Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 13)

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Introduction: Methanol (methyl alcohol) poisoning accidents take place most frequently by drinking it in mistake for ethanol. Methanol poisoning is not due to the effect 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 metabolized 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. The accumulation of formic acid...

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Nội dung Text: Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 13)

1. 4 1.4 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 effect 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. The accumulation of formic acid in the body provokes severe acidosis, which is characteristic for methanol poisoning. The metabolic (oxidation) velocity for metha- nol is about 5–10 times slower than that for ethanol. This is the reason why the poisoning symptoms do not appear soon after its ingestion, but appear after a while. Formic acid can be detected from urine for about one week after 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., film 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 flow rate: 5.0 mL/min. © Springer-Verlag Berlin Heidelberg 2005
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 sulfatea are placed in a 4 mL volume glass vial, capped with a silicon- septum cap and mixed well. ii. The vial is heated at 60°C on an aluminum block heater with stirring with a small Teflon- coated magnetic bar b. After 15 min of heating, about 0.6 mL volume of the headspace va- por is drawn into a gas-tight syringe c. Just after the vapor volume in the syringe is adjusted to 0.3 mLd 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., film thickness 0.25 µm, Supelco, Bellefonte, PA,USA) SPME devices and fibers f: 75 µm Carboxen/polydimethylsiloxane fibers (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 flow rate: 0.7 mL/min. Injection is made in the splitless mode upon inserting the SPME fiber h; it is changed to the split mode after 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. The vial is heated at 60°C on an aluminum block heater with stirring with a small Teflon- coated magnetic bar. After 5 min of heating, the holder needle of SPME is inserted into the vial through the septum, and the SPME fiber is exposed to the headspace vapor and kept there with stirring and heating at 60°C for 10 min. iiii. After the exposure, the fiber 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 fiber 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.
Analysis of formic acid 125 ⊡ Figure 4.1 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. 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 different retention times within 5 min; a few small background impurity peaks appeared. The extraction efficiency (recovery) of methanol spiked was 0.29%. Good linearity was found in the range of 50–500 µg/0.5 mL. The 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. The extraction efficiencies (recoveries)j were 0.25 % for whole blood and 0.38 % for urine. The calibration curve showed good linearity in the range of 1.56–800 µg/0.5 mL for both whole blood and urine specimens. The 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 esterified [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.
126 Methanol and formic acid ⊡ 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. • 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 flow 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. After 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. The vial is incubated at 35°C for 15 min with mixing gently several times. After 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.
Analysis of formic acid 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 fibers: 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 flow rate: 0.7 mL/min. The SPME fiber is injected into GC in the splitless mode and the splitter is opened after 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. After 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. The vial is incubated at 35°C for 5 min on an aluminum block heater. Then, the needle of the SPME holder is inserted into the vial through the septum, and the SPME fiber is exposed to the headspace vapor and kept there with stirring and warming at 35°C for 10 min. iii. After the exposure, the fiber is withdrawn into the needle and taken out of the vial; it is immediately injected into GC to expose the fiber to the carrier gas at high temperature for GC analysis. The 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 esterification. The background was clean except for the methanol peak. The extraction ef- ficiency (recovery) of formic acid spiked was 0.33 %. The calibration curve showed good lin- earity in the range of 50–500 µg (in the form of free formic acid)/0.5 mL. The 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. The peaks of methyl formate and IS appeared as big peaks; but some impurity peaks were observed in the background. The extraction effi-
128 Methanol and formic acid ⊡ Figure 4.3 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. ciencies (recoveries) of formic acid were 1.55 % for whole blood and 1.24 % for urine. The 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. The 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 different 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.
Poisoning cases, and toxic and fatal concentrations 129 ⊡ Figure 4.4 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. The acute methanol poisoning symptoms are vertigo, debility feeling, headache, nausea, vomiting and others; in rare cases, visual disturbance appears at an early stage. These symptoms usually appear 12–24 h after the injgestion, but in severe cases they can appear in about 1 h after the intake. The 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-
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, fifty seven people fell into methanol poisoning; among them, two lost their eyesight and 17 died. The 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. The toxicity of formic acid, a metabolite of methanol, is very high and induces blindness and acidosis. The concentration of formic acid in blood in methanol poisoning cases were re- ported to be 90–2,270 µg/mL [13, 14]. Notes a) The addition of ammonium sulfate to the mixture is effective to increase the extraction efficiency by the salting-out effect. b) The use of a stirrer is effective for shortening the time of the headspace extraction; heating at 60 °C is also effective to enhance the extraction efficiency. c) The septum of a vial made of silicone/Teflon 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) The internal standard calibration method is employed. At least 5–6 concentrations of methanol are plotted to confirm the linearity of the curve. e) The 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 field of forensic toxicology nowadays [6–8, 16–21]. The 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. Therefore, SPME seems very useful for analysis of drugs and poisons in forensic toxicology. f) On the surface of an SPME fiber, a liquid phase or an adsorbent material of 7–100 µm thickness is coated. A drug or a poison is extracted into the coating. The polarity and reten- tion capacity is dependent on the material of a coating and its thickness. > Table 4.1 sum- marizes SPME fibers now commercially available. The most suitable fiber should be se- lected empirically and theoretically for each compound to be analyzed. g) The SPME fibers should be pre-conditioned (aging at a high temperature for a certain in- terval) for new fibers or ones, which were not used for a long period. To protect a fiber 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 fiber, it does not produce a large volume of gas and thus does not need a large space of injection cham- ber; this is quite different from usual GC analysis with an organic solvent injection. Espe- cially for volatile compounds extracted by SPME, a large space of an injection chamber
⊡ Table 4.1 Various kinds of SPME fibers commercially available and their characteristics* Fiber Use Film thickness Liquid Adsorbent Polarity Analyte example(s) phase coexisting PDMS GC, HPLC 7 µm, 30 µm, ● – Low Hydrophobic compounds 100 µm DVB/PDMS GC, HPLC 65 µm (GC), – Porous polymer Intermediate/low Amines with short chains 60 µm (LC) 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/ GC 50/30 µm – Carbon type/ Low/intermediate/ Nasty smell of water Carboxen/PDMS polymer low 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 131
132 Methanol and formic acid is not desirable, because it causes broadening of peaks in GC chromatograms. Therefore, 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) The SPME is very suitable for splitless injection, because it does not produce a large volume of gas; the analyte is rapidly desorbed from the fiber and introduced into a capillary column. j) The internal standard calibration method is also employed for the SPME-GC analysis. Th e linearity of the calibration curve should be confirmed using at least 5–6 plots at different concentrations of methanol. On this occasion, a single fiber 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 different lot of a fiber. k) Although the extraction efficiency (recovery) of the headspace SPME is usually low, the entire amounts of methanol and IS adsorbed to the fiber can be introduced into a column. This results in relatively high sensitivity of the SPME-GC analysis. l) For esterification of formic acid, the action of concentrated sulfuric acid is required. Upon addition of the acid to an aqueous mixture, heat is produced. Therefore, 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. There- 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 esterification 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 coefficient of methyl formate on the surface of the SPME fiber. Therefore, 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 putrefied blood, in which ethanol has been produced postmortem, ethyl formate can be also produced by the esterification 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 133 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 for- mate in biologic fluids: potential application 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. Pharma- ceutical Press, London, pp 744–745 12) Tonkabony SHE (1975) Post-mortem blood 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 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, Watanabe K, Sato K et al. (1995) Detection of cocaine in human urine by solid-phase microextrac- tion 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 Toxicol 13:211–215 18) Lee 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 immersion- solid phase micro 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