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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