γ-Hydroxybutyric Acid (GHB) [BSA]

GHB is a metabolite of γ-aminobutyric acid in mammals and humans. It is an endogenous biological substance and is considered a neurotransmitter in the field of medicine and pharmacy. It plays a very important role in self-balance adjustment and regular sleep. As a strong and fast-acting inhibitor on the central nervous system, GHB has a sedative effect. As early as the 1960s, it was used as an anesthetic and hypnotic in clinical practice, but it was later found to have great toxic side effects. Long-term use will produce dependence syndrome, causing headaches, nausea, vomiting, and even respiratory depression and coma. Moreover, when mixed with other drugs and alcohol, the toxic side effects are more obvious. Therefore, GHB is gradually replaced by other drugs. Now, GHB is still used as an additional anesthetic and hypnotic, and is also used in the treatment of alcohol dependence, opium withdrawal syndrome and sleepiness. In addition, because GHB can also stimulate the secretion of hormones in the body and increase euphoria, it has been abused in entertainment venues since the 1990s and has quickly become popular. Many European and American countries have listed GHB as a Class I drug for control. Given its strong sedative and amnesiac effects, GHB is often associated with sexual crimes, which has brought about serious social problems. Gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) are prodrugs of GHB, which are usually regarded as related substances that are converted into GHB after ingestion, and are also endogenous substances in the brains of mammals. GBL is quickly converted into GHB under the catalysis of enzymes or when the pH is increased (such as by adding NaOH), and in the human body, 1,4-BD can also be quickly metabolized into GHB.

Figure 1. The role of GABA and glutamate as neurotransmitters. (Sources: Kim K, et al. 2023)

The molecular formula of GHB is C₄H₈O₃, and the molecular weight is 104.1. GHB is usually used as its sodium salt. Its sodium salt is a white powder and is easily soluble in water. The molecular formula of GBL is C₄H₆O₂, and the molecular weight is 86.1. It is a cyclic compound obtained when GHB loses a water molecule. The pure product is a colorless volatile liquid with a caramel taste, a density of 1.12, a boiling point of 204°C, and is miscible with water and soluble in methanol, ethanol, acetone and ether. 1,4-BD is slightly different from GHB in structure. It is a straight-chain structure with a hydroxyl group at each end. The molecular formula is C₄H₁₀O₂, the molecular weight is 90.1, the density is 1.02, and the boiling point is 228°C. The pure product is a colorless viscous liquid that is soluble in water, dimethyl sulfoxide, acetone and ethanol. GHB has a special chemical structure, a small molecular weight, and two highly polar groups, hydroxyl and carboxyl. Therefore, GHB has strong polarity and water solubility, and it is difficult to separate it from the aqueous medium by conventional extraction techniques. Combined with different analytical methods, liquid-liquid extraction, solid phase extraction, solid phase microextraction and other technologies are widely used to extract and separate GHB from different samples such as beverages, blood, urine, liver, and hair. As a commonly used extraction and separation method, liquid-liquid extraction technology has the characteristics of high efficiency, rapidity, and convenience, and has been widely used in environmental analysis, drug analysis and other fields. Due to its high-water solubility, GHB is difficult to be directly separated and extracted from the aqueous phase by general liquid-liquid extraction. Generally, before liquid-liquid extraction, GHB is first converted into GBL in an acidic medium, and then extracted with an organic solvent. Solid phase extraction is a new type of separation method that appeared in the 1970s. Compared with liquid-liquid extraction, it has the advantages of simple and fast operation, high extraction rate, stable extraction rate, clean extraction solution, low cost, and easy automation. As a new extraction and separation technology developed recently, solid phase microextraction inherits the advantages of solid phase extraction and effectively overcomes its defects. It is simple to operate and has good reproducibility. No organic solvent is used from extraction to injection. Desorption is fast and complete. It integrates extraction, concentration and injection. It has been widely developed and applied so far.

GHB is converted to GBL under acidic conditions. GBL and hydroxylamine hydrochloride generate-hydroxybutyrylhydroxylamine under alkaline conditions. Hydroxybutyrylhydroxylamine reacts with ferric chloride under acidic conditions to generate a purple-red complex. Therefore, chemical colorimetric method can be used to detect whether the sample contains GHB. Gas chromatography is rarely used in the analysis of GHB and its related substances, mainly because GHB has a small molecular weight and contains two strong polar groups, a hydroxyl group and a carboxyl group, and has poor thermal stability. Generally, GHB should be pretreated before sampling and analysis. There are two main methods for pretreatment: one is to convert GHB into GBL under acidic conditions; the other is to perform derivatization treatment. Derivatization mainly uses silanization method. The silanization reagents used are N, O-bistrimethyl-trifluoroacetamide, trimethylchlorosilane (TMCS), N-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA), etc. Compared with gas chromatography, gas chromatography-mass spectrometry is more widely used in the analysis of GHB and its related substances. It is generally necessary to pre-treat before injection. The pre-treatment methods are mainly to convert GHB into GBL and derivatization. Liquid chromatography analysis is a method that has been widely used in the field of research and analysis in recent years. Especially with the continuous deepening of life science research, liquid chromatography has also become an extremely important and indispensable separation and analysis method. Liquid chromatography analysis overcomes the unfavorable factors such as thermal stability of gas chromatography analysis and is more suitable for the detection and analysis of GHB. However, since its analytical sensitivity is not as high as that of gas chromatography analysis, derivatization methods are often used for pre-treatment in order to improve its detection limit. Liquid chromatography-mass spectrometry technology organically combines the high separation efficiency of liquid chromatography with the high sensitivity and strong structural analysis ability of mass spectrometry. After the sample is separated by liquid chromatography, it is identified with the help of mass spectrometry analysis results, opening up a new world for the separation and detection of complex samples. In particular, the application in the field of forensic identification, which has developed rapidly in recent years, has been further developed, and its superiority has also been shown in the analysis of drugs such as GHB. Based on its special separation mechanism, capillary electrophoresis is particularly suitable for the analysis of polar components in aqueous solutions. Compared with the traditional GC method, its biggest advantage is that it avoids the tedious extraction and derivatization process. This method is simple in sample processing, fast, sensitive, and easy to operate. It can be used as a rapid screening method for GHB in beverages in related tests.

Gamma-Hydroxybutyric Acid
Gamma-Hydroxybutyrate
4-Hydroxybutanoic Acid
Sodium Oxybate
Sodium Gamma-Hydroxybutyrate