1. 4 1. 4 © Springer-Verlag Berlin Heidelberg 2005 II.1.4 Methanol and formic acid by Xiao-Pen Lee and Keizo Sato Introduction Methanol ( methyl alcohol) poisoning accidents take place most frequently by drinking it in mistake for ethanol. Methanol poisoning is not due to the e ect of methanol itself, but due to toxicity of its metabolites. Methanol is rapidly absorbed into human body through the airway mucous membranes, digestive tract mucous membranes or the skin; it is metabo- lized into formaldehyde ( formalin, HCHO) and then formic acid (HCOOH) by the actions of alcohol dehydrogenase and aldehyde dehydrogenase, respectively. Formic acid inhibits cytochrome oxidase in the optic nerves, and causes visual disturbances followed by the loss of eyesight. e accumulation of formic acid in the body provokes severe acidosis, which is characteristic for methanol poisoning. e metabolic (oxidation) velocity for metha- nol is about 5–10 times slower than that for ethanol. is is the reason why the poisoning symptoms do not appear soon a er its ingestion, but appear a er a while. Formic acid can be detected from urine for about one week a er methanol ingestion. It is possible to diagnose methanol poisoning by detecting methanol and/or formic acid from blood and urine specimens. For analysis of methanol and formic acid, GC methods with packed columns were employed [1–5]. In this chapter, GC methods for analysis of them in blood and urine using a wide-bore capillary column and using solid-phase microextraction (SPME) [6–9] are presented. Analysis of methanol Reagents and their preparation (in common with both wide-bore capillary GC and headspace SPME-GC) • Methanol standard solution: a 0.127 mL volume of methanol of special grade is dissolved in 100 mL distilled water to prepare 1 mg/mL solution. • Internal standard (IS) solution: a 0.128 mL volume of acetonitrile of special grade is dis- solved in 100 mL distilled water to prepare 1 mg/mL solution. Conditions for wide-bore capillary GC Column: an Rtx-BAC2 wide-bore capillary column (30 m × 0.53 mm i.d., lm thickness 2.0 µm, Restek, Bellefonte, PA, USA). GC conditions: a Shimadzu GC-14B gas chromatograph (Shimadzu Corp., Kyoto, Japan) with an FID was used. Column (oven) temperature: 30°C (1 min)→ 20°C/min→ 210°C; injec- tion and detector temperature: 240°C; carrier gas: He; its ow rate: 5.0 mL/min. 124 Methanol and formic acid Procedure for wide-bore capillary GC i. A 0.5 mL volume of whole blood, 80 µL of IS solution, 0.5 mL of distilled water and 0.6 g of solid ammonium sulfate a are placed in a 4 mL volume glass vial, capped with a silicon- septum cap and mixed well. ii. e vial is heated at 60°C on an aluminum block heater with stirring with a small Te on- coated magnetic bar b . A er 15 min of heating, about 0.6 mL volume of the headspace va- por is drawn into a gas-tight syringe c . Just a er the vapor volume in the syringe is adjusted to 0.3 mL d by pushing the plunger slowly, it is rapidly injected into GC. iii. Quantitation: various concentrations of methanol and 80 µL of IS solution are spiked to vials containing 0.5 mL blank whole blood, 0.5 mL distilled water and 0.6 g ammonium sulfate each, followed by the above procedure, to make a calibration curve with methanol concentration on the horizontal axis and with peak areas ratio of methanol to IS on the vertical axis. Using the calibration curve, methanol concentrations in specimens can be calculated e . Conditions for headspace SPME-GC Column: a Supelcowax 10 medium-bore capillary column (30 m × 0.25 mm i.d., lm thickness 0.25 µm, Supelco, Bellefonte, PA,USA) SPME devices and bers f : 75 µm Carboxen/polydimethylsiloxane bers (both from Supelco) GC conditions [9]: the same GC instrument with an FID as above was used. Column (oven) temperature: 35°C (6 min)→ 20°C/min→ 135°C; injection port g and detector temperature: 280°C; carrier gas: He; its ow rate: 0.7 mL/min. Injection is made in the splitless mode upon inserting the SPME ber h ; it is changed to the split mode a er 90 s. Procedure for headspace SPME-GC i. A 0.5 mL volume of whole blood or urine, 2 µL of IS solution, 0.5 mL of distilled water and 0.6 g of ammonium sulfate are placed in a 4 mL volume glass vial, capped with a silicone- septum cap and mixed well. ii. e vial is heated at 60°C on an aluminum block heater with stirring with a small Te on- coated magnetic bar. A er 5 min of heating, the holder needle of SPME is inserted into the vial through the septum, and the SPME ber is exposed to the headspace vapor and kept there with stirring and heating at 60°C for 10 min. iiii. A er the exposure, the ber is withdrawn into the needle, and the needle of the syringe is taken out of the vial and immediately injected into the GC port to expose the ber in it. iv. Quantitation: to vials containing the above components each, one of various amounts of methanol and 2 µL of IS were added and processed as above to construct a calibration curve for quantitation i . 125 Assessment of both methods > Figure 4.1 shows a wide-bore capillary gas chromatogram obtained from 0.5 mL whole blood, to which 400 µg methanol and 80 µg acetonitrile (IS) had been added. Excellent peaks of methanol and IS appeared at di erent retention times within 5 min; a few small background impurity peaks appeared. e extraction e ciency (recovery) of methanol spiked was 0.29%. Good linearity was found in the range of 50–500 µg/0.5 mL. e detection limit was about 10 µg/0.5 mL. > Figure 4.2 shows a headspace SPME-gas chromatogram obtained for 0.5 mL whole blood, to which 200 µg methanol and 2 µg of IS had been added. Both peaks were separated well and appeared within 10 min. e extraction e ciencies (recoveries) j were 0.25 % for whole blood and 0.38 % for urine. e calibration curve showed good linearity in the range of 1.56–800 µg/0.5 mL for both whole blood and urine specimens. e detection limits were 0.5 µg/0.5 mL for whole blood and 0.1 µg/0.5 mL for urine. Analysis of formic acid Formic acid cannot be analyzed by GC in its underivatized form; it should be esteri ed [10] prior to the analysis. Usually, formic acid is methylated to be detected as formic acid methyl ester. Reagents and their preparation (in common with both methods) • IS: a 0.128 mL volume of acetonitrile of special grade is dissolved in 100 mL distilled water to prepare 1 mg/mL solution. Detection of methanol from human blood by wide-bore capillary GC. To 0.5 mL blank blood, 400 µg methanol and 80 µg IS had been added. ⊡ Figure 4.1 Analysis of formic acid 126 Methanol and formic acid • Methanol: reagent of special grade. • Sodium formate: 10 mg of sodium formate of special grade is dissolved in 10 mL water to prepare 1 mg/mL solution. • Concentrated sulfuric acid: reagent of special grade containing 98 % of the compound. Conditions for wide-bore capillary GC Column: the same column as used in the methanol analysis ( Rtx-BAC2 wide-bore capillary column). GC conditions: the same GC instrument with an FID was used. Column (oven) tempera- ture: 30°C (2 min)→ 5°C/min→ 100°C; injection and detector temperature: 240°C; carrier gas: He; its ow rate: 5.0 mL/min. Procedure for wide-bore capillary GC i. A 0.5 mL volume of whole blood and 500 µL IS solution are placed in a 7.5 mL volume glass vial; to the mixture, 0.3 mL of concentrated sulfuric acid is gradually added and mixed well under cooling with ice k . A er cooling the vial with ice, 25 µL (corresponding to 20 mg) of methanol and 0.2 mL distilled water are added to the above mixture, rapidly capped with a silicone-septum cap and mixed well. ii. e vial is incubated at 35°C for 15 min with mixing gently several times. A er the incuba- tion, about 0.6 mL of the headspace vapor is drawn into a gastight syringe and the volume is adjusted to 0.3 ml, which is rapidly injected into GC for analysis. ⊡ Figure 4.2 Detection of methanol from human blood by headspace SPME-GC. To 0.5 mL blank blood, 200 µg methanol and 2 µg IS had been added. 127 iii. Quantitation: to vials containing 0.5 mL of blank whole blood and 500 µL of IS solution each, various amounts of sodium formate l were added, followed by the procedure de- scribed above to construct a calibration curve with peak area ratio of formic acid to IS on the vertical axis for quantitation. Conditions for headspace SPME-GC Column: the same Supelcowax 10 medium-bore capillary column as used in the methanol analysis. SPME devices and bers: the same ones as used for methanol analysis. GC conditions [9]: the same GC instrument with an FID as used above was used. Column (oven) temperature: 30°C (3 min)→ 25°C/min→ 105°C→ 10°C/min→ 145°C; injection and de- tector temperature: 280°C; carrier gas: He; its ow rate: 0.7 mL/min. e SPME ber is injected into GC in the splitless mode and the splitter is opened a er 90 s. Procedure for headspace SPME-GC i. A 0.5 mL volume of whole blood or urine and 20 µL of IS solution are placed in a 7.5 mL volume glass vial; to the mixture, 0.3 mL of concentrated sulfuric acid is gradually added and mixed well under cooling with ice. A er cooling the vial, 25 µL (corresponding to 20 mg) of methanol m and 0.2 mL distilled water are added to the above mixture, capped with a silicone-septum cap and mixed well. ii. e vial is incubated at 35°C for 5 min on an aluminum block heater. en, the needle of the SPME holder is inserted into the vial through the septum, and the SPME ber is exposed to the headspace vapor and kept there with stirring and warming at 35°C for 10 min. iii. A er the exposure, the ber is withdrawn into the needle and taken out of the vial; it is immediately injected into GC to expose the ber to the carrier gas at high temperature for GC analysis. e quantitation is made in the same manner as described above. Assessment of both methods > Figure 4.3 shows a wide-bore capillary gas chromatogram obtained from 0.5 mL of blank whole blood, to which 400 µg formic acid and 500 µg acetonitrile (IS) had been added, using an Rtx-BAC2 wide-bore column. Excellent peaks of methyl formate and IS appeared; however the former peak was close to but separable from the big methanol peak, which had been used for esteri cation. e background was clean except for the methanol peak. e extraction ef- ciency (recovery) of formic acid spiked was 0.33 %. e calibration curve showed good lin- earity in the range of 50–500 µg (in the form of free formic acid)/0.5 mL. e detection limit was 15 µg/0.5 mL. > Figure 4.4 shows a headspace SPME-gas chromatogram obtained from 0.5 mL blank whole blood, to which 54 µg of formic acid and 20 µg of acetonitrile (IS) had been added, using a Supelcowax 10 medium-bore capillary column. e peaks of methyl formate and IS appeared as big peaks; but some impurity peaks were observed in the background. e extraction e - Analysis of formic acid 128 Methanol and formic acid ciencies (recoveries) of formic acid were 1.55 % for whole blood and 1.24 % for urine. e calibration curve showed good linearity in the range of 1.56–500 µg (in the form of free formic acid)/0.5 mL for both whole blood and urine specimens. e detection limit was 0.6 µg/0.5 mL for both specimens. Poisoning cases, and toxic and fatal concentrations Poisoning doses of methanol varies markedly according to di erent individuals. However, it is considered that the intake of 10–20 mL methanol causes severe visual disturbance or the loss of eyesight; the fatal dose is 30–100 mL [11]. Blood methanol concentrations of surviving poi- soned patients were reported to be not lower than 100 µg/mL [11]; those in fatal poisoning cases 200–3,200 µg/mL [4, 11, 12]. When blood concentration is more than 4 mg/mL, the victim dies of anaesthetic paralysis. Detection of formic acid from human blood by wide-bore capillary GC. To 0.5 mL blank blood, 600 µg sodium formate (equal to 400 µg formic acid) and 500 µg IS had been added. The big peak appearing at 3.2 min of retention time is due to methanol, which had been added for methylation reaction of formic acid. ⊡ Figure 4.3 129 e acute methanol poisoning symptoms are vertigo, debility feeling, headache, nausea, vomiting and others; in rare cases, visual disturbance appears at an early stage. ese symptoms usually appear 12–24 h a er the injgestion, but in severe cases they can appear in about 1 h a er the intake. e symptoms of its chronic poisoning are considered to appear by inhalation of methanol gas for a long time, extensive contact of the skin with methanol or continuous in- Detection of formic acid from human blood by headspace SPME-GC. To 0.5 mL blank blood, 80 µg sodium formate (equal to 54 µg formic acid) and 20 µg IS had been added. The big peak appearing at 5.7 min is due to methanol, which had been used for methylation of formic acid. ⊡ Figure 4.4 Poisoning cases, and toxic and fatal concentrations 130 Methanol and formic acid gestion of its small amounts; they are disturbances of the central nervous system, liver and eyes. In the Vodka (disclosed to be the mixture of methanol and water later) Smuggling Incident taking place in Iran, 1975, y seven people fell into methanol poisoning; among them, two lost their eyesight and 17 died. e methanol concentrations in heart blood obtained at autop- sies were 230–2,680 µg/mL (average 1,205 µg/mL) [12]. In Japan, 8 correspondences about methanol poisoning were received by Japan Poison Information Center in 2000. e toxicity of formic acid, a metabolite of methanol, is very high and induces blindness and acidosis. e concentration of formic acid in blood in methanol poisoning cases were re- ported to be 90–2,270 µg/mL [13, 14]. Notes a) e addition of ammonium sulfate to the mixture is e ective to increase the extraction e ciency by the salting-out e ect. b) e use of a stirrer is e ective for shortening the time of the headspace extraction; heating at 60 °C is also e ective to enhance the extraction e ciency. c) e septum of a vial made of silicone/Te on sometimes causes leakage of headspace gas, when a usual needle of a gastight syringe is inserted into the vial through the septum. To prevent such leakage, the authors are using 23 G needles with a 90 cut at their tips. When the needle of the syringe is pulled out of the vial, care should be taken not to aspirate atmospheric air into the syringe. d) e internal standard calibration method is employed. At least 5–6 concentrations of methanol are plotted to con rm the linearity of the curve. e) e SPME method is a new extraction technique developed by Pawliszyn et al. [15] of Waterloo University of Canada in 1990. It has been being used mainly for analysis of envi- ronmental pollutants in water; it is also being applied in the eld of forensic toxicology nowadays [6–8, 16–21]. e advantages of SPME are that it does not require any organic solvent and that the extraction, condensation and injection into GC can be achieved with one-step procedure. Especially in the headspace SPME, the impurity peaks appearing in a GC chromatogram is very few. erefore, SPME seems very useful for analysis of drugs and poisons in forensic toxicology. f) On the surface of an SPME ber, a liquid phase or an adsorbent material of 7–100 µm thickness is coated. A drug or a poison is extracted into the coating. e polarity and reten- tion capacity is dependent on the material of a coating and its thickness. > Table 4.1 sum- marizes SPME bers now commercially available. e most suitable ber should be se- lected empirically and theoretically for each compound to be analyzed. g) e SPME bers should be pre-conditioned (aging at a high temperature for a certain in- terval) for new bers or ones, which were not used for a long period. To protect a ber from contamination, the needle tip of SPME should be capped by sticking it into a GC port sep- tum. h) When a needle of SPME is injected into an injection port of GC to expose the ber, it does not produce a large volume of gas and thus does not need a large space of injection cham- ber; this is quite di erent from usual GC analysis with an organic solvent injection. Espe- cially for volatile compounds extracted by SPME, a large space of an injection chamber 131 ⊡ Table 4.1 Various kinds of SPME fibers commercially available and their characteristics* Fiber Use Film thickness Liquid phase Adsorbent coexisting Polarity Analyte example(s) PDMS GC, HPLC 7 µm, 30 µm, 100 µm ● – Low Hydrophobic compounds DVB/PDMS GC, HPLC 65 µm (GC), 60 µm (LC) – Porous polymer Intermediate/low Amines with short chains StableFlex DVB/PDMS GC 65 µm – Porous polymer Intermediate/low Low molecules Intermediately to highly polar compounds Polyacrylate GC, HPLC 85 µm ● – Intermediate Polar compounds with intermediate boiling points Carboxen/PDMS GC 75 µm – Carbon type Low/low Gas Compounds with low boiling points StableFlex DVB/ Carboxen/PDMS GC 50/30 µm – Carbon type/ polymer Low/intermediate/ low Nasty smell of water Carbowax/DVB GC 65 µm – Porous polymer High/intermediate Organic solvents with intermediate boiling points Carbowax/TPR HPLC 50 µm – Porous polymer High/high Surfectants PDMS: polydimethylsiloxane; DVB: divinylbenzene; TPR, template resin. * Cited with modification from a catalog book of Supelco entitled “2001 Chromatography Products: Sample Handling”. Poisoning cases, and toxic and fatal concentrations 132 Methanol and formic acid is not desirable, because it causes broadening of peaks in GC chromatograms. erefore, when the SPME method is used, a glass insert liner tube with a small internal diameter (0.5–0.8 mm) should be used to get a sharp peak of a target compound; this results in the better S/N ratio, sensitivity and quantitativeness. i) e SPME is very suitable for splitless injection, because it does not produce a large volume of gas; the analyte is rapidly desorbed from the ber and introduced into a capillary column. j) e internal standard calibration method is also employed for the SPME-GC analysis. e linearity of the calibration curve should be con rmed using at least 5–6 plots at di erent concentrations of methanol. On this occasion, a single ber should be repeatedly used (including the construction of a calibration curve) in a set of experiments to avoid the variation of data due to a di erent lot of a ber. k) Although the extraction e ciency (recovery) of the headspace SPME is usually low, the entire amounts of methanol and IS adsorbed to the ber can be introduced into a column. is results in relatively high sensitivity of the SPME-GC analysis. l) For esteri cation of formic acid, the action of concentrated sulfuric acid is required. Upon addition of the acid to an aqueous mixture, heat is produced. erefore, the gradual mixing of sulfuric acid should be made under cooling with ice. Formic acid exists in a liquid form, which is relatively inconvenient for handling. ere- fore, solid sodium formate can be used in place of free formic acid. Upon quantitation, the values should be calculated to those of the free formic acid. m) To achieve esteri cation of formic acid completely, an excess amount of methanol should be added for the reaction. However, the addition of a large amount methanol can badly af- fect the partition coe cient of methyl formate on the surface of the SPME ber. erefore, the minimal amount of methanol meeting the complete reaction should be used. In these experiments, 25 µL (20 mg) methanol was optimal for the present concentration range of formic acid (1.56–500 µg/0.5 mL). In the putre ed blood, in which ethanol has been produced postmortem, ethyl formate can be also produced by the esteri cation reaction. References 1) Anthony RM, Sutheimer CA, Sunshine I (1980) Acetaldehyde, methanol, and ethanol analysis by headspace gas chromatography. J Anal Toxicol 4:43–45 2) Henderson MH (1982) Determination of formic acid in aqueous fermentation broth by head-space gas chroma- tography. J Chromatogr 236:503–507 3) Kuo T-L (1982) The effects of ethanol and methanol intoxication I. A simple headspace gas chromatography for the determination of blood formic acid. Jpn J Legal Med 36:669–675 4) Pla A, Hernandez AF, Gil F et al. (1991) A fatal case of oral ingestion of methanol. Distribution in postmortem tissues and fluids including pericardial fluid and vitreous humor. Forensic Sci Int 49:193–196 5) Osterloh JD, D’Alessandro A, Chuwers P et al. (1996) Serum concentrations of methanol after inhalation at 200 ppm. J Occup Environ Med 38:571–576 6) Hall BJ, Brodbelt JS (1997) Determination of barbiturates by solid-phase microextraction – SPME and ion trap gas chromatography-spectrometry. J Chromatogr A 777:275–280 7) Kumazawa T, Seno H, Lee X-P et al. (1999) Extraction of methylxanthines from human body fluids by solid-phase microextraction. Anal Chim Acta 387:53–60 8) Kumazawa T, Seno H, Watanabe-Suzuki K et al. (2000) Determination of phenothiazines in human body fluids by solid-phase microextraction and liquid chromatography/tandem mass spectrometry. J Mass Spectrom 35:1091–1099 [...]...Poisoning cases, and toxic and fatal concentrations 9) Lee X-P, Kumazawa T, Kondo K et al (1999) Analysis of methanol or formic acid in body fluids by headspace solid-phase microextraction and capillary gas chromatography J Chromatogr B 734:155–162 10) Abolin C, McRae JD, Tozer TN et al (1980) Gas chromatographic head-space assay of formic acid as methyl formate in biologic fluids: potential application... X-P, Kumazawa T, Sato K et al (1996) Detection of organophosphate pesticides in human body fluids by headspace solid-phase microextraction-SPME and capillary gas chromatography with nitrogen-phosphorus detection Chromatographia 42:135–140 19) Kumazawa T, Lee X-P, Seno H et al (1996) Extraction of local anaesthetics in human blood by direct immersionsolid phase micro extraction-SPME Chromatographia... 16) Kumazawa T, Watanabe K, Sato K et al (1995) Detection of cocaine in human urine by solid-phase microextraction and capillary gas chromatography with nitrogen-phosphorus detection Jpn J Forensic Toxicol 13:207– 210 17) Seno H, Kumazawa T, Ishii A et al (1995) Detection of meperidine (pethidine) in human blood and urine by headspace solid phase microextraction and gas chromatography Jpn J Forensic... to methanol poisoning Biochem Med 23:209–218 11) Moffat AC, Jackson JV, Moss MS et al (eds) (1986) Clark’s Isolation and Identification of Drugs, 2nd edn Pharmaceutical Press, London, pp 744–745 12) Tonkabony SHE (1975) Post-mortem blood concentration of methanol in 17 cases of fatal poisoning from contraband vodka Forensic Sci 6:1–3 13) Fraser AD, MacNeil W (1989) Gas chromatographic analysis of methyl... formate and application in methanol poisoning cases J Anal Toxicol 13:73–76 14) Tanaka E, Honda K, Horiguchi H et al (1991) Postmortem determination of the biological distribution of formic acid in methanol intoxication J Forensic Sci 36:936–938 15) Arthur CL, Pawliszyn W (1990) Solid phase microextraction with thermal desorption using fused silica optical fibers Anal Chem 62:2145–2148 16) Kumazawa T,... extraction-SPME Chromatographia 43:59–62 20) Lee X-P, Kumazawa T, Sato K et al (1997) Detection of tricyclic antidepressants in whole blood by headspace solid-phase microextraction and capillary gas chromatography J Chromatogr Sci 35:302–307 21) Lord HL, Pawliszyn J (1997) Method optimization for the analysis of amphetamines in urine by solid-phase microextraction Anal Chem 69:3899–3906 133 . concentration of methanol in 17 cases of fatal poisoning from con- traband vodka. Forensic Sci 6:1–3 13) Fraser AD, MacNeil W (1989) Gas chromatographic analysis of. Handling”. Poisoning cases, and toxic and fatal concentrations 132 Methanol and formic acid is not desirable, because it causes broadening of peaks in GC chromatograms.