# SPME-GC/MS Analysis of Methanol in Biospecimen by Derivatization with Pyran Compound
## 2 Results and Discussion
### Derivatization and GC/MS Analysis  
Analysis using gas chromatography-mass spectrometry (GC/MS) enables the identification of chemicals through the interpretation of the mass spectrum. In this analysis, methanol appears very quickly (Figure 2) and could be overlapped with other compounds due to its low molecular weight which is similar to oxygen. To prevent this, methanol was analyzed using GC/MS through derivatization.  
Derivatization of methanol was carried out using a pyran-based compound, 3_A_-dihydro-2_H_-pyran (DHP), also known for protecting alcohol groups in organic synthesis. The reaction proceeds easily and quickly and its product is stable enough to be commercially traded.  
The internal standard (IS) used for quantification was ethanol substituted with deuterium (C\({}_{2}\)D\({}_{5}\)OH). Deuterium-substituted ethanol is extremely unlikely to exist in the natural environment. Owing to this property, it could be applied to the quantitative analysis of ethanol as an internal standard by GC/MS analysis, particularly in the drinker's blood.  
Derivatization of methanol and IS was easily performed under acidic conditions, especially with concentrated hydrochloric acid, and it showed reliable results. Under the acidic condition, the reaction of DHP with methanol and IS proceeded through nucleophilic addition, which gave 2-methoxytetrahydrofuran and 2-d5-ethoxytetrahydrofuran, respectively, as shown in Figure 3.  
The derivatized methanol and IS appeared at the retention time of 5.9 and 7.3 min, respectively, at the given GC/MS condition. GC/MS chromatogram and the mass spectra for each component are shown in Figure 4A-C. Especially, the peak at the retention time of 6.9 min in Figure 4A seems to be from the fragmented DHP or derivatized compounds, which has the _m/z_ of 101 of molecular ion and _m/z_ 51 of base ion and is supposed to be from butyl formate.  
Figure 3: Reaction schemes of methanol (**A**) and deuterium substituted ethanol (**B**) with 3_A_-Dihydro-2H-pyran (DHP).  
Figure 2: Gas chromatography/mass spectrometry (GC/MS) chromatogram for methanol in the blood sample.  
The base ion for both derivatized products was an ion with _m/z_ 85 altogether. Molecular ion for derivatized methanol and IS was an ion with _m/z_ 115 (Figure 4B) and _m/z_ 134 (Figure 4C), respectively. The proposed structure for the base ion is shown in Figure 5A and quantitative ions were shown in Figure 5B,C.  
Figure 4: GC/MS chromatogram of derivatized methanol in blood sample (**A**), mass spectra of derivatized methanol (2-methoxytetrahydrofuran)(**B**) and internal standard (2-d5-ethoxytetrahydrofuran)(**C**).  
The results obtained using GC/MS analysis by standard addition of methanol within the range of 0-2000 \(\upmu\)g/mL to the blood of the victim are shown in Figure 6. The calibration curve was prepared by using the area ratio of the ion with _m/z_ 115 for methanol and the ion with _m/z_ 134 for IS, as shown in Figure 7. The concentration of methanol in the blood of the victim was found to be 1218 \(\upmu\)g/mL by calculating the slope and intercept of the curve.  
For the linearity of the calibration curve, the \(r^{2}\) value was 0.9999, the detection limit was 0.56 \(\upmu\)g/mL, and the quantification limit was 1.7 \(\upmu\)g/mL. For the detection limit and quantification limit, S/N ratios of 10 blank samples were calculated with concentrations exceeding 3 and 10.  
Figure 5: The proposed structure for the ion with _m/z_ 85 for both derivatives (**A**), molecular ions with _m/z_ 115 for derivatized methanol (**B**) and _m/z_ 134 for derivatized internal standard (**C**).  
Figure 6: GC/MS chromatograms for the blood of the victim with standard methanol solution.  
Figure 7: Calibration curve derived by plotting the area ratio of the ion with _m/z_ 115 (derivatized methanol) and the ion with _m/z_ 134 (derivatized internal standard) against the concentration.