Quantitative Analysis of Vecuronium by HPLCESI MS - PowerPoint PPT Presentation
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Quantitative Analysis of Vecuronium by HPLCESI MS
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Office of the Armed Forces Medical Examiner. Division of Forensic Toxicology ... CV 10% within-day 8% between-day. LOD 0.005 mg/L. AFIP, Forensic Toxicology ... – PowerPoint PPT presentation
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Title: Quantitative Analysis of Vecuronium by HPLCESI MS
1
Quantitative Analysis of Vecuronium by
HPLC/ESI()-MS
- Shawn P. Vorce
- Office of the Armed Forces Medical Examiner
- Division of Forensic Toxicology
- Armed Forces Institute of Pathology
- Washington, DC 20306
2
Overview
- Case background
- What is vecuronium
- Analytical method
- Instrumentation
- Results
- Conclusions
3
Case Background
- 20 year old white male
- Camp medic assigned to FOB in Iraq
- Found dead (kneeling over cot)
- Needle mark upper arm
- Nubain, morphine injection, diazepam, xylocaine
- Syringe (10 mL) with yellowish liquid 0.7 mL
- Vial of vecuronium bromide
- Past positive drug test
4
Specimens
- lt 1 mL bile
- lt 1 mL gastric
- 86 g lung
- USACIL
- Vial Vecuronium Br
- 10-mL Syringe yellow liquid
- lt 6 mL blood
- 2 mL vitreous
- 19 mL urine
- 30 g brain
- 60 g kidneys
- 114 g liver
- 67 g spleen
5
Initial Testing Result
- Screening
- Drugs of abuse, forensic volatiles, spots, and a
base screen (GC/MS) - Confirmations
- 23 mg/dL blood ethanol
- 0.45 mg/L doxylamine (blood) urine confirmed
- 1.8 mg/L pseudoephedrine (blood) urine confirmed
6
Vecuronium
- Norcuron
- Aminosteroidal non-depolarizing neuromuscular
blocking agent - Synthetic quaternary amine
7
Vecuronium
- Paralytic agent (muscle relaxant)
- Inhibits acetylcholine from binding to receptors
- Prevent coughing, gagging, biting
- Does not impact
- Consciousness
- Pain
- Anxiety
8
Vecuronium
- Intravenous administration
- Intermediate duration
- 2-3 minute onset
- 25-40 minute duration of paralysis
- Respiratory assistance compulsory
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Vecuronium
- Indications
- Eliminate spontaneous breathing and promote
mechanical ventilation - Pharmacological restraint
- Decreases oxygen consumption
10
Pharmacokinetics
- 60 80 protein bound
- T½ 4 75 minutes
- Metabolite (3-desacetyl-vecuronium)
- 50-70 as potent as parent
- T½ 2 5 hours
- Prolong paralysis from 6 hrs 7 days
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Toxicity
- LD50 rats, intravenous 0.20 mg/kg
- KCN LD50 rats, intravenous 2.5 mg/kg
- TDLO woman, oral 0.13 mg/kg
- Enhances respiratory depression effects of
narcotics analgesics , thiobarbs, inhalation
anesthetics and other CNS depressants
12
Analytical Challenges
- No screening (base screen, immunoassay)
- No confirmatory method (GC/MS, LC/MS)
- Stability (high pH, temp, light sensitive)
- Reference standard unstable
- No metabolic profile or standard
- Time period
- Limited sample
13
Structure
14
Structure
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Ion-pairing Extraction
- 1.0 ml extraction (blood, urine, tissue)
- 1.0 ml 0.8 M phosphate buffer pH 5.5
- 1.0 ml KI (saturated solution)
- Equilibrate 15 minutes
- 5 ml methylene chloride (DCM)
- Mix, centrifuge, transfer, dry _at_ 45oc under N2
16
SPE Extraction
- Weak cation exchange column
- (Carboxylic acid pka 4.8)
- MeOH, H2O, buffer (pH ? 7.2)
- Load Sample
- Buffer (pH ? 7.2)
- MeOH/buffer (pH ? 7.2)
- MeOH
- 1HCl in MeOH
17
HPLC/ESI-MS
- Agilent 1100 LC/MSD SL
- Mobile phase
- 0.1 formic acid/methanol/0.01 TEA
- LC column
- Gemini 5 mm C18 (150 x 3.0 mm)
- Hybrid particle
- Stable pH 1-12
18
LC Gradient
- 0.1 formic acid/methanol/0.01 TEA
- 85/15 o minute
- 80/20 1 minute
- 60/40 6 minute
- 40/60 8 minute
- 30/70 9 minute
- Hold 3 minutes
- Post time 7 minutes
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Chromatogram
20
Method Parameter
- Linearity 0.010 1.000 mg/L
- R2 lt0.9994
- CV lt 10 within-day
- lt 8 between-day
- LOD 0.005 mg/L
21
Tissue Distribution
- blood 0.070 mg/L
- kidney 0.224 mg/kg
- liver 0.045 mg/kg
- spleen 0.080 mg/kg
- urine lt 0.010 mg/L
- vitreous none detected
22
Fatal Reported Values
- Vecuronium
- 1.2 mg/L blood
- 0.6 mg/L urine
- 3-desacetyl-vecuronium
- 4.4 mg/L blood
- 0.7 mg/L urine
23
Conclusions
- Vecuronium levels were therapeutic
- No respiratory assistance available
- Time of death concentration
- Likely higher
- Instability of compound in solution and In Vitro
- Urine lt LOD (pH stability issues)
- Urine turns basic overtime
- Sample urine pH 8.0
24
Conclusions
- Method could be applied to all aminosteroidal
neuromuscular blocking agents - pancuronium, rocuronium, atracurium, etc
- Method of extracting quaternary amine compounds
- Method simple and robust
- Final report pending
25
Future Considerations
- Add 3-desacetyl-vecuronium
- standard from AKZO NOBEL
- Check stability of vecuronium in urine at
different pHs - Full tissue distribution of vecuronium and
3-desacetyl-vecuronium
26
Acknowledgment
- Joe Magluilo
- Karoline Shannon
- American Registry of Pathology (ARP)
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