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...
• 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
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-
Protzek® Multi-drug screen test pannel Check 24/317
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)
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
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
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
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
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)
Data processing, patient 3p g, p
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)
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
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)
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
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)
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)
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)
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
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
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)
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
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 !
Acknowledgementsg
Principal pharmacist Yannick LecompteFrench Military Health ServiceFrench Military Health Service
Chief pharmacist Martine Perrin
Acknowledgementsg
Camille Richeval Luc Humbert CHRUCamille Richeval, Luc Humbert, CHRU
Shahriar Jahanbakht ACD / LabsShahriar Jahanbakht, ACD / Labs
LilleLille
Xevo G2 QTof
