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

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 speci

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© 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 eff 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 Th e accumulation of formic acid in the body provokes severe acidosis, which is characteristic for methanol poisoning Th e metabolic (oxidation) velocity for metha-nol is about 5–10 times slower than that for ethametha-nol Th is is the reason why the poisoning symptoms do not appear soon aft er its ingestion, but appear aft er a while Formic acid can be detected from urine for about one week aft 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., fi 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 fl ow rate: 5.0 mL/min

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 Th e vial is heated at 60°C on an aluminum block heater with stirring with a small Tefl on-coated magnetic bar b Aft er 15 min of heating, about 0.6 mL volume of the headspace va-por is drawn into a gas-tight syringe c Just aft er 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., fi lm thickness 0.25 µm, Supelco, Bellefonte, PA,USA)

SPME devices and fi bers f: 75 µm Carboxen/polydimethylsiloxane fi 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 fl ow rate: 0.7 mL/min Injection is made in the splitless mode upon inserting the SPME fi ber h; it is changed to the split mode aft 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 Th e vial is heated at 60°C on an aluminum block heater with stirring with a small Tefl on-coated magnetic bar Aft er 5 min of heating, the holder needle of SPME is inserted into the vial through the septum, and the SPME fi ber is exposed to the headspace vapor and kept there with stirring and heating at 60°C for 10 min

iiii Aft er the exposure, the fi 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 fi 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

Assessment of both methods

blood, to which 400 µg methanol and 80 µg acetonitrile (IS) had been added Excellent peaks

of methanol and IS appeared at diff erent retention times within 5 min; a few small background impurity peaks appeared Th e extraction effi ciency (recovery) of methanol spiked was 0.29% Good linearity was found in the range of 50–500 µg/0.5 mL Th e detection limit was about

10 µg/0.5 mL

blood, to which 200 µg methanol and 2 µg of IS had been added Both peaks were separated well and appeared within 10 min Th e extraction effi ciencies (recoveries)j were 0.25 % for whole blood and 0.38 % for urine Th e calibration curve showed good linearity in the range of 1.56–800 µg/0.5 mL for both whole blood and urine specimens Th 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 esterifi 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

• 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 temperatempera-ture: 240°C; carrier gas: He; its fl 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 Aft 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 Th e vial is incubated at 35°C for 15 min with mixing gently several times Aft 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.

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 fi 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 fl ow rate: 0.7 mL/min Th e SPME fi ber is injected into GC in the splitless mode and the splitter is opened aft 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 Aft 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 Th e vial is incubated at 35°C for 5 min on an aluminum block heater Th en, the needle of the SPME holder is inserted into the vial through the septum, and the SPME fi ber is exposed to the headspace vapor and kept there with stirring and warming at 35°C for 10 min

iii Aft er the exposure, the fi ber is withdrawn into the needle and taken out of the vial; it is immediately injected into GC to expose the fi ber to the carrier gas at high temperature for

GC analysis Th e quantitation is made in the same manner as described above

Assessment of both methods

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 esterifi cation Th e background was clean except for the methanol peak Th e extraction

ef-fi ciency (recovery) of formic acid spiked was 0.33 % Th e calibration curve showed good lin-earity in the range of 50–500 µg (in the form of free formic acid)/0.5 mL Th e detection limit was 15 µg/0.5 mL

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 Th e peaks of methyl formate and IS appeared

as big peaks; but some impurity peaks were observed in the background Th e extraction effi

-Analysis of formic acid

ciencies (recoveries) of formic acid were 1.55 % for whole blood and 1.24 % for urine Th 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 Th 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 diff 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

Th e acute methanol poisoning symptoms are vertigo, debility feeling, headache, nausea, vomiting and others; in rare cases, visual disturbance appears at an early stage Th ese symptoms usually appear 12–24 h aft er the injgestion, but in severe cases they can appear in about 1 h aft er the intake Th 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

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, fi ft y seven people fell into methanol poisoning; among them, two lost their eyesight and 17 died Th 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

Th e toxicity of formic acid, a metabolite of methanol, is very high and induces blindness and acidosis Th 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) Th e addition of ammonium sulfate to the mixture is eff ective to increase the extraction effi ciency by the salting-out eff ect

b) Th e use of a stirrer is eff ective for shortening the time of the headspace extraction; heating

at 60 °C is also eff ective to enhance the extraction effi ciency

c) Th e septum of a vial made of silicone/Tefl 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) Th e internal standard calibration method is employed At least 5–6 concentrations of methanol are plotted to confi rm the linearity of the curve

e) Th 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 fi eld of forensic toxicology nowadays [6–8, 16–21] Th 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 Th erefore, SPME seems very useful for analysis of drugs and poisons in forensic toxicology

f) On the surface of an SPME fi 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 Th e polarity and reten-tion capacity is dependent on the material of a coating and its thickness > Table 4.1

sum-marizes SPME fi bers now commercially available Th e most suitable fi ber should be se-lected empirically and theoretically for each compound to be analyzed

g) Th e SPME fi bers should be pre-conditioned (aging at a high temperature for a certain in-terval) for new fi bers or ones, which were not used for a long period To protect a fi 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 fi 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 diff 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

Liquid phase

Poisoning cases, and toxic and fatal concentrations

is not desirable, because it causes broadening of peaks in GC chromatograms Th 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) Th 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 fi ber and introduced into a capillary column j) Th e internal standard calibration method is also employed for the SPME-GC analysis Th e linearity of the calibration curve should be confi rmed using at least 5–6 plots at diff erent concentrations of methanol On this occasion, a single fi 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 diff erent lot of a fi ber

k) Although the extraction effi ciency (recovery) of the headspace SPME is usually low, the entire amounts of methanol and IS adsorbed to the fi ber can be introduced into a column

Th is results in relatively high sensitivity of the SPME-GC analysis

l) For esterifi cation of formic acid, the action of concentrated sulfuric acid is required Upon addition of the acid to an aqueous mixture, heat is produced Th 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 Th 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 esterifi 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 coeffi cient of methyl formate on the surface of the SPME fi ber Th 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 putrefi ed blood, in which ethanol has been produced postmortem, ethyl formate can be also produced by the esterifi 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