Transcript A Practical method for the Solid Phase Extraction and GC

A Practical Method for the
Solid Phase Extraction and
GC/MS Analysis of 7 Opiates in
Urine
Paul Crowley
Ventura Co. Sheriff’s Forensic Sciences Laboratory
Thanks to:
Debra Mittelbrun
Co-developer
Sarah Christiansen, Student Intern
References
Broussard LA, et al. “Simultaneous identification and
quantitation of codeine, morphine, hydrocodone, and
hydromorphone in urine as trimethyl and oxime
derivatives by gas chromatography-mass
spectrometry”. Clin Chem 43:6 1997 pp.1029-1032
Metherall R. “GC-MS Confirmation of Codeine,
Morphine, 6-Acetylmorphine, Hydrocodone,
Hydromorphone, Oxycodone, and Oxymorphone in
Urine”. J Anal Tox. 23 May/June 1999 pp.177-186
Why are we concerned with the
confirmation Hydrocodone/
Hydromorphone and
Oxycodone/Oxymorphone?
Prolific and widely abused. Illegal to possess w/o a
prescription.
23152 V.C. (DUI)
Often detected in addition to codeine, morphine, and
monoacetylmorphine in 11550 H.S. (under the
influence/controlled substance) cases.
Statistics
Screens
Total Screens for 01/0512/05: 4133
Opiates Positives: 580
Oxycodone Positives: 27
*started screening for oxy 06/05.
Confirmations
%
Codeine: 322
55.5
Morphine: 434
74.8
MAM: 273
47.0
Hydrocodone: 130 22.0
Hydromorph: 63
10.8
Prior Analysis Scheme
Screen for opiates by immunoassay.
Confirmation for free codeine, morphine, and
monoacetylmorphine by GC/MS.
Re-confirm with a second GC/MS method
when hydrocodone, and hydromorphone are
detected as interferents in the first
confirmation.
Challenges
2 confirmations if hydrocodone and hydromorphone
are suspected.
Poor cross-reactivity for oxycodone with opiates
immunoassay.
No SIMS method for new oxycodone immunoassay
(neutral/basic liquid-liquid extraction and GC/MS scan
mode).
Less than optimal chromatography/instrument
response for morphine with old derivatizing scheme
(1% Hydroxylamine in pyridine and MtBSTFA).
Goals
Incorporate all 7 opiates into a single SIMS method.
Improve chromatography/instrument response for
morphine.
LOD low enough for detection of free opiates. (no
hydrolysis)
Keep run time reasonable.
(< 20 min./inj.)
Instrumentation
Agilent 6890/5973N GC/MS
SIM mode/Chemstation
J & W Scientific DB-1 capillary column
(12 m x 0.20 mm x 0.33 um)
Equipment/Materials
Cerex System 48 Pressure Processor
(Other extractors will work, but must be compatible
with 6cc SPE tubes.)
SPEware Trace-B Polymer SPE columns
(hydrophilic polymer/cation exchanger)
 Good recovery for all analytes (including morphine).
 Consistent flow rates/no channeling.
Sample Preparation
To 2 mL urine add 50 uL of 10 ug/mL
dueterated internal standard.
(Cod, mor, mam, and oxc – d6)
(Hyc, hym, and oxm – d3)
Buffer to pH~5 with 2 mL Na Acetate
buffer.
Extraction
Load extractor with SPE columns and transfer
samples directly to the columns.
*No column pretreatment required.
Allow samples to soak on column for 5
minutes. (critical step)
Extract at 1 mL/minute.
Wash
1 mL H2O
1 mL 0.1M HCl
1 mL MeOH
1 mL ethyl acetate (Dry for 2 min.)
*All wash steps were done at 1 mL/min.
Elute
2 mL 3% Ammonium Hydroxide/EtAc
• 3% more consistent recovery than 2% soln.
• Morphine most susceptible to poor recovery.
*Avoid*
Old NH4OH (> 30 days).
Mix well.
Derivatization
Dry eluant under N2 at 37oC.
(No need to transfer before drying/reconstituting)
Reconstitute with 50 uL of a 1%
Hydroxylamine in Pyridine soln and incubate
at 45oC for 30 minutes.
Add 50 uL of MSTFA w/1% TMCS and
incubate at 65oC for additional 20 min.
Why 2-step process ?
Hydroxylamine in pyridine soln converts ketoopiates to oximes prior to MSTFA
derivatization.
• Eliminates codeine and morphine
interferents.
• Increases silyl derivative efficiency.
• Reduces isomeric derivatiztion products.
Derivatization Procedures that did
not work.
Aqueous methoxyamine soln added before
sample extraction.

Incomplete conversion of keto-opiates to oximes.
(Produced isomers)
BSTFA not as efficient as MSTFA.
Over derivatizing.
Heating at 75oC or higher also gave
rise to isomeric products.
Instrument Conditions
DB-1 (12 m)
Constant flow
(0.5 mL/min)
1.0 uL injection volume/splitless
Oven conditions: 100oC for 1 min., ramp to
270oC at 20oC/min., hold 4 min., ramp
40oC/min. to 300oC, hold 0.75 min.
(Total run time 15.0 min.)
Data Acquisition
Elution order
Codeine
Morphine
Hydrocodone
MAM
Hydromorphone
Oxycodone
Oxymorphone
Target, Qualifier Ions, and R.T.s
NIDA Protocol
Analyte: 1 quant ion, 2 qualifier ions
ISTD: 1 quant, 1 qualifier
Analyte/ISTD
Codeine
Codeine-d6
Morphine
Morphine-d6
6-MAM
6-MAM-d6
Hydrocodone
Hydorcodone-d3
Hydromorphone
Hydromorph-d3
Oxycodone
Oxycodone-d6
Oxymorphone
Oxymorphone-d3
Quant. Q1 Q2
371
377
429
435
399
405
297
300
355
358
474
480
532
535
343 356
349
414 401
420
400 340
406
386 371
389
444 429
447
385 459
391
517 533
520
RT(min)
8.2
8.5
8.8
8.7
8.9
9.2
9.4
*(12 m column)
Acquisition Parameters
SIM Analysis Mode/Chemstation
5 Groups (Hyc, MAM, Hym in same
group)
*Adjust dwell time to 15 or 20 to improve
chromatography.
Curve and Acceptance Criteria
3 point curve (40 ng/mL, 100 ng/mL and 500
ng/mL)
Quantitated by linear regression analysis,
forced through zero.
Peak well resolved from baseline and
Gaussian in shape.
I.R. +/- 20% and R.T. 1%.
Study Criteria
Precision and Accuracy
LOD/Sensitivity
Linearity
Carryover
Interference
Correlation
Precision and Accuracy
Prepared samples at 3 levels. (40 ng/mL, 1270
ng/mL, and 2500 ng/mL)
*MAM at two levels 6 and 12 ng/mL.
Analyzed samples in triplicate, 2 times/day for 10
days. (NCCLS/EP-10A)
C.V.’s for all analytes < 10%.
Bias at all levels was < 20%.
*Oxymorphone out at conc. > 1000 ng/ml.
P & A Data
Analyte n
Codeine 54
Level
Low
Mid
High
AVE
43.4
1379
2826
STDev. C.V. (%) Bias(%)
3.54
8.2
-8.5
32.5
2.3
-8.5
45.5
1.6
13.0
Morphine 28
Low
Mid
High
43.0
1477
3017
2.53
33.5
91.5
5.8
2.2
3.0
7.5
16.3
20.6
Hydroc
54
Low
Mid
High
40.5
1217
2307
2.56
31.1
35.3
6.3
2.5
1.5
1.3
-4.2
-7.7
MAM
54
Low
Mid
High
N/A
5.54
10.5
N/A
0.18
0.25
N/A
3.2
2.4
N/A
-11.4
-16.0
P & A Cont’d
Analyte
n
Hydromorphone 54
Level
Low
Mid
High
AVE
41.6
1480
2413
STDev.
1.6
28.2
103
C.V.(%)
3.9
1.91
3.83
Bias(%)
3.9
16.5
-3.5
Oxycodone
54
Low
Mid
High
40.8
1359
2794
3.9
31.9
6.5
9.5
2.3
1.6
2.0
7.0
11.8
Oxymorphone
54
Low
Mid
High
48.0
1200
1922
3.59
20.0
80.9
7.49
1.66
4.21
20.0
-5.5
-23.1*
L.O.D.’s
Sets of 5 replicates at each level (40,
30, 20, 10, and 5 ng/mL).
cod: 30
mor: 20
mam: 6
hyc: 20
hym: 20
oxc: 20
oxm: 20
• Goal < 40 ng/mL – 6 ng/mL for MAM.
Linearity
With the exception of Oxymorphone all
analytes demonstrated linearity out to 2500
ng/mL. ( < 20% target conc.)
Oxymorphone within 20% of target conc. up
to 1000 ng/mL. Bias was > than 20% and
was directly related to concentration.
Carryover
Did experience carryover at concentrations >
2500 ng/mL.
Most significant for codeine, followed by
oxymorphone and morphine.
(Might be a problem for total opiates)
Interference Study
Two Groups of Compounds:
1.) Possible interferents
2.) Often found in with opiates.
Group 1
Methadone Cis-tramadol
Naloxone
Meperidine
Natrexone
Papverine
Nalorphine
Buprenorph.
Norcodeine Dextrometh.
Normorphine Thebaine
Fentanyl
Levorphanol
Tramadol
Propox.
Norpropox.
Group 2
Amphetamine
Oxaz.
Methamp
Loraz.
MDMA
Fluraz.
MDA
Alpraz.
Cocaine
Temaz.
B.E.
Diaz.
Nordiaz
*No interference was
observed from compounds
from either group.
Correlation Study
40 specimens
Ante mortem and post mortem
All results correlated. (no false positives or
negatives)
Hydromorphone demonstrated expected loss
in concentration due to no hydrolysis.
No way to correlate oxycodone and
oxymorphone.
Final Analysis
Suitable P & A, LOD, Linearity, and Carryover
characteristics for the qualitative GC/MS confirmation
of codeine, morphine, MAM, hydrocodone,
hydromorphone, oxycodone, and oxymorphone urine.
Good for ante mortem and post mortem urine.
LOD’s are compatible for analysis without hydrolysis.
Extraction provided good analyte recovery.
Derivatization method yielded suitable
chromatography. (especially morphine)
Thank You for your time.
Any questions?
References
1.) Broussard LA, Presley LC, Pittman T, Clouette R, and Wimbish GH.
Simultaneous identification and quantitation of codeine, morphine, hydrocodone,
and hydromorphone in urine as trimethylsilyl and oxime derivatives by gas
chromatography-mass spectrometry. Clinical Chem 43:6 1997 pp.1029-1032.
2.) Meatherall R. GC-MS Confirmation of Codeine, Morphine, 6-Acetylmorphine,
Hydrocodone, Hydromorphone, Oxycodone, and Oxymorphone in Urine. J Anal
Tox Vol. 23 May/June 1999 pp.177-186.
3.) Meatherall R. GC-MS Confirmation of Codeine, Morphine, 6-Acetylmorphine,
Hydrocodone, Hydromorphone, Oxycodone, and Oxymorphone in Blood. J Anal
Tox Vol. 29 July/August 2005 pp.301-308.
4.) Smith ML, Hughes RO, Levine B, Dickerson S, Darwin WD, and Cone EJ.
Forensic Drug Testing for Opiates. VI. Urine Testing for Hydromorphone,
Hydrocodone, Oxymorphone, and Oxycodone with Commercial Opiate
Immunoassays and Gas Chromatography-Mass Spectrometry. J Anal Tox Vol. 19
January/February 1995 pp.18-26.
Reference’s Cont’d
Moore KA, Addison J, Levine B, and Smialek JE. Applicability of Opiate Cutoffs to
Opiate Intoxication Cases. “Letter to the Editor” J Anal Tox Vol. 25 October 2001 pp.657658.
5.)
Trihn A, Marlatt M, and Bell DS. Controlling SPE Selectivity through pH and Organic
Modifier Manipulation. “TheReporter” Sigma-Aldrich publication pp.8-9.
6.)
7.)
Derivatization Reagents. “Analytix” Sigma-Aldrich publication. March 2002 pp.1-7
8.)
Buddha DP, Shimomura ET, and Smith ML. A Practical Approach to Determine Cutoff
Concentrations for Opiate Testing with Simultaneous Detection of Codeine, Morphine, and
6-Acetylmorphine in Urine. Clinical Chem 45 1999 pp.510-519.
Moore KA, Addison J, Levine B, and Smialek JE. Applicability of Opiate Cutoffs to
Opiate Intoxication Cases. “Letter to the Editor” J Anal Tox Vol. 25 October 2001 pp.657658.
9.)
10.) National Committee for Clinical Laboratory Standards. Preliminary Evaluation of
Quantitative Clinical Laboratory Methods; Approved Guideline. EP10-A Vol. 18 No. 6 1998.