# SPME-GC/MS Analysis of Methanol in Biospecimen by Derivatization with Pyran Compound
## 1 Introduction  
Methanol is a highly versatile chemical used in the manufacture of solvents, pharmaceuticals, pesticides, fuels, resins, and electronics. It is easily accessible from our surroundings, but accidental or intentional ingestion can lead to life-threatening health conditions [1]. Methanol, when absorbed into the body, is metabolized to produce highly toxic formaldehyde and formate [2], which can cause blindness and death. Methanol is converted to formaldehyde by alcohol degrading enzyme (ADH) and to formate by aldehyde degrading enzyme (ALDH) in the liver [3]. The toxic effects of formate can damage the optic nerves and prevent the energy metabolism of the mitochondria, which eventually destroys the living body [4]. The amount of poisoning and lethal dose of methanol varies between individuals, but generally, the acute ingestion of 10 mL of methanol can cause permanent blindness and 100-200 mL can be fatal to most adults [5]. It was reported that the blood methanol levels from 504 to 1286 \(\upmu\)g/mL through inhalation exposure resulted in altered vision [6]. For the fatal cases by methanol intoxication, the concentration in blood showed a wide range from 150 to 4820 \(\upmu\)g/mL [7]. Methanol poisoning may be caused by the consumption of bootleg liquor and pesticides, misuse of medicines, or intake of chemicals containing methanol for suicide. First-aid measures include gastric lavage and administration of ethanol to slow down the metabolism of methanol [8; 9]. Therefore, in the case of methanol poisoning, ethanol administration is an important method of first aid [10]. Methanolis generally analyzed by a gas chromatography-flame ionization detector (GC-FID) [11; 12]. However, this method has some weakness due to low molecular weight and low boiling point (65 \({}^{\circ}\)C) of methanol which causes retention time overlap with other volatile organic compounds appearing at the beginning of the analysis [13]. Moreover, in the case of complex matrix samples, such as blood, the analytes with early retention time could be confused as a methanol peak. For this reason, the GC-FID, which depends on only retention time, could be difficult for qualitative and quantitative analysis of methanol in complex matrix samples. In order to overcome this drawback, analysis using the gas chromatography-mass spectrometry (GC/MS) could be a possible alternative. However, the molecular weight of methanol is similar to that of oxygen, and column bleeding may occur due to moisture from the injected volume of the headspace in the sample [14]. To solve the flaw of GC/MS in this study, methanol was derivatized in the sample and analyzed using GC/MS. Conventional pretreatment techniques for instrumental analysis include solvent extraction methods such as liquid-liquid extraction [15] and solid-phase extraction [16]. These processes not only require large amounts of solvents with high purity but also consume lots of time and effort resulting in high analysis costs. Consequently, derivatization reagents were added to the vial-containing sample, reaction products were extracted with solid-phase micro extraction (SPME) and directly analyzed by GC/MS [17; 18; 19]. In order to rule out external influences, deuterium-substituted ethanol (C\({}_{2}\)D\({}_{5}\)OH) was used as an internal standard (IS) and standard addition method was employed to avoid the matrix effect of the blood sample on the quantification of methanol [20]. Derivatization of methanol and IS was carried out with 3,4-dihydro-2H-pyran (DHP), a pyran-based compound under acidic conditions, and extraction was carried out using SPME fiber of carboxen/PDMS (CAR/PDMS) which possesses the best analytical properties among commercial SPME fibers [21; 22]. Moreover, this study investigated the cause of death in a victim who consumed veterinary skin-care medicine by analyzing the metabolite, formate, as well as methanol levels in the blood.