Endogenous Levels of

in Urine Over Time

Negin Aghdaie, M.S.LAPD – Scientific InvestigationDivision

Observations on

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)

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

Structures of Related Compounds

• GHB • GBL

• GABA • 1,4-Butanediol

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 ingestionin blood ~ 8 hours after ingestion

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

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 intra-individual GHB variation

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 daysonly the morning first void was collected

Extraction

• Liquid / Liquid • Conversion of GHB to GBL via acid hydrolysis

+ H2SO4 + H2O

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 H2O spiked at 18µg/mL– GBL = DI H2O spiked at 18µg/mL

Results of First 48 Hours

Urine GHB Concentrations Over 48 Hours

0

2

4

6

8

10

12

0 10 20 30 40 50

Hour* zero ho ur = midnight

GH

B C

once

ntra

tion

(ug/

mL)

F-1F-2F-3F-4M-1

Summary of Results (First 48 Hours)

3.75Overall

10< 2.5 - 4.851.552.29Male-1

124.14 - 9.891.655.68Female-4

111.86 - 7.832.014.42Female-3

6< 2.5—< 2.5Female-2

111.12 - 9.182.484.15Female-1

of Samples(µg/mL)(+/-)(µg/mL)

NumberRangeS.D. Mean

GHB concentrations from all voids during first 48 hours

First Void Results

Female-1 (28 days)

0123456789

10

0 5 10 15 20 25 30 35

Day

GH

B a

s GB

L (u

g/m

L)

First Void Results

Female-2 (30 days)

0123456789

10

0 5 10 15 20 25 30 35

Day

GH

B a

s GB

L (u

g/m

L)

First Void Results

Female-3 (32 days)

0123456789

10

0 5 10 15 20 25 30 35

Day

GH

B a

s GB

L (u

g/m

L)

First Void Results

Female-4 (32 days)

0123456789

10

0 5 10 15 20 25 30 35

Day

GH

B a

s GB

L (u

g/m

L)

First Void Results

Male-1 (25 days)

0123456789

10

0 5 10 15 20 25 30 35

Day

GH

B a

s GB

L (u

g/m

L)

Summary of Results (First Voids)

3.25Overall

251.24 - 4.880.872.73Male-1

323.09 - 8.631.455.02Female-4

320 - 4.611.072.66Female-3

300 - 4.101.09< 2.5Female-2

281.65 - 7.431.513.94Female-1

of Samples(µg/mL)(+/-)(µg/mL)

NumberRangeS.D. Mean

GHB concentrations from first void over one month

Visual Comparison

Male-1 (25 days)

0123456789

10

0 5 10 15 20 25 30 35

Day

GH

B a

s GB

L (u

g/m

L)

Female-4 (32 days)

0123456789

10

0 5 10 15 20 25 30 35

Day

GH

B a

s GB

L (u

g/m

L)

Female-3 (32 days)

0123456789

10

0 5 10 15 20 25 30 35

Day

GH

B a

s GB

L (u

g/m

L)

Female-2 (30 days)

0123456789

10

0 5 10 15 20 25 30 35

Day

GH

B a

s GB

L (u

g/m

L)

Female-1 (28 days)

0123456789

10

0 5 10 15 20 25 30 35

Day

GH

B a

s GB

L (u

g/m

L)

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

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

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

Random Population Study

• Analysis of 96 urine samples• Collected from random individuals

– assumed non-users• Same liquid / liquid extraction method

Random PopulationBackground GHB

n = 96

33

8

17

14

8

6

32 2 2

10

5

10

15

20

25

30

35

0 2 4 6 8 10 12 14 16

ug/mL GHB (Random Urine)

Num

ber

of S

ampl

es

FemaleFemale Background GHB

n = 38

19

45

1

4

21 1 1

0

2

4

6

8

10

12

14

16

18

20

0 2 4 6 8 10 12 14 16

ug/mL GHB (Random Urine)

Num

ber

of S

ampl

es

MaleMale Background GHB

n = 16

2

4

7

1 1 1

0

1

2

3

4

5

6

7

8

0 1 2 3 4 5 6

ug/mL GHB (Random Urine)

Num

ber

of S

ampl

es

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

History of GHB Cases at LAPD

• 194 Sexual Assault Cases (2000 – 2004) • 104 analyzed for GHB (2000 – 2004)

– 9 cases above 20µg/mL• Alcohol content analyzed in

175 Sexual Assault cases – urine alcohol– 23 cases ≥ 0.2 grams % alcohol

Distribution of GHB Concentration

53

4 4

3 2 0 0 0 1 0 0 0 1 0 0 0 0 1 0 0 1 0 0 0 1 1 1 0

0102030405060

0 30 60 90 120 150 180 210 2300

GHB Conc. ug/mL

Num

ber

of S

ampl

es

Sexual Assaults

Five Cases 1) Received both blood and urine from same person

blood – 190µg/mLurine – 2300µg/mL

2 & 3 are different victims from the same case2) urine – 69µg/mL3) urine – 108µg/mL4) urine – 740µg/mL 5) urine – 160µg/mL

Note all levels are well above 10µg/mL

Alcohol Content of SAE

0

10

20

30

40

50

60

0 0.03 0.06 0.09 0.12 0.15 0.18 0.21 0.24 0.27 0.3 0.33

grams % alcohol

Num

ber

of S

ampl

es

Other Drugs Confirmed inSexual Assault Cases

Barbiturates-3Opiates-6Benzodiazepines-14Marijuana-24Amphetamines-27Cocaine-49Drug CategoryNumber of Cases

GHB in DUI CasesTotal of Nine Cases:

Urine Blood141 110724 119880 176

120016321680

• High levels due to early collection• In such cases, police officers often find driver

passed out in car on the side of the roadNote - all results in µg/mL

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

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

Results• urine sample analyzed by LAPD & LACME

- LAPD Screen: Opiate, Amp, THC

GHB - 9µg/mLApril `02acetone 0.065G%ethanol 0.11G%GHB - 7µg/mLMarch `02

ethanol 0.09% BAMDMA, MDAAugust `01

MDMA, MDAGHB -12µg/mLGHB - 16µg/mLJuly `01LACMELAPD

Second Sample

• ‘Background Sample” received January `02– LAPD: Non-detected (below LOD)– LACME: 3.1µg/mL

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Acknowledgements

• LAPD Toxicology SectionTodd Swanton, Kristina Takeshita, Tanisha Paige-Smith, Tatiana Garcia, Henry Tuazon, Jose Gonzalez

• CSULA Criminalistics Department• California Association of Criminalists

endowment committee