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CLINICAL/FORENSICS ARTICLE

Fast Screening and Confirmation for Gamma-Hydroxybutyrate (GHB)

Using Restek Columns in Headspace GC or GC/MS Systems

By Kristi Sellers, Clinical/Forensic Innovations Chemist
  • Use an Rtx®-BAC1 column or Rtx®-BAC2 column for GHB screening.
  • Confirm and quantify positive GHB screens by using an Rxi™-5ms column.
  • Fast, reliable screening; accurate confirmation and quantification.

In the last ten years, gamma-hydroxybutyrate (GHB) and its related products 1,4-butanediol and gamma-butyrolactone (GBL) have been identified as abused substances in cases of driving under the influence and drug facilitated sexual assault. Currently, GHB is regulated as a federally controlled Schedule I drug. The rise in use of GHB and GHB-type products as recreational drugs is primarily due to their euphoric and sedative properties. 1,4-butanediol and GBL are quickly metabolized to GHB after ingestion and are analyzed as such. Because GHB is endogenous in humans, and has a half-life of one hour or less after injection, it is very important to collect biofluids (typically blood or urine) quickly for toxicological investigation. Analytical methods for GHB usually employ gas chromatography and mass spectrometry for quantification and confirmation. The methodology described here establishes a headspace GC-FID screening procedure followed by confirmation and quantification by headspace GC/MS, and was developed by the FBI Chemistry Unit.(1) We have adapted Rtx®-BAC1 and Rtx®-BAC2 columns—with court-tested and proven performance in blood alcohol analyses—and new, highly inert Rxi™-5MS columns to the methods.

A typical headspace GC-FID blood alcohol system, using an Rtx®-BAC1 column or an Rtx®-BAC2 column, can be adapted for GHB screening. For the analysis, GHB is converted to gamma-butyrolactone (GBL) to improve chromatography, and alpha-methylene-gamma-butyrolactone (AMGB) is used as the internal standard. Figure 1 illustrates the conversion reaction of GHB to GBL. Figure 2 shows that either Restek column is suitable for GHB screening, providing Gaussian peak shapes, baseline resolution, and an analysis time of less than 5 minutes.

Figure 1  Conversion of GHB to GBL.

A sample yielding positive screening results requires confirmation and quantification by GC/MS. The confirmation and quantification analysis incorporates the same headspace and GC conditions, including conversion of GHB to GBL, but GBL-d6 is the required internal standard. To illustrate GBL and GBL-d6 separation and peak shape on an Rxi™-5ms column we analyzed 1µL of a standard, using GC/MS (Figure 3). This typical liquid injection shows the two compounds are partially resolved on the Rxi™-5ms column, and positively identified using full scan. Then, extracted ion data (EI) were obtained (Figure 4). After positive identification, GHB is quantified by comparing the areas of the deuterated and undeuterated GBL extracted ions.

Because this methodology for analyzing GHB in biofluids employs sample introduction through a headspace technique, the need for injector and column maintenance is dramatically reduced. The use of an existing headspace GC system for blood alcohol analysis eliminates the need for additional equipment and allows rapid and reliable screening, using the same Rtx®-BAC1 or Rtx®-BAC2 column. For positive results, an Rxi™-5ms column in a GC/MS system provides accurate confirmation and quantification of GHB.


Figure 2   Symmetric peak for GHB, and baseline resolution from an internal standard in less than 5 minutes, using an Rtx®-BAC1 or Rtx®-BAC2 Column.

  1. gamma-hydroxybutyrate (GHB) / gamma-butyrolactone (GBL)
  2. alpha-methylene-gamma-butyrolactone (AMGBL)

Rtx®-BAC1 column


Rtx®-BAC2 column


Column:

Rtx®-BAC1 30m, 0.32mm ID, 1.8µm (cat.# 18003) and
Rtx®-BAC2 30m, 0.32mm ID, 1.2µm (cat.# 18002),
connected via universal “Y” Press-Tight® connector (cat.# 20405)

Sample:

GHB, GBL, α-methylene-γ-butyrolactone (AMGBL), 10µg/mL each in water

Inj.:

1.0mL headspace, split (split ratio 1:10), 1mm split inlet liner (cat.# 20972)

Inj. temp.:

200°C

Carrier gas:

helium, constant pressure

Linear velocity:

44cm/sec. @ 50°C

Oven temp.:

50°C (3 min.) to 150°°C @ 20°C/min. (hold 7 min.)

Det:

FID @ 240°C

Headspace autosampler:

Teledyne/Tekmar HT3

Sample/platen temp.:

100°C

Sample equilibration:

15 min.

Mixing time:

5 min.

Vial pressure:

10psig

Vial pressurization time:

2 min.

Loop fill time:

2 min.

Transfer line temp.:

120°C


Figure 3   An Rxi™-5ms column provides the resolution needed for reliable confirmation of GHB.

  1. GBL-d6 (m/z 92)
  2. GBL (m/z 86)

Column:

Rxi™-5ms 30m, 0.25mm ID, 0.25µm (cat.# 13423)

Sample:

50µg/mL gamma-butyrolactone (GBL) and
25µg/mL gamma-butyrolactone-d6 (GBL-d6) in methanol

Inj.:

1µL split (1:10), 4mm Siltek® treated single gooseneck inlet liner (cat.# 20798)

Inj. temp.:

250°C

Carrier gas:

helium, constant flow

Flow rate:

1mL/min.

Oven temp.:

40°C (3 min.) to 300°C @ 25°C/min. (hold 5 min.)

Det:

MS

Transfer line temp.:

280°C

Scan range:

35-200 amu

Ionization:

EI

Mode:

scan

Figure 4  Overlay of extracted ion chromatograms for GBL and GBL-d6.

  1. GBL-d6 (m/z 92)
  2. GBL (m/z 86)

Column:

Rxi™-5ms 30m, 0.25mm ID, 0.25µm (cat.# 13423)

Sample:

50µg/mL gamma-butyrolactone (GBL) and
25µg/mL gamma-butyrolactone-d6 (GBL-d6) in methanol

Inj.:

1µL split (1:10), 4mm Siltek® treated single gooseneck inlet liner (cat.# 20798)

Inj. temp.:

250°C

Carrier gas:

helium, constant flow

Flow rate:

1mL/min.

Oven temp.:

40°C (3 min.) to 300°C @ 25°C/min. (hold 5 min.)

Det:

MS

Transfer line temp.:

280°C

Scan range:

35-200 amu

Ionization:

EI

Mode:

scan

References

  1. LeBeau, M.A., M.A. Montgomery, M.L Miller, and S.G. Burmeister, J. Anal. Toxicol. 24 (6): 421-428 (Sept. 2000).