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A new approach is described for imaging mass spectrometry (IMS) of methamphetamine (MA) incorporated into human hair using matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) and MALDI-Fourier transform ion cyclotron resonance (FTICR). A longitudinal section of a lengthwise manually-cut single human hair shaft from a chronic MA user was directly analyzed by MALDI-TOF-IMS after deposited with α-Cyano-4-hydroxycinnamic acid matrix. A barcode-like image, which was most probably generated with repeated intakes of MA, was for the first time obtained by monitoring MA-specific product ion in the selected reaction monitoring mode. Laser beam scan lengthwise-cut hair shafts gave only poor mass spectra of MA, probably due to the loss of MA and/or the thermal denaturation of hair. The identity of MA detected in hair was further confirmed by MALDI-FTICR mass spectrometry. A combination with ultra-high resolution mass spectrometry by FTICR provided indisputable identification of MA. The MALDI-FTICR-IMS of another hair shaft from the same MA user also provided a barcode-like image by monitoring the protonated molecule of MA with ultra-high resolution. The two barcode-like images exhibited a close resemblance. Thus, MALDI-IMS can offer a new perspective: 'imaging hair analyses for drugs'.

An automated column-switching liquid chromatographic-atmospheric pressure chemical ionization-mass spectrometric (LC-APCI-MS) method for the simultaneous determination of frequently encountered benzodiazepine hypnotics and their relevant metabolites in urine has been established. A column-switching online solid-phase extraction technique was employed to enhance sensitivity and eliminate tedious sample pretreatment. The combination of an N-vinylacetamide-containing hydrophilic polymer online extraction column and a C18 semi-micro LC column provided the successful separations and MS determinations of the 11 benzodiazepines and 11 relevant metabolites tested in this study. The detection limits ranged 2-10 ng/mL and 10-50 ng/mL in the SIM and full-scan modes, respectively, using 50 microL of urine. The calibration curves were linear up to 500 ng/mL for all these analytes in the SIM mode. The present method provided successful forensic applications for the proof of benzodiazepine administration.

In order to discriminate selegiline (SG) use from methamphetamine (MA) use, the urinary metabolites of SG users have been investigated using high-performance liquid chromatography (HPLC)-electrospray ionization mass spectrometry (HPLC-ESI-MS). Selegiline-N-oxide (SGO), a specific metabolite of SG, was for the first time detected in the urine, in addition to other metabolites MA, amphetamine (AP) and desmethylselegiline (DM-SG). A combination of a Sep-pak C18 cartridge for the solid-phase extraction, a semi-micro SCX column (1.5 mm I.D.x 150 mm) for HPLC separation and ESI-MS for detection provided a simple and sensitive procedure for the simultaneous determination of these analytes. Acetonitrile-10 mM ammonium formate buffer adjusted to pH 3.0 (70:30, v/v) at a flow-rate of 0.1 ml/min was found to be the most effective mobile phase. Linear calibration curves were obtained over the concentration range from 0.5 to 100 ng/ml for all the analytes by monitoring each protonated molecular ion in the selected ion monitoring (SIM) mode. The detection limits ranged from 0.1 to 0.5 ng/ml. Upon applying the scan mode, 10-20 ng/ml were the detection limits. Quantitative investigation utilizing this revealed that SGO was about three times more abundant (47 ng/ml, 79 ng/ml) than DM-SG in two SG users' urine samples tested here. This newly-detected, specific metabolite SGO was found to be an effective indicator for SG administration.

In order to prove heroin (DAM) use, a simple, rapid and sensitive analytical method has been established by combining semi-microcolumn HPLC, a column switching technique and electrospray ionization mass spectrometry (ESI-MS). Urine samples were directly introduced to the system, and endogenous urinary constituents were removed by using on-line column switching solid-phase extraction with a strong cation-exchange (SCX) cartridge column (2.0 mm I.D. x 10 mm). Heroin and its metabolites enriched on the top of the column were then successfully analyzed with excellent separation by use of a SCX semi-microcolumn (1.5 mm I.D. x 150 mm), accompanied by ESI mass spectral detection. The proposed conditions are as follows: mobile phase, 10 mM ammonium acetate (pH 6.0)-acetonitrile (30:70, v/v) (for main separation) and 30 mM ammonium acetate (for trapping); flow-rates, 120 microl/min (for main separation) and 200 microl/min (for trapping); capillary voltage, +4.5 kV; cone voltage, 50 V. Linear calibration curves were obtained in the selected ion monitoring (SIM) mode using protonated molecular ions (m/z 370 for DAM, m/z 328 for MAM and m/z 286 for MOR) over the concentration ranges from 10 to 1000 ng/ml for morphine (MOR) and 1-100 ng/ml for DAM and 6-acetylmorphine (MAM). The detection limits were 0.1-3 ng/ml. Upon applying the scan mode, 2-30 ng/ml were the detection limits. The present HPLC-ESI-MS method was successfully applied to the determination of opiates in users' urine samples.

A simple, rapid, and sensitive method which allows us to simultaneously determine bromvalerylurea (BVU) and its three metabolites (3-methylbutyrylurea [MVU], alpha-(cystein-S-yl)isovalerylurea [CVU], and alpha-(N-acetylcystein-S-yl)isovalerylurea [AcCVU]) was investigated by frit-fast atom bombardment liquid chromatography-mass spectrometry (frit-FAB LC-MS). The LC-MS analysis was performed after the solid-phase extraction from tissue and urine samples with a Sep-Pak C18 cartridge. Tissue homogenates and urine were adjusted to pH 4.0 and applied to the cartridges. The retained BVU and its metabolites were eluted from the cartridge with 2 mL of acetonitrile/10 mM ammonium acetate buffer (pH 3.5, 50:50, v/v). The eluate was analyzed by LC-MS, which employs a semimicro type L-column ODS column. The proposed conditions are as follows: mobile phase A, 0.4% glycerol in acetonitrile/10 mM ammonium acetate buffer (pH 3.5) (5:95, v/v); mobile phase B, 0.4% glycerol in acetonitrile; elution mode, linear gradient, 100% A (5 min) to 100% B in 15 min; flow rate, 0.2 mL/min; split ratio, 1:40. Extraction recoveries of BVU and its metabolites were 91.90-97.79% from the spiked liver homogenate and 89.68-96.13% from the spiked urine. The detection limits ranged from 10 to 25 ng/g in selected ion monitoring mode.

For the verification of the use of chemical warfare agents (CWA), sarin, soman and VX, a simple rapid and accurate method which allows us to simultaneously determine their degradation products, isopropyl methylphosphonic acid (IPMPA), pinacolyl methylphosphonic acid (PMPA), ethyl methylphosphonic acid (EMPA) and methylphosphonic acid (MPA), in human serum, was explored by indirect photometric detection ion chromatography (IPD-IC) which employs an anion-exchange column. IC analysis was performed after sample preparation with an Ag+-form cation-exchange resin cartridge, and the four methylphosphonic acids could be separated well. The proposed conditions are as follows: eluent, 0.5 mM phthalic acid-0.1 mM Tris (hydroxymethyl) aminomethane-5% acetonitrile; flow-rate, 1.0 ml/min; temperature, 50 degrees C; UV detector, 266 nm. All four methylphosphonic acids were eluted within 30 min with hardly any disturbance by impurities in the serum. Linear calibration curves were obtained for MPA, EMPA and IPMPA in the concentration range from 50 ng/ml to 1 microg/ml and for PMPA from 100 ng/ml to 1 microg/ml. The relative standard deviation for the methylphosphonic acids ranged from 3.8 to 6.9% at 500 ng/ml and the detection limits were 40 ng/ml for MPA, EMPA and IPMPA and 80 ng/ml for PMPA. The method would be suitable for analysis of human serum samples.

For the unequivocal proof of the use of a nerve agent O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate (VX), a rapid, accurate and sensitive method which allows us to identify its main hydrolysis product ethyl methylphosphonic acid (EMPA) in human serum was explored by GC-MS. GC-MS analysis was performed after solvent extraction with acetonitrile in acidic conditions from the serum sample, which was previously deproteinized by micro-ultrafiltration, and subsequent tert.-butyldimethylsilyl derivatization with N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) with 1% tert.-butyldimethylsilyl chloride (t-BDMSC). Linear calibration curves were obtained in the concentration range from 50 to 500 ng/ml for EMPA in the full-scan EI mode and from 5 to 50 ng/ml for EMPA in the SIM EI mode. The relative standard deviation obtained at a sample concentration of 50 ng/ml was 8.4% in the full-scan mode and 7.3% in the SIM mode. Upon applying the full-scan EI and CI mode, 40 ng/ml and 80 ng/ml were the detection limits. Using the SIM-EI mode, in which the ion at m/z 153 was chosen, the limit was 3 ng/ml.

A rapid and convenient extraction method has been developed for the determination of various drugs and metabolites of forensic interest in blood by modifying the dispersive solid-phase extraction method "QuEChERS". The following 13 analytes with various chemical properties were used for the method development and its validation: amphetamine, methamphetamine, zolpidem, the carboxylate-form major metabolite of zolpidem M-1, flunitrazepam, 7-aminoflunitrazepam, phenobarbital, triazolam, α-hydroxytriazolam, brotizolam, α-hydroxybrotizolam, chlorpromazine, and promethazine. The modification of the QuEChERS method includes the use of relatively large amounts of inorganic salts in order to coagulate blood, which allows easy isolation of the organic extract phase. A combination of 100 mg anhydrous magnesium sulfate as a dehydrating agent, 50mg sodium chloride as a salting-out agent, and 500 μL acetonitrile containing 0.2% acetic acid as the organic solvent provided the optimum conditions for processing a 100 μL whole blood sample. The recoveries of the analytes spiked into whole blood at 0.5 μg/mL ranged between 59% and 93%. Although the addition of the graphitized carbon Envi-carb for cleanup decreased the recoveries of zolpidem and its carboxylate-form metabolite M-1, it was very effective in avoiding interferences by cholesterol. The present method can provide a rapid, effective, user-friendly, and relatively hygienic method for the simultaneous extraction of a wide range of drugs and metabolites in whole blood specimens.

Cross-reactivities of 76 kinds of phenethylamine-type designer drugs and related compounds to the urine drug tests Instant-View ™ (IV) (the Methamphetamine (MA) test, the Amphetamine 300 test, and the MDMA test) have been investigated. An on-site urine test kit consisting of these three IV tests has been evaluated for the on-site screening of MA users, and the kit has been found to have satisfactory specificity for drug enforcement purposes by separately detecting both MA and its metabolite amphetamine. The cross-reactivity profiles of Emit® II Plus Amphetamines Assay, Emit® II Plus Ecstasy assay, and Emit® d.a.u.® Amphetamine Class assay have also been investigated and discussed.

High-performance liquid chromatography/mass spectrometry enabled us not only to measure bromvalerylurea rapidly, < or = 2 h following accumulation of human bone marrow, but also to improve sensitivity without interference. The method can be widely and effectively used in the fields of pharmacy and forensic sciences.

1. The metabolism of selegiline (SG) has been studied by investigating the time-course of urinary excretion of SG and its metabolites using high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS) in combination with solid-phase extraction. 2. The excretion profiles of SG and its four major metabolites, selegiline-N-oxide (SGO), N-desmethylselegiline (DM-SG), methamphetamine (MA) and amphetamine (AP), were investigated in six healthy volunteers after oral administrations of SG hydrochloride in a single dose of 2.5 or 7.5mg, and a repeat twice-daily dose of 5.0 mg day(-1) (for 3 days). 3. The cumulative amount of SGO excreted within approximately the first 8-12h was comparable with MA, and the amount in the first 72 h was 2.0-7.8 times larger (2.8-13.2% of the dose) than that of DM-SG. 4. These results demonstrate that SGO can be used in place of DM-SG, which is known to be a main specific metabolite of SG, as a new indicator for the discrimination of SG use compared with MA abuse.

For proof of the presence of chemical warfare agents sarin, soman and VX, a rapid, accurate and sensitive method which allows us to determine their hydrolysis products ethyl methylphosphonic acid, isopropyl methylphosphonic acid and pinacolyl methyl phosphonic acid was explored by using continuous flow frit fast atom bombardment (FAB) LC-MS and LC-MS-MS. After derivatization of analytes with p-bromophenacyl bromide, LC-MS-MS analyses for screening were performed by a flow injection method. The three alkyl methylphosphonic acids (AMPAs) were eluted within 5 min, and the detection limits for the three AMPAs ranged from 1 to 5 ng/ml. For confirmation of the screening results, LC-MS-MS analysis with chromatographic separation was conducted by using a narrow bore column. The three AMPAs were all eluted with excellent separation within 25 min, and the detection limits ranged from 1 to 20 ng/ml. Quantitative measurement was performed by LC-MS in selected ion monitoring (SIM) mode with chromatographic separation. Linear calibration curves were obtained for the three AMPAs and the detection limits ranged from 0.5 to 3 ng/ml. The relative standard deviation for peak area ranged from 3.4 to 6.0% at 50 ng/ml for the three AMPAs.

A human serum sample collected from a victim of the Osaka VX incident was analyzed according to our developed technique for metabolites of VX. Gas chromatography-mass spectrometry (GC-MS) in full-scan electron impact and chemical ionization modes were used, and, for more reliable confirmation, GC-MS-MS was also employed. In the serum sample, both ethyl methylphosphonic acid and 2-(diisopropylamino-ethyl)methyl sulfide were detected. These results indicated that the techniques using GC-MS and GC-MS-MS were applicable to biological samples such as serum. These results also provide the first documented, unequivocal identification of the specific metabolites of VX in victim's serum and, furthermore, clarify a part of the metabolic pathway of VX in the human body.

Using ion mobility spectrometry (IMS), a simple, sensitive and rapid screening for methamphetamine (MA) incorporated in user's hair has been developed. To completely unbind MA from hair matrix and to achieve its effective vaporization for the IMS detection, the hair sample was digested in 5 M NaOH (methanol-water, 4:1, v/v) solution prior to IMS measurement. MA in hair was semi-quantitatively detected by monitoring the digested hair sample employing dibenzylamine (DBA) as internal standard. The minimum amount of hair sample required was 2 mg and its digested sample was ample for four IMS measurements. The detection limit of MA in hair was 0.5 ng mg(-1). This proposed method was applicable to the semi-quantitative detection of MA in users' hair samples, and to the sectional analysis for MA in a limited amount of user's hair. The IMS results obtained were in good agreement with their GC-MS determination.

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).

The method for simultaneous determination of cocaine and its four metabolites (benzoylecgonine, ecgonine methyl ester, ecgonine and norcocaine) in urine by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS) was studied. The mass spectra showed the quasi-molecular ions, [M+H]+ as the base peak. LC/APCI-MS analysis was performed by focusing the characteristic ions at m/ = 186, 290, 200, 304 and 290 for ecgonine, benzoylecgonine, ecgonine methyl ester, cocaine and norcocaine, respectively. Cocaine and its four metabolites were well separated by high performance liquid chromatography (HPLC). The recoveries of cocaine and its metabolites from the spiked urine were 40.3-94.7% by solid-phase extraction with two type cartridges (Bond Elut Certify and Bond Elut SCX).

A rapid and simple method of utilizing thin-layer chromatography (TLC) and pyrolysis gas chromatography (PyGC) for the identification and determination of methylbenactyzium bromide in human urine was studied in this report. Methylbenactyzium bromide was extracted from urine with ODS-cartridge (Sep-Pak C18), then spotted onto a silica gel 60 F254 TLC plate. After development, the separated spot of methylbenactyzium bromide was scraped and wrapped with a ferromagnetic foil without extraction by any organic solvents. The sample was applied into PyGC analysis. The optimum temperature for pyrolysis was 590 degrees C. The main degradation product of methylbenactyzium bromide was identified as diphenylmethane in this procedure by gas chromatography/mass spectrometry (GC/MS). A calibration graph prepared by absolute calibration method showed a good linearity over the concentration range of 1-75 micrograms/spot for methylbenactyzium bromide. The coefficient of variation obtained for eleven replicate analyses of the 3 micrograms/spot of standard methylbenactyzium bromide was 3.8%. The detection limit of this compound by this procedure was 0.1 micrograms/spot.