Simultaneous Detection of GHB_ Ketamine and Flunitrazepam Using by dfhdhdhdhjr

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									Simultaneous Detection of
   GHB, Ketamine and
Flunitrazepam Using Hair
         Samples
      Samantha Olson
        CHEM 4101
     December 13,2010
             The Problem
I was at a bar one night where it was
suspected that a friend of mine was drugged
with a “date rape drug.” She made it home
safely without an assault, but she did not go to
the police until a few days later. I would like to
attempt to qualitatively and quantitatively
detect three date rape drugs in her system.
            The Hypothesis
Based on her behavior, I hypothesize that
GHB was placed in her drink, the dosage of
which is between endogenous and lethal
levels.
                  The Analytes
 Since I do not know what was put in the drink, three
 major date rape drugs will be analyzed.
                                        O
         OH                     N
                                                   Cl




                  O2N               N



              O
                                        F

    HO                                             NH
                                                        O

     GB
     H                  flunitrazepam           ketamine
                         (Rohypnol)
Effects of these drugs include: drowsiness, loss of
consciousness, impaired judgment, impaired motor
skills and the inability to remember events that
occurred while under the influence. This is why they
                                            6
       Analyte Complications
•   These drugs are quickly Metabolized.5
•   Endogenous levels of GHB exist.6
•   It’s usually a low, one time dosage.5
•   Drug facilitated sexual assault cases
    are often not reported right away. 5
    Hair was chosen for sample collection,
    because drugs linger in the hair much
    longer than blood and urine and
    endogenous levels of GHB can be
    detected by analyzing several
    segments of the hair.5
 Techniques Considered
  Method          Advantages              Disadvantages

                                          Need extensive
                                       separation technique,
                Fast Runs, fairly
   FTIR                               overlap in simultaneous
                  inexpensive
                                       measurements, poor
                                         for quantification
                                         Different pH’s are
             Good LODs : 50pg/mL
                                           preferred for the
             - 1.8µg/mL, faster run
MEKC-UVvis                             different analytes, UV
             times (15 mins), good
                                       detection is poor with
                   resolution
                                                 GHB
              Good LOD (pg-ng),
                                       Derivitization must be
             instruments currently
  GC-MS                                  done to increase
               used in labs, good
                                               volatility
                  separation,
                                          Drugs must be
              More sensitive than       derivatized ($), and
             Method Chosen:
              HPLC/MS/MS
• There is less sample preparation
• Quantification and Identification are achieved
  simultaneously.
• The reproducibility is good.
• The limit of detection is good.
• These instruments are already used in many labs,
  so it’s cost effective.


                               Qu ic kT i me ™ a nd a
                     T IFF (Un comp ress ed) de comp re ss or
                        are n eed ed to se e th is p i cture.
     Specific HPLC and MS/MS
             Instruments
Waters 2795 Alliance HT         Micromass Quattro Ultima
  HPLC1                         MS 7
• Fairly affordable model   •   Can use CID to get
                                parent/daughter ion
• Autosampler provides
                                pairs10
  more accurate, faster
  injections                •   Sensitive enough to
                                detect low concentrations
• Common brand used             of this problem’s analytes
  throughout labs
                            •   Good mass resolution
• An Atlantis dC18          •   Uses ESI
  column may be
  adequate for the drugs
                 9
     Proposed Sample Preparation
• 4-5 Weeks after being drugged, hair samples taken from the
  vertex posterior (back of the head)
• Cut hair strands into 2 cm segments and place in separate
  containers and decontaminated
• Cut into small pieces and incubate roughly 20 mg overnight in
  phosphate buffer (pH 7) in presence of diazepam-d5 (internal
  standard)
• Extract with methylene chloride/diethyl ether (90/10 v/v)
      Proposed LC/MS/MS Method
• Calibration standards must be prepared and run
• Internal standard is added and a 20 µL sample is injected and
  elution is run
• Gradient is started at 5:95 acetonitrile:formic acid and
  increased to 95:5 acetonitrile:formic acid after 15 minutes with
  eluent flow of 0.2mL/min
• Mass analysis is done on the positive ion mode
                                           Results



          Qu ic kT i me ™ a nd a
T IFF (Un com press ed ) de com press or
   are n eed ed to se e th i s pi cture.
 Anticipated Figures of Merit
           Flunitrazepa           GHB           Ketamine
                 m
              2 pg/mg   10     0.4 ng/mg   9    0.1ng/mg    3
 LOQ
             0.5 pg/mg   8     0.2 ng/mg   9   0.18 ng/mg       3
 LOD
Precisio    Intra-day CV of    CV of 5.2% at   Intra-day % CV
                 3.58 8        2 ng/mg and          of 4 8
   n                            7.4% at 10
                                 ng/mg 9
Accurac       Within batch       97% for         % Relative
             precision: 12.1   2ng/mg and      Accuracy of 7.2
   y       (n=6, 10pg/mg 10    94% for 10             8

                                ng/mg 9
                Conclusions
• Hair samples were chosen due to the longer
  retention of drugs and the ability to determine
  endogenous GHB
• HPLC/MS/MS can be used to separate, identify and
  quantify all three analytes.
• Instrumentation was chosen for its affordability,
  accuracy and its ability to solve the problem.
• Seeing as victims of DFSA don’t always come
  forward in the time allotted for traditional sample
  analysis (i.e.: urine), this technique would be useful
  to prove that date rape drugs were consumed.
                                   References
1. Alliance HPLC High Thoroughput System. Waters Corporation, Oct 2008. Web.
     <http://www.waters.com/webassets/cms/library/docs/720000781en.pdf>.
2. Cheng, Pai-Sheng, Chien-Yu Fu, Choung-Huei Lee, Chiareiy Liu, and Chun-Sheng Chien. "GCミMS
     Quantification of Ketamine, Norketamine, and Dehydronorketamine in Urine Specimens and Comparative
     Study Using ELISA as the Preliminary Test Methodology." Journal of Chromatography B 852.1-2 (2007):
     443-49. Print.
3. Harun, Norlinda, and Robert A. Anderson. "Analysis of Ketamine and Norketamine in Hair Samples Using
     Molecularly Imprinted Solid-phase Extraction (MISPE) and Liquid Chromatography-Tandem Mass
     Spectrometry (LC-MS/MS)." Anal Bioanal Chem 396 (2010): 2249-459. Web.
4. Hsin-Yu, Bai. "Characterization and Evaluation of Two-Dimensional Microfluidic Chip-HPLC Coupled to
     Tandem Mass Spectrometry for the Quantitative Analysis of 7-aminoflunitrazepam in Human Urine." Analyst
     135 (2010): 2737-742. Web.
5. Kintz, Pascal, Marion Villain, and Vincent Cirimele. "Chemical Abuse in the Elderly: Evidence From Hair
     Analysis." Ther Drug Monit 30.2 (2008): 207-11.
6. Maxwell, James Carlisle. "Party Drugs: Properties, Prevalence, Patterns and Problems." Substance Use &
     Misuse 40 (2005): 1203-240. Print
7. ."Micromass Quattro Ultima Pt Mass Spectrometer Operator's Guide." Waers, 2003. Web.
     <http://www.waters.com/webassets/cms/support/docs/quattro_ultima_pt_guide_reva.pdf>.
8. Negrusz, Adam. "Detection of "Date-Rape" Drugs in Hair and Urine, Final Report." (2003). Print.
9.Stout, Phillip A., Kelsie D. Simons, and Sarah Kerrigan. "Quantitative Analysis of Gamma-Hydroxybutyrate at
     Endogenous Concentrations in Hair Using Liquid Chromatography Tandem Mass Spectrometry." Journal of
     Forensic Sciences 55.2: 531-37. Web.
10.Villain et al, Marion. "Screening Method for Benzodiazepines and Hypnotics in Hair at Pg/mg Level By Liquid
     Chromatography-Mass Spectrometry/Mass Spectrometry." Journal of Chromatography B 825 (2005): 72-78.

								
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