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Clinincal, Forensics

• Page 3

Accurate Mass LC-MS/MS Profiling of SyntheticCannabinoids

• Page 9

Screening analysis for drugs of abuse by LC-MS/MS enables fast polarity switching MRM triggered product ion scanning on the fly

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Identification of triazolam, etizolam and theirmetabolites in biological samples by liquid chromatography tandem mass spectrometry

Ben Figard1; Christopher L. Pennington2; Jeff Dahl3;

Susan Leonard4; Jorge Smith1, 1Shimadzu Scientific Instruments, Houston, TX; 2Rice University, Houston, TX; 3Shimadzu Scientific Instruments, Columbia, MD; 4Shimadzu Scientific Instruments, Marlborough, MA.

Accurate Mass LC-MS/MS Profiling of Synthetic Cannabinoids 　

IMSC 2012 PTu-089

PO-CON0000E

2

IntroductionNew synthetic designer drugs of abuse are being produced as readily available consumer products such as bath salts and incense. The uncontrolled and unregulated synthesis of these drugs causes numerous variations in formula, structure, and stereochemistry. These variations cause the

analysis of the compounds of interest to be very difficult and extremely time consuming. In this study we describe a new simple and efficient method by which these new synthetic drugs of abuse can be rapidly identified and quantified.

Materials and MethodThe samples received were herbal in nature. It was decided that aliquots of each sample would be extracted in acidic

aqueous, basic aqueous and various organic solvents. The resulting extracts were then analyzed by LC-MS.

For each extraction 224 – 248 mg of sample was weighed into a 20 mL scintillation vial, 5 mL of extraction solvent was added, samples were vortexed and sconicated for 60 minutes. Following sonication the extraction solvent was removed to another vial and (with the exception of aqueous solvents) evaporated to dryness under nitrogen. Solvents used for extraction were as follows: CHCl3,

Acetone, IPA, MeOH, ACN, 0.1 % NH4OH in water and 0.1 % formic acid in water. For LC-MS analysis samples were reconstituted with the addition of 2 mL of 0.1% formic acid in water followed by 3 mL of acetonitrile. Following solvent addition samples were vortexed, sonicated and analyzed by LC-MS. Aqueous extracts were analyzed as is.

Sample Preparation

InstrumentColumnMobile Phase

Flow RateInjection Volume

Gradient Program:

: Shimadzu XR system: Supelco Ascentis Express C-18 150 mm × 2.1 mm, 2.7 µm : A: 0.1% Formic Acid in Water B: 0.1% Formic Acid in ACN: 0.4 mL/min: 25 µL

Chromatography

Accurate Mass LC-MS/MS Profiling of Synthetic Cannabinoids

Time (min)

0

35

49

54

55

60

%B

5

20

90

90

5

5

Time (min)

0

45

54

55

60

%B

30

90

90

30

30

Gradient A Gradient B

3

Results

Mass SpectrometryInstrument : Shimadzu LCMS-IT-ToF Mass Spectrometer Ionization : ESIPolarity : PositiveMass Range: 100 – 500 m/z

Data AnalysisESI-MS accurate mass data was used to predict chemical formulas which were searched in databases such as ChemSpider (http://www.chemspider.com/) and MassBank (http://www.massbank.jp).ESI-MS accurate mass data and MS/MS data were also searched using METLIN (http://metlin.scripps.edu/). MassFrontier 5.0 (ThermoFisher) was used to help evaluate MS/MS data and propose chemical structures.

Late eluting compoundsbetter resolved by gradient B.

LC-MS Chromatogram:Chloroform extract (Gradient A)

Amino acids, metabolites & othersmall molecules. Methylone & related compounds could not be identified.

LC-MS Chromatogram:0.1% Formic Acid in H2O extract(Gradient A)


0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0(x10,000,000)

1:BPC (1.00)

9.12

7

16.3

67

17.7

72

19.2

05

20.1

8820

.833

24.0

78

26.9

57

LC-MSChromatogram: Chloroform extract (Gradient B)

B356.1997C25H25NO

Δppm =-3.37

D374.1905 C25H24NOF

Δppm =-2.67

E414.2071 C27H27NO3

Δppm =-2.67

F372.1954 C25H25NO2

Δppm =-1.07

C354.1843 C25H23NO

Δppm =-2.54

I400.2274 C27H29NO2

Δppm =0.75

H388.2085C26H26NOF

Δppm = 3.61

G390.1614

C25H24NOCl Δppm =-1.28

A370.2181 C26H27NO

Δppm = 4.32

4

LC Peaks A & B: The proposed structures for LC peaks A and B are known ethyl and methyl napthyl analogues of synthetic cannabinoid JWH-018. The structures for these two compounds were proposed based on the ability to match accurate mass and MS/MS data with an existing

entry in the METLIN database. The ability to match structures A and B with existing database entries greatly expedited the process of identifying other similar compounds present in the herbal sample extract.

LC Peak C: The MS/MS data for LC Peak C suggested that the compound was a methyl napthyl compound similar to LC peak B (JWH-122). The MS/MS data further suggested that the difference between compounds B and C was the addition of a double bond or ring (Δ = 2 Da) to the

nitrogen R group. A ChemSpider similarity search of the two structures yielded one compound with a chemical formula of C25H23NO (JWH-150). MS/MS data did not match this structure, however a third structure could be proposed based on this.


METLIN: Fragment Ion Metabolite SearchLC Peak A: Proposed Match JWH-210

METLIN: Fragment Ion Metabolite SearchLC Peak B: Proposed Match JWH-122

140 150 160 170 180 190 200 210 220 m/z

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Inten.(x1,000,000)

183.0800

214.1216 153.0704

144.0450 155.0852

MS/MS m/z 370 (Peak A) Proposed Compound

WH-210

125.0 150.0 175.0 200.0 m/z

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

169.0653

214.1217

141.0697

144.0447 115.0560

Proposed Compound JWH-122

Inten. (x1,000,000)

125.0 150.0 175.0 200.0 m/z 0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

3.75Inten.(x1,000,000)

169.0647

141.0696

212.1056

115.0542 158.0600

144.0441

Based on the observed accurate mass and MS/MS data two structures wereproposed for m/z 354.1843

Proposed structure double bond position not determined.

*

* C25H23NO353.1780

C10H8NO+

158.0600

C14H14NO+ 212.1070

ConclusionFrom the herbal sample chloroform extract 13 observed compounds had accurate mass and MS/MS data consistent with synthetic cannabinoids. All compounds gave either a m/z 169 or m/z 183 fragment ion, which suggests that they are either methyl or ethyl napthyl analogues to JWH-018. Proposed chemical formulas also suggest that some compounds are halogenated. Of the compounds observed two (JWH-210 & JWH-122) were identified using the METLIN metabolite data base accurate mass & MS/MS ion search. These two compounds were “certified” by the vendor to not be present in this product. It should be noted that this study was not quantitative in nature, and information regarding vendor method detection limits were not available. JWH-210 & JWH 122 may be present at levels less than the PDL of the vendor’s method.From a detection stand point; the results indicate that LC-MS/MS precursor ion scanning on a QqQ or QqToF may be a viable option to screen for classes of synthetic cannabinoids.

AcknowledgementsThe authors would like to thank and acknowledge Rice University and the Rice Shared Equipment Authority (SEA) for their support.

LC Peaks D & G: The proposed structures for compounds D & G are structurally similar to known halogenated analogues of synthetic cannabinoid JWH-018.


5

150.0 175.0 200.0 225.0 m/z 0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

2.50

2.75

3.00

3.25

3.50

3.75

4.00

169.0641

232.1119 141.0695

144.0437

MS/MS m/z 374 (Peak D)

Proposed structure fluorine position not determined.

**

Inten. (x10,000,000)

390.0 391.0 392.0 393.0 394.0 395.0 m/z 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

390.1614

392.1597 391.1648

393.1645

The observed isotope pattern is consistent with a chlorine containing compound.

Proposed structure chlorine position not determined.

* *

MS Spectrum Peak G Inten. (x100,000) MS/MS m/z 390 (Peak G)

125.0 150.0 175.0 200.0 225.0 250.0 275.0 m/z 0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

169.0650

141.0693

248.0826

250.0792

144.0449 212.1054

Inten. (x1,000,000)

For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.

© Shimadzu Corporation, 2012

First Edition: September, 2012

www.shimadzu.com/an/

PO-CON1218E

Toshikazu Minohata1, Ichiro Hirano1, Junko Iida1,

Keiko Kudo2, Noriaki Ikeda2, Kei Zaitsu3,

Noriaki Shima3, Munehiro Katagi3,

Hitoshi Tsuchihashi4, Koichi Suzuki4

1Shimadzu Corporation, Kyoto (Japan), 2Dep. of Forensic Pathology and Sciences, Kyushu

University, Fukuoka (Japan) , 3Forensic Science Lab., Osaka Prefectural Police,

Osaka (Japan), 4Dep. of Legal Medicine, Osaka

Medical Collage, Takatsuki (Japan)


IMSC 2012 PTh-192

2

IntroductionIn recent years the need for forensic, toxicological and clinical analyses have increased, and as a consequence of sample complexity, analysis has become increasingly challenging due to a growing trend in the use of illicit drugs and non-medicinal prescription drugs. Screening applications requires rapid and unambiguous results that can be achieved using a generic analysis method designed

for a large number of target compounds. To meet this need, a universal high speed MRM triggered product ion scanning method with fast polarity switching was applied to simultaneously screen, quantitate and confirm by reference to an MS/MS data base containing the majority of drugs of abuse available in Japan.

Materials and Methods


[Abused Drugs]　Amphetamine　Benzoyl ecgonine　Cocaine　Codeine　Dihydrocodeine　Ecgonine methyl ester　Ephedrine　Ketamine　MDA　MDMA　Methamphetamine　Methylephedrine　Methylphenidate　Morphine　Sildenafil　THC　THC-COOH

[Psychotropic Drugs]　Amitriptyline　Amoxapine　Aripiprazole　Chlorpromazine　Clomipramine　Dosulepin　Fluvoxamine　Haloperidol　Imipramine　Levomepromazine　Maprotiline　Mianserin　Mirtazapine　Nortriptyline　Olanzapine　Paroxetine　Promethazine　Quetiapine　Risperidone　Sertraline　Sulpiride　Trazodone　Zotepine

[Hypnotic Drugs]　7-Aminoflunitrazepam　7-Aminonimetazepam　7-Aminonitrazepam　8-Hydroxyetizolam　Allylisopropylacetylurea　alpha-Hydroxybrotizolam　alpha-Hydroxytriazolam　Alprazolam　Amobarbital (neg)　Barbital (neg)　Bromazepam　Bromovalerylurea　Brotizolam　Diazepam　desmethyldiazepam　Estazolam　Ethyl loflazepate　Etizolam　Flunitrazepam　Flurazepam　Hydroxyzine　Lorazepam　Lormetazepam　Midazolam　Nimetazepam　Nitrazepam　Oxazepam　Pentobarbital (neg)　Phenobarbital (neg)　Quazepam　Temazepam　Thiamylal (neg)　Triazolam　Zolpidem　Zopiclone

[Medical Drugs]　Acetaminophen　Acetylpheneturide　Atropine　Biperiden　Bupivacaine　Carbamazepin　Chlorpheniramine　Clonazepam　Dextromethorphan　Diclofenac　Diltiazem　Diphenhydramine　Diprophyline　Ethenzamide　Glibenclamide　Glimepiride　Ibuprofen (neg)　Lidocaine　Loxoprofen (neg)　Mepivacaine　Mexiletine　Pancuronium　Pentazocine　Salicylic_acid (neg)　Trihexyphenidyl　Vecuronium　Warfarin

[Pesticides]　Diquat　Fenitrothion (MEP)　Glufosinate　Malathion　Methomyl　Paraquat

[Natural Toxines]　Aconitine　Colchicine　Tetrodotoxin

Table 1 List of compounds for Forensic method.

3

Analysis of several drugs was performed using fast polarity switching and high speed data acquisition LC/MS/MS. This was achieved using Synchronized Survey Scan® which refers to the execution of MS/MS scanning triggered by survey

scan signals (in this case, MRM). Therefore, during the elution of a peak in MRM analysis, a full-product ion mass spectrum can also be obtained.

Samples were analyzed using a Nexera UHPLC system coupled to a LCMS-8030 triple quadrupole mass spectrometer (Shimadzu Corporation, Japan) with LC/MS/MS Method Package for Forensic Toxicology. Database contains product ion scan spectra for 286 forensic and toxicology-related compounds such as 87 Abused drugs, 105 Psychotropic drugs, 70 Hypnotic drugs and others. This library provides Synchronized Survey Scan parameters (product ion spectral data acquisition

parameters based on the MRM intensity as threshold) optimized for screening analysis. The simple quantitative method included the most frequently analyzed 111 components of Abused drugs, Psychotropic drugs and Hypnotic drugs for method validation (Table 1).Samples were separated using a Shim-pack FC-ODS using a gradient elution with ammonium formate and methanol.

MRM parameter Product Ion Scan parameter

Positive

Negative

Fig. 1 User Interface of MRM-Product Ion Scan setting at LabSolutions software.

Analytical Conditions

HPLC (Nexera UHPLC system) Column

Mobile Phase A

Mobile Phase B

Gradient Program

Flow Rate

Column Temperature

Injection Volume

: Shim-pack FC-ODS (2.0 mmI.D. × 150 mmL., 3 um)

: 10 mM ammonium formate

: Methanol

: 5%B (0 min) - 95%B (15-20 min) - 5%B (20.01 - 30 min)

: 0.3 mL / min

: 40ºC

: 5 uL

Mass (LCMS-8030 triple quadrupole mass spectrometry)

Ionization

Polarity

Probe Voltage

Nebulizing Gas Flow

Drying Gas Pressure

DL Temperature

BH Temperature

: ESI

: Positive & Negative

: +4.5 kV (ESI-Positive mode);

-3.5 kV (ESI-Negative mode)

: 1.5 L / min

: 10 L / min

: 250ºC

: 400ºC


4

Fig. 2 MRM - Product Ion Scan screening data about 4 compounds.

ResultsMS/MS Library MatchingMRM chromatograms of four compounds (each 1000 ng/mL) spiked into urine and analyzed by Nexera coupled to LCMS-8030 following sample preparation (Fig. 2). These product ion scans were searched against the MS/MS library and the four previously identified peaks were assigned a high hit score. The assay generates both MRM and Product

Ion Scan data (MS/MS) due to the speed of data acquisition from the LC/MS/MS system. This results in quantitative data and library searching / product matching data to help with product ion confirmation. Fast polarity switching helps to provide information rich product ion spectra resulting in better detection and identification for each compound.

Allylisopropylacetylurea

Diclofenac

Amobarbital (neg)

Thiamylal (neg)

10.0 12.4

0.0

1.0

2.0

3.0

4.0

(x100,000)

185.00>55.05(+)

12.5 10.1

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

(x100,000)

296.00>214.00(+)

296.00>215.05(+)

10.0 12.5

0.0

1.0

2.0

3.0

4.0

5.0

6.0

(x1,000)

225.15>42.00(-)

12.5 10.3

0.00

0.25

0.50

0.75

1.00

(x10,000)

253.00>101.00(-)

253.00>58.10(-)


5

Fig. 3 Registration of 1st coefficient and intersection by calibration curve.

Table 2 The calculated results of 12 compounds in whole blood using LC-MS/MS (n=2 average).

Simple Quantitative Method for Forensic analysesBased on the chromatogram obtained by injection of a fixed volume of individual reference standard solutions, the ratio of peak area of the reference standard was calculated and compared to that of the internal standard (Diazepam-d5). The resulting calibration curve was

prepared by plotting the ratios of the amount of the reference standard to that of the internal standard. 1st coefficient and intersection were calculated from the calibration curve and were registered to the LCMS method(Fig. 3).

The method was validated using 12 of 111 compounds, between 0,05 ng/mL and 5 ng/mL, spiked into whole blood and treated with solid phase extraction (Table 2). The

results from this method indicated a high quantitative performance and could prove useful as rapid confirmationand simple quantitative analysis.

0.05 ng/uL

Compounds

Diazepam-d5 (IS)AlprazolamAripiprazoleAtropineBrotizolamColchicineEstazolamEthyl loflazepateEtizolamFlunitrazepamHaloperidolRisperidoneTriazolam

12.98711.85715.5927.225

11.9879.794

11.46413.06812.09211.22912.01111.77811.728

396,803 114,210 59,975

327,992 42,175 21,970

128,563 85,673 73,746 77,218

616,938 783,134 34,424

[0.500]0.0380.0250.0840.0440.0150.0480.0280.0320.0600.0480.0380.042

342,441 918,575 700,323

3,105,470 325,945 159,050

1,078,497 489,250 575,984 545,933

5,378,666 6,811,884

283,935

[0.500]0.525 0.205 0.935 0.502 0.270 0.639 0.272 0.421 0.649 0.610 0.510 0.550

77,460 2,497,911 7,120,340

17,445,635 1,043,787

696,217 5,580,490 1,012,405 2,519,896 1,816,819

28,654,837 30,675,213

746,810

[0.500]6.72 3.07

10.92 7.49 3.72 5.49 2.68 5.67 7.12 7.10 6.72 6.78

R.T Area Conc.Area Conc.Area Conc.

0.5 ng/uL 5 ng/uL






ConclusionA high speed LC/MS/MS data acquisition system was applied to drug screening in forensic, toxicological and clinicalanalysis. To achieve a highly specific and sensitive detection method in screening and quantitation, an MRM triggered production scanning method using a polarity switching speed of 15msec and a scan speed of 15,000u/sec was applied to 111components including illicit drugs, psychotropics, hypnotics, pesticides and other substances. As the MRM acquisition time was very fast, this enabled product ion spectra to be generated in both positive andnegative ionization mode which could be matched against a user library of compounds as an automated aid toscreening and compound identification.


PO-CON1220E

Mayumi Matsui1, Toshikazu Minohata1,

Noriko Shoji2, Naohiro Kuriyama2, Chie Yokoyama2,

Keiko Matsumoto1, Jun Watanabe1, Junko Iida1

1Shimadzu Corporation, Kyoto, Japan, 2YMC Co., LTD., Komatsu, JAPAN

Identification of triazolam, etizolam and their metabolites in biological samples by liquid chromatography tandem mass spectrometry

IMSC 2012 PTh-196

2

IntroductionBenzodiazepines are one of the mostly widely prescribed groups of drugs because of their sedative, hypnotic, anxiolytic, antiepileptic and muscle relaxant properties. This class of compounds and their associated metabolites are also frequently present in clinical and forensic samples. For this reason, the analysis of benzodiazepines in biological fluids is of great importance to clinicians and forensic toxicologists. A key analytical challenge in the analysis of benzodiazepines is to identify etizolam, triazolam, and their

metabolites (alpha-hydroxyetizolam, 8-ethylhydroxyetizolam, alpha-hydroxytriazolam and 4-Hydroxytriazolam) as a mixture, because of their very similar chemical structure, molecular weight and fragmentation during mass spectrometry. In this study we report a new high resolution separating method for the simultaneous analysis of etizolam, triazolam and their metabolites.


Fig. 1 Structure of etizolam, triazolam and their metabolites

Fig. 2 LCMS-8030 triple quadrupole mass spectrometer

N

NN

NCH3

Cl

S

CH3 N

NN

NCH3

Cl

S

OH

N

NN

NCH3

Cl

Cl N

NN

NCH3

Cl

Cl

OH

N

NN

N

Cl

Cl

OH

EtizolamM.W. 342.07059

8-Ethylhydroxye tizolamM.W. 358.06550

Tria zolamM.W. 342.04389

4-Hydroxytria zolamM.W. 358.03881

alpha-Hydroxytria zolamM.W. 358.03881

N

NN

NCH3

Cl

S

CH3

OH

alpha-Hydroxye tizolamM.W. 358.06550

3

Materials and MethodsThree samples were prepared: A) mixture of all standards (alpha-hydroxytriazolam, 4-hydroxytriazolam, triazolam andetizolam), B) blank metabolized matrix using human liver S9 and C) metabolised matrix of triazolam and etizolam.

Triazolam, etizolam, and NADPH regeneration systemsolution (NADP+, glucose-6-phosphate, MgCl2 in H2O),NADPH regeneration system solution (glucose-6-phosphatedehydrogenase in sodium citrate buffer), human liver S9were mixed in 100 mM phosphate buffer (pH 7.4). Themixture was incubated at 37 deg C overnight (approx. 18

hrs). The control sample was prepared without triazolamand etizolam added. [Final concentration in incubationmixture; 40 µM triazolam and etizolam, 1.6 mM NADP, 3.3mM glucose-6-phosphate, 0.4 U/mL glucose-6-phosphatedehydrogenase, 3.3 mM MgCl2, 3.2 mg/mL S9 protein]

Extraction of incubation mixture:

In vitro metabolism of triazolam and etizolam in human liver S9:

The incubation mixture was extracted as follows and injected ontoLC/MS/MS. 1. Add 500 µL of ice-cold acetonitrile 2. Centrifuge at 2,000 rpm for 15 min and collect the supernatant 3. Dry the supernatant in a vacuum evaporator and resolve it in 150 µL of water 4. Filter the solution through 0.2 µm syringe filter (YMC Duo-Filter)

Samples were analyzed with UHPLC and a triple quadruple mass spectrometer using following conditions.

Analytical Conditions

HPLC: Nexera UHPLC system (Shimadzu Corporation, Japan)

Column: YMC-Triart C18 column, 1.9 µm, 12 nm (150 × 2 mm) Mobile phase: (A) 10 mM formic acid (B) 10 mM formic acid / acetonitrile (1/1) Flow rate: 0.3 mL/min Time program: B conc. 40%(0 min)-65%(40 min)-40%(40.01-60 min) Injection volume: 1 µL Column temperature: 40°C

Mass spectrometer: LCMS-8030 (Shimadzu Corporation, Japan)

Ionization: Electrospray ionization, Positive Scan type: multiple-reaction-monitoring mode (MRM) MRM triggered automatic MS/MS data acquisition


4


ResultsThe simultaneous analysis of drugs of abuse in clinical and forensic laboratories requires highly specific methods. The developed method in this study contained not only optimized MRM transition parameters and chromatographic conditions, but also product ion scanning which is automatically triggered once an MRM exceeds a specified threshold. The method was applied to the analysis of benzodiazepines; including etizolam, triazolam, and their known metabolites. In this experiment three samples were prepared (as described above).

Firstly, sample (A) was analyzed with the method of 12 MRM transitions, which were quantitative and qualitative transitions for etizolam, triazolam, and their known metabolites and it resulted in excellent separation for all four compounds. Next, sample (C) (blank matrix) was analyzed and no peaks were observed; therefore highlighting the excellent selectivity of the method. Sample (B) (metabolized drug) was then analyzed and three new peaks were found (in addition to the four peaks in sample (A)).

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min

0.00

0.25

0.50

0.75

1.00 (x100,000)

6:TIC(+) 5:TIC(+) 4:TIC(+) 3:TIC(+) 2:TIC(+) 1:TIC(+)

(C)

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min

0.0

2.5

5.0

7.5

(x10,000)

6:TIC(+) 5:TIC(+) 4:TIC(+) 3:TIC(+) 2:TIC(+) 1:TIC(+)

(A)

Etizolam

Triazolam

4-Hydroxytriazolam

alpha-Hydroxytriazolam

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min

0.0

0.5

1.0

1.5

(x100,000)

6:TIC(+) 5:TIC(+) 4:TIC(+)(5.00) 3:TIC(+)(5.00) 2:TIC(+)(5.00) 1:TIC(+)(5.00)

(B) Peak 1 8-Ethylhydroxyetizolam

Peak 3 alpha-Hydroxyetizolam

Peak 5

CEQualitativeCEQunatitativecompounds

-39359.05>111.20-22359.05>341.104-Hydroxytriazolam

-18359.05>341.15-27359.05>176.20alpha-Hydroxytriazolam

-27359.05>287.20-28359.05>286.20alpha-Hydroxyetizolam (M-VI)

-20359.05>315.25-24359.05>305.058-Hydroxyetizolam (M -III)

-27343.05>315.00-24343.05>308.20Triazolam

-37343.05>138.15-28343.05>314.10Etizolam

CEQualitativeCEQunatitativecompounds

-39359.05>111.20-22359.05>341.104-Hydroxytriazolam

-18359.05>341.15-27359.05>176.20alpha-Hydroxytriazolam

-27359.05>287.20-28359.05>286.20alpha-Hydroxyetizolam (M-VI)

-20359.05>315.25-24359.05>305.058-Hydroxyetizolam (M -III)

-27343.05>315.00-24343.05>308.20Triazolam

-37343.05>138.15-28343.05>314.10Etizolam

Peak 2

Peak 4

Peak 6

Peak 7

Fig. 3 12 MRM transitions for 6 drugs and metabolites (above) and MRM chromatograms for sample (A), (B), (C).

5


Two of the three unknown peaks were identified as 8-Ethylhydroxyetizolam and alpha-Hydroxyetizolam as they are known metabolites. However the third unknown peak, which was detected the same MRM transition as that of metabolites of these two compounds, was not identified.Next, sample (B) was re-acquired with MRM triggered

automatic MS/MS and product ion scans. These product ion scan spectra were searched against a hypnotics MS/MS library and the six previously identified peaks were assigned a high hit score. In the same manner as described here, this method is highly applicable to the screening of drugs of abuse in biological samples.

Loop time < 1 sec.

50 100 150 200 250 300 350 m/z0.0

0.5

1.0

1.5

2.0

Inten.(x10,000)

341.0

314.0272.9

111.1249.8183.1 358.4

Peak 4: 4 -Hydroxytriazolam

50 100 150 200 250 300 350 m/z0.0

2.5

5.0

7.5

Inten.(x10,000)

176.1

359.0313.0

277.0149.1 341.0266.0243.0204.1

Peak 2: alpha -Hydroxytriazolam

50 100 150 200 250 300 350 m/z0.0

2.5

5.0

Inten.(x10,000)

282.1 341.1315.1 359.1

247.1179.1154.1 204.057.2

331.1258.1232.096.0 291.1

Peak 1: 8 -Ethylhydroxyetizolam

50 100 150 200 250 300 350 m/z0.0

1.0

2.0

3.0

4.0

Inten.(x10,000)

286.1

287.1341.1305.1269.173.2 236.0137.3 330.1176.3209.4

Peak 3: alpha -Hydroxyetizolam

50 100 150 200 250 300 m/z0.0

1.0

2.0

3.0Inten.(x100,000)314.1

138.1 259.1

224.1 295.1245.1171.1123.157.2148.1 343.1

Peak 7: Etizolam

50 100 150 200 250 300 m/z0.0

2.5

5.0

Inten.(x100,000)

308.1

343.1

239.1165.1138.1 275.1

Peak 6: Triazolam

50 100 150 200 250 300 350 m/z0.0

0.5

1.0

Inten.(x10,000)

341.1

238.0273.0300.0314.0258.1

223.062.4 135.5110.5 331.3

Peak 5: ?

Fig. 4 MRM triggered MS/MS method and MS/MS spectra of Peak 1 to Peak 7

ConclusionsMetabolite analysis using LC/MS/MS with small particle size column achieved high resolution separation for the simultaneous analysis of etizolam, triazolam and their metabolites. The metabolites were detected and confirmed with MRM triggered automatic MS/MS data acquisition.

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