1. Simultaneous Detection of GHB, Ketamine and Flunitrazepam Using Hair Samples Samantha Olson CHEM 4101 December 13,2010 Samantha Olson CHEM 4101 December 13,2010

2. 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.

3. The Analytes Since I do not know what was put in the drink, three major date rape drugs will be analyzed. 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 are used in drug facilitated sexual assault.

4. Analyte Complications These drugs are quickly Metabolized. Endogenous levels of GHB exist. It’s usually a low, one time dosage. Drug facilitated sexual assault cases are often not reported right away. These drugs are quickly Metabolized. Endogenous levels of GHB exist. It’s usually a low, one time dosage. Drug facilitated sexual assault cases are often not reported right away. 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 MethodAdvantagesDisadvantages FTIR Fast Runs, fairly inexpensive Need extensive separation technique, overlap in simultaneous measurements, poor for quantification MEKC Good LODs : 50pg/mL - 1.8µg/mL, faster run times (15 mins), good resolution Different pH’s are preferred for the different analytes, UV detection is poor with GHB GC-MS Good LOD (pg-ng), instruments currently used in labs, good separation Derivitization must be done to increase volatility Fluorescence More sensitive than UV-vis, fast detection Drugs must be derivatized ($), and different derivatives are ideal for different drugs

6. 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. 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.

7. Specific HPLC and MS/MS Instruments Agilent 1220 Infinity LC System Very affordable Autosampler provides more accurate injections Common brand used throughout labs An Agilent ZORBAX Rx-C18 Column is adequate for this analysis Agilent 1220 Infinity LC System Very affordable Autosampler provides more accurate injections Common brand used throughout labs An Agilent ZORBAX Rx-C18 Column is adequate for this analysis Agilent 6500 Series Accurate-Mass Q- TOF LC/MS 2-4ppm mass accuracy (eliminate false positives) Femtomole sensitivity In-spectrum dynamic range of up to 5 orders of magnitude Agilent 6500 Series Accurate-Mass Q- TOF LC/MS 2-4ppm mass accuracy (eliminate false positives) Femtomole sensitivity In-spectrum dynamic range of up to 5 orders of magnitude

8. 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 Decontaminate Cut into small pieces and incubate roughly 20 mg overnight in phosphate buffer (pH 7) in presence of diazepam-d 5 (internal standard) Extract with methylene chloride/diethyl ether (90/10 v/v) Inject sample 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 Decontaminate Cut into small pieces and incubate roughly 20 mg overnight in phosphate buffer (pH 7) in presence of diazepam-d 5 (internal standard) Extract with methylene chloride/diethyl ether (90/10 v/v) Inject sample

9. Results These MS spectrums can help to chose which parent ions may be chosen GHBketamine flunitrazepam

10. The LOD using similar techniques and separate elution gave samples is 0.5 ng/mg, 0.1 ng/mg and 0.2 ng/mg for GHB, ketamine and flunitrazepam respectfully. I assume these values will be similar to the simultaneous determination of all drug concentrations.

11. 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. 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.

12. References Agilent 1220 Infinity LC: Infinitely More Affordable. Agilent Technologies, 1 July 2010. Web.. Agilent 6200 Series TOF and 6500 Series Q-TOF LC/MS System. Agilent Technologies, 1 July 2010. Web.. 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. 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. Kintz, Pascal, Marion Villain, and Vincent Cirimele. "Chemical Abuse in the Elderly: Evidence From Hair Analysis." Ther Drug Monit 30.2 (2008): 207-11. Maxwell, James Carlisle. "Party Drugs: Properties, Prevalence, Patterns and Problems." Substance Use & Misuse 40 (2005): 1203-240. Print