Determination of cocaine, norcocaine, benzoylecgonine and ecgonine methyl ester in rat plasma by high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatographic method with ultraviolet detection at 235 nm is described for the determination of cocaine and its metabolites benzoylecgonine, norcocaine and ecgonine methyl ester in rat plasma, collected during toxicity studies. Following simultaneous solid-phase extraction of all analytes and the internal standard tropacocaine, cocaine, benzoylecgonine and norcocaine were separated on a C₁₈ column. Ecgonine methyl ester and cocaine were separated on coupled cyanopropyl and silica columns, following derivatization of ecgonine methyl ester to p-fluorococaine. The extraction efficiencies of these compounds from plasma ranged from 78 to 87%, while the limits of detection ranged from 35 to 90 ng/ml. The assay was linear from 300 to 5000 ng/ml, and the within-day precision 2 to 8% over this concentration range.     

Quantitative analysis of cocaine and its metabolites in whole blood and urine by high-performance liquid chromatography coupled with tandem mass spectrometry

AIM: In forensic toxicology it is important to have specific and sensitive analysis for quantification of illicit drugs in biological matrices. This paper describes a quantitative method for determination of cocaine and its major metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine and ethylene cocaine) in whole blood and urine by liquid chromatography coupled with tandem mass spectrometry LC/MS/MS. METHOD: The sample pre-treatment (0.20 g) consisted of acid precipitation, followed by centrifugation and solid phase extraction of supernatant using mixed mode sorbent columns (SPEC MP1 Ansys Diag. Inc.). Chromatographic separation was performed at 30 degrees C on a reverse phase Zorbax C18 column with a gradient system consisting of formic acid, water and acetonitrile. The analysis was performed by positive electrospray ionisation with a triple quadropole mass spectrometer operating in multiple reaction monitoring (MRM) mode. Two MRM transitions of each analyte were established and identification criteria were set up based on the retention time and the ion ratio. The quantification was performed using deuterated internal analytes of cocaine, benzoylecgonine and ecgonine methyl ester. The calibration curves of extracted standards were linear over a working range of 0.001-2.00 mg/kg whole blood for all analytes. The limit of quantification was 0.008 mg/kg; the interday precision (measured by relative standard deviation-%RSD) was less than 10% and the accuracy (BIAS) less than 12% for all analytes in whole blood. Urine samples were estimated semi-quantitatively at a cut-off level of 0.15 mg/kg with an interday precision of 15%. CONCLUSION: A liquid chromatography mass spectrometric (LC/MS/MS) method has been developed for confirmation and quantification of cocaine and its metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine and ethylene cocaine) in whole blood and semi-quantitative in urine. The method is specific and sensitive and offers thereby an excellent alternative to other methods such as GC-MS that involves derivatisation.     

Solid-phase micro-extraction-gas chromatography-mass spectrometry and headspace-gas chromatography of tetrahydrocannabinol,amphetamine, methamphetamine,cocaine and ethanol in saliva samples

In the present work, a method was developed aiming at the serial detection of tetrahydrocannabinol (THC), amphetamine, methamphetamine, cocaine and ethanol in saliva. Saliva samples were submitted to an initial headspace procedure for ethanol determination by gas chromatography/flame ionization detector (GC-FID). After this step, two consecutive solid-phase micro-extractions (SPME) were carried out: THC was extracted by submersing a polydimethylsiloxane fiber (100 micro m) in the vial for 20 min; amphetamine, methamphetamine and cocaine were subsequently extracted after alkalinization. Derivatization of the amphetamines was carried out directly in the solution by adding 2 micro l of butylchloroformate. Gas chromatography-mass spectrometry (GC-MS) was used to identify the analytes in selected ion monitoring (SIM) mode. Confidence parameters of validation of the method were: recovery, linearity, intra- and inter-assay precision as well as limits of detection and quantification of the analytes. The limits of quantification (LOQ) obtained were: ethanol (0.010 g/l); amphetamine (5.0 ng/ml); methamphetamine (0.5 ng/ml); cocaine (5 ng/ml) and THC (5 ng/ml). The method proved to be highly precise (coefficient of variation<8%) for all detected substances.     

Rapid assay of cocaine, opiates and metabolites by gas chromatography—mass spectrometry

The simultaneous assay of cocaine, opiates and metabolites in small biological samples continues to be a difficult task. This report focuses upon tabulation of important techniques (extraction, derivatization, chromatographic conditions, detection mode, data acquisition) reported over the last decade that were used in the development of assays for these analytes. The most prevalent procedures for extraction of cocaine, opiates and metabolites were liquid—liquid and solid-phase extraction isolation methods. Following extraction analytes were derivatized and analyzed by gas chromatography—mass spectrometry. The technique most often used for chromatographic separation was fused-silica capillary column gas chromatography. Detection generally was performed by selected ion monitoring in the positive-ion electron-impact ionization mode, although full-scan acquisition and positive- and negative-ion chemical ionization methods have been used. It was apparent from the review that there is a continuing need for greater sensitivity and selectivity in the assay of highly potent opiates and for cocaine and metabolites.     

Determination of cocaine, benzoylecgonine and cocaethylene in human hair by solid-phase microextraction and gas chromatography-mass spectrometry

The present work describes a highly precise and sensitive method developed to detect cocaine (COC), benzoylecgonine (BE, its main metabolite) and cocaethylene (CE, transesterification product of the coingestion of COC with ethanol) in human head hair samples. The method was based on an alkylchloroformate derivatization of benzoylecgonine and the extraction of the analytes by solid-phase microextraction (SPME). Gas chromatography-mass spectrometry (GC-MS) was used to identify and quantify the analytes in selected ion monitoring mode (SIM). The limits of quantification and detection (LOQ and LOD) were: 0.1 ng/mg for COC and CE, and 0.5 ng/mg for BE. Good inter- and intra-assay precision was observed. The dynamic range of the assay was 0.1-50 ng/mg. The method is not time consuming and was shown to be easy to perform.     

Simultaneous assay of cocaine, heroin and metabolites in hair, plasma, saliva and urine by gas chromatography—mass spectrometry

As part of an ongoing research program on the development of drug detection methodology, we developed an assay for the simultaneous measurement of cocaine, heroin and metabolites in plasma, saliva, urine and hair by solid-phase extraction (SPE) and gas chromatography—mass spectrometry (GC—MS). The analytes that could be measured by this assay were the following: anhydroecgonine methyl ester; ecgonine methyl ester; ecgonine ethyl ester; cocaine; cocaethylene; benzoylecgonine; cocaethylene; norcocaethylene; benzoylnorecgonine; codeine; morphine; norcodeine; 6-acetylmorphine; normorphine; and heroin. Liquid specimens were diluted, filtered and then extracted by SPE. Additional handling steps were necessary for the analysis of hair samples. An initial wash procedure was utilized to remove surface contaminants. Washed hair samples were extracted with methanol overnight at 40°C. Both wash and extract fractions were collected, evaporated and purified by SPE. All extracts were evaporated, derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) and analyzed by GC—MS. The limit of detection (LOD) for cocaine, heroin and metabolites in biological specimens was approximately 1 ng/ml with the exception of norcodeine, normorphine and benzoylnorecgonine (LOD = 5 ng/ml). The LOD for cocaine, heroin and metabolites in hair was approximately 0.1 ng/mg of hair with the exception of norcodeine (LOD = 0.3 ng/mg) and normorphine and benzoylnorecgonine (LOD = 0.5 ng/mg). Coefficients of variation ranged from 3 to 26.5% in the hair assay. This assay has been successfully utilized in research on the disposition of cocaine, heroin and metabolites in hair, plasma, saliva and urine and in treatment studies.     

Detection of cocaine and cocaethylene in sweat by solid-phase microextraction and gas chromatography/mass spectrometry

In the present work, a semi-quantitative method was developed to detect simultaneously cocaine (COC) and cocaethylene (CE) (transesterification product of the coingestion of COC with ethanol) in sweat. Sweat samples were collected by means of a non-occlusive sweat patch device supplied by PharmChek. The method was based on the dissolution of COC and CE incorporated into the patch, with 0.2 M sodium acetate buffer (pH 5.0) and the extraction of the analytes by solid-phase microextraction (SPME). Gas chromatography/mass spectrometry (GC-MS) was used to detect the analytes in selected ion monitoring mode (SIM). The method showed to be very simple, rapid and sensitive. The limits of detection were 5 ng/ml for COC and CE (12.5 ng/patch). Good inter and intra-assay precision was also observed (coefficient of variation <8%) with the use of deuterated internal standards.     

Development and validation of a liquid chromatography-mass spectrometry assay for the determination of opiates and cocaine in meconium

A procedure based on liquid chromatography-mass spectrometry (LC-MS) is described for determination of 6-monoacetylmorphine, morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, cocaine, benzoylecgonine and cocaethylene in meconium using nalorfine as the internal standard. The analytes are initially extracted from the matrix by methanol (6-monoacetylmorphine, morphine, codeine, cocaine, benzoylecgonine and cocaethylene) or 0.01 M ammonium hydrogen carbonate buffer (morphine-3-glucuronide, morphine-6-glucuronide). Subsequently a solid-phase extraction with Bondelut Certify columns (6-monoacetylmorphine, morphine, codeine, cocaine, benzoylecgonine and cocaethylene) or ethyl solid-phase extraction columns (morphine-3-glucuronide, morphine-6-glucuronide) was applied. Chromatography was performed on a C(8) reversed-phase column using a gradient of acetic acid 1%-acetonitrile as a mobile phase. Analytes were determined in LC-MS single ion monitoring mode with atmospheric pressure ionisation-electrospray (ESI) interface. The method was validated in the range 0.005-1.00 microg/g using 1 g of meconium per assay and applied to analysis of meconium in newborns to assess fetal exposure to opiates and cocaine.     

Application of a validated high-performance liquid chromatography-mass spectrometry assay to the analysis of m- and p-hydroxybenzoylecgonine in meconium

A high-performance liquid chromatography (HPLC)-mass spectrometry (MS) assay, already validated for opiates and cocaine in meconium, has been re-applied for determination of m- and p-hydroxybenzoylecgonine, using nalorphine as the internal standard. Methodology included an initial extraction from the matrix by methanol and then a solid-phase extraction (SPE). A reversed-phase chromatography was used with a gradient of 1% acetic acid-acetonitrile coupled to atmospheric pressure ionization electrospray-mass spectrometry single ion monitoring mode. This method, validated in the range 0.005-1.00 microg analytes/g meconium, proved useful to identify and quantify these two metabolites in meconium samples, already tested for the presence of cocaine, benzoylecgonine and cocaethylene. A positivity of range of concentrations varied between 0.007 and 0.338 microg/g, confirming the importance of these two hydroxylated derivatives to monitor fetal exposure to cocaine.     

The development and validation of liquid chromatography method for the simultaneous determination of metformin and glipizide, gliclazide, glibenclamide or glimperide in plasma

This article describes the development of SPE and HPLC methods for the simultaneous determination of metformin and glipizide, gliclazide, glibenclamide or glimperide in plasma. Several extraction and HPLC methods have been described previously for the determination of each of these analytes in plasma separately. The simultaneous determination of these analytes is important for the routine monitoring of diabetic patients who take combination medications and for studying the pharmacokinetics of the combined dosage forms. In addition this developed method can serve as a standard method for the plasma determination of these analytes therefore saving time, effort and money. The recoveries of the developed methods were found to be between 76.3% and 101.9%. The limits of quantification were between 5 and 22.5 ng/ml. The intraday and interday precision (measured by coefficient of variation, CV%) was always less than 9%. The accuracy (measured by relative error %) was always less than 12%. Stability analysis showed that all analytes are stable for at least 3 months when stored at -70 degrees C.     

Subnanogram on-line column-switching liquid chromatographic-tandem mass spectrometric quantification method for nelfinavir and methoxyphenol metabolite M1 in rat plasma

A new on-line, rapid and sensitive column-switch LC/MS/MS method to measure nelfinavir (NFV), an HIV-1 protease inhibitor, and its major metabolite (M1) in rat plasma was developed. Rat plasma containing the analytes and the internal standard was treated with acetonitrile and the supernatant was processed through an on-line extraction and an analytical columns, with a column-switch device. ESI-LC/MS with multiple reaction monitors for appropriate analytes was performed. This assay gave a limit of quantitation (LOQ) of <1 ng/mL for the analytes with 5 min run time. The within-run and between-run precisions were <12 and <10%, respectively. This analytical method was successfully applied to a study to correlate changes in maternal and placental NFV plasma concentrations in rats following NFV exposure in utero.     

Determination of dapoxetine, an investigational agent with the potential for treating depression, and its mono- and di-desmethyl metabolites in human plasma using column-switching high-performance liquid chromatography

A column-switching high-performance liquid chromatographic (HPLC) method is described for the determination of dapoxetine, and its mono- and di-desmethyl metabolites in human plasma. The analytes, including an internal standard, were extracted from plasma at basic pH with hexane—ethyl acetate. The organic extract was evaporated to dryness and the residue reconstituted with acetonitrile. The analytes were separated from late-eluting endogenous substances on a Zorbax RX-C₈ pre-column. The front-cut fraction containing the analytes was further separated on a second RX-C₈ column. The analytes were detected by their native fluorescence, using excitation and emission wavelengths of 230 and 330 nm, respectively. The limit of quantitation was determined to be 20 ng/ml, and the response was linear from 20 to 200 ng/ml. The method has been successfully applied to human plasma samples in a Phase I study.     

Detection, quantification and confirmation of anabolic steroids in equine plasma by liquid chromatography and tandem mass spectrometry

Anabolic androgenic steroids are related to the male sex hormones and are abused in equine sports. In an effort to deter the abuse of anabolic steroids, a sensitive LC-MS/MS method was developed for detection, quantification and confirmation of eight major anabolic steroids (testosterone, normethandrolone, nandrolone, boldenone, methandrostenolone, tetrahydrogestrinone (THG), trenbolone, and stanozolol) in equine plasma. Formation of solvent adduct ions of the analytes was observed under electrospray ionization (ESI) conditions, and desolvation of the solvent adduct ions by source collision-induced decomposition (CID) increased the abundance of the [M+H]+ ions as well as the multiple-reaction monitoring (MRM) signals. ESI (+) and APCI (+) were compared with respect to sensitivity for the analytes and the former provided better sensitivity. The matrix effect on ion suppression or enhancement was evaluated, and was negligible. Confirmation of the analytes was performed using criteria of three ion transitions and LC retention time of each analyte. The limit of detection (LOD) and quantification (LOQ) was 25 pg/mL. The limit of confirmation (LOC) was 25 pg/mL for boldenone; 50 pg/mL for normethandrolone, nandrolone, and methandrostenolone; and 100 pg/mL for testosterone, THG, trenbolone, and stanozolol. The analytes were evaluated for stability and found to be stable in plasma for 24h at room temperature, 13 days at 4 degrees C, and 34 days at -20 and -70 degrees C. The method was successfully applied to analyses of equine plasma samples for pharmacokinetics study. This method is sensitive and useful for detection, quantification and confirmation of these anabolic steroids in equine plasma.     

Simultaneous determination of cortisol and prednisolone in body fluids by using HPLC-DAD coupled with second-order calibration based on alternating trilinear decomposition

A novel method for simultaneous determination of cortisol and prednisolone in body fluids has been developed in this paper. Three-way data recorded by high-performance liquid chromatography with a diode array detector (HPLC-DAD) have been analyzed by second-order calibration based on the alternating trilinear decomposition (ATLD) algorithm. The chemometric methodology selected exploits the second-order advantage of the three-way data arrays, which allows one to obtain concentrations of individual calibrated analytes even in the presence of interferences not present in the calibration samples (e.g. background in urine or plasma). It was applied to simultaneous determination of cortisol and prednisolone in both plasma and urine samples. Though the chromatographic and spectral peaks of the analytes were heavily overlapped and interferents coeluted with the compounds studied, good recoveries of the analytes could be obtained with HPLC-DAD coupled with second-order calibration based on ATLD. Sample preparation was based on solvent extraction (SE), and quantification can be carried out with simple mobile phase. The time required for the quantification process is shorter than other methods.     

Determination of xanomeline (LY246708 tartrate), an investigational agent for the treatment of Alzheimer's disease, in rat and monkey plasma by capillary gas chromatography with nitrogen-phosphorus detection

A GC method is described for the determination of xanomeline (LY246708 tartrate) and selected metabolites in rat and monkey plasma. The analytes, including an internal standard, were extracted from plasma at basic pH with hexane. The organic extract was evaporated to dryness and the residue was reconstituted in hexane. The analytes were separated from metabolites and endogenous substances using a DB1701 capillary column. The analytes were detected using nitrogen-phosphorus detection (NPD). The limit of quantitation was determined to be 8 ng/ml, and the response was linear from 8 to 800 ng/ml. The method has been successfully applied to rat and monkey samples pursuant to the development of xanomeline as an agent for the symptomatic treatment of Alzheimer's disease.     

A sensitive and selective HPLC/ESI-MS/MS assay for the simultaneous quantification of 16-dehydropregnenolone and its major metabolites in rabbit plasma

A sensitive, selective and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous quantification of 16-dehydropregnenolone (DHP) and its five metabolites 4,16-pregnadien-3, 20-dione (M(1)), 5-pregnene-3beta-ol-20-one (M(2)), 5-pregnene-3beta, 20-diol (M(3)), 5-pregnene-3beta-ol-16, 17-epoxi-20-one (M(4)) and 5,16-pregnadien-3beta, 11-diol-20-one (M(5)) in rabbit plasma using dexamethasone as internal standard (IS). The analytes were chromatographed on Spheri-5 RP-18 column (5 microm, 100 mm x 4.6 mm i.d.) coupled with guard column using acetonitrile:ammonium acetate buffer (90:10, v/v) as mobile phase at a flow rate of 0.65 ml/min. The quantitation of the analytes was carried out using API 4000 LC-MS-MS system in the multiple reaction monitoring (MRM) mode. The method was validated in terms of linearity, specificity, sensitivity, recovery, accuracy, precision (intra- and inter-assay variation), freeze-thaw, long-term, auto injector and dry residue stability. Linearity in plasma was observed over a concentration range of 1.56-400 ng/ml with a limit of detection (LOD) of 0.78 ng/ml for all analytes except M(3) and M(5) where linearity was over the 3.13-400 ng/ml with LOD of 1.56 ng/ml. The absolute recoveries from plasma were consistent and reproducible over the linearity range for all analytes. The intra- and inter-day accuracy and precision method were within the acceptable limits and the analytes were stable after three freeze-thaw cycles and their dry residues were stable at -60 degrees C for 15 days. The method was successfully applied to determine concentrations of DHP and its putative metabolites in plasma during a pilot pharmacokinetic study in rabbits.     

Liquid chromatography-tandem electrospray mass spectrometry method for determination of serial chiral novel anticholinergic compounds of phencynonate in rat plasma

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of serial chiral novel anticholinergic compounds of phencynonate in rat plasma. After a simple protein-precipitation using methanol, the post-treatment samples were separated on a CAPCELL UG120 column with a mobile phase of a mixture of methanol and water (35:65) containing 0.1% formic acid. The serial chiral analytes and internal standard (IS) were all detected by the use of selected reaction monitoring mode (SRM). The method of all serial chiral analytes developed was validated in rat plasma with a daily working range of 0.5-100 ng/ml with correlation coefficient, R(2) > or = 0.99 and a sensitivity of 0.5 ng/ml as lower limit of quantification, respectively. This method was fully validated for the accuracy, precision and stability studies for all serial chiral analytes. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of serial chiral novel anticholinergic compounds of phencynonate in rat plasma.     

Cocaine detection via rolling circle amplification of short DNA strand separated by magnetic beads

A novel and sensitive fluorescence biosensor based on aptamer and rolling circle amplification for the determination of cocaine was developed in the present work. Here cocaine aptamers immobilized onto Au nanoparticles modified magnetic beads hybridized with short DNA strand. In the presence of cocaine, the short DNA strand was displaced from aptamer owing to cocaine specially binding with aptamer. Next, the short DNA strand was separated by magnetic beads and used to originate rolling circle amplification as primer. The end products of rolling circle amplification were detected by fluorescence signal generation upon molecular beacons hybridizing with the end products of rolling circle amplification. With rolling circle amplification and the separation by magnetic beads reducing the background signal, the new strategy was suitable for the detection of as low as 0.48 nM cocaine. Compared with reported cocaine sensors, our method exhibited excellent sensitivity. Our new strategy may provide a platform for numerous proteins and low molecular weight analytes to highly sensitively detect by DNA amplification.     

The presence of cocaine and benzoylecgonine in rat cerebrospinal fluid after the intravenous administration of cocaine.

Cocaine hydrochloride, in doses of 0.5, 1.0, 2.0 and 4.0 mg/kg, iv, was administered to male Sprague-Dawley rats. Cerebrospinal fluid (CSF) was collected from the cisterna magna over a 20 min period and blood samples were obtained at 20 min after cocaine administration. In addition, blood samples for the 1 mg/kg dose of cocaine were collected at 2, 10, 20 and 30 min following drug injection. Gas chromatography/mass spectrometry was used for the analysis of cocaine and its metabolites in plasma and CSF. The disappearance of cocaine (1 mg/kg) from the plasma exhibited first order kinetics with a half-life of 18.11 +/- 3.22 min. Cocaine and benzoylecgonine were found in CSF and the concentrations of cocaine and benzoylecgonine increased in CSF as the doses of cocaine were increased. CSF flow rates were not altered by the iv administration of cocaine or benzoylecgonine. The CSF-to-plasma ratios for cocaine were quite similar to each other over the dosage range of cocaine that was administered; however, the CSF-to-plasma ratios for benzoylecgonine decreased as the concentrations of benzoylecgonine increased in plasma and CSF. When benzoylecgonine (2 mg/kg, iv) was given, the compound was detected in CSF indicating that benzoylecgonine can enter into the central nervous system from the peripheral blood. This investigation shows that cocaine and benzoylecgonine can be assayed in CSF and that the plasma levels of these compounds correlate with their concentrations in CSF.     

Simultaneous determination of five antipsychotic drugs in rat plasma by high performance liquid chromatography with ultraviolet detection

A significant percentage of psychiatric patients who are treated with antipsychotics are treated with more than one antipsychotic drug in the clinic. Thus, it is advantageous to use a rapid and reliable assay that is suitable for determination of multiple antipsychotic drugs in plasma in a single run. A simple and sensitive HPLC-UV method was developed and validated for simultaneous quantification of olanzapine, haloperidol, chlorpromazine, ziprasidone, risperidone and its active metabolite 9-hydroxyrisperidone in rat plasma using imipramine as an internal standard (I.S.). The analytes were extracted from rat plasma using a single step liquid-liquid acid solution back extraction technique with wash procedure, which provided the very clear baseline for blank plasma extraction. The compounds were separated on an Agilent Eclipse XDB C8 (150 mm x 4.6 mm i.d., 5 microm) column using a mobile phase of acetonitrile/30 mM ammonium acetate including 0.05% triethylamine (pH 5.86 adjusted with acetic acid) with gradient elution. All of the analytes were monitored using UV detection. The method was validated and the linearity, lower limit of quantitation (LLOQ), precision, accuracy, recoveries, selectivity and stability were determined. The LLOQ was 2.0 ng/ml and correlation coefficient (R(2)) values for the linear range of 2.0-500.0 ng/ml were 0.998 or greater for all the analytes. The precision and accuracy for intra-day and inter-day were better than 7.44%. The recovery was above 74.8% for all of the analytes. This validated method has been successfully used to quantify the plasma concentration of the analytes for pharmacological and toxicological studies following chronic treatment with antipsychotic drugs in the rat.