Quantitative Analysis of Vecuronium by HPLCESI(+)-MS by fsd65350

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									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



AFIP, Forensic Toxicology
                              Overview
 Case background
 What is vecuronium
 Analytical method
 Instrumentation
 Results
 Conclusions


  AFIP, Forensic Toxicology
               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
  AFIP, Forensic Toxicology
                           Specimens
   < 6 mL blood                 < 1 mL bile
   2 mL vitreous                < 1 mL gastric
   19 mL urine                  86 g lung
   30 g brain
   60 g kidneys                USACIL
   114 g liver                  Vial “Vecuronium Br”
   67 g spleen                  10-mL Syringe – yellow
                                  liquid
    AFIP, Forensic Toxicology
         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


  AFIP, Forensic Toxicology
                      Vecuronium
 Norcuron®
 Aminosteroidal non-depolarizing
 neuromuscular blocking agent
 Synthetic quaternary amine



  AFIP, Forensic Toxicology
                      Vecuronium
 Paralytic agent (muscle relaxant)
 Inhibits acetylcholine from binding to
  receptors
 Prevent coughing, gagging, biting
 Does not impact
      Consciousness

      Pain

      Anxiety

  AFIP, Forensic Toxicology
                      Vecuronium
 Intravenous administration
 Intermediate duration
   2-3 minute onset
   25-40 minute duration of paralysis

 Respiratory assistance compulsory


  AFIP, Forensic Toxicology
                      Vecuronium
 Indications:
     Eliminate spontaneous breathing and
      promote mechanical ventilation
     Pharmacological restraint
     Decreases oxygen consumption



  AFIP, Forensic Toxicology
             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

  AFIP, Forensic Toxicology
                 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
   AFIP, Forensic Toxicology
           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


     AFIP, Forensic Toxicology
                                 Structure
                                 O


                             O



                                     N+
N                                         Androstane-based                                  O



O                                                                                18    O

                                                                           12         17
                                                             19       11        13
     O
          vecuronium                             N
                                                 N                              14
                                                                                       16
                                                                                      15
                                                                                                N+
                                                                                                 +


            C34H57N2O4+                                  1            9
                                                     2           10        8
           Mol. Wt.: 557.8                           3       5             7
                                                         4            6
                                                HO
                                                HO


                                                  3-desacetyl vecuronium
                                                          C32 55 2O3+
                                                          C32H55N2O3+
    AFIP, Forensic Toxicology                            Mol. Wt.: 515.8
                                Structure
                                O                                       O



                            O                                       O



                                    N+                                      N+
N                                        N+




O                                        O



     O
          vecuronium                          O
                                                  pancuronium
           C34H57N2O4+                            C35H60N2O42+
          Mol. Wt.: 557.8                         Mol. Wt.: 572.9
                                                   m/z 286.4



    AFIP, Forensic Toxicology
       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 @ 45oc under N2


  AFIP, Forensic Toxicology
                     SPE Extraction
 Weak cation exchange column
    (Carboxylic acid pka 4.8)


   MeOH, H2O, buffer (pH  7.2)        O

                                   Si
   Load Sample                         O-   N+   O

   Buffer (pH  7.2)                                 O

   MeOH/buffer (pH  7.2)         Si
                                        O



   MeOH                                O-

   1%HCl in MeOH

     AFIP, Forensic Toxicology
                   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


  AFIP, Forensic Toxicology
                       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

  AFIP, Forensic Toxicology
                                      Chromatogram




                                                      4.323
        MSD1 API-ES, Pos, SIM, Frag: Var, "SIM"

45000
                                     pancuronium
40000                                                                                        m/z     voltage




                                                              5.382
                                                                                       vec   557.3    120
35000
                                                                                             279.3    250
30000                                                                 vecuronium             398.3    250
25000                                                                                  pan   286.4    120
                                                                                             340.4    250
20000


15000


10000


5000


   0

                                2                 4                    6           8           10              min



         AFIP, Forensic Toxicology
              Method Parameter
   Linearity                   0.010 – 1.000 mg/L
                                R2 = <0.9994

   %CV                         < 10% within-day
                                < 8% between-day

   LOD                         0.005 mg/L

    AFIP, Forensic Toxicology
              Tissue Distribution
   blood                       0.070 mg/L
   kidney                      0.224 mg/kg
   liver                       0.045 mg/kg
   spleen                      0.080 mg/kg
   urine                       < 0.010 mg/L
   vitreous                    none detected


    AFIP, Forensic Toxicology
         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




    AFIP, Forensic Toxicology
                         Conclusions
   Vecuronium levels were therapeutic
      No respiratory assistance available
   Time of death concentration
      Likely higher
      Instability of compound in solution and In Vitro
   Urine < LOD (pH stability issues)
      Urine turns basic overtime
      Sample urine pH 8.0


    AFIP, Forensic Toxicology
                         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

    AFIP, Forensic Toxicology
        Future Considerations
   Add 3-desacetyl-vecuronium
       standard from AKZO NOBEL
   Check stability of vecuronium in urine at
    different pH’s
   Full tissue distribution of vecuronium and 3-
    desacetyl-vecuronium


    AFIP, Forensic Toxicology
             Acknowledgment

Joe Magluilo

Karoline Shannon

American Registry of Pathology (ARP)


AFIP, Forensic Toxicology

								
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