Simultaneous determination of amphetamine,methamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine in human hair by gas chromatography-mass spectrometry

A procedure is presented for the simultaneous identification and quantification of amphetamine (AP), methamphetamine (MA), methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine (MDMA) in human hair. The method involves decontamination of hair with dichloromethane and warm water, heat-alkaline hydrolysis in the presence of deuterated internal standards, liquid-liquid extraction and gas chromatography-mass spectrometry after derivatization with pentafluoropropionic anhydride-pentafluoropropanol. The limit of detection for AP, MA and MDA was 0.05 ng/mg using a 50-mg hair sample; for MDMA it was 0.1 ng/mg. Coefficients of variation ranged from 7 to 18%. This assay has been successfully utilized in the evaluation of the deposition of the drugs in hair obtained from various parts of the anatomy of a stimulant abuser.     

Validation of a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair using LC-MS/MS and its application to human and rat hair

Benzodiazepines and zolpidem are controlled in many countries due to their inherent adverse effects of a high degree of tolerance and dependence. Recently, as some of these drugs have become distributed illegally and available through media such as the Internet, their abuse is becoming a serious social problem. Hair is a useful specimen to prove chronic drug use. In the present study, a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair was established and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The drugs and their metabolites in hair were extracted using methanol, filtered and injected on the LC-MS/MS. The following validation parameters of the method were satisfactory: selectivity, linearity, matrix effect, recovery, process efficiency, intra- and inter-assay precision and accuracy and processed sample stability. The limit of detection (LOD) and the limit of quantification (LOQ) were the total drug detected from the sample. The LODs ranged from 0.005 ng (zolpidem) to 0.5 ng (bromazepam and chlordiazepoxide) and the LOQs were 0.25 ng in every analyte except for bromazepam and chlordiazepoxide, for which they were 0.5 ng. The developed method was successfully applied to five legal cases involving use of benzodiazepines and zolpidem and to an animal study on drug incorporation into hair. Diazepam and its three metabolites, as well as lorazepam, were detected in hair from both the multiple- and single-dose administration groups of lean Zucker rats. The concentration of diazepam was higher than those of its metabolites in both dark grey and white hair from the multiple-dose administration groups, with the mean concentration ranges from 0.16 to 0.51 ng/mg and from 0.10 to 0.24 ng/mg, respectively. The mean concentration ranges of lorazepam were from 0.05 to 0.37 ng/mg in dark grey hair and from 0.11 to 0.45 ng/mg in white hair from the multiple-dose administration groups. Hair pigmentation did not have any significant effect on the degree of the deposition of drugs and their metabolites in hair.     

Screening method for benzodiazepines and hypnotics in hair at pg/mg level by liquid chromatography-mass spectrometry/mass spectrometry

A procedure is presented for the screening of 16 benzodiazepines and hypnotics in human hair by LC-MS/MS (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, clobazam, diazepam, lorazepam, lormetazepam, midazolam, nordiazepam, oxazepam, temazepam, tetrazepam, triazolam, zaleplon and zolpidem). The method involves decontamination of hair with methylene chloride, hair cut into small pieces, incubation of 20 mg in phosphate buffer (pH 8.4) in the presence of 1 ng diazepam-d5 used as internal standard, liquid-liquid extraction with diethyl ether/methylene chloride (10/90) and separation using liquid chromatography-tandem mass spectrometry. The limits of quantification for all benzodiazepines and hypnotics range from 0.5 to 5 pg/mg using a 20-mg hair sample. Linearity is observed from the limit of quantification of each compound to 200 pg/mg (r2 > 0.99). Coefficients of variation measured on six points and at two concentrations (10 and 50 pg/mg) range from 5 to 20% for all drugs but one. Extraction recovery, measured at the two same concentrations range from 32 to 76%. These results were found suitable to screen for 16 benzodiazepines in hair and detect them at very low concentrations, making this method suitable to monitor single dose.     

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.     

Integration of GC/EI-MS and GC/NCI-MS for simultaneous quantitative determination of opiates, amphetamines, MDMA, ketamine, and metabolites in human hair

In this paper, the possibility of using a multiple ionization mode approach of GC/MS was developed for the simultaneous hair testing of common drugs of abuse in Asia, including amphetamines (amphetamine, AP; methamphetamine, MA; methylenedioxy amphetamine, MDA; methylenedioxy methamphetamine, MDMA; methylenedioxy ethylamphetamine, MDEA), ketamine (ketamine, K; norketamine, NK), and opiates (morphine, MOR; codeine, COD; 6-acetylmorphine, 6-AM). This strategy integrated the characteristics of gas chromatography-mass spectrometry (GC-MS) using electron impact ionization (EI) and negative chemical ionization (NCI). Hair samples (25 mg) were washed, cut, and incubated overnight at 25 degrees C in methanol-trifluoroacetic acid (methanol-TFA). The samples were extracted by solid phase extraction (SPE) procedure, derivatized using heptafluorobutyric acid anhydride (HFBA) at 70 degrees C for 30 min, and the derivatives analyzed by GC-MS with EI and NCI. The limit of detection (LOD) with GC/EI-MS analysis obtained were 0.03 ng/mg for AP, MA, MDA, MDMA, and MDEA; 0.05 ng/mg for K, NK, MOR, and COD; and 0.08 ng/mg for 6-AM. The LOD of GC/NCI-MS analysis was much lower than GC/EI-MS analysis. The LOD obtained were 30 pg/mg for AP and MDA in GC/EI-MS and 2 pg/mg in GC/NCI-MS. Therefore, the sensitivity of AP and MDA in GC/NCI-MS was improved from 15-fold compared with EI. The sensitivity of AP, MA, MDA, MDMA, MDEA, MOR, and COD was improved from 15- to 60-fold compared with EI. In addition, the sensitivity of 6-AM increased 8-fold through selection of m/z 197 for the quantitative ion. Moreover, K and NK could dramatically improve their sensitivity at 200- and 2000-fold. The integration of GC/EI-MS and GC/NCI-MS can obtain the high sensitivity and complementary results of drugs of abuse in hair. Six hair samples from known drug abusers were examined by this new strategy. These results show that integrating the characteristics of GC/EI-MS and GC/NCI-MS were not only enhancement of the sensitivity but also avoid wrong results and wrong interpretations of correct results.     

Screening and quantitation of multiclass drugs of abuse and pharmaceuticals in hair by fast liquid chromatography electrospray time-of-flight mass spectrometry

In this work, an automated screening method for the simultaneous identification and quantitation of 30 representative multiclass drugs (including opiates, cocaine and its main metabolite, cannabinoids, amphetamines and other stimulants in hair samples) has been developed using fast liquid-chromatography time-of-flight mass spectrometry (LC-TOFMS). The identification and quantitation of the drugs were carried out by liquid chromatography using a C(18) column (4.6×50 mm) with 1.8 μm particle size. Accurate mass measurements of ions of interest (typically [M+H](+)) by electrospray time-of-flight mass spectrometry in the positive ionization mode were used for unambiguous confirmation of the targeted species. Three sample preparation methodologies were evaluated: (a) direct methanolic extraction by sonication, (b) acidic extraction, and (c) alkaline digestion. Direct methanolic extraction showed better recoveries and cleaner extracts. The limits of detection obtained in hair matrix were as low as 5 pg mg(-1) for cocaine and cannabidiol, ranging from 5 to 75 pg mg(-1) for the studied species while the LOQ ranged from 15 to 250 pg mg(-1). The method has been applied to six hair samples from drug consumer volunteers, where the presence of at least one drug was confirmed by accurate mass measurements within 2 ppm (mass error) in most cases. The present study demonstrates the usefulness of LC-TOFMS for both screening and quantitation purposes in drug testing in hair. In addition, the possibility of non-target or a posteriori data analysis of samples or the extension of the procedure for testing for additional compounds offers interesting features for forensic analysis.     

Development of a two-step injector for GC-MS with on-column derivatization, and its application to the determination of amphetamine-type stimulants (ATS) in biological specimens

A two-step auto-injector has been developed for the automated on-column derivatization and subsequent GC-MS of amine-type drugs and metabolites. To effectively derivatize such analytes, this injector has been designed to inject the derivatization reagent several seconds after the sample has been injected. Eleven kinds of amphetamine-type stimulants (ATS) and their typical metabolites were examined, using the trifluoroacetylation reagent N-methyl bis(trifluoroacetamide) (MBTFA). Although the quantitative derivatization of the hydroxyl groups was difficult, this technique was successfully applied to the determination of ATS in urine, blood, and hair specimens. The detection limits of methamphetamine and amphetamine in hair were 0.2 and 0.1 ng/mg hair, respectively, in the full-scan mode, when a 10 mg hair sample is analyzed.     

Detection of sulphamethazine residues in cattle and pig hair by HPLC-DAD

An HPLC method with diode array detection (DAD) is proposed for the detection of sulphamethazine (SMZ) residues in pig and cattle hair. Hair samples were extracted under alkaline conditions (NH4OH 0.2M for calf samples and NaOH 0.1M for piglet samples) and purified with a dual solid-phase extraction (SPE) cartridge system (reverse phase/strong-cation exchange). Recovery of SMZ in fortified samples varied from 70 to 85%, with a limit of quantification of 0.155 ng/mg. Residues of SMZ (7.2-59.2 ng/mg) were detected both in calf and piglet hairs after a therapeutic treatment with SMZ, while no interfering peak was observed in samples from untreated animals.     

A sensitive assay for the quantitative analysis of vinorelbine in mouse and human EDTA plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry

A sensitive, specific and fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay for the determination of vinorelbine in mouse and human plasma is presented. A 200 microL aliquot was extracted with solid-phase extraction (SPE) using Bond-Elut C(2) cartridges. Dried extracts were reconstituted in 100 microL 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) containing the internal standard vintriptol (100 ng/mL) and 10 microL volumes were injected onto the HPLC system. Separation was achieved on a 50 mm x 2.0 mm i.d. Gemini C(18) column using isocratic elution with 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) at a flow rate of 0.4 mL/min. HPLC run time was only 5 min. Detection was performed using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay quantifies vinorelbine from 0.1 to 100 ng/mL using human plasma sample volumes of 200 microL. With this method vinorelbine can be measured in mouse plasma samples when these samples are diluted eight times in control human plasma. Calibration samples prepared in control human plasma can be used for the quantification of the drug. The lower limit of quantification in mouse plasma is 0.8 ng/mL. This assay is used to support preclinical and clinical pharmacologic studies with vinorelbine.     

Simultaneous,quantitative determination of opiates,amphetamines, cocaine and benzoylecgonine in oral fluid by liquid chromatography quadrupole-time-of-flight mass spectrometry

A method using liquid chromatography coupled to tandem mass spectrometry is described for the determination of drugs of abuse in oral fluid. The method is able to simultaneously quantify amphetamines (amphetamine, methamphetamine, MDA, MDMA and MDEA), opiates (morphine and codeine), cocaine and benzoylecgonine. Only 200 micro of oral fluid is spent for analysis. The sample preparation is easy and consists of mixed mode phase solid-phase extraction. Reversed-phase chromatography is carried out on a narrow bore phenyl type column at a flow-rate of 0.2 ml/min. A gradient is applied ranging from 6 to 67.6% methanol with ammonium formate (10 mM, pH 5.0) added to the mobile phase. The column effluent was directed into a quadrupole-time-of-flight instrument by electrospray ionization, without the use of a splitter. A validation study was carried out. Recovery ranged from 52.3 to 98.8%, within-day and between-day precision expressed by relative standard deviation were less than 11.9 and 16.8%, respectively, and inaccuracy did not exceed 11.6%. The limit of quantification was 2 ng/ml (0.66 x 10(-5)-1.48 x 10(-5) M) for all compounds. Internal standards were used to generate quadratic calibration curves (r(2)>0.999). The method was applied to real samples obtained from suspected drug users. An interference was observed from the device used to sample the oral fluid, consequently this was excluded from the method which was validated on oral fluid obtained by spitting in a test-tube.     

Determination of amiodarone and desethylamiodarone in horse plasma and urine by high-performance liquid chromatography combined with UV detection and electrospray ionization mass spectrometry

A rapid method for the quantification of amiodarone and desethylamiodarone in animal plasma using high-performance liquid chromatography combined with UV detection (HPLC-UV) is presented. The sample preparation includes a simple deproteinisation step with acetonitrile. In addition, a sensitive method for the quantification of amiodarone and desethylamiodarone in horse plasma and urine using high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is described. The sample preparation includes a solid-phase extraction (SPE) with a SCX column. Tamoxifen is used as an internal standard for both chromatographic methods. Chromatographic separation is achieved on an ODS Hypersil column using isocratic elution with 0.01% diethylamine and acetonitrile as mobile phase for the HPLC-UV method and with 0.1% formic acid and acetonitrile as mobile phase for the LC-MS/MS method. For the HPLC-UV method, good linearity was observed in the range 0-5 microg ml(-1), and in the range 0-1 microg ml(-1) for the LC-MS/MS method. The limit of quantification (LOQ) was set at 50 and 5 ng ml(-1) for the HPLC-UV method and the LC-MS/MS method, respectively. For the UV method, the limit of detection (LOD) was 15 and 10 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs of the LC-MS/MS method in plasma were much lower, i.e. 0.10 and 0.04 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs obtained for the urine samples were 0.16 and 0.09 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The methods were shown to be of use in horses. The rapid HPLC-UV method was used for therapeutic drug monitoring after amiodarone treatment, while the LC-MS/MS method showed its applicability for single dose pharmacokinetic studies.     

Simultaneous determination of 13 amphetamine related drugs in human whole blood using an enhanced polymer column and gas chromatography-mass spectrometry

Metamphetamine (MA) is one of the most frequently encountered abused drugs in Japan and the Triage immunoassay kit is often used to screen for this drug. However, immunoassay screening also gives positive results with other structurally related compounds, such as 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), p-methoxyamphetamine (PMA), an ephedrine metabolite and beta-phenethylamine (PEA). Therefore, it is important to develop a simple and reliable method which can determine these drugs simultaneously. This paper describes a simple method for simultaneous identification and quantification of 13 amphetamine related drugs in human whole blood. The method consists of a solid phase extraction using a new polar-enhanced Focus column followed by acetylation and gas chromatography-mass spectrometry in the scan mode. Tetradeuterated MA and trideuterated methylephedrine (ME) were used as internal standards. As the Focus column required only simple extraction steps and provided a clean extract, identification of each drug was feasible even at low concentrations. The calibration curves were linear over the concentration range from 50 to 5000 ng/ml for all drugs with correlation coefficients that exceeded 0.99. The lower limits of detection of the drugs were 5-50 ng/ml. The absolute recoveries for the drugs were 65-95% and 64-89% at concentrations of 100 and 1000 ng/ml, respectively. Accuracy and precision data were satisfactory when using 2 internal standards. The applicability of the assay was proven by the analysis of blood samples in forensic cases. This method should be most useful for confirmation of positive immunoassay results for amphetamines and related drugs.     

Simple and sensitive determination of Delta(9)-tetrahydrocannabinol, cannabidiol and cannabinol in hair by combined silylation, headspace solid phase microextraction and gas chromatography-mass spectrometry

A new method for determination of Delta(9)-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in hair based on alkaline hair hydrolysis, extraction by iso-octane, combined derivatization with N,O-bis-(trimethylsilyl)-trifluoroacetamide and headspace solid phase microextraction of the extract residue, and gas chromatography-mass spectrometry was developed and evaluated. The limits of detection of the three compounds were 0.01-0.02 ng/mg. The method was routinely applied to more than 250 hair samples. In 77 positive samples, the concentrations ranged from LOD to 4.2 ng/mg for THC (mean 0.49 ng/mg), to 12.1 ng/mg for CBD (mean 0.37 ng/mg) and to 0.85 ng/mg for CBN (mean 0.12 ng/mg) using a sample amount of 30 mg. The frequently observed increase of the segmental drug concentrations from proximal to distal is explained by progressive accumulation in the hair shaft from sebum or side stream smoke.     

Anion exchange SPE and liquid chromatography-tandem mass spectrometry in GHB analysis

In this study, the extraction of γ-hydroxybutyrate (GHB) from urine using solid-phase extraction (SPE) is described. SPE was performed on anion exchange columns after samples of urine had been diluted with de-ionized water. After application of the diluted samples containing GHB-d(6) as an internal standard, the sorbent was washed with deionized water and methanol and dried. The GHB was eluted from the SPE column with a solvent consisting of methanol containing 6% glacial acetic acid. The eluent was collected, evaporated to dryness, and dissolved in mobile phase (100 μL) for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in negative multiple reaction monitoring (MRM) mode. Liquid chromatography was performed in gradient mode employing a biphenyl column and a mobile phase consisting of acetontitrile (containing 0.1% formic acid) and 0.1% aqueous formic acid. The total run time for each analysis was less than 5 min. The limits of detection/quantification for this method were determined to be 50 and 100 ng/mL, respectively. The method was found to be linear from 500 ng/mL to 10,000 ng/mL (r(2)>0.995). The recovery of GHB was found to be greater than 75%. In this report, results of authentic urine samples analyzed for GHB by this method are presented. GHB concentrations in these samples were found to be range from less than 500 ng/mL to 5110 ng/mL.     

Hair analysis for drugs of abuse XIX. Determination of ephedrine and its homologs in rat hair and human hair

A sensitive GC-MS method was developed for the quantitative analysis of ephedrine (EP), phenylpropanolamine (PPA) and methylephedrine (ME) in animal and human hair. After washing with 0.1% sodium dodecyl sulfate, hair samples (10 mg) were added with deuterated internal standards, extracted by 1-h sonication and over night soaking in 2 ml of 5 M HCl-methanol (1:20) at room temperature. Following evaporation of the liquid phase, the residue was dissolved in phosphate buffer solution (pH 6.0) and purified using a solid-phase extraction procedure with Bond Elut Certify columns. Two types of derivatization were compared - using trifluoroacetic anhydride (TFAA) and pentafluoropropionic anhydride (PFPA) - for discrimination of EP and methamphetamine (MA). Derivatized extracts were analyzed by GC-MS in the EI mode using a capillary column (OV-1 equivalent). From the results comparing three GC-MS conditions, PFP-derivatives separated with a temperature gradient of 20°C/min from 60°C to 280°C gave the best resolution between EP and MA. ME was analyzed as a trimethylsilyl derivative using N,O-bis-trimethylsilyl acetamide at the above GC condition. The assay was linear from 0.5 to 50 ng/mg (r=0.998) and capable of detecting less than 50 pg of derivatized EP, PPA and ME on-column. Intra-assay precision was characterized by C.V. values from 5 to 16% in the concentration range of 1–10 ng/mg hair. The method was used for the quantitative determination of EP, PPA and ME in the hair obtained from three rats with dark brown hair after ten intraperitoneal injections (5 mg/kg/day) of the three drugs and from three male and one female volunteers with black hair after an oral dose of 50 mg/day of EP-HCl for three days. Hair samples were collected by shaving from the back of rats and cutting from the scalp of humans 28 days after the first dose. The incorporation rates of EP, PPA and ME into hair (the ratios of [hair concentration] to [AUC]) obtained from the animal experiment were 0.10, 0.07 and 0.03, respectively, which are a little lower than those (0.14, 0.10 and 0.04) of their desoxy-compounds, MA, amphetamine and dimethylamphetamine. EP was detected at an average of 2.25 ng/mg (n=4) in human scalp hair and at a range of 1–29 ng/mg (n=3) in human beard hair until day 14, but its metabolite (PPA) was at a trace level in the hair of the four subjects. The method was successfully used for detection of ME and EP in the hair of a neonate and its mother who was abusing Bron syrup containing ME during the pregnancy.     

Modified method of nalbuphine determination in plasma: validation and application to pharmacokinetics of the rectal route

A solid-phase extraction (SPE) procedure was developed for the quantification of nalbuphine in a small volume (500 μl) of human plasma with subsequent assay by high-performance liquid chromatography (HPLC) and electrochemical detection using 6-monoacetylmorphine as internal standard. Plasma was extracted using Bond Elute certified extraction columns (LCR: 10 ml, 130 mg) after conditioning with methanol and 0.2 M Tris buffer (pH 8). Elution was performed with a CH₂Cl₂-isopropanol-NH₄OH (79:20:, v/v). The organic phase was evaporated to dryness and resuspended in HPLC mobile phase containing 2% isopropanol. Linearity was assessed over the 5–100 ng/ml concentration range and a straight line passing through the origin was obtained. Experiments with spiked plasma samples resulted in recoveries of 95±5.4% and 98±6.2% for nalbuphine and 6-monoacetylmorphine, respectively. The optimal pH conditions for the SPE were found at pH 8. The intra-day coefficients of variation (C.V.) for 5, 40, and 100 ng/ml were 5.3, 3.0 and 2.3% (n=8) and the inter-day C.V.s were 7.7, 3.2 and 3.5% (n=10), respectively. The detection limit for 500 μl plasma sample was 0.02 ng/ml and the limit of quantification 0.1 ng/ml (C.V.=12.4%). The ease of the proposed method of analysis, as well as its high accuracy and sensitivity allow its application to pharmacokinetic studies. A preliminary kinetic profile of nalbuphine after rectal administration in a pediatric patient is presented.     

LC-MS analysis of trimethoxyamphetamine designer drugs (TMA series) from urine samples

A sensitive liquid chromatography-mass spectrometric (LC-MS) method for quantification of an active psychedelic hallucinogenic drugs (trimethoxyamphetamines) in human urine after solid-phase extraction (SPE) with C(18) cartridge was developed and validated. Chromatographic separation was achieved on reversed-phase Phenomenex 3.0 microm Polar Plus column (150 mm x 2.1 mm) with acetonitrile -0.2% acetic acid as mobile-phase and the step gradient elution resulted in a total run time of about 20 min. The analytes were detected by using an electrospray positive ionization mass spectrometry in selected ion monitoring (SIM) mode. In the evaluated concentration range (10-200 ng/mL) (R(2) > or = 0.998) a good linear relationship was obtained. The lower limits of detection (LLODs) and quantification (LLOQs) ranged from 4.26 to 9.12 ng/mL and from 13.18 to 29.22 ng/mL, respectively. Average recoveries ranged from 68.52 to 97.90% in urine at the concentrations of 25, 50 and 100 ng/mL. Intra- and inter-day relative standard deviations were 3.70-10.77% and 7.63-12.94%, respectively. This LC-MS method proved to be robust and reliable, and suitable for the use as a confirmation method in clinical urine drug testing.     

Determination of donepezil, an acetylcholinesterase inhibitor, in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantification of donepezil, a centrally and selectively acting acetyleholinesterase inhibitor, in human plasma. After sample alkalinization with 0.5 ml of NaOH (0.1 M), the test compound was extracted from I ml of plasma using isopropanol-hexane (3:97, v/v). The organic phase was back-extracted with 75 microl of HCl (0.1 M) and 50 microl of the acid solution was injected into a C18 STR ODS-II analytical column (5 microm, 150x4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.02 M, pH 4.6), perchloric acid (6 M) and acetonitrile (59.5:0.5:40, v/v) and was delivered at a flow-rate of 1.0 ml/min at 40 degrees C. The peak was detected using a UV detector set at 315 nm, and the total time for a chromatographic separation was approximately 8 min. The method was validated for the concentration range 3-90 ng/ml. Mean recoveries were 89-98%. Intra- and inter-day relative standard deviations were less than 7.3 and 7.6%, respectively, at the concentrations ranging from 3 to 90 ng/ml. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it could be successfully applied for pharmacokinetic studies and therapeutic drug monitoring.     

Simultaneous determination of glycyrrhizin, a marker component in radix Glycyrrhizae, and its major metabolite glycyrrhetic acid in human plasma by LC-MS/MS

Glycyrrhizin (GLY) which has been widely used in traditional Chinese medicinal preparation possesses various pharmacological effects. In order to investigate the pharmacokinetic behavior of GLY in human after oral administration of GLY or licorice root, a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of GLY and its major metabolite glycyrrhetic acid (GA) in human plasma. The method involved a solid phase extraction of GLY, GA, and alpha-hederin, the internal standard (IS), from plasma with Waters Oasis MCX solid phase extraction (SPE) cartridges (30 mg) and a detection using a Micromass Quattro LC liquid chromatography/tandem mass spectrometry system with electrospray ionization source in positive ion mode. Separation of the analytes was achieved within 5min on a SepaxHP CN analytical column with a mobile phase of acetonitrile:water (50:50, v:v) containing 0.1% formic acid and 5mM ammonium acetate. Multiple reaction monitoring (MRM) was utilized for the detection monitoring 823--> 453 for GLY, 471--> 177 for GA and 752--> 456 for IS. The LC-MS/MS method was validated for specificity, sensitivity, accuracy, precision, and calibration function. The assay had a calibration range from 10 to 10,000 ng/mL and a lower limit of quantification of 10 ng/mL for both GLY and GA when 0.2 mL plasma was used for extraction. The percent coefficient of variation for accuracy and precision (inter-run and intra-run) for this method was less than 11.0% with a %Nominal ranging from 87.6 to 106.4% for GLY and 93.7 to 107.8% for GA. Stability of the analytes over sample processing (freeze/thaw, bench-top and long-term storage) and in the extracted samples was also tested and established.     

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.