Observations on Endogenous Levels of. in Urine Over Time

Transcription

1 Observations on Endogenous Levels of in Urine Over Time Negin Aghdaie, M.S. LAPD Scientific InvestigationDivision

2 Introduction CNS depressant Endogenous compound in Urine Naturally occurring neurotransmitter Byproduct of GABA metabolism Effects include: euphoria, confusion, dizziness, headache, vomiting, amnesia, unconsciousness, and even death GBL is present at low levels in foods Highest level in red wine (5µg/mL)

3 History Synthesized in 1960 Shortly after used as an anesthetic in Europe 1970 s present: clinically used as treatment for alcohol withdrawal and narcolepsy 1980 s: perceived growth hormone 1990 s: increased use as rave/party drug FDA bans over the counter sale 2000 s: DEA Schedule I substance Xyrem approved for Narcoleptics w/cataplexy DEA Schedule III

4 Structures of Related Compounds GHB GBL GABA 1,4-Butanediol

5 Metabolism rapid onset (15-30 minutes) peak urine concentrations observed within 4 hours of ingestion effects lasts 3-4 hours (dose dependant) detection: in urine ~ 12 hours after ingestion in blood ~ 8 hours after ingestion

6 GHB and Drug Facilitated Sexual Assaults Due to its CNSD activity: Detectives routinely ask for analysis in sexual assault cases Interpretation of results difficult because: GHB is an endogenous compound found in the human body including urine rapid elimination > 24hr little chance for detection delay in reporting sexual assaults

7 Objectives of Study Measure urine GHB of non-users, over one month period Observe fluctuations of urine GHB concentration throughout the day determine pattern, if any Compare inter-individual as well as intraindividual GHB variation

8 Experimental Factors Specimens were collected from five volunteers (non-ghb users) for ~30 consecutive days 4 females & 1 male date & time of sample collection noted stored in polypropylene tubes at ~4 C for 3-8 months before analysis Study was divided into two parts: 1. First 48 hours every urine void collected 2. Next 28 days only the morning first void was collected

9 Extraction Liquid / Liquid Conversion of GHB to GBL via acid hydrolysis + H2SO4 + H2O

10 Standards and Controls Internal Standard = GVL 25µg/mL Linearity Standards = GHB 2.5, 5, 10, 25, 50µg/mL ratio of responses GHB(as GBL) : GVL were plotted against calibrator conc. to generate calibration curve Negative Controls: DI water & pooled negative urine Positive Controls: GHB = DI H 2 O spiked at 18µg/mL GBL = DI H 2 O spiked at 18µg/mL

11 Results of First 48 Hours Urine GHB Concentrations Over 48 Hours GHB Concentration (ug/ml) F-1 F-2 F-3 F-4 M Hour * zero hour = midnight

12 Summary of Results (First 48 Hours) GHB concentrations from all voids during first 48 hours Mean S.D. Range Number (µg/ml) (+/-) (µg/ml) of Samples Female Female-2 < 2.5 < Female Female Male < Overall 3.75

13 First Void Results Female-1 (28 days) GHB as GBL (ug/ml) Day

14 First Void Results GHB as GBL (ug/ml) Female-2 (30 days) Day

15 First Void Results Female-3 (32 days) GHB as GBL (ug/ml) Day

16 First Void Results Female-4 (32 days) GHB as GBL (ug/ml) Day

17 First Void Results Male-1 (25 days) GHB as GBL (ug/ml) Day

18 Summary of Results (First Voids) GHB concentrations from first void over one month Mean S.D. Range (µg/ml) (+/-) (µg/ml) Female Female-2 < Female Female Male Overall 3.25 Number of Samples

19 Visual Comparison GHB as GBL (ug/ml) Female-1 (28 days) Day Male-1 (25 days) GHB as GBL (ug/ml) Female-2 (30 days) Day GHB as GBL (ug/ml) Female-3 (32 days) Day GHB as GBL (ug/ml) Day GHB as GBL (ug/ml) Female-4 (32 days) Day

20 Overall Summary Total of 147 samples from 5 individuals Maximum concentration - 9.8µg/mL Average of all samples - 3.4µg/mL Intra- and inter-individual variation was observed in both sections of the study Possible slight rise & fall trend on 48 hr. study

21 Discussion Intra-individual variation of urine GHB observed in this study reveals that a determining a set background GHB per individual is unfeasible Pattern of fluctuation Out of 147 samples, 10 contained 7µg/mL GHB

22 Future Studies Larger sample pool Collect all voids over a longer period of time Compare male vs. female levels Re-analyze samples after prolonged storage

23 Random Population Study Analysis of 96 urine samples Collected from random individuals assumed non-users Same liquid / liquid extraction method

24 Random Population Background GHB n = Number of Samples ug/ml GHB (Random Urine)

25 Female Female Background GHB n = Number of Samples ug/ml GHB (Random Urine)

26 Male Male Background GHB n = Number of Samples ug/ml GHB (Random Urine)

27 Summary of Results (random population) Maximum level detected 14µg/mL GHB Female population range (0-14µg/mL) Male population range (0-5µg/mL) 7 samples contained 6µg/mL GHB

28 History of GHB Cases at LAPD 194 Sexual Assault Cases ( ) 104 analyzed for GHB ( ) 9 cases above 20µg/mL Alcohol content analyzed in 175 Sexual Assault cases urine alcohol 23 cases 0.2 grams % alcohol

29 Distribution of GHB Concentration Number of Samples GHB Conc. ug/ml

30 Sexual Assaults Five Cases 1) Received both blood and urine from same person blood 190µg/mL urine 2300µg/mL 2 & 3 are different victims from the same case 2) urine 69µg/mL 3) urine 108µg/mL 4) urine 740µg/mL 5) urine 160µg/mL Note all levels are well above 10µg/mL

31 Alcohol Content of SAE Number of Samples grams % alcohol

32 Other Drugs Confirmed in Sexual Assault Cases Number of Cases Drug Category Cocaine Amphetamines Marijuana Benzodiazepines Opiates Barbiturates

33 GHB in DUI Cases Total of Nine Cases: Urine Blood High levels due to early collection In such cases, police officers often find driver passed out in car on the side of the road Note - all results in µg/ml

34 2001 Sexual Assault Case Study Information from report provided by SA nurse ~ 1hr after ingestion: victim lost consciousness & assault occurred ~ 3hr: woke up and tried to vomit ~ 3.5hr: left premises returned home, upon arrival collapses ~ 4hr: roommates drive victim to hospital ~ 8-9hr: urine for sexual assault exam is collected

35 Case Study continued One day later, follow-up report from detective: No use of drugs or alcohol before assault Needed assistance leaving premises Unknown male helped her

36 Results urine sample analyzed by LAPD & LACME - LAPD Screen: Opiate, Amp, THC July `01 August `01 March `02 April `02 LAPD GHB - 16µg/mL MDMA, MDA ethanol 0.09% BA GHB - 9µg/mL LACME GHB -12µg/mL MDMA, MDA GHB - 7µg/mL ethanol 0.11G% acetone 0.065G%

37 Second Sample Background Sample received January `02 LAPD: Non-detected (below LOD) LACME: 3.1µg/mL????????

38 Acknowledgements LAPD Toxicology Section Todd Swanton, Kristina Takeshita, Tanisha Paige-Smith, Tatiana Garcia, Henry Tuazon, Jose Gonzalez CSULA Criminalistics Department California Association of Criminalists endowment committee