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Fast Screening and Confirmation of
Gamma-Hydroxybutyrate (GHB) in Urine
Maximize your analytical options with this versatile GHB extraction method. No derivatization
means faster sample preparation. Extracts are amenable to both liquid injection GC/FID and
headspace GC/MS methods.
By Amanda Rigdon, Pharmaceutical Innovations Chemist and Kristi Sellers, Clinical/Forensic Innovations Chemist
• 16 •
2008 vol. 3
Gamma-hydroxybutyrate (GHB) and its precursor, gamma-butyrolactone (GBL), are controlled substances associated with drug-
facilitated sexual assault. Criminal cases often hinge on lab results, which can include screening urine samples and then quantifying
GHB using GC/MS. In its native state, GHB is extremely difficult to chromatograph and must be analyzed as a trimethylsilyl deriv-
ative or converted to GBL. The headspace (HS) procedure described here (adapted from an FBI Chemistry Unit method) eliminates
time-consuming derivatization.
1
This procedure reduces sample preparation time and minimizes both column contamination from
derivatization reagents and contamination from sample matrix caused by liquid injections.
Eliminate Derivatization and Reduce
System Contamination
Samples were spiked in urine and extracted accord-
ing the procedure in Table I, using alpha-methyl-
ene-gamma-butyrolactone (AMGB) as an internal
standard. GHB is converted to GBL with sulfuric
acid, eliminating the need for derivatization
(Figure 1). Note the unconverted sample shows
comparable levels of GBL and AMGB, whereas
GBL levels in the converted sample are significant-
ly higher, due to the conversion of GHB to GBL.
Reliably Screen Samples Using
Existing Blood Alcohol Testing Set-Up
Headspace injections (using the total vaporization
technique) of the final urine extracts were screened
by GC/FID using an Rtx®-BAC1 column in a blood
alcohol headspace GC system. This system is com-
Figure 1
GHB can be converted to GBL for analysis, saving time by
eliminating derivatization.
1. Label two screw top test tubes per specimen.
One for total GHB, the other for GBL only.
2. Add 1mL of sample (urine) to each tube.
3. Add 50µL of AMGB to each tube.
4. Add 150µL concentrated sulfuric acid only to
tubes used for analysis of total GHB.
5. Vortex all tubes and allow them to sit 5 minutes.
6. Add 5mL methylene chloride to each tube.
Shake 10 minutes to extract.
7. Centrifuge samples at 3,000 rpm for 5 minutes.
8. Transfer bottom (methylene chloride) layer to a
clean test tube for drying.
9. Concentrate samples to ~100µL at 30°C under
nitrogen.
10. For headspace analysis, inject 15µL of sample into
a capped headspace vial. Or, for liquid injection,
transfer extract to a limited volume insert.
Table I
Extraction procedure for GHB and GBL.
1. GBL
2. AMGB (IS)
Column:
Rtx
®
-BAC1, 30m, 0.32mm ID,
1.8µm (cat.# 18003)
Sample:
50µg/mL GHB, GBL, and
AMGB (IS) in urine
A: unconverted
B: converted with sulfuric acid
Inj.:
1mL headspace injection, split (10:1),
1mm split inlet liner (cat.# 20972)
Inj. temp.:
200°C
Carrier gas:
helium, constant flow
Flow rate:
1.0mL/min.
GC_CF01039B
GC_CF01039
B. Acid Converted
Change in peak area
due to conversion of
GHB to GBL
A. Unconverted
Similar peak areas
for GBL and IS in
unconverted sample
Headspace conditions:
Equilibration temp.: 100°C
Equilibration time: 10 min.
Injection volume: 1mL
Oven temp.:
50°C (hold 3 min.) to 150°C @
20°C/min. (hold 7 min.)
Det.:
FID @ 240°C
Hydrogen:
40mL/min.
Air:
400mL/min.
Makeup:
40mL/min.
Sample: urine spiked with 50µg/mL each GHB, GBL, and AMGB (IS), extracted according to procedure in
Table I, and analyzed using headspace (total vaporization technique).
Clinical/Forensic/Toxicology
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