Download LC/MS for Clinical Toxicology

High resolution MS for Clinical
Toxicology
Definitive and slow (48-72 h)
vs.
Presumptive and fast (1-8 h)?
Society for Forensic Toxicology Workshop
September 27, 2011
Alan H.B. Wu
San Francisco General Hospital
University of California, San Francisco
TOF MS for unknown toxicology analysis
• Measurement of mass to 4 decimal places.
• Distinguishes compounds with the same
nominal mass but different exact mass.
• Soft ionization = molecular ion
• Development of protocols for all intended drugs
by LC/MS/MS will be labor intensive.
• TOF-MS may enables presumptive detection of
compounds without prior experience by the
testing lab
Clinical vs. forensic tox needs
• Clinical toxicology needs are less stringent than forensic
toxicology needs. Exact assignment of unknown
intoxicant may be unnecessary.
• TATs for reporting of results is more important than
exact drug determination.
• Reporting of a list of candidate compounds is
acceptable, i.e., clinical history and toxicology knowledge
is used by clinicians to cull down list.
• Presence of isomers of compounds without exact
identification of unknown is acceptable, i.e., clinical
management approach is unchanged.
• Serum will be examined as a specimen of choice for
potential impairment assessment
MS-TOF
Nanospray
Tip
MCP
DETECTOR
PUSHER
HEXAPOLE
HEXAPOLE
COLLISION
CELL
QUADRUPOLE
ION
SOURCE
REFLECTRON
SKIMMER
HEXAPOLE
Time measured
from ionization
to detector
Measurement of
mass to 4 decimal
places
TOF
Thermo Fisher Orbitrap MS
Mass accuracy
Accuracy is measured as mass error (in ppm)
Mass error (ppm) = I MWmeas- MWtheo I · 1 x 106
MWtheo
= I 250.0005 – 250.0000 I · 1 x 106
250.0000
= 2 ppm
2 ppm accuracy translates to:
MW
amu error
100
0.0002
200
0.0004
300
0.0006
400
0.0008
Example for TOF-MS
H2N
Morphine, C17H19NO3
MW = 285.3377
7-aminoclonazepam
C15H12ClNO3
MW = 285.7283
Pentazocine:
C19H27NO
MW=285.2093
For M/Z = 285.1365
At 10ppm accuracy
At 5ppm accuracy
At 3ppm accuracy
5 possible formula
C17H18NO3, C15H26N4O2, C20H16N2
C17H18NO3
Example #1: unknown acidosis?
CASE 1
27yo F, Hx of chronic abdominal pain and vomiting, chronic
use of APAP. pH= 7.12, AG= 22, APAP= 3g/mL. Lactate,
ethanol, methanol, ethylene glycol= neg
CASE 2
38yo F, Hx of depression and chronic pain, possible O/D of
Vicodin
pH= 6.9, AG= 33, Osm gap= 24. APAP= 371mcg/mL, ALT=
8045U/L, AST=8026 U/L, lactate, ethanol, methanol,
ethylene glycol = neg
Cause of acidosis?
Pyroglutamic acid accumulation
•Chronic ingestion and
acute intoxication of
APAP depletes the body
of glutathione  loss of
negative feedback to γglutamylcysteine
synthetase
•Accumulation of γglutamylcysteine and
overproduction of 5oxoproline (pyroglutamic
acid)
LC-TOF for oxoproline
Name
Score (MFG)
5-oxoproline
83.91
5-oxoproline
97.98
5-oxoproline
97.03
RT
0.674
0.688
0.68
Mass
Formula (MFG)
129.0433
C5 H7 N O3
129.0425
C5 H7 N O3
129.0432
C5 H7 N O3
Patient 1: Serum 5-OP= 35.8 mmol/L
Urine 5-OP= 0.65 mmol/mmol Cr
Patient 2: Urine 5-OP= 0.1 mmol/mmol Cr
Height
431995
1778980
3215050
Example #2: herbal supplements?
Herbal medicine use in the US
•CDC Survey: about 18.9% of adult US population
•Herbals are regulated as dietary supplements by the FDA
•Manufacturers of products falling into this category are not
required to prove the safety or efficacy, but the FDA may
withdraw a product if harmful
•Structural analogues as dopants present in herbal medicines
produced in Asia has concerned US distributors
AIM: Evaluate the presence of synthetic chemical drugs and
their analogues in herbal medications for distribution
Analysis of herbal supplements
Herbal X
• Claims to increase libido
• Most ingredients have been studies for their role in supporting healthy
sexual performance and increased libido
• Listed ingredients- Acorus rhizome, Cornus, Alpinia, Lycium, Cherokee
Rose fruits, Black walnut seed, Cimicifuga rhizome, Cordyceps mycelium,
Poria sclerotium, and Glycyrhhiza, Panax Ginseng, and Rehmania roots
MS-TOF Qualitative Analysis
• Methanol extract of Herbal X contains very high formula matches for
•acetildenafil (structural analogue of Viagra)
•hydroxyhomosildenafil (derivative of Cialis)
• Both have been reported as dopants in traditional Chinese medicine
Analysis of herbal supplements
Name
Score (MFG)
Acetildenafil
84.05
Hydroxyhomosildenafil
91.08
RT
Mass Diff (MFG, ppm) Formula (MFG) Ions
4.147 466.2700
-1.7
C25 H34 N6 O3
4
4.602 488.2208
-0.53 C23 H32 N6 O4 S
7
Sildenafil: R=H
Acetildenafil
Homosildenafil: R=CH3
Hydroxyhomosildenafil: R= CH2OH
Height
1548390
1118384
Example #3: drug-induced seizure
ED patient w/ multiple seizures and was comatose.
Family claims pt took herbal supplement. At 2:30 pm,
tox fellow called SFGH for seizure panel and herbal
capsule to r/o possible intoxication from supplement.
Samples arrived at 3PM. By 4PM the results were
obtained:
Drug-related seizures
Pesola et al. J Emerg Med 2002;22:235-9
• SFGH sees 2-3 seizing patients in the ED every day
– Many are unexplained
• 6% of new onset seizures are estimated to be drugrelated
• No way to quickly determine if a seizure is drug-related
• A seizure panel could improve patient care and cut costs
2003 CA Poison Control Study
Thundivil et al. J Med Toxicol 2007;3:15-19.
Analyzed 386 drug-related seizures
• 3.6% status epilepticus
• 66% involved suicide attempts
• 15% resulted from drug abuse
• 7 deaths
 Identified the most common drugs and drug
classes that caused the seizures
Breakdown of seizures by drug type
Other 12.4%
Buproprion 23.0%
Tramadol 7.5%
Isoniazid 5.9%
TCAs 7.7%
Diphenhydramine
8.4%
Venlafaxine 5.9%
MDMA 3.4%
Amphetamines 6.9%
Cocaine 4.9%
Antidepressantsother 9.3%
Antipsychotics 4.7%
Development of TOF seizure panel
Compound Name
Bupropion
Citalopram
Cocaine
Diphenhydramine
Isoniazid
Lamotrigine
Methamphetamine
MDMA
Quetiapine
Tramadol
Venlafaxine
Patient
SP1- ED
SP2- DM
SP3- LP
SP4- RA
SP5-JH
SP6- LV
Mass
239.1092
324.1638
303.1471
255.1623
137.0589
255.0079
149.1205
193.1103
383.1690
263.1885
277.2042
RT
3.512
3.780
3.162
3.842
0.755
3.194
2.631
2.643
4.003
3.172
3.689
Medication
Quetiapine
Citalopram
Citalopram
Isoniazid
Bupropion
Isoniazid
Diphenhydramine
Lamotrigine
Accuracy (ppm)
2.75
4.00
4.75
2.00
4.50
3.00
1.75
4.25
5.00
5.00
4.00
%CV
0.50
2.50
2.20
0.75
1.30
1.50
0.20
2.25
2.20
2.10
1.20
LC/MS-TOF Detection
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Example #3: results
By 4PM the following results were obtained:
Formula and retention time match: diphenhydramine, serum level ~
550ng/mL (therapeutic: 20-50)
Herbal supplement extract: No matches for seizure-causing drugs and
7000 drugs/pesticides on herbal database
Retrospect: patient’s presentation was consistent w/ diphenhydramine
intoxication. Pt managed accordingly and recovered. What about the
herbals:
Example #4: Green Pill
15 y-old male w/ vfib arrest after smoking THC
and intake of green pill. Pt was defibed, had
inferior lead ST  MI. He was intubated and
sedated but became hypertensive while waking
up. He continued to have labile intermittent blood
pressures, requiring pressors and
antihypertensives. Can THC cause this?
Serum (d0)
Urine (d2)
Pill
MDMA
87 ng/mL
4
2.3 mg/125
MDA
19
12
ND
MDEA
61
5
440 ng/125
mg
Subsequent genetic testing demonstrated a novel RYR2 mutation in human
cardiac ryanodine receptor gene suggesting catecholaminergic polymorphic
ventricular tachycardia.
DEA complication
Dear Registrant: This letter is being sent to every DEA
registered analytical lab. DEA has received questions
regarding testing for potency and contaminants.
A DEA registered analytical lab is only authorized to received
controlled substances for analysis from another DEA
registrant or an entity that is specifically exepmted from
registration pursuant to 21CFG 1301.23 or 4.
Joseph T. Rannazzisi, Deputy Chief of Operations, DEA.
Example #5: mistaken identity
Patient presented after taking 0.5 mL clomifene (SERM, black
market) sublingually. Pt. c/o chest discomfort, palpitations,
tremor, anxiety, 5 min after taking drug. EKG: narrow complex
tachycardia, HR rate 150. Pt was on testosterone for body
building. He self-prescribed clomifene to counteract the
hirsutism and gynecomastia). The clomifene taken by patient
was tested. Attending was suspicious that the bottle actually
contains clenbuterol (decongestant, bronchodilator).
Analysis: 28 mg/mL clenbuterol, no clomifene found.
Recommended dosage: <120 g/d. Patient took 14.2 mg.
Case of mistaken identity (part 2)
Case 1
57yo non-diabetic male with hx of benzodiazepine abuse found
unresponsive at rehab facility. Fingerstick glucose = 41mg/dL. Upon ED
arrival, pt was administered with naloxone and continuous glucose infusion.
Pt was unresponsive to naloxone. Admission glucose= 33 mg/dL
Octeotride and glucagon was given, ED suspicious of sulfonylurea
intoxication. On HD2, pt’s glucose rebounded to normal range w/o
supplemental glucose. He was extubated and mental status became clear.
Case 2
48yo F found unresponsive at home; GCS=5, glucose was undetectable as
per paramedics report. Administration of glucose, pt’s GCS improved to 7.
Upon ED arrival, patient became alert upon administration of second
ampule of glucose and admitted she ingested 2 pills of “street valium”
Patient’s recovered on HD 2; glucose level rebounded to normal.
Case of mistaken identity (part 2)
Serum analysis revealed formula
match for GLYBURIDE. Confirmed by
retention time match to reference
standard.
Patient
Serum level
(ng/mL)
#1 T0
1198
T0 + 4.5 hours
693
T0 + 8 hours
590
T0 + 12.5 hours
undetectable
#2 T0
647
Therapeutic dose: 30-350 ng/mL
Toxic: > 600ng/mL
Glyburide
Diazepam
Internet pharmacy: example 6
•
•
•
•
22 y-o Russina femal
Chief complaint: lerthargy
S/P ingestion of “Adderall” from India
PE: lethargy, hypertension (143/83),
tachycardia (HR 124)
• Negative urine for amphetamine (and all
other DAU screens)
Adde Cure 30 ingredients?
Amphetamine toxicity?
Hypertension √
Tachycardia √
Agitation
Seizures
Hyperthermia
(+) Utox
Zolpidem 9.7 mg
Manufacturer’s address in India
Example 7: Torsades
Case
54 yo M with complex medical history had a suicide attempt. In the ER, his mental
status precipitously diminished leading to intubation with need for sedation/
paralysis. Patient had AMI; while being resuscitated, patient developed Torsades de
Pointes which resolved after CPR, IV MG, and IV NaHCO3. EKG showed a
prolonged QT >600msec
List of Medications
Aspirin
Fluoxetine
Metoprolol
Atorvastatin
Levothyroxine
Trazodone
Buspirone
Metformin
Which medication did he overdose on that may have caused torsades?
Example 7: Drugs causing Torsades
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Zomig (for migranes, especially)
Mycins & Mycetins
Floxacins
Antifungals & conazoles
Tricyclics and other antipsychotics
Gastrointestinal drugs
Anti-Parkinson drugs
Anti-anginal, anti-cancer
Narcotics
CNS stimulants
Anti-convulsants
Cholinesterase inhibitors
Sedatives
Diuretics
Bronchodilators
Catecholamines
Appetite suppressants
Phosphodiesterase inhibitors
Torsades de Pointes
Example 7: Torsades
Drugs with formula and RT matches in patient’s serum:
Aspirin, Fluoxetine, Metformin, Metoprolol, Trazodone
Drugs not detected in patient’s serum:
Atorvastatin, Buspirone, Levothyroxine
Sample
Trazodone
(mcg/mL)
Metformin
(ng/mL)
02/08 1215H
4.06
159.78
20.11
11.0
10.9
02/08 2120H
4.09
101.69
34.07
23.7
7.1
02/10 0420H
2.74
16.97
39.86
31.4
15.5
02/11 0505H
2.47
9.41
40.97
32.5
7.7
02/16 0445H
0.0276
4.76
22.50
13.8
8.9
Therapeutic level
0.5-2.5
1000-4000
150-500
100-600
4.0
5000
>1300
650
Toxic level
Fluoxetine Norfluoxetine Metoprolol
(ng/mL)
(ng/mL)
(ng/mL)
Example 8: Valproic acid toxicity
1000
ug/mL VPA
CASE
50 yo male O/D with
valproic acid. Pt was
comatose,
hypernatremic,
hypocalcemic, initial AG=
18
800
600
400
200
0
0
Monitor metabolites,
pathways, and therapy
relevant in VPA toxicity
10
20
30
40
50
Time after Admission (hr)
60
VPA intoxication
3.510 0 6
06
Peak Area (AU)
2.510 0 6
2.010 0 6
1.510 0 6
1000000
1.310 0 6
1000000
750000
500000
500000
250000
0
0
0
20
40
60
80
100
120
Adipic acid
Suberic acid
1.510 0 6
VP-carnitine
2en/4en-VPA
0
140
20
40
60
80
1.410 7
Acetylcarnitine
1.210 7
1.010 7
8.010 6
6.010 6
4.010 6
2.010 6
0
0
100
120
Time after Admission (hr)
Time after Admission (hr)
Peak Area (AU)
Peak Area (AU)
3.010
1.810 0 6
20
40
60
80
100
120
140
Time after Admission (hr)
Therapeutic approach: carnitine supplementation
140
Example 9: SPICING it up with
Synthetic Cannabis
Spice… A little History
1990’s Synthetic cannabinoids are synthesized in the
lab of John W Huffman (JWH) at Clemson
University
JWH-018 (1998)
2002
2004
2006
2008
Spice first appeared in Asia as herbal incense
Spice became available in Europe
First incidences of Spice intoxication called in to
Poison Control Centers in Europe
Japan and Germany started analyzing herbal
incense samples bought from headshops/ internet
Feb, 2009 First published articles on SPICE
being adulterated with synthetic
cannabinoids
Apr, 2009 German lab quantified adulterants
in SPICE; more JWH compds found
April, 2010 JWH-018 metabolites identified
UCSF cases
Two 17yo were brought in by ambulance after smoking Spike
Maxx
Patient 1- F, vomiting, slightly confused, T=36.3, HR=110,
RR=20, K=3.1 mmol/L, no urine drug screen
Patient 2- M, vomiting, very altered/confused, dilated pupils,
cool skin, excess salivation, HR= 120, K= 3.0 mmol/L, Lactate=
4.4 mmol/L, NEGATIVE urine drug screen
Both patients were treated with antiemetics, IV fluid and were
discharged 3, and 6hrs post-presentation
Synthetic cannabinoids
Classical
cannabinoids
HU- 120
Cyclohexylphenols
Naphthoylindoles
CP 47,497
JWH- 018
Naphthoylpyrroles
Phenylacetylindoles
THC
JWH- 250
JWH-030
Why are they more potent than THC?
Structure-activity correlation and behavioral studies in rats
have established requirements for pharmacological activity
Preliminary lab results
Spike Maxx
Preliminary results suggest presence of
JWH – 007
JWH – 073
JWH – 398
Blood sample results
Both patients have 11-OH-THC
Girl has a formula match for JWH-018
JWH- 018
JWH- 007
Legal Status of SPICE in the US
Illegal
Kansas (02/2010)
North Dakota (02/25/2010)
Mississippi (04/2010)
Kentucky (04/13/2010)
Alabama (04/25/2010)
Tennessee (07/01/2010)
Louisiana ((07/18/2010)
Hawaii (08/01/2010)
Georgia (08/15/2010)
Missouri (08/28/2010)
Temporary Ban/ Legislation Proposed
Arkansas
Florida
Illinois
Iowa
Maryland
Michigan
New York
Oklahoma
Utah
Municipal Restriction
Indiana
Minnesota
Texas
Wisconsin
Conclusions
Spice and other herbal incense products contain synthetic
cannabinoids
For lack of toxicological testing, these group of compounds
are potentially toxic
The lack of regulation on the availability of these herbal
products pose danger especially to the younger population
There is a need to develop a clinical assay for these group
of compounds
LC-MS/TOF is a promising platform to use as it can respond
to changes in the type of synthetic cannabinoids used to
adulterate these herbal products
Example 10:
•11 Patients distributed in 4 hospitals
•3 referred to SFGH
•AM- 23yo male, critically ill; (admitted 5/30 0021H, died
5/30 2030H
•JMT-19yo female condition progressively became worse
•LS- 24yo female doing better
•Both critically ill patients presented with hyperthermia,
seizure, acute onset of internal bleeding and kidney failure
Labs- Patients
JMT (serum sample obtained 5/30 0430H, 3 hrs ai)
•Compds with RT, Formula matches
•MDMA- 792 ng/mL, MDEA- 12ng/mL
•MDA, Midazolam, Lidocaine, Acetaminophen
•Compds with Formula matches
•Trimethoxyamphetamine
LS
•MDMA (05/30 0130H)- 410ng/mL
•MDMA (05/30 1951H)- 193ng/mL
ECSTASY
Recreational use: 100-250ng/mL
Reported lethal dose: 500-10,000ng/mL
Compounds screened
Serum/Urine Samples screened for compds with Retention Times: 298
Amphetamines (23)
Stimulants (7)
Opioids (29)
Sedatives/Hypnotics (7)
Benzodiazepines (34)
Anesthetics (9)
Barbiturates (14)
Muscle Relaxants (6)
Psychotropic Alkaloids (9)
Analgesics (7)
Antihistamines (12)
Antidepressants (24), Anticonvulsants (32), Antipsychotics (16)
Cardiovascular (44), Respiratory (6)
Antidiabetics (6), Anorectics (8)
Others (4)
Labs- Patient 1 (AM)
Sample
Date/Time Drawn
MDMA (ng/mL)
S1
05/30 0021H
534
S2
05/30 0333H
487
S3
05/30 0600H
363
S4
05/30 1010H
298
S5
05/30 1445H
94
Sample
Other Drugs Detected (Formula, RT Matches)
S1
Meperidine, Phencyclidine
Midazolam, Phenytoin/Oxcarbamazepine
Theophylline/Theobromine, Caffeine
S2
Meperidine
Midazolam, Phenytoin/Oxcarbamazepine
Theophylline/Theobromine, Caffeine
Lidocaine, Acetaminophen
S3
Midazolam, Phenytoin/Oxcarbamazepine
Theophylline/Theobromine, Caffeine
Lidocaine, Acetaminophen
S4
Midazolam, Phenytoin/Oxcarbamazepine
Theophylline/Theobromine, Caffeine
Acetaminophen
S5
Midazolam, Phenytoin/Oxcarbamazepine
Acetaminophen
Labs- Patient 2 (JMT)
Sample
Date/Time Drawn
CK (U/L)
MDMA
(ng/mL)
S1
05/30 0135H
694
1300
S2
05/30 0433H
6473
792
S7
05/31 0005H
18253
119
S8
06/01 0400H
15921
72
S9
06/03 0324H
13152
17
S10
06/05 0400H
26934
nd
S11
06/07 0430H
35051
nd
Example #7: Rave or adulterant?
Labs- Capsule analysis
Fifteen capsules/pills were submitted by the DCPD for analysis
CASE 34A- XZ- White Capsule (135mg)
•Compds with RT, Formula matches
•MDMA ~110 mg, MDEA~ 40ug
•Ketamine
•Compds with Formula matches
•DMA, TMA
CASE 34B- VT- Light Yellow Capsule (275mg)
•Compds with RT, Formula matches
•MDMA ~270mg, MDEA~25ug
•Compds with Formula matches
•TMA, Methylenedioxy-2-methoxyamphetamine
Dosage of Regular Ecstasy in the Black Market: 80-120mg/ pill
Ecstasy is regularly sold as a pill NOT AS A CAPSULE; apparently,
the two samples above are suspiciously just being given away
instead of being sold.
Example #11
 12 yo F at CHO with an unknown poisoning. She was found
unresponsive, down at home and had waxing/waning mental status
with intermittent agitation and myoclonic jerking.
 K+ of 2.9, anion gap 19
 No medications
 Only family medications – diabetic
Thermo Exactive Orbitrap
Patient sample
Total Ion Chromatogram
Carbamazepine
(237.1022)
Extracted Ion Chromatogram
(237.1024 m/z)
Carbamazepine standard (in drug free serum)
Carbamazepine
Extracted Ion Chromatogram
(237.1024 m/z)
 Accurate mass and retention time match
 Medical team advised to order serum carbamazepine
level
 Carbamazepine level – Toxic -18.5 ug/mL
(reference 5-12 ug/mL)
 Unexpected finding: unclear where patient obtained
carbamazepine
High resolution mass spectrometry
applications
• Investigate toxicology cases on a stat basis
• Examine medications and herbals for stated ingredients,
dopants, and clinically significant contaminants
• Investigate atypical pharmacokinetic pathways, e.g.,
alternate pathways due to overdose, co-morbidities, or
genetic variances
Current model for clinical toxicology: ED
Symptoms suggestive of drug
toxicity or overdose.
“Drug screen” order placed.
Immunoassay screening (1 h TAT)
cocaine, opiates, THC, barbs, benzos
PCP, amphet, methadone, etc.
GC/MS confirmation analysis, if available (1-7 d TAT)
What about toxicity to other compounds, e.g., therapeutic drugs?
Current model for clinical toxicology: ICU
Unexplained comatose, renal, liver,
pulmonary, or muscle disease
Comprehensive drug screen order placed
Sample received from ICU, no clinical information provided
Sent to reference lab for “blinded” comprehensive drug screen,
TAT 5-7 d
Results received too late for real-time therapeutic
Implementation. Results used for documentation purposes only
PCC-centric operation for regional
toxicology lab
ER, hosp. #1
ER, hosp. #2
Rehab clinic
PCC
Regional tox lab
ICU, hosp. #1
ICU, hosp. #2
Clin tox testing strategy?
Definitive and slow (24-48 h)
vs.
Presumptive and fast (1 h)?
UCSF/SFGH Tox Team
Analytical: SFGH
Alan Wu, Ph.D.
Kara Lynch, Ph.D.
Deborah French, Ph.D.
Roy Gerona, Ph.D.
Matt Petrie, Ph.D.
Sarah Shugarts, Ph.D.
Tab Toochinda, MD
Shannon Kastner, BS
Clinical: CA PCC
Kent Olson, MD
Craig Smollin, MD
Patil Armenia, MD
Thanjira Jiranantakan, MD
Derek Leung, MD