Phase I metabolites (organic acids) of gamma-hydroxybutyric acid -validated quantification by GC-MS and description of endogenous concentration ranges.

Gamma hydroxybutyric acid (GHB) is known as a sedative drug used in Drug Facilitated Crimes. Its detection window is very short. GHB undergoes intensive phase I metabolism to organic acids (glycolic acid, succinic acid, dihydroxy butyric acids). These could be potential analytical targets to broaden the detection window. The aim of the present study was to enable the detection of endogenous levels of these metabolites in biological samples (blood and urine). A gas chromatographic mass spectrometric method using liquid liquid extraction and derivatization wth MSTFA for the quantification was developed. Validation results were in accordance with international guidelines and the method was able to quantify endogenous levels of the substances in both urine and blood. Endogenous concentrations were shown to be < 0.03-4.92 mg/L for glycolic acid, < 0.03 to 1.28 mg/L for GHB, < 0.28-18.1 mg/L for succinic acid, < 0.12-1.38 mg/L for 2,4-dihydroxy butyric acid and < 0.13-2.59 mg/L for 3,4-dihydroxy butyric acid, respectively, in serum samples of 101 volunteers. Urinary endogenous concentrations were shown to be 1.30-400 mg/L for glycolic acid, < 0.03 to 1.94 mg/L for GHB, 1.17-273 mg/L for succinic acid, 0.72-26.2 mg/L for 2,4-dihydroxy butyric acid and 1.88 - 122 mg/L for 3,4-dihydroxy butyric acid, respectively, in urine samples of 132 volunteers. These endogenous concentrations represent a basis to which concentrations after the intake of GHB can be compared to in order to proof the intake of this substance.

now widely used in the treatment of sleep disorders. 12,13 However, in nontherapeutic use, GHB is frequently used by youth and bodybuilders or as a weight-loss supplement. 14,15 The effects of GHB include sedation, drowsiness, forgetfulness, and muscle relaxation. In addition, because it is colorless and nearly odorless, its detection is difficult, and thus, it is used as a knockout drug in cases of drug-facilitated sexual assaults. [16][17][18] Therefore, GHB is currently a highly controlled substance worldwide, and its possession, sale, import, and export are prohibited. 19,20 Because GHB is an endogenous substance, it is important to distinguish the uptake from endogenous concentrations in biological samples in forensic cases. 21,22 However, because of the relatively short half-life, detection windows in blood and urine are maximum 6 and 12 h, respectively. 23,24 In addition, the lack of useful metabolites as biomarkers leads to difficulties in detection of this substance. 25 Recent results from measuring concentrations of both GHB phase II metabolites, GHB-glucuronide and GHB-4-sulfate, indicate that these markers are not reliable and appropriate for expanding the detection window after GHB use or forensic toxicology. [26][27][28] GHB is quickly metabolized to succinic semialdehyde by gammahydroxybutyric acid-dehydrogenase and to succinic acid, "a mediator of the citric acid cycle," by succinic semialdehyde dehydrogenase. 25 It has been observed that the levels of the number of alpha and beta metabolites of GHB increase after GHB consumption, 29,30 namely, organic acids 2,4-dihydroxybutyric acid, 3,4-dihydroxybutyric acid, and 4,5-dihydroxyhexanoic acid that have been described as part of phase I GHB metabolism. Chemical structures of these organic acids are shown in Table 1.
In clinical laboratories, GHB metabolites are already used for the diagnosis of hereditary illnesses like fumarase deficiency, 31 succinate semialdehyde dehydrogenase deficiency 29 (succinic acid), or primary hyperoxaluria type II 32 (glycolic acid). Overall, approximately 50 diseases have been described in which an inherited single-enzyme defect causes a high concentration of acidic metabolites in the blood or urine. 33 However, the determination of glycolic acid can also have a value in ethylene glycol poisonings. 34 Currently, the phase I metabolites of GHB have not been quantitatively determined in blood and urine samples in forensic laboratories. The aim of the present study was to enable the detection of these metabolites in biological samples and to describe endogenous reference ranges. Therefore, a routine method for the quantification of GHB and its isomers 2-hydroxybutyrate, 3-hydroxybutyrate, and 3-hydroxyisobutyrate, and of organic acids within GHB metabolism (glycolic acid, succinic acid, 2,4-dihydroxybutyrate, and 3,4-dihydroxybutyrate) using gas chromatography-mass spectrometry (GC-MS) had to be developed and validated.
All standard stock solutions (10 mg/mL) were prepared using methanol.
The deuterated internal standard (GHB-d 6

| Liquid-liquid extraction
Urine or serum (1 mL) was added to 40 μL of the internal standard GHB-d 6 (500 μg/mL). An amount of 100 μL of 6M hydrochloric acid was added to adjust the pH to 1-2. Finally, the analytes were extracted using 5 mL of ethyl acetate. The mixture was thoroughly shaken for 1 min and centrifuged at 3000 rpm for 3 min. The supernatant was carefully transferred to another glass test tube. Then, 100 μL of HCl (0.1 mol/L in 2-propanol) was added, and the supernatant was dried under a gentle stream of nitrogen at room temperature.
The dried residue was derivatized and trimethylsilylated using 500 μL of MSTFA at 60 C for 60 min. The reaction mixture was transferred to an autosampler vial, and 2 μL was injected into the GC-MS system.

| Assay validation for serum and urine analyses
The GC-MS procedure was validated for the quantification of the aforementioned organic acids in accordance with an international guideline. 35 For drawing the calibration curves and for performing the quantitative measurements, the ratios of the peak area of the target ion of the organic acids to the peak area of the internal standard GHB-d 6 were applied.
The linearity and sensitivity of the method were tested in water.
The comparison of calibration curves within the matrix water and those within the matrices serum or urine revealed the validity of the matrix water (comparable incline of the calibration curves). The method's selectivity and specificity, accuracy, precision, and stability of the analytes were evaluated in both serum and urine.

| Selectivity
Because every single analyte detected by the described method is endogenously present in serum and urine samples, usual selectivity studies are not possible. Nevertheless, six different sources of serum and urine samples were analyzed for peaks interfering (peak shoulders, wrong ratio of peak area of the target to peak area of the qualifier ion) with the signals of the analytes or the internal standard.

| Linearity
Linearity was studied by analyzing a seven-point calibration using the concentrations of 1, 2.5, 5, 7.5, 10, 15, and 20 mg/L of all analytes in water. The curve was created six times on different days.

| Accuracy and precision
Quality control samples were prepared using pooled blank serum or urine and spiked to provide two final concentrations (2 and

| Stability
Stability was tested in the extracted samples within the autosampler (at room temperature) and in serum and urine samples.
The stability of the extracts within the autosampler was evaluated for 40 h after extraction. Six control samples each for low (2 mg/L) and high (17 mg/L) concentrations were extracted, connected, and aliquoted again into six extracts. These extracts were injected into the device at 0, 4, 10, 15, 24, and 40 h after extraction and derivatization and were analyzed using the validated method.
In a second step, the stability of the analytes in serum and urine samples was tested by storing 20 measured real serum and urine samples at −20 C. After 1 month, the samples were reextracted, analytes were reexamined, and the observed results were compared.

| Detection of endogenous concentrations
After the method was validated, it was used to determine the endogenous levels of the organic acids: GHB, succinic acid, glycolic acid, 2,4-dihydroxybutyric acid, and 3,4-dihydroxybutyric acid.
Authentic serum (n = 101) and urine (n = 132) samples taken from a mixed population (serum: 55 men, 46 women; urine: 77 men, 55 women) were evaluated. The intake of GHB or related substances was excluded among these patients using a questionnaire after informed consent. After the samples were received, the serum was quickly separated from the red blood cells, and serum and urine samples were stored at −20 C until further analysis, which was a maximum of 7 days after blood or urine sampling. concentration range detected in real serum samples. However, urine samples showed concentrations >20 mg/L for some analytes. In these cases, samples had to be diluted before extraction. The method's imprecision and bias were always less than 15% and compliant with the guideline. Bias and precision data were reported in RSD% and are presented in Table 2.

| RESULTS AND DISCUSSION
The analytical limits of the method determined by a calibration curve in the low-concentration range according to DIN 32646 are shown in Table 3. The method showed limits of detection that were  According to their findings, Busardo et al. recommended the analysis of GHB in blood and urine within 3 days of sampling and the storage at −20 C or 4 C to avoid instability issues. 41 During validation experiments, we tested GHB stability at −20 C for 1 month. GHB concentrations were always less than LoQ in serum.
The hydroxylated metabolites of GHB seemed to be very stable and did not change a lot after storage at −20 C for a month. Overall, the validation showed suitable selectivity, sensitivity, accuracy, precision, and linearity, which were in the range from the LoQ to 20 mg/L; all the mentioned parameters indicate the reproducibility and repeatability of this method and its suitability for quantitative analysis. The stability of succinic acid in serum as well as in urine samples seemed to be a problem; however, the stability of all other analytes over 1 month at −20 C was acceptable.
To confirm the applicability of the method and to determine endogenous concentrations of the parameters in human serum and urine, the levels of GHB, 3,4-dihydroxybutyrate, 2,4-dihydroxybutyrate, succinic acid, and glycolic acid were measured using the validated method in the collective. Table 4 presents the minima, maxima, median, and mean values of the concentrations of the organic acids detected in serum samples (n = 101). Table 5   A lack of the enzyme SSADH, called succinic semialdehyde dehydrogenase deficiency (SSADHD), leads to increased concentrations of GHB in urine and other body fluids. 54 Shinka et al. 30 and Brown et al. 29 received urine samples from one and three patients, respectively, suffering from this disease. An increased excretion of 3,4-dihydroxybutyric, 4-hydroxy-3-oxobutyrate, 54 and glycolic acid, a further product of β-oxidation of 3,4-dihydroxybutyric, has been shown in these patients. Metabolites representing α-oxidation of GHB (2,4-dihydroxybutyric acid) have also been found increased, however, to a lesser extent. 29 Oxidation of 2,4-dihydroxybutyric acid to 2-oxoacid and oxidative decarboxylation led to 3-hydroxypropionic acid, which was also sometimes found in the urine samples. 29  However, a quantification of these substances and a description of their endogenous concentration range have, to the best of our knowledge, never been conducted at least in forensic laboratories.
We concentrated on glycolic acid, succinic acid, 2,4-dihydroxybutyric acid, and 3,4 hydroxybutyric acid because these substances are commercially available and could be potential target analytes after the intake of GHB.
In our study, 2,4-dihydroxybutyrate and 3,4-dihydroxybutyrate could be detected in serum at concentrations ranging from less than LoD to 1.38 mg/L and less than LoD to 2.59 mg/L, respectively.
It could be confirmed that β-oxidation of GHB is preferred over α-oxidation. 29

| CONCLUSIONS
A method was developed and validated to quantify GHB and its phase I metabolites, namely succinic acid, glycolic acid, 2,4-dihydroxybutyric acid, and 3,4 dihydroxybutyric acid, in human serum and urine. The endogenous concentration ranges for these analytes in urine and serum were defined. In a future study, these metabolites must be determined in samples of patients who chronically or uniquely take GHB for medical reasons or in forensic cases after the known intake of GHB to evaluate if these parameters could be useful biomarkers with regard to the detection of a GHB intake.