DRUGS OF ABUSE IN SERUM BY UHPLC-MSIMS 75

VALI QUANTITATIVE MULTAN DETERMINATION COCAIN AND AMPHETAMINES IN SERUM SV U PLC COUPLED

TANDEM MASS

N Dubois 1 B Ph Hubert23 C

words Cocaine UlJlclltgt -nnhtnninc UHPLC-MSMS nnthnrl validation total errer

1 Service Meacuteclicoleacutegalle de (Environnement et en I=ntrritgt

CHU Sart-Tilman 2 Service de Chimie Analvti~]ue

Deacutepartement de Pharmacie Universiteacute de

3 CIRM Universiteacute de

Address for Corres~)on(jenle N Dubois Service de lEnvironnement et en I=ntrltgtlwi

CHU Sart-Tilman B35 Tour Il +5 B-4000

+3243667680 Fax + 32 4 366 88 89 E-mail nathalieduboischuulgacbe

The use worldwide of Eushy

would have used cocaine year (1) Ber1zovLeCQclnIcircrle is one of the major metabolites of cocaine formed by either spolnteumlmelDus hJllrnI1CIC

Acta Clinica 2010 65-)uPt)lerrlent

76 DRUGS OF ABUSE IN SERUI1 BY UHPLC-I1SI1S

(MBDB) are

opioid users is estimated at in Europe andbetween 12

650000 of them would have received substitution treatment in 2007 Heroin accounts for the Qreatest share of in the

which

to

very rapidly rnrlo1or1

matic esterase to form n-~rptvlrTInrnhlinp

is into morphine esterase Morphine is quickly to its

meJaDOtne morphine-3-glucuronide (M3G) somewhat more to amounts morshy

lnICHy-ho

use of different

Dhlnemiddotmiddoto-slUlunClnide (M6G)According to the report Centre for Drugs

2009 (1) polydrug norm in Europe the comshy

is responsible CornplICates most of the problems faces

Knc)wteCHJe only a few allowed

buprenorphine Ilaloxone and two narcotic antag()n were quantified Gas chromatography to mass spectrometry had been widely used for many (78) liquid chromatoQraDhv

(UHPlC-MSMS) It reDliICed techniques used

MATERIALS AND

materials for aU compounds and intershy - were from lGC Url1rYlrmiddotnorn

(Molsheim France) of pu-rity upper than 99

water were from Biosolve Netherlands) citric acid least analytical (Steinheim ven Belgium) Oasis MCX (Zellik

For the preparation of the stock solution of internai commercial solutions of benzoshy

were prepared solution containing

analytes was of rYlnhlYlio

(MAMP) MDMA MDA n-~rptJlmlnrnI1inCcedill (6MAM) (MOR) oxycoshydone (OCOD) hydromorphone (HMOR) the secshy

of cocaine (COc) berlzolecgorline codeine (COD) norcodeine

(EMOR) pholcodine (PHOl) M3G and naltrexorle

one (BUP) and norlbuorenc)rshyphine (NBUP) Group constitution l1oran1c

3ngtlu7o-l in dUI)licate

establlish the calibration curves

Acta Cinica

77 DRues OFABUSE IN SERUM BY UHPLC-MSMS

3 they were in tri~)licate were to estimate the validation f~r~negttegtlc

the method limits An extract was n tln~lrct1 for each run

CT)nngt n stock ~VlULIJII

1ml ammonia in rT1C1rn_

evalpOlratEd to

were injected to the column

Instrumentation Analysis was ntflrnltt1

pIed to a mass CfamplrtrmiddotfIITegttl)1

(Waters Belgium) The chromlatc)grcphic CQongtr_

tion was on an Acquit Y T3 column (100 x 21mm size 18lm Washyters) an filter at 40degC elution was performed at a constant flow of min using a mixture SmM ammonium formate in

Table 1 UPlC A ammonium formate SmM pH 3 B methanol pH 3

00 1000 00

10 1000 00

20 925 75

55 890 110

160 100 900

170 100 900

180 1000 00

190 1000 00

3 with forshy

flow was set at and gas flow was set at 800Llh The MS method was divided into 5 functions rlPf1pnrlirlo on the retention times of multiple reaction mOllecule for identification and

v fragmentation aU()WE~d

Method validation I1rrmiddot r1nn to 15017025 and the

DhrIrotir01 Sciences and Tech-

Elution was carried out using a cegtnrnamplntt1

of 19 minutes To column contamination with matrix highly Ictunampgtt1 r rnf1I1

tion with 1hlo

linearity in The trueness agreement

mean value obtained from the valldeacuteltlcm the value which is either as a

conventional true value or an accepted legttIPregt value

Acta Cfinica 20 10 65-~uPJ)enlent

76 DRUGS OF ABUSE IN SERUI1 BY UHPLC-I1SI1S

(MBDB) are

opioid users is estimated at in Europe andbetween 12

650000 of them would have received substitution treatment in 2007 Heroin accounts for the Qreatest share of in the

which

to

very rapidly rnrlo1or1

matic esterase to form n-~rptvlrTInrnhlinp

is into morphine esterase Morphine is quickly to its

meJaDOtne morphine-3-glucuronide (M3G) somewhat more to amounts morshy

lnICHy-ho

use of different

Dhlnemiddotmiddoto-slUlunClnide (M6G)According to the report Centre for Drugs

2009 (1) polydrug norm in Europe the comshy

is responsible CornplICates most of the problems faces

Knc)wteCHJe only a few allowed

buprenorphine Ilaloxone and two narcotic antag()n were quantified Gas chromatography to mass spectrometry had been widely used for many (78) liquid chromatoQraDhv

(UHPlC-MSMS) It reDliICed techniques used

MATERIALS AND

materials for aU compounds and intershy - were from lGC Url1rYlrmiddotnorn

(Molsheim France) of pu-rity upper than 99

water were from Biosolve Netherlands) citric acid least analytical (Steinheim ven Belgium) Oasis MCX (Zellik

For the preparation of the stock solution of internai commercial solutions of benzoshy

were prepared solution containing

analytes was of rYlnhlYlio

(MAMP) MDMA MDA n-~rptJlmlnrnI1inCcedill (6MAM) (MOR) oxycoshydone (OCOD) hydromorphone (HMOR) the secshy

of cocaine (COc) berlzolecgorline codeine (COD) norcodeine

(EMOR) pholcodine (PHOl) M3G and naltrexorle

one (BUP) and norlbuorenc)rshyphine (NBUP) Group constitution l1oran1c

3ngtlu7o-l in dUI)licate

establlish the calibration curves

Acta Cinica

77 DRues OFABUSE IN SERUM BY UHPLC-MSMS

3 they were in tri~)licate were to estimate the validation f~r~negttegtlc

the method limits An extract was n tln~lrct1 for each run

CT)nngt n stock ~VlULIJII

1ml ammonia in rT1C1rn_

evalpOlratEd to

were injected to the column

Instrumentation Analysis was ntflrnltt1

pIed to a mass CfamplrtrmiddotfIITegttl)1

(Waters Belgium) The chromlatc)grcphic CQongtr_

tion was on an Acquit Y T3 column (100 x 21mm size 18lm Washyters) an filter at 40degC elution was performed at a constant flow of min using a mixture SmM ammonium formate in

Table 1 UPlC A ammonium formate SmM pH 3 B methanol pH 3

00 1000 00

10 1000 00

20 925 75

55 890 110

160 100 900

170 100 900

180 1000 00

190 1000 00

3 with forshy

flow was set at and gas flow was set at 800Llh The MS method was divided into 5 functions rlPf1pnrlirlo on the retention times of multiple reaction mOllecule for identification and

v fragmentation aU()WE~d

Method validation I1rrmiddot r1nn to 15017025 and the

DhrIrotir01 Sciences and Tech-

Elution was carried out using a cegtnrnamplntt1

of 19 minutes To column contamination with matrix highly Ictunampgtt1 r rnf1I1

tion with 1hlo

linearity in The trueness agreement

mean value obtained from the valldeacuteltlcm the value which is either as a

conventional true value or an accepted legttIPregt value

Acta Cfinica 20 10 65-~uPJ)enlent

77 DRues OFABUSE IN SERUM BY UHPLC-MSMS

3 they were in tri~)licate were to estimate the validation f~r~negttegtlc

the method limits An extract was n tln~lrct1 for each run

CT)nngt n stock ~VlULIJII

1ml ammonia in rT1C1rn_

evalpOlratEd to

were injected to the column

Instrumentation Analysis was ntflrnltt1

pIed to a mass CfamplrtrmiddotfIITegttl)1

(Waters Belgium) The chromlatc)grcphic CQongtr_

tion was on an Acquit Y T3 column (100 x 21mm size 18lm Washyters) an filter at 40degC elution was performed at a constant flow of min using a mixture SmM ammonium formate in

Table 1 UPlC A ammonium formate SmM pH 3 B methanol pH 3

00 1000 00

10 1000 00

20 925 75

55 890 110

160 100 900

170 100 900

180 1000 00

190 1000 00

3 with forshy

flow was set at and gas flow was set at 800Llh The MS method was divided into 5 functions rlPf1pnrlirlo on the retention times of multiple reaction mOllecule for identification and

v fragmentation aU()WE~d

Method validation I1rrmiddot r1nn to 15017025 and the

DhrIrotir01 Sciences and Tech-

Elution was carried out using a cegtnrnamplntt1

of 19 minutes To column contamination with matrix highly Ictunampgtt1 r rnf1I1

tion with 1hlo

linearity in The trueness agreement

mean value obtained from the valldeacuteltlcm the value which is either as a

conventional true value or an accepted legttIPregt value

Acta Cfinica 20 10 65-~uPJ)enlent

78 DRues OF ABUSE IN SERUM BY UHPLC-MSMS

Table 2 - Retention times and MRM transitions of each analyte and internal standard (15)

Compound Internai std Ret time Cone MRMl Collision MRM2 Collision (min) voltage (V) energy energy

(V) (V)

M3G-d3

M6G-d3

286 329

39 46510gt28920 33

M3G M6G

M3G-d3

M6G-d3

288331 39 46225gt46225 12 46225gt28625 33

Pholcodine Morphine-d3

300 35 39920gt39920 10 3992gt 11410 31

Morphine-d3

340 35 28905gt20120 28

Morphine Morphine-d3

341 35 28610gt28610 12 28610gt20110 29

Hydromorphone-d3

418 35 28910gt 18510 28

Hydromorphone Hydromorphone-d3

420 35 28610gt28610 12 28610gt18500 31

Norcodeine Codeine-d3

642 30 28620gt28625 10 28620gt26815 20

Dihydrocodeine-d6

642 30 30815gt20215 32

Dihydrocodeine Dihydrocodeacuteine-d6

644 30 30215gt30215 9 30215gt19915 32

Codeine-d3

650 35 30315gt21515 25

Codeine Codeine-d3

652 35 30015gt30015 13 30015gt21515 25

Naloxone Codeine-d3

662 27 32810gt32830 8 32810gt11410 20

Oxycodone-d3

701 28 31910gt30125 18

Oxycodone Oxycodone-d3

703 28 31625 gt29830 19 31625gt24125 30

Hydrocodone-d3

733 35 30315gt21515 25

Hydrocodone Hydrocodone-d3

735 39 30015gt30015 13 30015gt21515 25

Amphetamine-da 685 16 14400gt12710 8

Amphetamine Amphetamine-da 687 16 13600gt11910 8 13600gt9100 16

Methamphetamine-da 734 19 15800gt 12405 11

Methamphetamine Methamphetamine-da 736 19 15000gt 11910 11 15000gt9100 20

Naltrexone Codeine-d3

742 27 34215gt34215 10 34215gt32415 21

MDA-ds 747 15 18500gt 16805 11

MDA 749 15 18000gt 16310 11 18000gt10500 23

6-MAM-d3

761 39 33120gt211 10 24

6-MAM 6-MAM-d 3

763 39 32820gt21110 24 32820gt 16510 40

MDMA-ds 770 20 19905gt 16505 13

MDMA MDMA-d s 772 20 19405gt 16305 13 19405gt 10500 24

Ethylmorph ine Codeine-d3

819 35 31425gt31430 12

Benzoylecgonine-d3

888 26 29300gt17110 18

Benzoylecgonine Benzoylecgonine-d3

890 26 29020gt 16815 18 29020gt 10510 30

MBDB-ds 898 18 21310gt17910 11

MBDB MBDB-ds 900 18 20820gt 17715 11 20820gt13510 11

Acetylcodeine Codeine-d3

943 40 34230gt34230 11 34230gt22525 28

Cocaine-d3

955 27 30710gt18510 21

Cocaine Cocaine-d3

957 27 30420gt18215 21 30420gt8215 32

Cocaethylene-d3 1043 30 32115gt19915 19

Cocaethylene Cocaethylene-d3

1045 30 31825gt19620 19 31825gt8215 30

Buprenorphine-d4

1198 60 47220gt40020 42

Buprenorphine Buprenorphine-d 4 1200 60 46815gt41415 36 46815gt39615 42

Norbuprenorphine Buprenorphine-d4

1114 60 41440gt101 10 35 41440gt8310 65

EDDP-d 3

1157 35 28115gt24920 24

EDDP 1159 35 27815gt24920 24 27815gt23410 32

Methadone-d3

1282 25 31320gt26820 15

Methadone Methadone-d3

1284 25 31020gt31020 5 31020gt26520 15

Acta Clinica Belgica 2010 65-Supplement 1

DRUGS OF ABUSE IN SERUM BY UHPLC-MSMS 79

FUNCTION 1 FUNCTION 2

DlHYDROCODEfNE

fi PHOLCODINE CODEINE

MORPHINE

HYDROMORPHONE

FUNCTION 3 FUNCTION 4

ACETYLCODEINE

BENZOYLECGON1NE

ETHYLM ORPHINE

FUNCTION 5

Trueness is in terms of relative bias atic (11) Trueness was ail comshypounds since were than 19 for concentrations lower

nnltPintpti in nrlgtrlClnn was determined by computing the

1-rImiddotrI Deviations (RSDs) for rIgtITIt-il

inT n1iid~ nlrlririrln at concentration level

BUPRENORPHfNE

of the validation CTIln~I~c

20 concentrations concentrations upper

ity they did not exceed 30 concentrations lower than 20f-lgL and 15 for concentrations

for RSDs are n cnT~

the (ngtll-lI be attributed to the

Acta Clinica 2010 65-5uflplement

80 DRues OF ABUSE IN SERUM BY UHPLC-MSMS

AMP

uncertainty of measurement lower- upper- limacirct of quantitation

MAMP MDMA MDA MBDB

000 1151 1534 215

-979 -211 190 -398

-013 145 139 006

058 163 051 430

285 122 -227 282

605 578 552 463

447 485 732 490

251 640 671 269

669 286 659 571

414 292 234 285

450 258 511 352

409 473 544 449

284 219 334 401

477 304 341 312

109 186 230 241

221 378 419 369 ------------------------~-------

4192 1443 1089 2699

2274 423 1072 1962

1068 258 680 874

156 473 910 1266

790 706 865 1267

477 452 647 725

570 204 432 273

1028 853 1487 1307

HMOR

-1218

-872

-242

-214

-330

683

232

652

724

557

839

390

162

195

111

523

9767

5073

3491

2242

1563

423

665

828

2254

1110

8022

5165

3607

960

1508

1849

()

I1g1L

10

20

40

60

100

300

600

1000

10

20

40

60

100

300

600

40

60

100

300

600

1000

10

20

40

60

100

300

600

1000

277

-387

170

367

062

681

881

487

1065

315

509

678

217

233

189

283

1402

850

518

835

1007

543

495

567

6MAM

1843

150

177

321

-179

525

428

526

719

450

247

364

266

167

181

356

1199

450

287

364

531

176

181

544

MOR

-258

-821

-358

-040

-041

343

736

924

1087

590

337

347

234

162

205

538

4871

3347

1072

825

527

599

308

632

OCOD

787

179

342

626

370

238

14

-085

188

565

615

056

135

161

077

224

978

873

707

713

536

350

657

9659 3321 2437 6210

5238 938 2466 4523

2429 544 1495 1991

2641 998 2037 2894

1804 1617 1973 2902

1005 1002 1460 1647

1311 439 974 589

2367 1937 3414 2997

3079

1941

1096

1820

2317

1234

1128

1283

2685

949

620

767

1207

376

382

1211

11200

7710

2454

1878

1197

1375

685

1381

2251

1945

1525

2353

1642

1229

804

1501

10 26 10 10 20 10 32 10 73LQL (iJglL)

1000 706 894 665 786 1000 985 1000 858UQL (l1gL)

amphetamine MAMP =methamphetamine 6MAM 6-acetylmorphine MOR morphineOCOD oxycodone HMOR hydromor-

Acta Clinica 2010 65--SuDplement

81 DRues OF ABUSE IN SERUM BY UHPLC-MSM5

5000 i

50 100 300 1500 3000 5000

50 100 300 1500 3000

415 365 490 355 477

3348 888 779

1067

749

318 409 362

1136 318 436 362 559

6714 2571 687 931 762

COCET

-209 -729 -224 016

250 349 588 285

418 626 588 340 244

2872 937 1409 1240

737

COD

284 112 257

275 458 211 151

458 560 542 842 3335 1547 966 1277 1244

-313 -346 -267 -351

531 299 659 762 640

719 933 640

2996 1541 2032 1348

-295 -644 235 -654

1026 583 495 638

5Q3 638

1230 1063 1378

-1221 479 918 662

461 372 430 072 089

116 142

941 1198 259 317

COC =cocaine BZE = benzoylecgonine COCET =cocaethylene COD codeine NCOD =norcodeine 6ACOD dihydrocc)deine HCOD hydrocodone EMOR =ethylmorphine PHOl pholcodine

577 241 -367 068 -014

259 321 579 122 304

579 155 368

1068 1221 338 800

486 -280 125 392

609 655 421 515

443 694

6328 2441 944 1528

PHOL

1677 632 817 -085

1291 649 384 485

513

1529 1382 1100

1500

300

551 715

1375 1213 1532 635

674 542

1738 1423 1156 1277

1096 427 736

2456 929 1579 919

714 1375 705

1552 3049 1538 1963

896 648 689

1993 1385 1462 1300

860 891 1046 661 313

5406 1965 1915 2301 1428

300 1500 3000 5000

Intra-assay predshy 50 sion 100

300 1500 3000 5000

Inter-assay precishy 50 sion 100

IntelCme(jiate 300

1500 3000

3

-599 -695 -190

1036 596 409 656 301 586

1953 643

EDDP

-747 -102 -011 351 385

1354 628 669 51 602

M3G

1378 -480 -463

-213 339

1194 655 347

666 436

M6G

133 -725 123 -041 -301

2091 593 943

585 704

711 -433 165 266 -167

1195 589 600 689 611

1364 239 -242

005 322

1354 295 796 782 574

05 10 30 150 300 500 05 10 30 150 300

BUP

1511 -935 -769 -364 261

880 784 1118

611 575

500

NBUP

1010 -612 691 -814 216

946 813 757

500

be~nd METHA =methadone M3G = morphine-3-glucuronide M6G morphine-6-glucuronide BUP = buprenorphine NBUP =norbuprenorphine

Acta Clinica 2010 6gt-)ucplemiddotment

82 DRUeS OF ABUSE IN SERUM BY UHPLC-MSMS

Aeeuracy Profile A(curacy Profile Accuncy PTOme

Metharnphetamiumlne

Accuracy frofllf

Morphine

AccurICt Profil il ACClITampcy Accuracy Prame

Accuracy froflle Alaquouracy Fronle Accuracy prome

Accuracy Profile

Eth)1 morphine Pholcodine

Acta Clinica 2010 6-)UPI)len1ent

83 DRues OF ABUSE IN SERUM BY UHPLC-MSMS

Accuracv prom Accuruy Proflle

Mltlhadone M3G

Actuucy Proflfe

The dOits represent the relative t)adr-calculatE~dconcentraUonsam1 an ptlJttE~dlilgtlith respect to their talraetedCOnCel1ltral~ion [S-expeCtation tolerance timits and the dotted curves represent the acceptance

measurand The cgtvlrlvltt1

i 1 around represents an

the u nknown true rnrtit1in~ level of 95

concentrations Values each analyte are presented in Table 2

The total error the of the ot-hrt1

contain 875 acshyce[)talnce Umits were set at plusmn 20 for concentrations upper than and at plusmn 50 for concentrations

to accu rate timation of a is fi t1nlt~nt-1

validity of the method Total error is ness and is clearly a results accuracy The accuracy CgtVIUccgtc

of between the

or an accepted otmiddot nr

The accuracy tremes of the 875 In1onl1

exshywill

the tOTIC

range The intersections between

the lower the profile and

the of

tation (LQL) as weil as the upper limit UQL of(1

important to have a as be to measure two MRM by n1l1

19 MRM rngtnnHC

Acta Clinica 2010 65SUl)plE~ment

84 DRUGS OF ABUSE IN SERUM BY UHPLC-MSMS

time function MS method was dishyvided in five time functions and MRM dwell times were adius1ted to maximize cocitilitl

anltiteacuteltie determination urnhtrnln~ in serum answered to our

rocnnnCcgtc were established for each

OXfroldorlP and hydromorphone from for buprenorphine norbuprenorphine Bishy

concentrations upper than 20flglL 8 for

did not ex-20flgL and

than 20flglL for inter-lower

30 for concentrations 15 for concentrations

ases were crrIlllor than 19 for concentrations than 20l-lgL than 12 for concentrations than For RSDs did not exceed 20 concentrations lower than

tolerance Umit did not ov-11

(20 for concentrations lower concentrations upper than 20flgL) at 875 The lower limit of quantitation was 71lglL for

compound which was than obtained by Ge-MS technology

CONCLUSION

ln to gtI-lIIII narcotic subshyaddictia~n andor intoxication

most common

REFERENCES

1 Observatoire europeacuteen des drogues et des toxicomanies Rapshyport annuel Office des de lUnion Eungtpeacuteenne 2009

2 E MA LeBeau MA Sibum M An aushytomated SPELCMSMS method for the of cocaine and metabolites in whole B 2008 87415-20

3 Chegraveze M Deveaux M Martin c Lhermitte M G Simulshytaneous determination of six and in hair blood and urine LC-ESI-MSMS to the deshytermination of MDMA after low ecstasy intake Forensic Sei ln t 2007 170 100-104

4 MK Nielsen MKK Markussen LO Klinke HB Unnet K Determination of 19 of abuse and metabolites in whole blood liquid rhrmrtrar~nhmiddotI-tnrm

mass spectrometry Anal Bioanal Chem 2009 in press 5 M Bafile E D Curini R DAscenzo G Ro-

FS Multiclass of illicit in and oral Huids LC-MSMSAnal 8ioanal Chem 2009 393 709-718

6 Lacroix c Saussereau E Bodin G Goulleacute JP Quantification des et par rhrOrrrtr

phie liquide haute 1 spectromeacutetrie de masse en tandem de leacutechantillon ATA 2008

20(1) 25-38 7 Paterson S Lintzeris N Mitchell TB Cordera R Nestor L

J Validation of to detect iIIiumlcit heroiumln use nrpcrrilr pheacute1rrriacieutical heroin for the manageshy

ment of dependenceAddiction 2005 100 1832-1839 8 Kudo K Ishida T Hara K Kashimura 5 A Ikeda N 5ishy

multaneous determination of 13 related drugs in human whole blood an enhanced polymer columnand gas spectrometry J B 2007 855 115-120

9 Rook EJ Hillebrand MJ H et aL The qualntilatile of heroin methadone and their metabolites and the

simultaneousdetection of cocaiumlne and their metabolites in human liquid

with tandem mass spectr()mEtrv Chrom B 2005 824 213middot221

10 Hubert Ph J-J B et aL Harmonizashytion of for the validation of oUeacutemtitalive anecircllytical proceltlunes A SFSTP Part 1 j Pharm Biomed Ana 2004 36 579-586

11 Hubert Ph Nguyen-Huu J-J Boulanger B et aL Harmonizashytion of for the validation of owmtitalive rnllvtirr1

procedures A 5FSTP Part Il J pharm 8iomed Anal 2007 45 70-81

12 Hubert Ph NClIlVn-~-l11l1 J-J B et aL Harmonizashytion of strltlte~~ies for the validation of analytical procedlures A SFSTP - Part III J Pharm Biomed Anal 2007 45 82-96

13 Rozet E Ceccato A Hubert C et aL Analysis of recent pharshymaceutical documents on method vaUshy

20071158111-125

Acta Clinica 2010 65-5ulpeuroment

DRUGS OF ABUSE IN SERUM BY UHPLC-MSMS 79

FUNCTION 1 FUNCTION 2

DlHYDROCODEfNE

fi PHOLCODINE CODEINE

MORPHINE

HYDROMORPHONE

FUNCTION 3 FUNCTION 4

ACETYLCODEINE

BENZOYLECGON1NE

ETHYLM ORPHINE

FUNCTION 5

Trueness is in terms of relative bias atic (11) Trueness was ail comshypounds since were than 19 for concentrations lower

nnltPintpti in nrlgtrlClnn was determined by computing the

1-rImiddotrI Deviations (RSDs) for rIgtITIt-il

inT n1iid~ nlrlririrln at concentration level

BUPRENORPHfNE

of the validation CTIln~I~c

20 concentrations concentrations upper

ity they did not exceed 30 concentrations lower than 20f-lgL and 15 for concentrations

for RSDs are n cnT~

the (ngtll-lI be attributed to the

Acta Clinica 2010 65-5uflplement

80 DRues OF ABUSE IN SERUM BY UHPLC-MSMS

AMP

uncertainty of measurement lower- upper- limacirct of quantitation

MAMP MDMA MDA MBDB

000 1151 1534 215

-979 -211 190 -398

-013 145 139 006

058 163 051 430

285 122 -227 282

605 578 552 463

447 485 732 490

251 640 671 269

669 286 659 571

414 292 234 285

450 258 511 352

409 473 544 449

284 219 334 401

477 304 341 312

109 186 230 241

221 378 419 369 ------------------------~-------

4192 1443 1089 2699

2274 423 1072 1962

1068 258 680 874

156 473 910 1266

790 706 865 1267

477 452 647 725

570 204 432 273

1028 853 1487 1307

HMOR

-1218

-872

-242

-214

-330

683

232

652

724

557

839

390

162

195

111

523

9767

5073

3491

2242

1563

423

665

828

2254

1110

8022

5165

3607

960

1508

1849

()

I1g1L

10

20

40

60

100

300

600

1000

10

20

40

60

100

300

600

40

60

100

300

600

1000

10

20

40

60

100

300

600

1000

277

-387

170

367

062

681

881

487

1065

315

509

678

217

233

189

283

1402

850

518

835

1007

543

495

567

6MAM

1843

150

177

321

-179

525

428

526

719

450

247

364

266

167

181

356

1199

450

287

364

531

176

181

544

MOR

-258

-821

-358

-040

-041

343

736

924

1087

590

337

347

234

162

205

538

4871

3347

1072

825

527

599

308

632

OCOD

787

179

342

626

370

238

14

-085

188

565

615

056

135

161

077

224

978

873

707

713

536

350

657

9659 3321 2437 6210

5238 938 2466 4523

2429 544 1495 1991

2641 998 2037 2894

1804 1617 1973 2902

1005 1002 1460 1647

1311 439 974 589

2367 1937 3414 2997

3079

1941

1096

1820

2317

1234

1128

1283

2685

949

620

767

1207

376

382

1211

11200

7710

2454

1878

1197

1375

685

1381

2251

1945

1525

2353

1642

1229

804

1501

10 26 10 10 20 10 32 10 73LQL (iJglL)

1000 706 894 665 786 1000 985 1000 858UQL (l1gL)

amphetamine MAMP =methamphetamine 6MAM 6-acetylmorphine MOR morphineOCOD oxycodone HMOR hydromor-

Acta Clinica 2010 65--SuDplement

81 DRues OF ABUSE IN SERUM BY UHPLC-MSM5

5000 i

50 100 300 1500 3000 5000

50 100 300 1500 3000

415 365 490 355 477

3348 888 779

1067

749

318 409 362

1136 318 436 362 559

6714 2571 687 931 762

COCET

-209 -729 -224 016

250 349 588 285

418 626 588 340 244

2872 937 1409 1240

737

COD

284 112 257

275 458 211 151

458 560 542 842 3335 1547 966 1277 1244

-313 -346 -267 -351

531 299 659 762 640

719 933 640

2996 1541 2032 1348

-295 -644 235 -654

1026 583 495 638

5Q3 638

1230 1063 1378

-1221 479 918 662

461 372 430 072 089

116 142

941 1198 259 317

COC =cocaine BZE = benzoylecgonine COCET =cocaethylene COD codeine NCOD =norcodeine 6ACOD dihydrocc)deine HCOD hydrocodone EMOR =ethylmorphine PHOl pholcodine

577 241 -367 068 -014

259 321 579 122 304

579 155 368

1068 1221 338 800

486 -280 125 392

609 655 421 515

443 694

6328 2441 944 1528

PHOL

1677 632 817 -085

1291 649 384 485

513

1529 1382 1100

1500

300

551 715

1375 1213 1532 635

674 542

1738 1423 1156 1277

1096 427 736

2456 929 1579 919

714 1375 705

1552 3049 1538 1963

896 648 689

1993 1385 1462 1300

860 891 1046 661 313

5406 1965 1915 2301 1428

300 1500 3000 5000

Intra-assay predshy 50 sion 100

300 1500 3000 5000

Inter-assay precishy 50 sion 100

IntelCme(jiate 300

1500 3000

3

-599 -695 -190

1036 596 409 656 301 586

1953 643

EDDP

-747 -102 -011 351 385

1354 628 669 51 602

M3G

1378 -480 -463

-213 339

1194 655 347

666 436

M6G

133 -725 123 -041 -301

2091 593 943

585 704

711 -433 165 266 -167

1195 589 600 689 611

1364 239 -242

005 322

1354 295 796 782 574

05 10 30 150 300 500 05 10 30 150 300

BUP

1511 -935 -769 -364 261

880 784 1118

611 575

500

NBUP

1010 -612 691 -814 216

946 813 757

500

be~nd METHA =methadone M3G = morphine-3-glucuronide M6G morphine-6-glucuronide BUP = buprenorphine NBUP =norbuprenorphine

Acta Clinica 2010 6gt-)ucplemiddotment

82 DRUeS OF ABUSE IN SERUM BY UHPLC-MSMS

Aeeuracy Profile A(curacy Profile Accuncy PTOme

Metharnphetamiumlne

Accuracy frofllf

Morphine

AccurICt Profil il ACClITampcy Accuracy Prame

Accuracy froflle Alaquouracy Fronle Accuracy prome

Accuracy Profile

Eth)1 morphine Pholcodine

Acta Clinica 2010 6-)UPI)len1ent

83 DRues OF ABUSE IN SERUM BY UHPLC-MSMS

Accuracv prom Accuruy Proflle

Mltlhadone M3G

Actuucy Proflfe

The dOits represent the relative t)adr-calculatE~dconcentraUonsam1 an ptlJttE~dlilgtlith respect to their talraetedCOnCel1ltral~ion [S-expeCtation tolerance timits and the dotted curves represent the acceptance

measurand The cgtvlrlvltt1

i 1 around represents an

the u nknown true rnrtit1in~ level of 95

concentrations Values each analyte are presented in Table 2

The total error the of the ot-hrt1

contain 875 acshyce[)talnce Umits were set at plusmn 20 for concentrations upper than and at plusmn 50 for concentrations

to accu rate timation of a is fi t1nlt~nt-1

validity of the method Total error is ness and is clearly a results accuracy The accuracy CgtVIUccgtc

of between the

or an accepted otmiddot nr

The accuracy tremes of the 875 In1onl1

exshywill

the tOTIC

range The intersections between

the lower the profile and

the of

tation (LQL) as weil as the upper limit UQL of(1

important to have a as be to measure two MRM by n1l1

19 MRM rngtnnHC

Acta Clinica 2010 65SUl)plE~ment

84 DRUGS OF ABUSE IN SERUM BY UHPLC-MSMS

time function MS method was dishyvided in five time functions and MRM dwell times were adius1ted to maximize cocitilitl

anltiteacuteltie determination urnhtrnln~ in serum answered to our

rocnnnCcgtc were established for each

OXfroldorlP and hydromorphone from for buprenorphine norbuprenorphine Bishy

concentrations upper than 20flglL 8 for

did not ex-20flgL and

than 20flglL for inter-lower

30 for concentrations 15 for concentrations

ases were crrIlllor than 19 for concentrations than 20l-lgL than 12 for concentrations than For RSDs did not exceed 20 concentrations lower than

tolerance Umit did not ov-11

(20 for concentrations lower concentrations upper than 20flgL) at 875 The lower limit of quantitation was 71lglL for

compound which was than obtained by Ge-MS technology

CONCLUSION

ln to gtI-lIIII narcotic subshyaddictia~n andor intoxication

most common

REFERENCES

1 Observatoire europeacuteen des drogues et des toxicomanies Rapshyport annuel Office des de lUnion Eungtpeacuteenne 2009

2 E MA LeBeau MA Sibum M An aushytomated SPELCMSMS method for the of cocaine and metabolites in whole B 2008 87415-20

3 Chegraveze M Deveaux M Martin c Lhermitte M G Simulshytaneous determination of six and in hair blood and urine LC-ESI-MSMS to the deshytermination of MDMA after low ecstasy intake Forensic Sei ln t 2007 170 100-104

4 MK Nielsen MKK Markussen LO Klinke HB Unnet K Determination of 19 of abuse and metabolites in whole blood liquid rhrmrtrar~nhmiddotI-tnrm

mass spectrometry Anal Bioanal Chem 2009 in press 5 M Bafile E D Curini R DAscenzo G Ro-

FS Multiclass of illicit in and oral Huids LC-MSMSAnal 8ioanal Chem 2009 393 709-718

6 Lacroix c Saussereau E Bodin G Goulleacute JP Quantification des et par rhrOrrrtr

phie liquide haute 1 spectromeacutetrie de masse en tandem de leacutechantillon ATA 2008

20(1) 25-38 7 Paterson S Lintzeris N Mitchell TB Cordera R Nestor L

J Validation of to detect iIIiumlcit heroiumln use nrpcrrilr pheacute1rrriacieutical heroin for the manageshy

ment of dependenceAddiction 2005 100 1832-1839 8 Kudo K Ishida T Hara K Kashimura 5 A Ikeda N 5ishy

multaneous determination of 13 related drugs in human whole blood an enhanced polymer columnand gas spectrometry J B 2007 855 115-120

9 Rook EJ Hillebrand MJ H et aL The qualntilatile of heroin methadone and their metabolites and the

simultaneousdetection of cocaiumlne and their metabolites in human liquid

with tandem mass spectr()mEtrv Chrom B 2005 824 213middot221

10 Hubert Ph J-J B et aL Harmonizashytion of for the validation of oUeacutemtitalive anecircllytical proceltlunes A SFSTP Part 1 j Pharm Biomed Ana 2004 36 579-586

11 Hubert Ph Nguyen-Huu J-J Boulanger B et aL Harmonizashytion of for the validation of owmtitalive rnllvtirr1

procedures A 5FSTP Part Il J pharm 8iomed Anal 2007 45 70-81

12 Hubert Ph NClIlVn-~-l11l1 J-J B et aL Harmonizashytion of strltlte~~ies for the validation of analytical procedlures A SFSTP - Part III J Pharm Biomed Anal 2007 45 82-96

13 Rozet E Ceccato A Hubert C et aL Analysis of recent pharshymaceutical documents on method vaUshy

20071158111-125

Acta Clinica 2010 65-5ulpeuroment

80 DRues OF ABUSE IN SERUM BY UHPLC-MSMS

AMP

uncertainty of measurement lower- upper- limacirct of quantitation

MAMP MDMA MDA MBDB

000 1151 1534 215

-979 -211 190 -398

-013 145 139 006

058 163 051 430

285 122 -227 282

605 578 552 463

447 485 732 490

251 640 671 269

669 286 659 571

414 292 234 285

450 258 511 352

409 473 544 449

284 219 334 401

477 304 341 312

109 186 230 241

221 378 419 369 ------------------------~-------

4192 1443 1089 2699

2274 423 1072 1962

1068 258 680 874

156 473 910 1266

790 706 865 1267

477 452 647 725

570 204 432 273

1028 853 1487 1307

HMOR

-1218

-872

-242

-214

-330

683

232

652

724

557

839

390

162

195

111

523

9767

5073

3491

2242

1563

423

665

828

2254

1110

8022

5165

3607

960

1508

1849

()

I1g1L

10

20

40

60

100

300

600

1000

10

20

40

60

100

300

600

40

60

100

300

600

1000

10

20

40

60

100

300

600

1000

277

-387

170

367

062

681

881

487

1065

315

509

678

217

233

189

283

1402

850

518

835

1007

543

495

567

6MAM

1843

150

177

321

-179

525

428

526

719

450

247

364

266

167

181

356

1199

450

287

364

531

176

181

544

MOR

-258

-821

-358

-040

-041

343

736

924

1087

590

337

347

234

162

205

538

4871

3347

1072

825

527

599

308

632

OCOD

787

179

342

626

370

238

14

-085

188

565

615

056

135

161

077

224

978

873

707

713

536

350

657

9659 3321 2437 6210

5238 938 2466 4523

2429 544 1495 1991

2641 998 2037 2894

1804 1617 1973 2902

1005 1002 1460 1647

1311 439 974 589

2367 1937 3414 2997

3079

1941

1096

1820

2317

1234

1128

1283

2685

949

620

767

1207

376

382

1211

11200

7710

2454

1878

1197

1375

685

1381

2251

1945

1525

2353

1642

1229

804

1501

10 26 10 10 20 10 32 10 73LQL (iJglL)

1000 706 894 665 786 1000 985 1000 858UQL (l1gL)

amphetamine MAMP =methamphetamine 6MAM 6-acetylmorphine MOR morphineOCOD oxycodone HMOR hydromor-

Acta Clinica 2010 65--SuDplement

81 DRues OF ABUSE IN SERUM BY UHPLC-MSM5

5000 i

50 100 300 1500 3000 5000

50 100 300 1500 3000

415 365 490 355 477

3348 888 779

1067

749

318 409 362

1136 318 436 362 559

6714 2571 687 931 762

COCET

-209 -729 -224 016

250 349 588 285

418 626 588 340 244

2872 937 1409 1240

737

COD

284 112 257

275 458 211 151

458 560 542 842 3335 1547 966 1277 1244

-313 -346 -267 -351

531 299 659 762 640

719 933 640

2996 1541 2032 1348

-295 -644 235 -654

1026 583 495 638

5Q3 638

1230 1063 1378

-1221 479 918 662

461 372 430 072 089

116 142

941 1198 259 317

COC =cocaine BZE = benzoylecgonine COCET =cocaethylene COD codeine NCOD =norcodeine 6ACOD dihydrocc)deine HCOD hydrocodone EMOR =ethylmorphine PHOl pholcodine

577 241 -367 068 -014

259 321 579 122 304

579 155 368

1068 1221 338 800

486 -280 125 392

609 655 421 515

443 694

6328 2441 944 1528

PHOL

1677 632 817 -085

1291 649 384 485

513

1529 1382 1100

1500

300

551 715

1375 1213 1532 635

674 542

1738 1423 1156 1277

1096 427 736

2456 929 1579 919

714 1375 705

1552 3049 1538 1963

896 648 689

1993 1385 1462 1300

860 891 1046 661 313

5406 1965 1915 2301 1428

300 1500 3000 5000

Intra-assay predshy 50 sion 100

300 1500 3000 5000

Inter-assay precishy 50 sion 100

IntelCme(jiate 300

1500 3000

3

-599 -695 -190

1036 596 409 656 301 586

1953 643

EDDP

-747 -102 -011 351 385

1354 628 669 51 602

M3G

1378 -480 -463

-213 339

1194 655 347

666 436

M6G

133 -725 123 -041 -301

2091 593 943

585 704

711 -433 165 266 -167

1195 589 600 689 611

1364 239 -242

005 322

1354 295 796 782 574

05 10 30 150 300 500 05 10 30 150 300

BUP

1511 -935 -769 -364 261

880 784 1118

611 575

500

NBUP

1010 -612 691 -814 216

946 813 757

500

be~nd METHA =methadone M3G = morphine-3-glucuronide M6G morphine-6-glucuronide BUP = buprenorphine NBUP =norbuprenorphine

Acta Clinica 2010 6gt-)ucplemiddotment

82 DRUeS OF ABUSE IN SERUM BY UHPLC-MSMS

Aeeuracy Profile A(curacy Profile Accuncy PTOme

Metharnphetamiumlne

Accuracy frofllf

Morphine

AccurICt Profil il ACClITampcy Accuracy Prame

Accuracy froflle Alaquouracy Fronle Accuracy prome

Accuracy Profile

Eth)1 morphine Pholcodine

Acta Clinica 2010 6-)UPI)len1ent

83 DRues OF ABUSE IN SERUM BY UHPLC-MSMS

Accuracv prom Accuruy Proflle

Mltlhadone M3G

Actuucy Proflfe

The dOits represent the relative t)adr-calculatE~dconcentraUonsam1 an ptlJttE~dlilgtlith respect to their talraetedCOnCel1ltral~ion [S-expeCtation tolerance timits and the dotted curves represent the acceptance

measurand The cgtvlrlvltt1

i 1 around represents an

the u nknown true rnrtit1in~ level of 95

concentrations Values each analyte are presented in Table 2

The total error the of the ot-hrt1

contain 875 acshyce[)talnce Umits were set at plusmn 20 for concentrations upper than and at plusmn 50 for concentrations

to accu rate timation of a is fi t1nlt~nt-1

validity of the method Total error is ness and is clearly a results accuracy The accuracy CgtVIUccgtc

of between the

or an accepted otmiddot nr

The accuracy tremes of the 875 In1onl1

exshywill

the tOTIC

range The intersections between

the lower the profile and

the of

tation (LQL) as weil as the upper limit UQL of(1

important to have a as be to measure two MRM by n1l1

19 MRM rngtnnHC

Acta Clinica 2010 65SUl)plE~ment

84 DRUGS OF ABUSE IN SERUM BY UHPLC-MSMS

time function MS method was dishyvided in five time functions and MRM dwell times were adius1ted to maximize cocitilitl

anltiteacuteltie determination urnhtrnln~ in serum answered to our

rocnnnCcgtc were established for each

OXfroldorlP and hydromorphone from for buprenorphine norbuprenorphine Bishy

concentrations upper than 20flglL 8 for

did not ex-20flgL and

than 20flglL for inter-lower

30 for concentrations 15 for concentrations

ases were crrIlllor than 19 for concentrations than 20l-lgL than 12 for concentrations than For RSDs did not exceed 20 concentrations lower than

tolerance Umit did not ov-11

(20 for concentrations lower concentrations upper than 20flgL) at 875 The lower limit of quantitation was 71lglL for

compound which was than obtained by Ge-MS technology

CONCLUSION

ln to gtI-lIIII narcotic subshyaddictia~n andor intoxication

most common

REFERENCES

1 Observatoire europeacuteen des drogues et des toxicomanies Rapshyport annuel Office des de lUnion Eungtpeacuteenne 2009

2 E MA LeBeau MA Sibum M An aushytomated SPELCMSMS method for the of cocaine and metabolites in whole B 2008 87415-20

3 Chegraveze M Deveaux M Martin c Lhermitte M G Simulshytaneous determination of six and in hair blood and urine LC-ESI-MSMS to the deshytermination of MDMA after low ecstasy intake Forensic Sei ln t 2007 170 100-104

4 MK Nielsen MKK Markussen LO Klinke HB Unnet K Determination of 19 of abuse and metabolites in whole blood liquid rhrmrtrar~nhmiddotI-tnrm

mass spectrometry Anal Bioanal Chem 2009 in press 5 M Bafile E D Curini R DAscenzo G Ro-

FS Multiclass of illicit in and oral Huids LC-MSMSAnal 8ioanal Chem 2009 393 709-718

6 Lacroix c Saussereau E Bodin G Goulleacute JP Quantification des et par rhrOrrrtr

phie liquide haute 1 spectromeacutetrie de masse en tandem de leacutechantillon ATA 2008

20(1) 25-38 7 Paterson S Lintzeris N Mitchell TB Cordera R Nestor L

J Validation of to detect iIIiumlcit heroiumln use nrpcrrilr pheacute1rrriacieutical heroin for the manageshy

ment of dependenceAddiction 2005 100 1832-1839 8 Kudo K Ishida T Hara K Kashimura 5 A Ikeda N 5ishy

multaneous determination of 13 related drugs in human whole blood an enhanced polymer columnand gas spectrometry J B 2007 855 115-120

9 Rook EJ Hillebrand MJ H et aL The qualntilatile of heroin methadone and their metabolites and the

simultaneousdetection of cocaiumlne and their metabolites in human liquid

with tandem mass spectr()mEtrv Chrom B 2005 824 213middot221

10 Hubert Ph J-J B et aL Harmonizashytion of for the validation of oUeacutemtitalive anecircllytical proceltlunes A SFSTP Part 1 j Pharm Biomed Ana 2004 36 579-586

11 Hubert Ph Nguyen-Huu J-J Boulanger B et aL Harmonizashytion of for the validation of owmtitalive rnllvtirr1

procedures A 5FSTP Part Il J pharm 8iomed Anal 2007 45 70-81

12 Hubert Ph NClIlVn-~-l11l1 J-J B et aL Harmonizashytion of strltlte~~ies for the validation of analytical procedlures A SFSTP - Part III J Pharm Biomed Anal 2007 45 82-96

13 Rozet E Ceccato A Hubert C et aL Analysis of recent pharshymaceutical documents on method vaUshy

20071158111-125

Acta Clinica 2010 65-5ulpeuroment

81 DRues OF ABUSE IN SERUM BY UHPLC-MSM5

5000 i

50 100 300 1500 3000 5000

50 100 300 1500 3000

415 365 490 355 477

3348 888 779

1067

749

318 409 362

1136 318 436 362 559

6714 2571 687 931 762

COCET

-209 -729 -224 016

250 349 588 285

418 626 588 340 244

2872 937 1409 1240

737

COD

284 112 257

275 458 211 151

458 560 542 842 3335 1547 966 1277 1244

-313 -346 -267 -351

531 299 659 762 640

719 933 640

2996 1541 2032 1348

-295 -644 235 -654

1026 583 495 638

5Q3 638

1230 1063 1378

-1221 479 918 662

461 372 430 072 089

116 142

941 1198 259 317

COC =cocaine BZE = benzoylecgonine COCET =cocaethylene COD codeine NCOD =norcodeine 6ACOD dihydrocc)deine HCOD hydrocodone EMOR =ethylmorphine PHOl pholcodine

577 241 -367 068 -014

259 321 579 122 304

579 155 368

1068 1221 338 800

486 -280 125 392

609 655 421 515

443 694

6328 2441 944 1528

PHOL

1677 632 817 -085

1291 649 384 485

513

1529 1382 1100

1500

300

551 715

1375 1213 1532 635

674 542

1738 1423 1156 1277

1096 427 736

2456 929 1579 919

714 1375 705

1552 3049 1538 1963

896 648 689

1993 1385 1462 1300

860 891 1046 661 313

5406 1965 1915 2301 1428

300 1500 3000 5000

Intra-assay predshy 50 sion 100

300 1500 3000 5000

Inter-assay precishy 50 sion 100

IntelCme(jiate 300

1500 3000

3

-599 -695 -190

1036 596 409 656 301 586

1953 643

EDDP

-747 -102 -011 351 385

1354 628 669 51 602

M3G

1378 -480 -463

-213 339

1194 655 347

666 436

M6G

133 -725 123 -041 -301

2091 593 943

585 704

711 -433 165 266 -167

1195 589 600 689 611

1364 239 -242

005 322

1354 295 796 782 574

05 10 30 150 300 500 05 10 30 150 300

BUP

1511 -935 -769 -364 261

880 784 1118

611 575

500

NBUP

1010 -612 691 -814 216

946 813 757

500

be~nd METHA =methadone M3G = morphine-3-glucuronide M6G morphine-6-glucuronide BUP = buprenorphine NBUP =norbuprenorphine

Acta Clinica 2010 6gt-)ucplemiddotment

82 DRUeS OF ABUSE IN SERUM BY UHPLC-MSMS

Aeeuracy Profile A(curacy Profile Accuncy PTOme

Metharnphetamiumlne

Accuracy frofllf

Morphine

AccurICt Profil il ACClITampcy Accuracy Prame

Accuracy froflle Alaquouracy Fronle Accuracy prome

Accuracy Profile

Eth)1 morphine Pholcodine

Acta Clinica 2010 6-)UPI)len1ent

83 DRues OF ABUSE IN SERUM BY UHPLC-MSMS

Accuracv prom Accuruy Proflle

Mltlhadone M3G

Actuucy Proflfe

The dOits represent the relative t)adr-calculatE~dconcentraUonsam1 an ptlJttE~dlilgtlith respect to their talraetedCOnCel1ltral~ion [S-expeCtation tolerance timits and the dotted curves represent the acceptance

measurand The cgtvlrlvltt1

i 1 around represents an

the u nknown true rnrtit1in~ level of 95

concentrations Values each analyte are presented in Table 2

The total error the of the ot-hrt1

contain 875 acshyce[)talnce Umits were set at plusmn 20 for concentrations upper than and at plusmn 50 for concentrations

to accu rate timation of a is fi t1nlt~nt-1

validity of the method Total error is ness and is clearly a results accuracy The accuracy CgtVIUccgtc

of between the

or an accepted otmiddot nr

The accuracy tremes of the 875 In1onl1

exshywill

the tOTIC

range The intersections between

the lower the profile and

the of

tation (LQL) as weil as the upper limit UQL of(1

important to have a as be to measure two MRM by n1l1

19 MRM rngtnnHC

Acta Clinica 2010 65SUl)plE~ment

84 DRUGS OF ABUSE IN SERUM BY UHPLC-MSMS

time function MS method was dishyvided in five time functions and MRM dwell times were adius1ted to maximize cocitilitl

anltiteacuteltie determination urnhtrnln~ in serum answered to our

rocnnnCcgtc were established for each

OXfroldorlP and hydromorphone from for buprenorphine norbuprenorphine Bishy

concentrations upper than 20flglL 8 for

did not ex-20flgL and

than 20flglL for inter-lower

30 for concentrations 15 for concentrations

ases were crrIlllor than 19 for concentrations than 20l-lgL than 12 for concentrations than For RSDs did not exceed 20 concentrations lower than

tolerance Umit did not ov-11

(20 for concentrations lower concentrations upper than 20flgL) at 875 The lower limit of quantitation was 71lglL for

compound which was than obtained by Ge-MS technology

CONCLUSION

ln to gtI-lIIII narcotic subshyaddictia~n andor intoxication

most common

REFERENCES

1 Observatoire europeacuteen des drogues et des toxicomanies Rapshyport annuel Office des de lUnion Eungtpeacuteenne 2009

2 E MA LeBeau MA Sibum M An aushytomated SPELCMSMS method for the of cocaine and metabolites in whole B 2008 87415-20

3 Chegraveze M Deveaux M Martin c Lhermitte M G Simulshytaneous determination of six and in hair blood and urine LC-ESI-MSMS to the deshytermination of MDMA after low ecstasy intake Forensic Sei ln t 2007 170 100-104

4 MK Nielsen MKK Markussen LO Klinke HB Unnet K Determination of 19 of abuse and metabolites in whole blood liquid rhrmrtrar~nhmiddotI-tnrm

mass spectrometry Anal Bioanal Chem 2009 in press 5 M Bafile E D Curini R DAscenzo G Ro-

FS Multiclass of illicit in and oral Huids LC-MSMSAnal 8ioanal Chem 2009 393 709-718

6 Lacroix c Saussereau E Bodin G Goulleacute JP Quantification des et par rhrOrrrtr

phie liquide haute 1 spectromeacutetrie de masse en tandem de leacutechantillon ATA 2008

20(1) 25-38 7 Paterson S Lintzeris N Mitchell TB Cordera R Nestor L

J Validation of to detect iIIiumlcit heroiumln use nrpcrrilr pheacute1rrriacieutical heroin for the manageshy

ment of dependenceAddiction 2005 100 1832-1839 8 Kudo K Ishida T Hara K Kashimura 5 A Ikeda N 5ishy

multaneous determination of 13 related drugs in human whole blood an enhanced polymer columnand gas spectrometry J B 2007 855 115-120

9 Rook EJ Hillebrand MJ H et aL The qualntilatile of heroin methadone and their metabolites and the

simultaneousdetection of cocaiumlne and their metabolites in human liquid

with tandem mass spectr()mEtrv Chrom B 2005 824 213middot221

10 Hubert Ph J-J B et aL Harmonizashytion of for the validation of oUeacutemtitalive anecircllytical proceltlunes A SFSTP Part 1 j Pharm Biomed Ana 2004 36 579-586

11 Hubert Ph Nguyen-Huu J-J Boulanger B et aL Harmonizashytion of for the validation of owmtitalive rnllvtirr1

procedures A 5FSTP Part Il J pharm 8iomed Anal 2007 45 70-81

12 Hubert Ph NClIlVn-~-l11l1 J-J B et aL Harmonizashytion of strltlte~~ies for the validation of analytical procedlures A SFSTP - Part III J Pharm Biomed Anal 2007 45 82-96

13 Rozet E Ceccato A Hubert C et aL Analysis of recent pharshymaceutical documents on method vaUshy

20071158111-125

Acta Clinica 2010 65-5ulpeuroment

82 DRUeS OF ABUSE IN SERUM BY UHPLC-MSMS

Aeeuracy Profile A(curacy Profile Accuncy PTOme

Metharnphetamiumlne

Accuracy frofllf

Morphine

AccurICt Profil il ACClITampcy Accuracy Prame

Accuracy froflle Alaquouracy Fronle Accuracy prome

Accuracy Profile

Eth)1 morphine Pholcodine

Acta Clinica 2010 6-)UPI)len1ent

83 DRues OF ABUSE IN SERUM BY UHPLC-MSMS

Accuracv prom Accuruy Proflle

Mltlhadone M3G

Actuucy Proflfe

The dOits represent the relative t)adr-calculatE~dconcentraUonsam1 an ptlJttE~dlilgtlith respect to their talraetedCOnCel1ltral~ion [S-expeCtation tolerance timits and the dotted curves represent the acceptance

measurand The cgtvlrlvltt1

i 1 around represents an

the u nknown true rnrtit1in~ level of 95

concentrations Values each analyte are presented in Table 2

The total error the of the ot-hrt1

contain 875 acshyce[)talnce Umits were set at plusmn 20 for concentrations upper than and at plusmn 50 for concentrations

to accu rate timation of a is fi t1nlt~nt-1

validity of the method Total error is ness and is clearly a results accuracy The accuracy CgtVIUccgtc

of between the

or an accepted otmiddot nr

The accuracy tremes of the 875 In1onl1

exshywill

the tOTIC

range The intersections between

the lower the profile and

the of

tation (LQL) as weil as the upper limit UQL of(1

important to have a as be to measure two MRM by n1l1

19 MRM rngtnnHC

Acta Clinica 2010 65SUl)plE~ment

84 DRUGS OF ABUSE IN SERUM BY UHPLC-MSMS

time function MS method was dishyvided in five time functions and MRM dwell times were adius1ted to maximize cocitilitl

anltiteacuteltie determination urnhtrnln~ in serum answered to our

rocnnnCcgtc were established for each

OXfroldorlP and hydromorphone from for buprenorphine norbuprenorphine Bishy

concentrations upper than 20flglL 8 for

did not ex-20flgL and

than 20flglL for inter-lower

30 for concentrations 15 for concentrations

ases were crrIlllor than 19 for concentrations than 20l-lgL than 12 for concentrations than For RSDs did not exceed 20 concentrations lower than

tolerance Umit did not ov-11

(20 for concentrations lower concentrations upper than 20flgL) at 875 The lower limit of quantitation was 71lglL for

compound which was than obtained by Ge-MS technology

CONCLUSION

ln to gtI-lIIII narcotic subshyaddictia~n andor intoxication

most common

REFERENCES

1 Observatoire europeacuteen des drogues et des toxicomanies Rapshyport annuel Office des de lUnion Eungtpeacuteenne 2009

2 E MA LeBeau MA Sibum M An aushytomated SPELCMSMS method for the of cocaine and metabolites in whole B 2008 87415-20

3 Chegraveze M Deveaux M Martin c Lhermitte M G Simulshytaneous determination of six and in hair blood and urine LC-ESI-MSMS to the deshytermination of MDMA after low ecstasy intake Forensic Sei ln t 2007 170 100-104

4 MK Nielsen MKK Markussen LO Klinke HB Unnet K Determination of 19 of abuse and metabolites in whole blood liquid rhrmrtrar~nhmiddotI-tnrm

mass spectrometry Anal Bioanal Chem 2009 in press 5 M Bafile E D Curini R DAscenzo G Ro-

FS Multiclass of illicit in and oral Huids LC-MSMSAnal 8ioanal Chem 2009 393 709-718

6 Lacroix c Saussereau E Bodin G Goulleacute JP Quantification des et par rhrOrrrtr

phie liquide haute 1 spectromeacutetrie de masse en tandem de leacutechantillon ATA 2008

20(1) 25-38 7 Paterson S Lintzeris N Mitchell TB Cordera R Nestor L

J Validation of to detect iIIiumlcit heroiumln use nrpcrrilr pheacute1rrriacieutical heroin for the manageshy

ment of dependenceAddiction 2005 100 1832-1839 8 Kudo K Ishida T Hara K Kashimura 5 A Ikeda N 5ishy

multaneous determination of 13 related drugs in human whole blood an enhanced polymer columnand gas spectrometry J B 2007 855 115-120

9 Rook EJ Hillebrand MJ H et aL The qualntilatile of heroin methadone and their metabolites and the

simultaneousdetection of cocaiumlne and their metabolites in human liquid

with tandem mass spectr()mEtrv Chrom B 2005 824 213middot221

10 Hubert Ph J-J B et aL Harmonizashytion of for the validation of oUeacutemtitalive anecircllytical proceltlunes A SFSTP Part 1 j Pharm Biomed Ana 2004 36 579-586

11 Hubert Ph Nguyen-Huu J-J Boulanger B et aL Harmonizashytion of for the validation of owmtitalive rnllvtirr1

procedures A 5FSTP Part Il J pharm 8iomed Anal 2007 45 70-81

12 Hubert Ph NClIlVn-~-l11l1 J-J B et aL Harmonizashytion of strltlte~~ies for the validation of analytical procedlures A SFSTP - Part III J Pharm Biomed Anal 2007 45 82-96

13 Rozet E Ceccato A Hubert C et aL Analysis of recent pharshymaceutical documents on method vaUshy

20071158111-125

Acta Clinica 2010 65-5ulpeuroment

83 DRues OF ABUSE IN SERUM BY UHPLC-MSMS

Accuracv prom Accuruy Proflle

Mltlhadone M3G

Actuucy Proflfe

The dOits represent the relative t)adr-calculatE~dconcentraUonsam1 an ptlJttE~dlilgtlith respect to their talraetedCOnCel1ltral~ion [S-expeCtation tolerance timits and the dotted curves represent the acceptance

measurand The cgtvlrlvltt1

i 1 around represents an

the u nknown true rnrtit1in~ level of 95

concentrations Values each analyte are presented in Table 2

The total error the of the ot-hrt1

contain 875 acshyce[)talnce Umits were set at plusmn 20 for concentrations upper than and at plusmn 50 for concentrations

to accu rate timation of a is fi t1nlt~nt-1

validity of the method Total error is ness and is clearly a results accuracy The accuracy CgtVIUccgtc

of between the

or an accepted otmiddot nr

The accuracy tremes of the 875 In1onl1

exshywill

the tOTIC

range The intersections between

the lower the profile and

the of

tation (LQL) as weil as the upper limit UQL of(1

important to have a as be to measure two MRM by n1l1

19 MRM rngtnnHC

Acta Clinica 2010 65SUl)plE~ment

84 DRUGS OF ABUSE IN SERUM BY UHPLC-MSMS

time function MS method was dishyvided in five time functions and MRM dwell times were adius1ted to maximize cocitilitl

anltiteacuteltie determination urnhtrnln~ in serum answered to our

rocnnnCcgtc were established for each

OXfroldorlP and hydromorphone from for buprenorphine norbuprenorphine Bishy

concentrations upper than 20flglL 8 for

did not ex-20flgL and

than 20flglL for inter-lower

30 for concentrations 15 for concentrations

ases were crrIlllor than 19 for concentrations than 20l-lgL than 12 for concentrations than For RSDs did not exceed 20 concentrations lower than

tolerance Umit did not ov-11

(20 for concentrations lower concentrations upper than 20flgL) at 875 The lower limit of quantitation was 71lglL for

compound which was than obtained by Ge-MS technology

CONCLUSION

ln to gtI-lIIII narcotic subshyaddictia~n andor intoxication

most common

REFERENCES

1 Observatoire europeacuteen des drogues et des toxicomanies Rapshyport annuel Office des de lUnion Eungtpeacuteenne 2009

2 E MA LeBeau MA Sibum M An aushytomated SPELCMSMS method for the of cocaine and metabolites in whole B 2008 87415-20

3 Chegraveze M Deveaux M Martin c Lhermitte M G Simulshytaneous determination of six and in hair blood and urine LC-ESI-MSMS to the deshytermination of MDMA after low ecstasy intake Forensic Sei ln t 2007 170 100-104

4 MK Nielsen MKK Markussen LO Klinke HB Unnet K Determination of 19 of abuse and metabolites in whole blood liquid rhrmrtrar~nhmiddotI-tnrm

mass spectrometry Anal Bioanal Chem 2009 in press 5 M Bafile E D Curini R DAscenzo G Ro-

FS Multiclass of illicit in and oral Huids LC-MSMSAnal 8ioanal Chem 2009 393 709-718

6 Lacroix c Saussereau E Bodin G Goulleacute JP Quantification des et par rhrOrrrtr

phie liquide haute 1 spectromeacutetrie de masse en tandem de leacutechantillon ATA 2008

20(1) 25-38 7 Paterson S Lintzeris N Mitchell TB Cordera R Nestor L

J Validation of to detect iIIiumlcit heroiumln use nrpcrrilr pheacute1rrriacieutical heroin for the manageshy

ment of dependenceAddiction 2005 100 1832-1839 8 Kudo K Ishida T Hara K Kashimura 5 A Ikeda N 5ishy

multaneous determination of 13 related drugs in human whole blood an enhanced polymer columnand gas spectrometry J B 2007 855 115-120

9 Rook EJ Hillebrand MJ H et aL The qualntilatile of heroin methadone and their metabolites and the

simultaneousdetection of cocaiumlne and their metabolites in human liquid

with tandem mass spectr()mEtrv Chrom B 2005 824 213middot221

10 Hubert Ph J-J B et aL Harmonizashytion of for the validation of oUeacutemtitalive anecircllytical proceltlunes A SFSTP Part 1 j Pharm Biomed Ana 2004 36 579-586

11 Hubert Ph Nguyen-Huu J-J Boulanger B et aL Harmonizashytion of for the validation of owmtitalive rnllvtirr1

procedures A 5FSTP Part Il J pharm 8iomed Anal 2007 45 70-81

12 Hubert Ph NClIlVn-~-l11l1 J-J B et aL Harmonizashytion of strltlte~~ies for the validation of analytical procedlures A SFSTP - Part III J Pharm Biomed Anal 2007 45 82-96

13 Rozet E Ceccato A Hubert C et aL Analysis of recent pharshymaceutical documents on method vaUshy

20071158111-125

Acta Clinica 2010 65-5ulpeuroment

84 DRUGS OF ABUSE IN SERUM BY UHPLC-MSMS

time function MS method was dishyvided in five time functions and MRM dwell times were adius1ted to maximize cocitilitl

anltiteacuteltie determination urnhtrnln~ in serum answered to our

rocnnnCcgtc were established for each

OXfroldorlP and hydromorphone from for buprenorphine norbuprenorphine Bishy

concentrations upper than 20flglL 8 for

did not ex-20flgL and

than 20flglL for inter-lower

30 for concentrations 15 for concentrations

ases were crrIlllor than 19 for concentrations than 20l-lgL than 12 for concentrations than For RSDs did not exceed 20 concentrations lower than

tolerance Umit did not ov-11

(20 for concentrations lower concentrations upper than 20flgL) at 875 The lower limit of quantitation was 71lglL for

compound which was than obtained by Ge-MS technology

CONCLUSION

ln to gtI-lIIII narcotic subshyaddictia~n andor intoxication

most common

REFERENCES

1 Observatoire europeacuteen des drogues et des toxicomanies Rapshyport annuel Office des de lUnion Eungtpeacuteenne 2009

2 E MA LeBeau MA Sibum M An aushytomated SPELCMSMS method for the of cocaine and metabolites in whole B 2008 87415-20

3 Chegraveze M Deveaux M Martin c Lhermitte M G Simulshytaneous determination of six and in hair blood and urine LC-ESI-MSMS to the deshytermination of MDMA after low ecstasy intake Forensic Sei ln t 2007 170 100-104

4 MK Nielsen MKK Markussen LO Klinke HB Unnet K Determination of 19 of abuse and metabolites in whole blood liquid rhrmrtrar~nhmiddotI-tnrm

mass spectrometry Anal Bioanal Chem 2009 in press 5 M Bafile E D Curini R DAscenzo G Ro-

FS Multiclass of illicit in and oral Huids LC-MSMSAnal 8ioanal Chem 2009 393 709-718

6 Lacroix c Saussereau E Bodin G Goulleacute JP Quantification des et par rhrOrrrtr

phie liquide haute 1 spectromeacutetrie de masse en tandem de leacutechantillon ATA 2008

20(1) 25-38 7 Paterson S Lintzeris N Mitchell TB Cordera R Nestor L

J Validation of to detect iIIiumlcit heroiumln use nrpcrrilr pheacute1rrriacieutical heroin for the manageshy

ment of dependenceAddiction 2005 100 1832-1839 8 Kudo K Ishida T Hara K Kashimura 5 A Ikeda N 5ishy

multaneous determination of 13 related drugs in human whole blood an enhanced polymer columnand gas spectrometry J B 2007 855 115-120

9 Rook EJ Hillebrand MJ H et aL The qualntilatile of heroin methadone and their metabolites and the

simultaneousdetection of cocaiumlne and their metabolites in human liquid

with tandem mass spectr()mEtrv Chrom B 2005 824 213middot221

10 Hubert Ph J-J B et aL Harmonizashytion of for the validation of oUeacutemtitalive anecircllytical proceltlunes A SFSTP Part 1 j Pharm Biomed Ana 2004 36 579-586

11 Hubert Ph Nguyen-Huu J-J Boulanger B et aL Harmonizashytion of for the validation of owmtitalive rnllvtirr1

procedures A 5FSTP Part Il J pharm 8iomed Anal 2007 45 70-81

12 Hubert Ph NClIlVn-~-l11l1 J-J B et aL Harmonizashytion of strltlte~~ies for the validation of analytical procedlures A SFSTP - Part III J Pharm Biomed Anal 2007 45 82-96

13 Rozet E Ceccato A Hubert C et aL Analysis of recent pharshymaceutical documents on method vaUshy

20071158111-125

Acta Clinica 2010 65-5ulpeuroment