Analysis of γ-hydroxybutyric acid, DL-lactic acid, glycolic acid, ethylene glycol and other glycols in body fluids by a direct injection gas chromatography-mass spectrometry assay for wide use

Abstract

Analysis of blood of severely intoxicated patients always requires prompt investigation. Diagnosis of intoxication with ethylene glycol, gamma-hydroxybutyric acid or D-lactic acid takes hours, since several different procedures are required. Rapid derivatization of the common hydroxyl function may resolve this analytical problem. Here we describe a fast method for the simultaneous measurement of ethylene glycol, glycolic acid, gamma-hydroxybutyric acid and racemic lactic acid. Only 20 microl of serum, plasma or urine are required for immediate derivatization at 70 degrees C with 750 microl of bis-N,O-trimethylsilyl trifluoroacetamide after adding 20 microl of internal standard solution (1,3-propylene glycol) and 20 microl of the catalyst dimethylformamide. After centrifugation an aliquot is transferred to a gas chromatographic system and analyzed with electron-impact mass spectrometry in selective ion monitoring mode. The derivatized acids and ethylene glycol are well separated and detected with a limit of detection ranging from 0.12 mg/l for ethylene glycol to 0.95 mg/l for gamma-hydroxybutyric acid, while the limit of quantification ranged from 0.4 mg/l for ethylene glycol to 3.15 mg/l for gamma-hydroxybutyric acid. The method is linear from 0.5 to 1800 mg/l blood for ethylene glycol, from 0.7 to 1200 mg/l for lactic acid, from 1.2 to 1800 mg/l for glycolic acid, and from 3.2 to 200 mg/l for gamma-hydroxybutyric acid, with analytical recoveries, accuracy, day-to-day and within-day precision well within the required limits. Total analysis time with one calibrator was 30 min, derivatization time included. This method is very suitable for emergency toxicology, since several toxic substances can be quantified simultaneously in a fast and sensitive manner.

Determination of gamma-hydroxybutyric acid in biofluids using a one-step procedure with “in-vial” derivatization and headspace-trap gas chromatography–mass spectrometry

“…Both the initial concentration and the concentration obtained by reanalysis using our described method varied from -4 to 4 % from their mean, well in line with the above mentioned requirement for ISR. Furthermore, analyzing the study samples by the use of HS-trap and by the use of the method by Van hee et al [12] led to similar GHB concentrations for the same sample. The difference between the two obtained results was within -9.4 to 16.7 % from the mean, calculated by dividing the difference between the two results (separately obtained by the two above-…”

“…Furthermore, for method comparison, we performed a cross-validation by analyzing an aliquot of the samples at the Laboratory of Toxicology of ZNA Stuivenberg Hospital in Antwerp, using the method of Van hee et al [12]. The difference between the result obtained by the newly developed HS-trap method and that obtained by the previously published method for the determination of GHB should be within 20 % of the mean for at least 67 % of the samples [25].…”

“…An aliquot of the sample was also analyzed using the method of Van hee et al [12]. Results are summarized in Table 4.…”

“…Several options have been described that allow to reduce the work-load associated with GC-MS-based GHB determination in biological fluids. These include the addition of excess derivatization reagents directly to biological samples, thus omitting the extraction step [12][13][14][15], the use of HS injection after conversion of GHB to gamma-butyrolactone (GBL) [16][17][18] or "in-vial" derivatization with for example hexylchloroformate prior to solid-phase microextraction (SPME) [19] or HS injection [20]. The available HS-based methods, either 'classical' static HS [16], SPME [17,20], or solidphase dynamic extraction (SPDE) [18], all start from 0.5-1 ml of biofluid (water, urine, plasma, serum or whole blood), providing sufficient sensitivity (LLOQ from 0.1 to 5.0 µg/ml).…”

“…Measured GHB concentrations (µg/ml) in real samples using the HS-trap GC-MS method (HS-trap) and the method of Van hee et al [12]. Urine samples (U) were frozen at -20 °C before reanalysis after 14 days; serum (S) samples were frozen at -20 °C before reanalysis after 30 days; whole blood sample was frozen at -20°C before reanalysis after 7 days.…”