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

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
.

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

> 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

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

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

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
Poisoning cases, and toxic and fatal concentrations