimadzu CL-770, the temperature-controlled spectrophotometer. A sample was diluted and mixed in an Emit stTM urine reagent vial with an Emit stTM dilutor. Thirty seconds after mixing, the mixture was transferred to the flow cell of the spectrophotometer kept at 37°C, and the absorbance change at 349 nm was measured for 30 s (60 s for CN). Calibration curves were examined over a range of 0 to 50 μg/ml for MA, morphine-3-glucuronide (M3G) and benzoylecgonine (BE), 0 to 0.5 μg/ml for Δ9-tetrahydrocannabinol-11-oic acid (THC-COOH), and were found to be relatively linear between 0 and 0.5 μg/ml for MA and M3G, 0 and 1 μg/ml for BE, 0 and 0.3 μg/ml for THC-COOH. Cutoff values were able to be set at the concentration of 0.5 μg/ml for MA, 0.5 μg/ml for M3G (corresponding to 0.2 μg/ml), 0.3 μg/ml for BE, 0.1 μg/ml for THC-COOH, and within-run coefficients of variation (C. V.) for the samples of the cutoff levels were less than 2% (n=10). The quantitative results from enzyme multiplied immunoassay technique (EMIT) assays for 100 urine samples were compared with those from GC, GC/MS and LC/MS, and the former showed an excellent correlation with the latter for MA and BE levels. Dihydrocodeine, however, showed a high cross-reactivity with MO, which often caused "false positive results."

Direct Quantitative Determination of Optically Active Absorbing Drugs in Human Urine by Circular Dichroism. Simultaneous Direct Determination of β-Ractam Antibiotics and Proteins

□ Over 200 urine samples from 61 subjects were analyzed by circular dichroism spectroscopy. The results proved that this technique can be applied to the direct determination of optically active absorbing drugs in urine of subjects under multiple drug administration, independently of the presence of proteins, which can simultaneously be determined. A list of noninterfering drugs is included. The validity of the present method was confirmed by analysis of variance, the β-Mactam antibiotics ampicillin, cefoxitin, and cephalexin being chosen as model drugs and human albumin as the analytical standard for protein determination. The results demonstrated that the proposed method is accurate and precise, the correlation coefficients being higher than 0.9996. A circular dichroism and HPLC data comparison was successfully performed. The principal advantages of this method are simplicity, quickness, and economy, no derivatization or chromatographic separation steps being needed.

Detection of hypoglycemic drugs in human urine using micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) is evaluated as a potential analytical method for the separation and detection of a series of sulfonylurea drugs used in the treatment of hyperglycemia. These drugs are often surreptitiously abused, producing extremely low blood glucose levels and symptoms indistinguishable from those associated with an insulin-secreting tumor. Separation buffer containing 50 mM sodium dodecyl sulfate (SDS) was found to be adequate for the MEKC separation of the third generation drugs (glipizide and glyburide) but not the second generation drugs (acetohexamide chlorpropamide, tolazamide, and tolbutamide). At a pH of 8.5 in the presence of 20 mM borate/20 mM phosphate and 150 mM SDS, all seven components were adequately resolved with an analysis time of 17 min. Altering the concentration of the buffering components to either 5 mM borate/5 mM phosphate or 40 mM borate alone reduced the analysis time to less than 10 min with no observable loss in resolution. A series of other micelle-forming surfactants were evaluated, and only sodium cholate provided an improvement over the SDS-based system. Optimal separation was obtained with 75 mM sodium cholate and led to complete analysis with baseline resolution of all seven components in less than 8 min. These conditions were shown to be adequate for the detection of the hypoglycemic drugs spiked into normal urine and in patients taking these drugs. The precision associated with nine consecutive injections of six samples (n = 54) was found to be acceptable with percent coefficient of variance for absolute migration times (MTabs) for all peaks averaging 0.89 with peak area and peak height being 8.49 and 8.26, respectively. The between-sample precision was found to average 0.92% for MTabs and 8.56% and 8.45%, respectively, for the relative peak area and peak height. With a detection limit for the drugs in urine (following extraction) in the 50 ng/mL range, the potential exists for an MEKC-based assay for the detection of sulfonylurea drugs in urine.

Coupled liquid-liquid extraction and column switching LC for the determination of a new antihistaminic H1 drug in human urine

Mizolastine (SL 85.0324) is a new antihistaminic H1 benzimidazole derivative which is excreted into urine almost completely metabolized; about 2% of the unchanged drug is excreted as conjugated compound which requires enzymatic deconjugation before analysis. Since the existing methods for plasma samples do not work on deconjugated human urine due to interferences, a new method was developed. The method is based on a diethyl-ether extraction of mizolastine and an internal standard from alkalinized urine. The ether extract is back-extracted with an aqueous buffer (pH=2.6), this extract is neutralized (pH=6.5) and an aliquot injected into a C18 pre-column where clean up and preconcentration take place. The analytes are then desorbed from the pre-column and transferred to the analytical column. The analytical column is a C18 type specially seactivated for basic compounds with an eluent of acetonitrile/phosphate solution (pH=4.5), 40/60, v/v, at a flow rate of 1 ml min−1. Detection is at 285 nm. The method is linear in the range 10–500 ng ml−1 with a lower limit of detection of 10 ng ml−1. The precision and accuracy, evaluated during intra-day and inter-day assays, are satisfactory for pharmacokinetic investigations.

Analysis of illicit drugs in human urine by micellar electrokinetic capillary chromatography with on-column fast scanning polychrome absorption detection

Using micellar electrokinetic capillary chromatography (MECC) with a borate/phosphate buffer containing 75 mM SDS (pH 9.1), common drugs of abuse and/or their metabolites, including opioids, benzoylecgonine, amphetamines, and methaqualone, can easily be analyzed. After solid-phase extraction of 5 mL of urine, drug concentrations down to about 100 ng/mL can be unambiguously monitored with on-column multiwavelength detection. Peak assignment is achieved through comparison of retention times and absorption spectra of eluting peaks with those of computer-stored model runs. The effectiveness of the approach is demonstrated with data obtained from different patient urines which tested positively for one or several drugs using nonisotopic immunoassays. Results suggest that MECC of illicit drugs is a highly specific and sensitive instrumental approach suitable for confirmation testing following a positive response of a toxicological screening procedure.

Identification of conjugated metabolites of pyrazolone drugs in human urine by pyrolysis-mass spectrometry

It is shown that a combination of pyrolysis electron ionization and soft ionization mass spectrometry represents a direct approach for the elucidation of the complex biotransformation pattern of two pyrazolone analgesics, antipyrine and dipyrone. In a first screening procedure crude extracts from biological material can readily be utilized. Urinary conjugates of structural hydroxy isomers formed from antipyrine can be differentiated by specific thermolytic reactions of the pyrazolone moiety. As a rule, sulpho conjugates are found to exhibit a lower stability against thermal stress than the corresponding gluco conjugates. Using this integrated mass spectrometric approach, investigation of the phase II metabolism of dipyrone succeeded in identifying five unknown metabolites.

High-performance liquid chromatographic assay for azatadine in human urine

A high-performance liquid chromatographic assay for the quantitative determination of azatadine and a base (1 M sodium hydroxide) hydrolyzable conjugate of azatadine in humna urine has been developed. Reversed-phase separation of azatadine and the internal standard, 8-chloroazatadine, was accomplished on a 300 ×3.9 mm I.D. μBondapak CN column. Following liquid—liquid extraction fro m urine, azatadine was quantitatively determined by UV detection at 214 nm. No interferences were observed in the extracts obtained from drug-free urine. Detector response (peak area ratio) was linear from 10 to 2500 ng/ml. This method has been shown to provide accurate and precise determinations of the unchanged and hydrolyzed drug in human urine, following the twice daily oral administration (1–2 mg) of azatadine maleate.

Methods of identification and confirmation of abusive drugs in human urine

A thin-layer chromatography (TLC) procedure is described to be used as the initial drug detection method for urine surveillance in a drug abuse treatment program. While the TLC method is sufficiently sensitive, it is prone to false-positive results. For this reason, two other drug detection methods (gas—liquid chromatography and radioimmunoassay) have been incorporated to confirm positive results obtained with TLC. The combined methodologies result in a urine surveillance procedure that is versatile, sensitive and highly reliable.