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Fast, Sensitive LC-MS/MS Analysis of GHB and Related Compounds in Human Blood for Forensic Testing

Featured Application: Gamma-Hydroxybutyrate (GHB) in Whole Blood on Force C18

  • Simultaneously analyze GHB, GBL, and 1,4-BD in whole blood samples in a fast, 5-minute cycle time.
  • Chromatographic separation of actual GBL and GBL produced from GHB in-source conversion.
  • Sufficient sensitivity to measure endogenous levels of GHB and to identify exogenous drug ingestion.

Recently, gamma-hydroxybutyrate (GHB) and its precursors gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) have been gaining popularity as recreational drugs because of their euphoric properties. In addition, due to its potent pro-sexual effects, GHB is increasingly implicated in drug-facilitated sexual assault. Because of their easy availability and potential for misuse, demand is growing for analytical determination of these drugs in both urine and blood samples. Analysis can be challenging because these compounds are small molecules that are difficult to retain, so many methods employ time-consuming derivatization and require long analysis times. Measurement is further complicated by both endogenous levels of GHB, which is present in all tissues as a naturally occurring neurotransmitter, and by the very rapid metabolism and elimination of ingested GHB.

The LC-MS/MS analysis of GHB and its precursors shown here uses a quick and simple protein crash procedure for whole blood samples. Excellent retention and chromatographic separations are reliably obtained in a fast, 5-minute analysis without the need for derivatization or extensive sample preparation. All compounds were chromatographically separated on a Force C18 column, which is critical for this analysis because GHB can convert to GBL in the instrument’s ion source. In addition, method sensitivity is sufficient to measure low endogenous levels of GHB and to identify exogenous ingestion of these drugs. This LC-MS/MS analysis of GHB and its precursors is a faster alternative to typical approaches found in the literature and provides an effective means of testing and reporting levels in biological specimens.

PeakstR (min)Precursor ionProduct ion
1.γ-Hydroxybutyric acid (GHB)1.42105.287.0
2.1,4-Butanediol (1,4-BD)1.4291.055.0
3.γ-Butyrolactone (GBL)1.7587.045.0
ColumnForce C18 (cat.# 963431E)
Dimensions:100 mm x 3.0 mm ID
Particle Size:3 µm
Pore Size:100 Å
Guard Column:Force C18 EXP guard column cartridge 5 mm, 3.0 mm ID, 3 µm (cat.# 963450253)
Temp.:30 °C
Conc.:500 ng/mL
Inj. Vol.:10 µL
Mobile Phase
A:0.5% Formic acid in water
B:0.5% Formic acid in methanol
Time (min)Flow (mL/min)%A%B
Ion Mode:ESI+
Sample Preparation100 μL of whole human blood was fortified at 50 μg/mL with GHB, GBL, 1,4-BD, and GHB-D6 (IS) using 5 μL of 1 mg/mL solutions. The blood was precipitated with 380 μL methanol. The sample was then vortexed at 1000 rpm for 10 seconds and centrifuged at 3000 rpm for 10 minutes at 10 °C. 50 μL of the supernatant was removed and diluted to 1 mL with water. The sample was then vortexed and subjected to LC-MS/MS analysis. (Internal standard not shown on chromatogram.)