Identification of Methoxyisoflavone Metabolites in Human ......1 2 LCMS 2012 Methoxyisoflavone uman...

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Montreux Identification of M Identification of M Metabolites in H UPLC/Q T Yannick Lecompte 1 , Camille 3 Martine Perrin 3 , 2 1 LCMS 2012 Methoxyisoflavone Methoxyisoflavone Human Urine by E TOF/MS E e Richeval 2 , Luc Humbert 2 , 4 Patrick Arpino 4 4 3

Transcript of Identification of Methoxyisoflavone Metabolites in Human ......1 2 LCMS 2012 Methoxyisoflavone uman...

Page 1: Identification of Methoxyisoflavone Metabolites in Human ......1 2 LCMS 2012 Methoxyisoflavone uman Urine by OF/MSE Richeval2, Luc Humbert2, Patrick Arpino 4 3 4 Cannabis druggg screening

• Montreux

Identification of MIdentification of MMetabolites in H

UPLC/QT

Yannick Lecompte1, Camille3Martine Perrin3,

21

LCMS 2012

MethoxyisoflavoneMethoxyisoflavone Human Urine by

ETOF/MSE

e Richeval2, Luc Humbert2, 4Patrick Arpino4

43

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Cannabis drug screeningg g

• Driving after consumption o

• Drug screening at the road sDrug screening at the road sUrine (2001) or saliva (2009) screchromatography kitschromatography kits

• If positive : confirmation by GC

• Example from the Parisian pControlled drivers : 4072

Positive : 1206 (ca. 30%)

Confirmed : 895 (22%)Confirmed : 895 (22%)

• 25% false positivehttp://www.prefecturedepolice.interieur.gouv.fr/Mediatheqcannabis

gg

of cannabis is prohibited !

sidesideeening using commercial immuno-

C/MS (TMS dervs)

police (2009)

que/Bonus/Quiz/Vrai-ou-faux-que-connaissez-vous-sur-le-

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Protzek® Multi-drug screen test pannel Check 24/317

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Known cross-reactivityy

• 2005-2009

62 135 medical fitness examinati

h dFrench Gendarmerie:

• 1004 positive1004 positive

• 80 % confirmed

• 20 % false positive

• Non-steroidal anti-inflammator

frequently involved in cross-reafrequently involved in cross rea

ion tests before admission to the

ry drug (NSAI), e.g. niflumic acid

actionsactions

Lecompte et al. (2011)

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Isoflavonoïds

O

O

Flavones

O

O

1

Isoflavones

87 2

O1

A C

56 3

4 1'2'

3'

A C

B6'

5'4'O

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Some common isoflavone

R

OR7

R7

OH

OH

OCH3

OR5R4'

aglyconesg y

R R ’R5 R4’

H OH Daidzein Soy flour; soybean paste

OH OH Genistein

OH OH Prunetin

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5-methyl-7methoxyisoflavoy y

7 OOCH CH3

5

OCH OCH3

Prepared by synthesis (US patent 416Negative reaction to a Cannabis dru

one

O

OO

3746, 1979)ug test

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Healthy volunteers(n = 3)

1-oral administrationt

Urine samples A

methoxyisoflavone 2 capsules, 150 mg

t0

2-oral administrationt + methoxyisoflavone

2 capsules, 150 mgt0 +12h

3-oral administrationt + methoxyisoflavone

2 capsules, 150 mg

t +

t0 +24h

t0 +30hUrine samples B

Cannabis screening

IRCGNParis

BSTFA GC/MSBSTFA; GC/MSAC2O; GC/MSLC/MS

Cannabis screening

CHRULille

Enzymatic hydrolysisExtraction pH4

UPLC/Q-TOF MS

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UPLC-QTOF MS conditioQ

• UPLCUPLC

Water Acquity HSS C18 150mm x 2

50°C; Gradient ACN/Water , 0.1%

Q TOF• Q-TOF

Model: Waters, XEVO-G2-QTOF

Acquisition modes: MSE and MS/M

• Waters Data Processing

MetaboLynx™; MassFragment™MetaboLynx ; MassFragment

• Further data processing

ACD/Labs MS Manager™; ACD/Sp

ons

2 mm, 1.8 µm

HCOOH, HCOONH4 buffer, pH 3

MS modes

ectrus ™ (beta version)

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Data processing, patient 3p g, p

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Results from ACD/Labs Int

TIC

6.56.05.55.04.54.03.53.02.52.01.51.00.5Retention T

Metabolites

telliXtract processingp g

13.012.512.011.511.010.510.09.59.08.58.07.57.0Time (min)

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ESI+ MS/MS of parent molep

retroOOCH3

retro

CH3 OH+

16

77.03991.055

103.055121.029

152.063

150140130120110100908070605040

ecule

o Diels Alder223.077

o Diels Alder(RDA)

C2H4O

CO2

224.083267.102

65.070 178.079181.066

211.112252.079

280270260250240230220210200190180170160

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No detectable trace of the pare

MC for : 267 1230±0 002 Da/z

p

MC for : 267.1230±0.002 Da/z 267.1015±0.002 Da/z

267.1230

OOCH3CH3

CH O+

CH3 OH

C17H15O3+= 267.10157 Da/z

267.1015

6.56.05.55.04.54.03.53.02.52.01.51.00.5Retention T

ent molecule in patient urinep

C14H19O5+=267.1232 Da/z ?

267.1228

13.012.512.011.511.010.510.09.59.08.58.07.57.0Time (min)

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Major metabolite Parent–CH3

OOH

1,3Aj 3

CH OCH3 O1,3B

m/z=253.0859

6 56 05 55 04 54 03 53 02 52 01 51 00 5 6.56.05.55.04.54.03.53.02.52.01.51.00.5Retention T

500 µL - Bglu/IRCGN_Patient_3_Acide07 May 2012 21:17:16Retention Time: 10.395Scan: 1351Scan: 1351

1 3B

151 039103 05577 039

1,3A1,3B

15014013012011010090807060504030m/

151.039141.070103.05577.039

67.055

3 MH+= 253.0859 Da/z OO

CH3

3

H+

O+

H

13 012 512 011 511 010 510 09 59 08 58 07 57 0 13.012.512.011.511.010.510.09.59.08.58.07.57.0Time (min)

253.086MH+

B

197.096182.073MNa+

B

270260250240230220210200190180170160m/z

275.068225.0914

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Parent – CH3 + (OH) MH+= 269.3 ( )

OOH

3'OH

4'CH3 OOH

OOOH

O

?

5

6.56.05.55.04.54.03.53.02.52.01.51.00.5Retention T

0808 Da/z

OOH

1O

OH

OOHOH

2'

O

OH

2 4

O

3

13.012.512.011.511.010.510.09.59.08.58.07.57.0Time (min)

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Parent – CH3 + 2(OH) MH+= 2853 ( )

3

1 2

OOH

1 2CH3 O

OH

OH

6.56.05.55.04.54.03.53.02.52.01.51.00.5Retention Tim

5.0757 Da/z

13.012.512.011.511.010.510.09.59.08.58.07.57.0me (min)

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Parent + (OH) MH+= 283.0964 ( )

OOCH3

2'3

CH OCH3 OOH

6.56.05.55.04.54.03.53.02.52.01.51.00.5R t ti TRetention T

Da/z

22

33'

14

13.012.512.011.511.010.510.09.59.08.58.07.57.0Ti ( i )Time (min)

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Parent + 2(OH) MH+= 299.0914( )

OOCH3C3

CH3 OOH

CH3 OOH

6.56.05.55.04.54.03.53.02.52.01.51.00.5Retention T

OOCH3

OOH

OH

4 Da/z

4

1

2 3

13.012.512.011.511.010.510.09.59.08.58.07.57.0Time (min)

OOOH

CH3 OOH

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Parent + HCOOH MH+= 297

O OH

OO

OCH3

6.56.05.55.04.54.03.53.02.52.01.51.00.5Retention T

72.080105.071

119.086

151.077 190.1242

.07575 Da/z

OO OOCH3

OOH O

13.012.512.011.511.010.510.09.59.08.58.07.57.0Time (min)

297.076 315.086

MH+ MH3O+

4203.071239.067

255.071

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Methoxyisoflavone major my j

TIC

P-CH

6.56.05.55.04.54.03.53.02.52.01.51.00.5Retention T

Metabolites

metabolitesP-CH3

H3+OH3

13.012.512.011.511.010.510.09.59.08.58.07.57.0Time (min)

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Conclusions

• Characterization of urinary meta

isoflavone oral absorption was b

• Large metabolite screening perfo

• 19 metabolites characterized by

by conventional GC/MS

Luc Humbert, Camille Richev

bolites following methoxy-

both rapid and effective.

ormed using MSE acquisition mode

UPLC/Q-TOF MS vs 4 metabolites

val, Ann Toxicol Anal. 2012; 24 (2): 87-96

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A better source of inform

• Incledon T Van Gammeren D Anto• Incledon T, Van Gammeren D, Anto

The Effects of 5-Methyl-7-Methoxyisofl

Performance in College-Aged Men. Med

33, S338.

• Wilborn CD, Taylor LW, Campbell B

Greenwood M et alGreenwood M, et al.

Effects of methoxyisoflavone, ecdyster

supplementation on training adaptation

Sports Nutr. 2006, 3, 19.

It dIt does no

mation

onio Jonio J.

avone on Body Composition and

dicine & Science in Sports & Exercise. 2001,

BI, Kerksick C, Rasmussen CJ,

one, and sulfo-polysaccharide

ns in resistance-trained males. J Int Soc

t k !ot work !

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Acknowledgementsg

Principal pharmacist Yannick LecompteFrench Military Health ServiceFrench Military Health Service

Chief pharmacist Martine Perrin

Page 28: Identification of Methoxyisoflavone Metabolites in Human ......1 2 LCMS 2012 Methoxyisoflavone uman Urine by OF/MSE Richeval2, Luc Humbert2, Patrick Arpino 4 3 4 Cannabis druggg screening

Acknowledgementsg

Camille Richeval Luc Humbert CHRUCamille Richeval, Luc Humbert, CHRU

Shahriar Jahanbakht ACD / LabsShahriar Jahanbakht, ACD / Labs

LilleLille

Xevo G2 QTof