volatile substance abuse—post-mortem diagnosis

Volatile substance abuse—post-mortem diagnosis

Sarah M.R. Wille, Willy E.E. Lambert*

Laboratory of Toxicology, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium

Abstract

A substantial number of children and adolescents world-wide abuse volatile substances with the intention to experience an

euphoric state of consciousness. Although the ratio of deaths to nonfatal inhalation escapades is low, it is an important and

preventable cause of death in young people. In the analytical investigation of volatile substances proper sample collection,

storage and handling are important in view of the volatile nature of the compounds. Volatile organic compounds in post-mortem

matrices such as blood, urine and tissues are generally determined by gas chromatography after extracting the compounds with

methods such as static and dynamic headspace or even with pulse-heating and solvent extraction. In post-mortem cases,

metabolites in urine seem less relevant, however, trichloroethanol and trichloroacetic acid were determined in several cases.

When interpreting qualitative and quantitative results, researchers should be aware of false conclusions. The main reason why

scepticism is necessary is the occurrence of losses of analytes during sampling, sample handling and storage, which results in

false quantitation.

# 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Volatile substance abuse; Post-mortem; Sample handling; Headspace analysis; Volatile organic compounds

1. Introduction

1.1. Prevalence

Epidemiological studies indicate that a substantial num-

ber of children and adolescents world-wide abuse volatile

substances with the intention to experience an euphoric state

of consciousness [1–3].

According to the National Institute on Drug Abuse [4,5]

use of inhalants in the US for 8th and 10th graders declined

from 21.6% in 1995 to 15.2% in 2002, while about 17%

of the adolescents have at least experimented with inhalants

[3–5]. On the contrary, inhalant abuse has emerged and

gradually increased in magnitude in most countries in Asia

and the Pacific region. In South-America about 10% of

Bolivian street youth is using inhalants. Approximately

80% of all Bolivian youth is at risk of becoming serious

inhalant abusers, which could lead to tremendous social

problems. Abuse of volatiles is also prevalent in Brazil

(24%) [2] and Mexico (22%) [5]. The prevalence of volatile

substance abuse in Colombia was about 1.7% in 1995, while

prevalence appears to be lower in the general population but

substantially higher among marginalized children in Peru. In

the UK and in Oslo, respectively, about 4–10% and 10% of

youth used volatile substances at least once [3]. In Poland

volatile substance abuse is second after heroin and morphine

abuse [3], while about 20% of the total number of registered

drug users in Budapest (Hungary) are inhalant abusers [5]. A

variety of products present at home and on working places

contain substances that can be inhaled, e.g. paints, glues,

correction fluid and gasoline. Volatile organic compounds

are appealing because they are inexpensive, legal, readily

available and easily concealed. The effect (high) occurs and

disappears relatively quickly, which forms an additional

advantage. Abuse of solvents is mostly seen in disrupted

families, economically disadvantaged groups and ethnic

minorities [5–7]. Use is not gender specific, but sustained

abuse is more common among males and 90% of deaths are

men [3]. About 70% of deaths occur under the age of 20,

with the highest number between the age of 14 and 19

[1,3,5]. Of all deaths in teenagers 5% are due to volatile

substance abuse and nearly one third of deaths resulted from

first time use [7]. More than 50% of the deaths were caused

by direct toxic effects of the substance (sudden sniffing

death), notably cardiac toxicity and respiratory depression.

Other causes included plastic bag asphyxia (21%), aspiration

Forensic Science International 142 (2004) 135–156

* Corresponding author. Tel.: þ32-9-264-81-35;

fax: þ32-9-264-81-83.

E-mail address: [email protected] (W.E.E. Lambert).

0379-0738/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.forsciint.2004.02.015

of stomach contents (18%) and trauma due to dangerous

behaviour (11%) [8]. Mostly the abuse of volatile organic

compounds is rapidly abandoned, while some degree of

inhalant use on a lifetime basis is also common. Sometimes

initiation of inhalant use precedes multiple substance abuse

[6].

Although the ratio of deaths to nonfatal inhalation esca-

pades is extremely low, it is an important and potentially

preventable cause of death in young people [1].

1.2. Modes of use

To achieve high concentrations in the lungs and the brains

for intensified and prolonged euphoria different methods

such as ‘sniffing’ (inhalation from an open container or

heated pan), ‘bagging’ (inhalation from a plastic bag) or

‘huffing’ (inhalation by using a piece of cloth soaked in a

volatile substance) can be used. Even oral use such as

drinking or squirting directly into the mouth has been

reported [6,9].

1.3. Classification

Volatile substances of abuse can be classified based on

their chemical structure [2,10], pharmacological and beha-

vioural effects [11] or even on their commercial use. As the

analytics are an important issue in this particular review,

chemical classification is of most interest. The chemical

structure of a compound will affect the whole analytical

process, e.g. sample preparation, chromatographic separa-

tion and detection. Inhaled volatile substances fall into

several chemical families, notably hydrocarbons, oxyge-

nated compounds and halogenated compounds (Table 1).

The hydrocarbons can be subdivided into aliphatic hydro-

carbons such as propane, butane, gasoline and aromatic

hydrocarbons such as toluene, xylene and ethyl benzene,

while oxygenated compounds are subdivided in ethers,

esters, alcohols, nitrites and ketones. Nitrites, ‘poppers’,

are a class of products mostly used in dancing clubs and

in the gay-community to enhance sexual activity. The group

of the halogenated compounds is a mixture of products,

which include several anaesthetics.

1.4. Toxicity

Because of the abuse of a wide variety of inhalants it is not

surprising to find a tremendous range of acute and chronic

physical problems [6]. The lipid solubility and volatility of

the compounds enhance their toxicity. Due to the extensive

capillary surface area of the lungs [6] blood levels peak

already within a few minutes after exposure. Because of their

lipid solubility volatiles easily cross lipid membranes and

distribute to well-perfused organs such as brains, liver, heart

and kidneys [10]. This condition will be retained if sudden

death occurs, but if exposure continues the compound will

slowly accumulate in poorly perfused tissues such as muscle

and fat [2]. The toxicity of volatiles depends on the com-

pound itself and on the magnitude, duration and the route of

exposure. Abused inhalants have different mechanisms for

their toxicological effects [11]. The most important toxico-

logical effects are those observed for the heart, lungs, kidney,

neurological system, liver and bone marrow.

1.4.1. Cardiovascular toxicity

Cardiac arrest is the most common cause of sudden death

in volatile substance abuse. Mostly it is caused by cardiac

arrhythmias due to sensitisation of the myocard to catecho-

lamines following exercise, fright or sexual activity. Stimu-

lation of the nervus vagus, due to spraying aerosol

propellants in the mouth, may lead to inhibition of the heart

and subsequent bradycardiac arrest, which is another cause

of sudden death [3]. Myocardial ischemia caused by cor-

onary vasospasm has also been hypothesised as a mechan-

ism. When nitrites are used a vasodilatation and pooling of

blood into the extremities can cause orthostatic hypotension

and syncope [3].

1.4.2. Neurological toxicity

Because of their lipophilic character, volatiles have a

serious impact on the brain and other parts of the nervous

Table 1

Volatile organic compounds classification

Hydrocarbons Oxygenated compounds Halogenated compounds

Aliphatic Aromatic Esters Alcohols Ethers Ketones Nitrites Anaesthetics Other

Propane Toluene Isoamylacetate Butanol tert-Butylether Acetone n-Butylnitrite Desflurane Chloral hydrate

Butane Ethylbenzene Butylacetate Propanol Dimethyl ether Butanone Amylnitrite Sevoflurane Tetrachloro-ethylene

Neopentane Xylene Pentylnitrite Isoflurane Trichloro-ethylene

Heptane Benzene Enflurane Methylene-chloride

Hexane Halothane Difluoroethane

Pentane Bromochlorodifluoromethane

Ethylchloride

Dichlorodifluoromethane

Trichlorofluoromethane

Adapted from [2,10,12].

136 S.M.R. Wille, W.E.E. Lambert / Forensic Science International 142 (2004) 135–156

system, especially in chronic users. Most volatiles, espe-

cially anaesthetics, act as a central nervous system depres-

sant [13]. The neurotoxic effects of prolonged inhalant abuse

include neurological syndromes that reflect damage to parts

of the brain involved in controlling cognition, movement,

vision and hearing. Cognitive abnormalities can range from

mild impairment to severe dementia [4].

1.4.3. Pulmonary toxicity

Volatile substances may evoke sudden death through

asphyxiation or they can cause direct damage to the pulmon-

ary tissue. Asphyxiation can be caused by oxygen displace-

ment, aspiration of vomit or suffocation in the used plastic

bag. Respiratory arrest may be caused by central nervous

respiratory depression or by vagal stimulation. Direct damage

of the pulmonary tissue can lead to physiologic and anatomic

pulmonary abnormalities. These abnormalities can be caused

by fire-breathing (filling the mouth with (butane) gas and

igniting it while exhaling), metabolites of the products or by

the products themselves [3].

1.4.4. Nephrotoxicity, hepatotoxicity and bone marrow

toxicity

Chronic exposure can produce significant damage not only

to heart and lungs but also to the liver and kidneys. There are a

variety of renal disorders due to the used compound or as a

result of metabolic transformation. Although metabolism

normally results in detoxification, enhanced toxicity may

also result. Carbon tetrachloride, chloroform, dichloro-

methane, n-hexane, trichloroethylene and possibly halothane

can cause the formation of toxic metabolites [2]. Free radicals

originated from metabolism can lead to hepatotoxicity by

causing peroxidation of the hepatocyte cell membranes [10].

Bone marrow suppression is a result of chronic toxicity of

volatile substances (e.g. benzene) and leads to several hae-

matological changes while abuse of nitrite inhalants is asso-

ciated with methemoglobinemia [14]. Other problems such as

peripheral neuropathy, peri-oral eczema, burns and gastro-

intestinal problems by swallowing the products can also occur

[3,6].

1.5. Strategy

This review will discuss the analytical methods to detect

volatile substances in post-mortem samples such as blood,

heart, kidney, brain, liver and urine. Volatiles are a wide-

spread group of compounds that can be subdivided in related

chemical structures (Table 1). However, it is more relevant to

discuss the analytical methods rather than the different

compounds, because one analytical method mostly detects

several compounds.

Analysis of a sample can be based on direct detection of

the compound itself in blood or tissues such as heart, liver,

kidney and brain, but also on detection of metabolites,

especially in urine [14]. Blood is the most interesting matrix,

but due to their lipophilic character volatile organic com-

pounds are detected also easily in brain tissue. Due to its high

fat content the brain is a reliable source of sampling, and is it

is more resistant (unless there is a severe trauma) to post-

mortem decomposition [15].

Analysis of urinary metabolites may extend the detection

window of inhalant abuse. However, of the compounds

commonly abused only the metabolites of toluene, trichlor-

oethylene, xylene and benzene i.e. hippuric acid, trichlor-

oacetic acid, methylhippuric acid and phenol can be used

[2,14,56]. In post-mortem analysis, however, metabolites

seem less important. In literature, only chloral hydrate,

tetrachloroethylene and trichloroethylene metabolites seem

worth to analyse [28,49,50,52–54].

2. Storage and sample handling

In the analytical investigation of volatile substances

proper sample collection, storage and handling are impor-

tant, in view of the chemical properties and volatile nature of

the compounds. However, useful qualitative results can also

be obtained if the conditions for sample storage and handling

are less ideal [2].

Loss of the volatile substances by evaporation is one of

the main causes of difficulties in quantitative analytical

procedures. Therefore analysis of the sample should start as

soon as possible. Volatile substances diffuse from the

sample into the atmosphere until equilibrium is reached.

Every time the container is opened, losses occur due to the

replacement of air. Losses may also occur if an aliquot of

the sample is taken without opening the container (piercing

through the septum) due to negative pressure, which will be

revealed after introduction of air. Every time fresh air

comes inside the container, diffusion of the compounds

will create a new equilibrium. Samples should therefore be

stored in gas-tight, good sealed containers with minimal

headspace. Addition of an internal standard to the sample

immediately after autopsy will minimise experimental

errors due to evaporation during storage or tissue homo-

genisation [16]. A good seal also protects the sample

against contamination from environmental and laboratory

sources of volatiles [17–19]. Storage, transport and hand-

ling of the sample should always occur at approximately�5

till 4 8C. Lower temperatures should be avoided for long-

term blood sample storage due to formation of n-hexanal

from degradation of fatty acids. n-Hexanal often leads to

interferences in the analysis of low toluene levels [20],

however, in intoxication cases the contribution remains

limited. Samples should only come in contact with inert

materials such as glass, teflon or aluminium foil. Soft

rubber stoppers should be avoided due to their high affinity

and permeability for toluene [21].

For non-post-mortem blood samples an anticoagulant

(lithium heparin) is required [19]. Tubes containing EDTA

and gel-separators should be avoided as false positive

results for xylene, ethylbenzene, toluene and 1-butanol

S.M.R. Wille, W.E.E. Lambert / Forensic Science International 142 (2004) 135–156 137

have been reported due to the presence of gel-separators,

while 1-butanol and 2-methyl-2-propanol have been

demonstrated in tubes coated with EDTA [19,22]. Addition

of sulphuric acid or sodium fluoride [2,20] is advised when

esters such as ethyl acetate are present in the sample to

abolish esterase activity. Sodium azide prevents growth of

micro-organisms [2,14,18,19,23].

3. Analytical techniques

3.1. Introduction

Volatile organic compounds are generally determined by

gas chromatography resulting in qualitative as well as

quantitative data. First, sampling, introduction of the sample

and the analytical separation will be discussed. Thereafter

the detection of the compounds will receive attention.

3.2. Sampling and introduction into the analytical column

3.2.1. Extraction techniques [16,23]

Today, sample handling and introduction aim to increase

sample load and thus sensitivity. It should be simple, effi-

cient, inexpensive and minimize the use of chemicals [24].

Therefore, headspace techniques have become very popular

in the past years. The headspace technique can be divided in

two modes: the static mode (headspace and variants (HS)

such as solid phase microextraction (HS-SPME), cryogenic

oven trapping (HS-COT), cryogenic focusing (HS-CF)) and

the dynamic mode, also referred to as purge and trap [16].

Other extraction techniques are pulse-heating and solvent

extraction. Modifications of ‘traditional’ headspace are

introduced to enhance sensitivity by concentration effects

(SPME) or increase of the volume injected into the column

(COT/CF). Changes are also made to improve quantification

by elimination of matrix effects (HS-multiple headspace

(MHE), HS-full evaporation technique (FET) and standard

addition (SAM)). These modifications will be discussed later

(see quantitation modes). Headspace techniques all have the

advantage of avoiding contamination of the chromato-

graphic system by non-volatile substances originating from

the sample matrix [25,26].

3.2.1.1. Static headspace (HS). The static mode refers to

partition and eventually equilibrium of the analyte between

the sample and the gas phase in a closed system. After

equilibrium is achieved, the vial is pressurised and an aliquot

of headspace air is sampled and injected into the gas

chromatograph. The headspace procedure is simple, mini-

mises the number of artefacts during analysis and can

measure water soluble compounds effectively. It is, however,

less sensitive for highly water soluble compounds [16]. If the

sample is a liquid, such as blood, urine or gastric content,

complete equilibrium is obtained relatively easy. The sam-

ple, mostly after addition of distilled water and an internal

standard solution, is heated for a period of time. Agitation of

the sample and addition of salt may be used to accelerate

evaporation. After heating, an aliquot of headspace air is

injected in the column either manually (needle) or with an

autosampler and injection loop. Tissues such as liver, kidney,

adipose tissue, lungs and brain are suitable matrices for the

determination of volatile organic compounds in post-mor-

tem sampling. They are analysed by following the same

procedures as the liquid samples. Sometimes, as in the

analysis of bromochlorodifluoromethane, butane and a

few other components, the tissues are treated with a pro-

teolytic enzyme such as Subtilisin A [20]. Homogenisation

using an Ultra-Turrax may also be necessary [27,28]. To

avoid evaporation of the compounds, homogenisation should

always occur at low temperature and in closed containers

[16] (Table 2).

3.2.1.2. HS-chemical (HS-c). El-Haj et al. [29] described a

chemical transformation of toluene and ethyl benzene to

relatively non-volatile products to overcome interferences in

the detection with flame ionisation detection (FID) or Four-

ier transform infrared detection (FTIR). Benzotrichloride is

almost quantitatively formed when toluene reacts with

chlorine gas under UV-light exposure. In the presence of

water, it is converted to benzoic acid and hydrochloric acid.

Under UV-light and in the presence of water ethyl benzene

reacts with chlorine to form benzoic acid, 1- and 2-phenyl

ethanol.

After addition of sodium chloride, headspace was per-

formed and 10 ml of headspace gas was introduced into a

chlorine gas containing vial. This was placed in the sun for

10 min and after addition of water and an evaporation step,

BSTFA (N,O-bis trimethylsilyl trifluoroacetamide) was

added. After heating the sample, an aliquot was injected

(Fig. 1).

3.2.1.3. HS-solid phase microextraction (HS-SPME). Solid

phase microextraction is a sampling and concentration

technique based on the principle of ‘likes dissolves likes’,

thus the affinity of the compound for a fibre and partitioning

of analytes between the solid phase (fibre) and the matrix

[30]. Sorption modes are direct immersion and headspace

sampling. The main considerations for mode selection are

the nature of the sample matrix, the analyte’s volatility and

the affinity for the matrix [31]. Direct immersion of the fibre

into the sample is not relevant here due to the physicochem-

ical characteristics of the analytes discussed in this review.

The time and temperature of conditioning, extraction and

desorption can vary. An overview of typical conditions is

given in Table 3. The most applied fibre is coated with a 100-

mm polydimethylsiloxane phase (PDMS) for absorption of

apolair compounds such as toluene and halothane. A poly-

acrylate 85-mm fibre was used for absorption of hydrolysed

n-butylnitrite [24,32] to overcome specific problems of

extracting polar analytes from a polar matrix. One case

described the use of a Carboxen/PDMS (75 mm) fibre to


Table 2

Analytical data for HS extraction

Reference Matrix Compounds Add./IS HS time

(min)

HS temp.

(8C)

Inj. Comments Inj. vol. (ml)

[20] Blood, urine,

tissue

Bromochlorodifluoromethane,

butane, FC11, FC12, isobutane,

propane, dimethylether

Tissue: Subtilisin A

IS: ethylbenzene,

1,1,2-trichloroethane

15 65 Manual Warm needle (10 min on

heating block)

0.10–0.30

[25] Blood Isoflurane IS: 1,4-dioxane 30 55 Autosampler Needle temperature: 120 8C 1.0

[32] Urine, gastric

content

Methylene chloride, volatile

petroleum contents, (ethanol)

Sodium chloride

(urine), IS: t-butanol

20 60 Manual 0.250

[40] Blood Hydrocarbons IS: toluene d8, cold water 20 60 Manual 2

[42] Blood Halothane, enflurane, isoflurane,

sevoflurane

IS: dichloromethane 15 55 Manual 1

[43] Blood Hydrocarbons, esters, aldehydes,

ketones, ethers, (alcohols)

Potassium fluoride,

sodium chloride

33 50 Autosampler Valve/loop temperature: 54 8C,

Aux. gas pressure: 130 kPa,

vial pressurisation time: 15 s,

carrier gas: He 70 ml/min

1

[58] Blood, lung,

gastric content,

urine, bile, brain,

liver, kidney,

vitreous humor

Methyl-2-pentane,

methyl-3-hexane,

methylcyclohexane, heptane

20 80 Autosampler Needle temperature: 90 8C,

loop temperature: 100 8C, vial

pressurisation time: 2 min,

carrier gas: He 10 psi,

pressurisation pressure: 17 psi

0.070

[59] Blood Desflurane, sevoflurane,

isoflurane, enflurane, halothane

Anticoagulant, IS:

1,4-dioxane

Autosampler

[63] Blood, urine,

tissue

Ethylchloride Sodium chloride,

IS: 1-propanol

5 37 0.7

[64] Blood, brain,

lung, liver, spleen,

kidney, muscle,

adipose tissue

Enflurane Distilled water,

IS: 1,4-dioxane

30 55 Autosampler Vial pressurisation time:

0.13 min, pressurisation:

124 kPa, transferline and loop

temperature: 100 8C, loop fill

time: 0.15 min, injection

time: 0.20 min, loop

equilibration time: 0.15 min

1

[65] Blood, urine Toluene Sodium citrate solution, IS:

isobutanol

20 55 Autosampler 0.250

[66] Blood, urine Difluoroethane IS: 1-propanol 15 37 Manual 0.3

[67] Blood, brain, lung Propane Fluoride (blood) Manual 0.5

[68] Blood, urine, lung Diethylether, hydrocarbons Sodium chloride 26 80 Autosampler Valve/loop temperature: 85 8C,

Aux. gas pressure: 130 kPa,

vial pressurisation time: 15 s,

sample loop V: 1 ml, vent loop

fill time: 1 s, injection time: 2 min

[69] Blood Hydrocarbons Cold water, IS 20 60 Manual

[70] Blood, urine Toluene Sodium citrate solution,

shaken, IS: 1,4-dioxane

20 60 Autosampler Valve and transferline

temperature: 140 8C1.0

S.M

.R.

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Table 2 (Continued )

Reference Matrix Compounds Add./IS HS time

(min)

HS temp.

(8C)

Inj. Comments Inj. vol. (ml)

[74] Brain, liver,

lung, blood

Chlorodifluoromethane IS: genetron 502, shaken 60 RT

[75] Heart, lung, brain,

liver, blood,

spleen, kidney

Trichlorofluoromethane IS: dichloromethane 30 40 Manual

[76] Blood, lung Methylchloroform 60 20

[77] Blood, brain, heart Dichloromethane, chloroform NaCl, IS: sec-butanol 20 55 0.250

Metabolites

[28] Blood, tissue Tetrachloroethylene,

trichloroethylene,

trichloroacetic acid

IS: chloroform

[52] Blood, lung,

liver, kidney,

stomach content

Trichloroethylene,


IS: n-butanol, NaCl,

water, vortex, sonication

35 80 Autosampler Loop temperature: 110 8C,

transferline temperature 120 8C

HS quantification

[44] Blood, tissue Acetone, 2-propanol, 2-butanone,

tert-butylether, benzene, toluene,

ethylbenzene, propyl-benzene,

o-, p-, m-xylene

28 130 (FET),

80 (MHE)

Aux. gas pressure: 210 kPa, not

more than 10 mg of compound

HS chemical

[29] Blood Toluene, ethylbenzene Distilled water 3–5 65 2

HS followed by COT

[35] Blood, urine Ethylacetate, benzene, butan-1-ol,

toluene, butylacetate, isoamyl-

acetate (¼thinner components)

Distilled water, stirring

bar, heparin sodium (in

blood), IS: ethylbenzene

30 90 Manual 5

[39] Blood o-, m-, p-Xylenes Distilled water,

IS: aniline

30 100 Manual 2

[60] Blood, lung Propane, isobutane, butane,

neopentane, n-pentane,

bromochlorodifluoromethane

10 50 0.100–0.250

[61] Blood Sevoflurane, isoflurane,

enflurane, halothane

Distilled water,

IS: halothane

15 55 Manual 10

[62] Blood Chloroform, methylene chloride Distilled water,

IS: methylenechloride,

chloroform

20 55 Manual 5

HS followed by Tenax-trap

[37] Blood n-Propane, isobutane, n-butane IS: t-butyl-methyl ether 20 60 Autosampler,

Tenax-trap

Vial pressurisation time: 1 min 0.5

Abbreviations: Add., addition to headspace vial; IS, internal standard; HS time, headspace heating time; Aux., auxiliary; HS temp., headspace heating temperature; FET, full evaporation

technique; Inj., injection mode; MHE, multiple extraction method; Inj. vol., injection volume; RT, room temperature.

14

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.M.R

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extract chloroform and methylene chloride from blood and

urine [73].

The extraction can be accelerated by agitation, addition of

salts or through heating (the rate of absorption decreases,

while the rate of release increases with higher temperature)

[30]. In case of highly volatile compounds equilibration

times are short even without agitating [33].

SPME can be applied to small sample volumes and is a

solvent-free, simple and rapid technique [15]. There is no

intense air peak in the chromatogram often observed

with headspace direct injection [30]. The clean extracts

enhance reproducibility [32], selectivity [31] and sensitiv-

ity [30].

However, the absorption capacity is more sensitive for

highly volatile and low molecular mass compounds. Some-

times a cooling device is applied to create better peak shapes

[34], thereby combining SPME and cryogenic oven trapping

[24,34].

3.2.1.4. HS-cryogenic trapping (HS-COT, HS-CF). HS-

cryogenic trapping is a combination of HS or HS-SPME

and a low temperature of the whole column (COT) or the

sample-introduction region at the inlet of the column (CF) to

trap volatile organic compounds [35]. A microcomputer-

controlled device can lower the oven temperature to less than

0 8C with use of liquid carbon dioxide or liquid nitrogen.

Fig. 1. Extracted ion chromatogram (A) and EI mass spectrum (B) of the TMS derivative of the benzoic acid obtained from the reaction of

headspace of a blood sample obtained from a glue abuser with chlorine/sunlight and then water. The molecular ion mass of 194 and the base

peak ion mass of 179 were used in the extraction of chromatogram. Source: [29] Fig. 4 (p. 393).


Table 3

Analytical data for HS-SPME extraction

Reference Matrix Compounds Cond. time/temp. HS time/temp. Ad. time/temp. Desorp. time/temp. Fibre Add.

[15] Blood, lung,

brain, fat

VOC, incl xylene, butane,

halothane, toluene,

petroleum residues

5 min/200 8C 1.5 h/80 8C 30 min/– 1 min/220 8C 100 mm PDMS Distilled water,

IS: cyclohexanone

[27] Blood, urine,

tissues

Tetrachloroethylene,

trichloroethylene,


5 min/60 8C 1 min/60 8C 15 s/250 8C 100 mm PDMS Ultra Turrax, IS:

carbontetrachloride

[32] Urine, gastric

contents

Methylenechloride,

volatile petroleum

products (ethanol)

5–10 min/260 8C 20 min/60 8C 10 min/60 8Curine, 5 min/

60 8C gastric

1 min/225 8Curine, 1 min/

250 8C gastric

100 mm PDMS,

85 mm PA

Sodium chloride in

urine, IS: t-butanol

[70] Blood, urine Toluene 20 min/60 8C 1 min/60 8C 1 min/250 8C 100 mm PDMS 0.1% sodium azide,

shaken (rpm 85), IS:

1,4-dioxane,

(iso-butanol)

[71] Blood, urine Thinner components:

toluene, benzene,

n-butylacetate, n-butanol,

n-isoamylacetate

1 h/250 8C 15 min/80 8C 5 min/80 8C 3 min/200 8C 100 mm PDMS Distilled water,

magnetic stirring bar,

IS: ethylbenzene

[72] Blood, liver,

kidney, brain,

urine, bile,

stomach contents

Halothane, isoflurane,

chloroform, diethylether

1 h/250 8C 15 min/100 8C 15 min/100 8C 2 min/250 8C 100 mm PDMS Ammonium

sulphate, sulphuric

acid solution

[73] Blood, urine Chloroform,

methylene chloride

10 min/30 8C 20 min/30 8C 2 min/240 8C 75 mm carboxen/

PDMS

Distilled water,

shaken 10 s IS:

methylene chloride,

chloroform

SPME followed by COT

[24] Blood, urine Hydrolysed n-butyl-nitrite 30 min/155 8C 1–3 h/20-23 8C 5–20 min/60 8C 1 min/240 8C 100 mm PDMS,

85 mm PA

IS: n-propanol

[34] Blood Hydrocarbons 2 min/�5 8C 300/�5 8C 3 min (1 min

sampling)/250 8C(2 min purging)

100 mm PDMS VOC-free water,

IS: toluene d8

Abbreviations: SPME, solid phase micro extraction; Ad. time, adsorption time; Cond. time, conditioning time; Ad. temp., adsorption temperature; Cond. temp., conditioning temperature;

Desorp. time, desorption time; HS time, headspace time before fibre introduction; Desorp. temp., desorption temperature; HS temp., headspace temperature; Add., addition in headspace vial;

VOC, volatile organic compounds; PA, polyacrylate; PDMS, polydimethylsiloxane.

14

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The temperature needed to trap the volatiles, depends on the

volatile in question [36] (Table 4). While in the headspace

technique only a maximum of 1 ml of headspace air could

be injected into narrow bore columns in the split mode,

injection of 2 till 10 ml of the headspace air was described

for medium bore columns in the splitless mode without any

loss using the HS-COT method [36] (Fig. 2).

Cryogenic trapping is more than ten times more sensitive

than SPME due to higher possible sample load and better

peakshape [36]. When carbon dioxide is used to cool the

column, one has to be aware of the danger for suffocation

due to large carbon dioxide leaks [36].

Not always the whole column is cooled (CF). Bouche et al.

[37] have described the determination of n-propane, n-

butane and isopropane in post-mortem blood by combination

of static HS with cold trapping on a Tenax sorbent followed

by flash desorption to enhance sensitivity, while Tytgat and

Daenens [24] cooled the inception of the column with liquid

carbon dioxide using SPME as extraction method for hydro-

lysed n-butyl nitrite.

3.2.1.5. Headspace dynamic mode: purge and trap. The

dynamic mode is accomplished by purging a carrier gas

constantly above the sample and trapping the evaporated

volatiles in a cryogenic and/or adsorbent trap [16,23].

Extensive heating afterwards releases the analytes from

the trap into the column. The dynamic procedure is effec-

tive for compounds with moderate to high water solubility

and the concentration step, ‘the trapping’, enhances sensi-

tivity. It is, however, difficult, costly and artefacts due to

impurities in the purging gas occur. Attention has to be paid

to contamination of the trap and to occurrence of leaks

[24]. Precise quantification suffers from incomplete recov-

ery after purging, trapping and desorption [16]. Recoveries

are increased by heating the sample and increasing purge

time [26]. For apolar compounds such as toluene lower

detection limits are obtained than for polar ones (propanol,

acetone, isopropanol) partly due to the fact that purging

and trapping of polar compounds is less efficient [38].

Although Watanabe-Suzuki and co-workers [36,39] sug-

gest that the purge-trapping technique is not suitable for

biological samples due to foaming, it seems to offer better

recoveries and lower detection limits in the analysis of

volatile organic compounds as compared to headspace

extraction [26]. Ojanpera et al. even applied the technique

to the analysis of tert-butylmethyl ether and 1,1,2-trichlor-

oethene in post-mortem blood samples after addition of

Antifoam B emulsion. A purge-trap concentrator equipped

with a cooled Tenax-trap (2,6-diphenyl-p-phenylene oxide

polymer) was used [38]. Despite the relatively low specific

surface area of the Tenax-trap, it has a high sorption

capacity and thermal stability. The retention of water is

relatively small, thus it is possible to trap compounds

efficiently from water saturated headspace gases [23].

Water can cause problems, especially if a cryogenic trap

is used. Morinaga et al. [40] described a purge and trap

method with a Tenax TA-trap combined with a dehydrata-

tion agent Chromosorb G for the analysis of toluene,

benzene, xylenes and ethylbenzene in blood. Tenax-traps

can also be used in combination with static headspace as

described by Bouche et al. [37] (Table 5).

3.2.1.6. Solvent extraction. Although headspace and solid

phase microextraction are the most commonly used sam-

pling techniques over the past few years, solvent extraction

is used for chloral hydrate, trichloroethylene and their

metabolites. Solvent extraction, however, is time consum-

ing, laborious and more difficult to automate. Interferences

Table 4

Analytical data for HS-COT/CF extraction

Reference Matrix Compounds Extr. method Cooling gas Oven temp. (8C) Comments

[24] Blood, urine Hydrolysed n-butyl nitrite SPME Liquid CO2 �5 to 5 inception

of the column

[34] Blood Hydrocarbons SPME Liquid CO2 �40

[35] Blood, urine Ethyl acetate, benzene,

butan-1-ol, toluene, butyl

acetate, isoamyl-acetate

HS Liquid CO2 5

[37] Blood n-Propane, n-butane,

isopropane

HS þ Tenax Liquid N2 �80 cooled

inlet-liner

(Tenax-trap)

Tenax-trap heated

to 200 8C for

6 min

[39] Blood o-, m-, p-Xylenes HS Liquid CO2 5

[60] Blood Propane, butane, isobutane HS NS 0–10



HS NS �40

[62] Blood Chloroform, methylene

chloride

HS Liquid CO2 �30

Abbreviations: HS-COT, headspace cryogenic oven trapping; Extr. method, extraction method; HS-CF, headspace cryogenic focussing; Oven

temp., oven temperature; HS, headspace; SPME, solid phase microextraction; NS, not specified.


Fig. 2. Left: Schematic diagram of the instrumentation used for cryogenic oven-trapping (COT)-capillary GC (A) and for cryo-focusing (B).

The COT conditions for analysis of chloroform or dichloromethane are shown. For cryo-focusing the chamber can be cooled to �180 8C(minimum) with gas from liquid N2 and down to �90 8C with gas from liquid CO2. Right: Headspace capillary GC for chloroform (peak 2)

and dichloromethane (peak 1) as a function of initial oven temperature. Each compound (5 mg) was added to 0.5 ml human whole blood for

headspace extraction. Source: [36] Fig. 1 (p. 76), Fig. 2 (p. 78).


Table 5

Solvent-, pulse-heated purge and trap extraction

Reference Matrix Compounds Add./IS Extraction

liquid

Vortex

time

Centrifuge

Solvent extraction

[49] Blood, liver,

kidney, stomach

contents

Chloral hydrate,

trichloroethanol,

trichoroacetic acid

Water, sulphuric

acid, IS: 1,3

dichloro-2-propanol

Diethylether 2 min 10 min,

2500 � g

[50] Blood, urine,

stomach content

Chloral hydrate,

trichloroethanol

IS:

tetrachloroethylene

Chloroform 30 s 30 s,

10000 rpm

[53] Blood, liver,

kidney, lung,

stomach contents

Trichloroethylene,

trichloroethanol,


Water, sulphuric

acid, IS: 1,3

dichloro-2-propanol


2500 � g

[54] Blood, urine Trichloroethanol

(chloral hydrate,

trichloroacetic acid)


2500 rpm

Reference Matrix Compounds Add./IS Heating

time (s)

Ferromag.

cond. temp.

Oven

temp. (8C)

Needle

temp. (8C)

Pulse-heated extraction

[42] Blood Halothane, enflurane,

isoflurane, sevoflurane

4 160 170 200

Reference Matrix Compounds Add./IS Gas flow

(ml/min)

Purge time

(min)

Dry purge

time (min)

Line/valve

temp. (8C)

Trap Extraction

temp. (8C)

Trap

temp. (8C)

Desorption

time/temp.

Sample

P (kPa)

Trap

P (kPa)

Purge-trap extraction

[38] Blood Aceton, isopropanol,

1-propanol,

1,1,1-trichloro-ethane,

isoflurane, diethylether,

tert-butanol, toluene,

isobutylmethylketone,

ethylacetate

Antifoam b He 35 10 6 Tenax

1/8 in. � 12 in.

�20 4 min/

225 8C140 40

[40] Blood Hydrocarbons Cold water,

IS: toluene d8

N2 500 Tenax TA

(60/80 mesh)

40 220 8C

[57] Blood 27 VOC’s Antifoam b, NaF,

IS: diethylketone

He 38 15 6 150 Tenax

1/8 in. � 12 in.

Room

temperature

�20 2 min/

225 8C140 50

Abbreviations: Add., addition; Ferromag. cond. temp., ferromagnetic conduction time; temp., temperature; IS, internal standard.

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in the chromatographic run can occur because of co-extrac-

tion of non-volatiles or due to the solvent front [16]. Precise

determination, especially of low boiling compounds, cannot

be expected [16] (Table 5).

3.2.1.7. Pulse-heating. Although post-mortem relevance

was not quite clear in the cited reference [26], pulse-heating

can be a possible detection method for volatiles such as

anaesthetics in post-mortem samples. Alcohol is not dis-

cussed in this review, however, pulse-heating was used for

the determination of alcohol in post-mortem blood, urine

and synovial fluid [41]. Pulse-heating of a biological sample

is performed by a curie point pyrolyser, which is a ferro-

magnetic coil quickly heated up by electromagnetic induc-

tion [26]. After introduction and heating of the sample in the

pyrolyser, volatiles can evaporate from the matrix directly

into the column, without interference of the macromolecules

present in the matrix. Advantages include the small sample

volume needed, short extraction time and especially the

elimination of matrix effects [26,42] (Table 5).

3.2.2. Sample introduction

The sample is mostly introduced in the splitless mode

because of the higher sensitivity as compared to the split

mode. However, also split and programmed temperature

vaporisation (PTV) are possible injection modes.

3.3. Separation

3.3.1. Columns

At first, packed columns were used to determine volatile

compounds [16]. Currently, almost all laboratories use

capillary columns due to practical advantages and improved

sensitivity (partially caused by a new sample introduction),

efficiency, reproducibility and reliability [20]. Therefore we

only discuss analytical methods using capillary columns. In

capillary columns the length, film thickness and stationary

phase all influence the retention and separation of the

compounds. The phases used (Table 6) vary from polar

(polyethylene glycol) to apolair (dimethylpolysiloxane),

due to the broad range of polarity (e.g. toluene versus

acetone) of the target compounds. The length is mostly

30 m, but can vary depending on separation requirements.

Increase of column length results in better resolution, but

also in an increased runtime and column bleed, which causes

lower sensitivity.

Volatiles are analysed on columns with film thickness

between 0.25 and 10 mm. On the latter, volatiles are retained

longer resulting in an enhanced resolution. However, also a

higher column bleed occurs. When selecting the internal

diameter of the column (0.25, 0.32 or 0.53 mm), attention

has to be paid to different aspects. The maximum sample

volume that can be loaded depends on the film thickness and

the column internal diameter. The smaller the diameter, the

faster the analysis and the better the resolution. A 0.25-mm

internal diameter is advised for MS use.

3.3.2. Temperature programme

The temperature programme is either isothermal or

(mostly) programmed depending on the number and the

type of compounds to be separated (Table 6).

3.4. Detection

Usually electron capture detection (ECD), flame ionisa-

tion detection and mass spectrometry (MS) (SIM-mode)

are used for quantitation purposes, while MS an Fourier

transform infrared are used for identification purposes

(Table 6).

3.4.1. Non-spectral techniques

3.4.1.1. ECD. Electron capture detection is applied for

compounds with high electron affinities such as halogenated

substances and compounds containing nitro- and keto-func-

tions: e.g. bromochlorodifluoromethane, trichloroethylene,

hexane-2,5-dione and amylnitrite. ECD is very sensitive, but

has a more narrow linear range as compared to FID [20].

3.4.1.2. FID. Flame ionisation detection is sensitive to

hydrocarbons in general. It has a wide linear range and

excellent baseline stability. As seen in Fig. 3, the selectivity

and sensitivity of detection largely depends on the com-

pound and detection technique used (ECD versus FID).

3.4.2. Spectral techniques

3.4.2.1. MS. Although low molecular compounds often give

less specific mass spectra due to background interferences

and similar spectra of related compounds (e.g. butane and 2-

methylpropane, Fig. 4A), GC–MS has become very popular

to detect volatile organic compounds. It is applied for

screening with identification based on relative retention

and spectral information, and for quantitation, especially

in the single ion monitoring (SIM) mode [43].

3.4.2.2. FTIR. FTIR often gives more informative spectra

especially for low molecular weight compounds (e.g.

butane, acetone, ethylacetate, Fig. 4B) but sensitivity is

lower and interferences due to water and carbon dioxide

can be troublesome [20].

3.5. Quantitation

Determination of the initial concentration of volatiles in

the sample matrix can be done through the measurement of

the equilibrated vapour phase concentration and the partition

coefficient.

However, the most common method for quantitation is

based on standard calibration curves and internal standardi-

sation [16]. The interaction of analytes with matrix compo-

nents can result in binding of the volatile organic compounds

thus becoming unavailable for determination in the gas


Table 6

Analytical data of separation and detection modes

Reference Matrix Compounds Extr.

method

Inj. temp.

(8C)

Inj. M Inj. vol. Column Gas

flow

Oven Detector Det.

limit

Quant.

limit

Derivat.

[20] Blood, urine,

tissue

Bromochlorodifluoromethane

butane, FC11, FC12,

isobutane, propane,

dimethylether (224 comp)

HS 150 Splitless 0.10–

0.30 ml

SPB-1, 60 m �0.53 mm � 5 mm

He

8.6 cm3/

min

40 8C (6 min)–5 8C/

min!80 8C;

80 8C–10 8C/

min!200 8C

FID/ECD

[25] Blood Isoflurane HS 100 Split

1:30

1.0 ml DB 5, 30 m �0.25 mm �0.25 mm

He 40

cm/s

35 8C (1.4 min)–

40 8C/min!80 8C(2 min)

MS 1.2 mg/ml 4.7 mg/ml

[28] Blood, tissue Tetrachloroethylene,

trichloroethanol,


HS 150 ECD: WCOT

SPSIL5CB,

25 m � 0.53 mm

N2 3 ml/

min

ECD: 65 8C(2 min)–10 8C/

min!130 8C(1.5 min)

ECD 0.25 mg/ml 0.36 mg/m1 BSTFA

MS: BP20, 25 m �0.22 mm � 0.25 mm

MS: 45 8C–10 8C/

min!200 8CMS

[42] Blood Halothane, enflurane,

isoflurane, sevoflurane

HS 120 1 ml DB 1, 30 m �0.53 mm � 5 mm

He

15 ml

/min

Isothermal: 60 8C MS

PH 170 GS-Q-GC, 30

mm � 0.53 mm

Isothermal: 160 8C 0.2 mg/ml

[43] Blood Hydrocarbons, esters,

aldehydes, ketones, ethers,

(alcohols)

HS 60 Split 1 ml DB 1, 30 m �0.25 mm � 0.1 mm

He

18 m1/

min

40 8C (4 min)–

10 8C/min!200 8C,

200 8C–50 8C/

min!250 8C

MS-ITD 0.03 till

0.74 mmol/l

[44] Blood, tissue Acetone, 2-propanol,

2-butanone, tert-butylether,

benzene, toluene,

ethylbenzene, propyl-benzene,

o-, p-, m-xylene

HS

(FET,MHE)

35 mg

150 DB 1, 30 m �0.25 mm � 1 mm

MS-ITD 0.4–1 nmol polar

voc (blood),

0.03–0.1 nmol

nonpolar (brain)

[52] Blood, lung,

liver, kidney,

stomach

content

Trichloroethylene,

trichloacetic acid

HS 220 2 ml 100%

dimethylsilicone,

30 m � 0.32 mm

� 1.0 mm

He

51713 Pa

40 8C (12 min)

20 8C/min!2408 C

MS

140 DB 1301, 30 m �0.53 mm � 1 mm

He

27580 Pa

FID

[58] Blood, lung,

gastric

content, urine,

bile, brain,

liver, kidney,

vitreous humor

Methyl-2-pentane,

methyl-3-hexane,

methylcyclohexane,

heptane

HS Splitless 0.070 m1 624CB, 30 m �0.25 mm

35 8C (5 min)–

15 8C/min!150 8C (5 min)

MS 0.0005 mg/ml

(blood,

heptane)

[59] Blood Desflurane,

sevoflurane, isoflurane,


HS 100 Split

1:30

XTI-5, 60 m �0.25 mm �0.24 mm

He

40 cm/s

35 8C (3.5 min)–

40 8C/min!120 8C (0.68 min)

MS 1 mg/ml Below

10 mg/ml

[63] Blood, urine,

tissue

Ethylchloride HS 250 0.7 m1 Innowax, 15 m �0.25 mm � 0.5 mm

He 1.05

ml/min

Isothermal: 50 8C FID

S.M

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method

Inj. temp.

(8C)


flow

Oven Detector Det.

limit

Quant.

limit

Derivat.

[64] Blood, brain,

lung, liver,

spleen, kidney,

muscle,

adipose tissue

Enflurane HS 150 Split

30:1

1 ml FID: DB-Wax,

30 m � 0.53 mm

� 1.0 mm

He 20

ml/min

Isothermal: 35 8C FID 200–

300 ng/

0.25 g

250 MS: HP-Wax,

30 m � 0.25 mm

� 0.25mm

He 1.0

ml/min

35 8C (5 min)–

10 8C/min!85 8CMS

[65] Blood, urine Toluene HS 220 0.250 m1 AT 5, 30 m �0.25 mm


[66] Blood, urine Difluoroethane HS 200 Split 0.3 ml RTX-BAC-1,

30 m � 0.32 mm

� 1.8mm

He 2

ml/min


[67] Blood, brain,

lung

Propane HS 40 DB 1, 30 m �0.32 mm


[68] Blood, urine,

lung

Diethylether, hydrocarbons HS 120 Split DB 1, 30 m �0.25 mm � 1 mm

He 18

ml/min

40 8C (4 min)–

10 8C/min!200 8CMS-ITD

200 8C–50 8C/

min!250 8C[69] Blood Hydrocarbons HS 100 2 ml DB 1, 2 � 30 m �

0.53 mm � 5 mm

He 30

m1/min

40 8C (4 min)–

8 8C/min!200 8CMS 0.001–

0.01 mg/ml

[74] Brain, liver,

lung, blood

Chlorodifluoromethane HS 150 Split

25:1

5 ml

(blood),

50 ml

(tissue)

100%

methylsilicone,

12 m � 0.25 mm

� 0.25 mm; glass

connector, 0.25 mm

He 5 psi 35 8C (2 min)–

20 8C/min!45 8CMS

PEG, 15 m �0.25 mm �0.25 mm

[75] Heart, lung,

brain, liver,

blood, spleen,

kidney

Trichlorofluoromethane HS 150 Split

1:70

0.1 ml HP 5 Ultra 2,

12 m � 0.2 mm

� 0.33 mm

He 1 psi Isothermal: 30 8C MS

[76] Blood, lung Methylchloroform HS 350 Split

10:1

DB 5, 15 m �0.32 mm � 1 mm

He 1

ml/min

40 8C (3 min)–

20 8C/min!300 8C (5 min)

FID

[77] Blood, brain,

heart

Dichloromethane,

chloroform

HS 250 0.250 ml DB 5, 30 m He 4 psi 70 8C (3 min)–

10 8C/min!180 8C

MS

[29] Blood Toluene, ethylbenzene HS-chem. 250 2 m1 HP5MS, 30 m �0.25 mm �0.25 mm

He 1

ml/min

70 8C (2 min)–

20 8C/min!230 8C(2 min)

FID

FTIR

BSTFA

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[70] Blood, urine Toluene HS-SPME 250 Split

30:1

FID: HP-Innowax,

30 m � 0.25 mm

� 0.25 mm

FID: 60 8C(6 min)–10 8C/

min!140 8C(3 min)

FID 0.001 mg/

ml SPME,

0.01 mg/ml

HS

HS MS: HP 5ms,

30 m � 0.25 mm

� 0.25 mm

MS: isothermal:

60 8CMS

[15] Blood, lung,

brain, fat

VOC, incl xylene, butane,

halothane, toluene, petrole

residues

HS-SPME 220 Splitless DE 1701,

30 m � 0.25 mm

� 0.25 mm

He

1 ml/min

35 8C (1 min)–

8 8C/min!270 8C(1 min)

MS

[27] Blood, urine,

tissues

Tetrachloroethylene,

trichloroethylene,


HS-SPME 250 SPB 1, 30 m �0.25 mm

N2

3 m1/min

ECD: 60 8C(1 min)– 10 8C/

min!130 8C(1.54 min)

ECD 3–5 ng/g 3-Methyl-

1-p-toyl

triazene in

diethyl

oxideMS: 45 8C–10 8C/

min!200 8CMS

[32] Urine, gastric

contents

Methylene chloride,

volatile petroleum

contents, (ethanol)

HS-SPME 225, 250 Splitless DB 1, 30 m �0.25 mm �0.25 mm

30 8C (5 min)–5 8C/

min!125 8C (5 min)

or 40 8C (5 min)–

5 8C/min!125 8C(9 min)

MS

[71] Blood, urine Thinner components:

toluene, benzene,

n-butylacetate, n-butanol,

n-isoamylacetate

HS-SPME 200 Splitless DB-Wax, 30 m �0.32 mm �0.25 mm

He

4 m1/min

35 8C (1 min)–

20 8C/min!230 8CFID 0.0022–

0.0048

mg/ml

[72] Blood, liver,

kidney, brain,

urine, bile,

stomach

contents

Halothane, isoflurane,

chloroform, diethylether

HS-SPME 250 Splitless HP 5ms, 30 m �0.25 mm �0.25 mm

He 1.0

ml/min

40 8C (2 min)–

30 8C!280 8CMS 0.004

mg/kg

[73] Blood, urine Chloroform,

methylene chloride

HS-SPME 240 Splitless RTX 624, 30 m �0.32 mm �1.80 mm

He

2 ml/min

35 8C (1 min)–

10 8C/min!105 8CFID 0.3 mg/ml

(blood),

0.2 mg/ml

(urine)

[34] Blood Hydrocarbons HS-

SPME-COT

250 Splitless XTI 5, 30 m �0.25 mm �0.25 mm

He 2.1

ml/min

�40 8C (1 min)–

30 8C/min!290 8CMS 0.01 mg/g

blood

[24] Blood, urine Hydrolysed n-butylnitrite HS-

SPME-CF

240 Splitless HP 1, 30 m �0.53 mm �2.64 mm

He

4 ml/min

50 8C (5 min)–

10 8C/min!260 8C(5 or 10 min)

FID 0.001

mg/ml

[35] Blood, urine Ethytacetate, benzene,

butan-1-ol, toluene,

butylacetate,

isoamylacetate

HS-COT 200 Splitless 5 ml DB624, 30 m �0.32 mm �1.80 mm

He

5 ml/min

5 8C (1 min)–

20 8C/min!100 8CFID 0.0005–

0.005

mg/ml

1008 C (3 min)!110 8C

[39] Blood o-, m-, p-Xylenes HS-COT 180 Splitless 2 ml AT-WAX, 30 m �0.32 mm � 0.5 mm

He 2.2

ml/min

5 8C (1 min)–

15 8C/min!180 8C(4 min)

FID 0.020

mg/ml

[60] Blood, lung Propane, isobutane,

butane, neopentane,

n-pentane,

bromochlorodifluoromethane

HS-COT 30 0.100–

0.250 m1

DB5 Isothermal: between

0 and 10 8CMS

S.M

.R.

Wille,

W.E

.E.

La

mb

ert/Fo

rensic

Scien

ceIn

terna

tiona

l1

42

(20

04

)1

35

–1

56

14

9



method

Inj. temp.

(8C)


flow

Oven Detector Det.

limit

Quant.

limit

Derivat.



HS-COT 150 Splitless 10 ml RTX-Votatile,

30 m � 0.32

� 1.5 mm

He 2

ml/min

�40 8C (1 min)–

10 8C/min!70 8CFID 0.010–

0.100

mg/ml70 8C–20 8C/min!250 8C (4 min)

[62] Blood Chloroform, methylene

chloride

HS-COT 250 Splitless 5 ml RTX-Volatile,

30 m � 0.32 mm

� 1.5 mm

He 3

ml/min

�30 8C (1 min)–

10 8C/min!100 8CFID 0.004

mg/ml

100 8C–20 8C/

min!280 8C[37] Blood n-Propane, isobutane,

n-butane

HS-CF/Tenax 200 Split

(0.7:1,

40:1)

0.5 ml WCOT CP624,

41 m � 0.25 mm

� 2.1 mm

He 1.0

ml/min

Isothermal: 35 8C MS 0.0013–

0.00301

mg/ml

0.0055–0.012 mg/ml

[38] Blood Aceton, isopropanol,

1-propanol, 1,1,1-trichloro-

ethane, isoflurane, diethylether,

tert-butanol, toluene, isobuty

methylketone, ethylacetate

PT Tenax 240 Split

1:4

PoraPLOT Q,

25 m � 0.32

� 10 mm

He 1.5

ml/min

30 8C (2 min)–

15 8C/min!240 8C (2 min)

FID

FTIR

0.05–

10 mg/ml

(low mol

alcohols:

100 mg/ml)

[40] Blood Hydrocarbons PT Tenax 250 2 ml DB 5, 15 m �0.53 mm � 15 mm

40 8C–4 8C/

min!90 8CMS 0.001–

0.01 mg/g

[57] Blood 27 VOC PT Tenax 250 Splitless PoraPLOT Q,

25 m � 0.32

� 10 mm

He 3.8

ml/min

30 8C (2 min)–

15 8C/min!250 8C (5 min)

FlD 0.01–

24 mg/ml

FTIR

[49] Blood, liver,

kidney,

stomach

contents

Chloral hydrate,

trichloroethanol,


Solvent 150 Splitless 0.001 m1 DB 1, 30 m �0.32 mm � 5 mm

80 8C (1 min)–

10 8C/min!110 8C,

110 8C (12 8C)–20

8C/min!250 8C

ECD Diazome-

thane

[50] Blood, urine,

stomach

content

Chloral hydrate,

trichloroethanol

Solvent 250 Splitless 100%

methylsilicone,

15 m � 0.25 mm

� 1.0 mm

He 1.5

ml/min


[53] Blood, liver,

kidney, lung,

stomach

contents

Trichloroethylene,

trichloroethanol,


Solvent 150 PTV 0.0001 ml ECD: DB 1, 30 m �0.32 mm � 5 mm

ECD: 80 8C(1 min)–10 8C/

min!110 8C,

110 8C (12 8C)–

20 8C/min!250 8C

ECD 5–10 pg 25 pg Diazome-

thane

0.001 ml FTIR: HP Ultra 1,

25 m � 0.32 mm �0.5 mm

FTIR: 40 8C(1 min)–10 8C/

min!250 8C

FTIR

Abbreviations: Extr. method, extraction method; PH, pulse-heating; PT, purge and trap; Inj. temp., injection temperature; He, helium; FID, flame ionization detection; Inj. M, injection mode; MS, mass spectrometer; FTIR,

Fourier transformation infrared detector; Inj. vol., injection volume; ITD, ion trap detection; ECD, electron capture detector; Det. limit, detection limit; COT, cryogenic oven trapping; FET, full evaporation technique; Quant.

limit, quantitation limit; CF, cryogenic focusing; MHE, multiple headspace; Derivat., derivatisation method; chem., chemical; SPME, solid phase microextraction; HS, headspace; PTV, programmed temperature vaporization.

Column phase: wax, innowax: crosslinked polyethylene glycol; 5: 5% diphenyl–195% dimethylsiloxane; 1: polydimethylsiloxane; 624: 6% cyanopropylphenyl–94% methlylpolysiloxane; Pora PLOT Q: styrene-divinyl benzene

porous polymer; RTX volatile: diphenyl-dimethylpolysiloxane; WCOT SPSIL 5CB: 100% polydimethylsiloxane; DB 1701: 14% cyanopropylphenyl–86% dimethylpolysiloxane; DB 1301: 6% cyanopropylphenyl–94% phenyl.

15

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phase. This matrix effect limits the use of direct headspace

as a simple quantitative method, especially for forensic

post-mortem samples [44]. The standard addition method

(SAM), multiple headspace extraction method (MHE) and

full evaporation technique (FET) [44] are quantitative

procedures that try to overcome this matrix effect [16].

The standard addition procedure compares the signal of

the sample with and without addition of a known amount

of the analyte in question. The assumption is made that the

dispersion in the matrix of the added analyte is the same as

for the original analyte. This is not always correct, espe-

cially when it involves heterogeneous biological samples

such as tissues [16,44]. The multiple headspace technique

is a stepwise extraction of volatile organic compounds into

the gas phase by using a series of regular headspace

analyses. Every time an aliquot is taken and analysed

re-equilibration occurs. The vapour concentration will

reduce logarithmically by each step and eventually it is

possible to extrapolate the original concentration from the

curves obtained after a few analyses, in combination with

internal of external standardisation or standard addition

[16,26,44,78].

The full evaporation technique approach is based on the

equilibration of a small amount of sample at high tempera-

ture so that the analyte is (almost) fully evaporated. The

main drawback here is the lower sensitivity as compared to

headspace analysis. FET is nevertheless also applicable to

forensic work if a sensitive detection system is used as

described by Schuberth [44] for the determination of ben-

zene, toluene and other volatiles in post-mortem blood and

tissues with a MS-ion trap detector.

4. Metabolites

4.1. Introduction

Elimination of volatile organic compounds can occur

unchanged in exhaled air or through metabolism occurring

mainly in the liver, resulting in elimination of more polair

and thus less volatile compounds in the exhaled air and

urine. Factors such as age, disease, dose and exposure may

effect the rate and extent of metabolism [2]. Analysis of

metabolites from volatiles are of importance in occupational

and environmental exposure and in monitoring of abuse as is

seen in the literature [45–48]. A variation of methods such as

high performance liquid chromatography, high performance

capillary electrophoresis and gas chromatography are used

for the determination of metabolites of volatiles in urine. In

post-mortem cases, metabolites in urine seem less relevant,

as the mother component in blood or tissues can give a direct

toxicological link. However, chloral hydrate, due to its fast

metabolism by alcohol dehydrogenase is unlikely to be

detected post-mortem, and thus determination of its active

metabolite in urine, trichloroethanol, is relevant [49–51].

Trichloroethanol and trichloroacetic acid, both metabolites

of trichloroethylene are determined together with their

mother compound in several post-mortem cases [52,53].

Phenol was analysed [56], however, not as metabolite of

benzene.

4.2. Sampling

Static headspace and solvent extraction are the methods

used for sampling of the compounds. The headspace

Fig. 3. Analysis of the qualitative standard mixture with detector

sensitivities (FSD) (a) FID 3.2 nA and (b) ECD 64 kHz. Column,

60 m � 0:53 mm i.d. SPB-1 (5 mm film); oven temperature, 40 8C(6 min), then to 80 8C at 5 8C min�1, then to 200 8C at

10 8C min�1; and injection volume, �0.010 cm3. Peaks: 1:

propane, 2: FC12, 3: dimethyl ether, 4: isobutane, 5: butane, 6:

BCF, 7: ethanol, 8: acetone, 9: propan-2-ol, 10: FC11, 11: FC113,

12: halothane, 13: butanone, 14: hexane, 15: chloroform, 16: 1,1,1-

trichloroethane, 17: carbon tetrachloride, 18: trichloroethylene, 19:

methyl isobutyl ketone, 20: 1,1,2-trichloroethane (internal stan-

dard), 21: toluene, 22: tetrachloroethylene, 23: 2,2,2-trichloroetha-

nol, and 24: ethylbenzene (internal standard). Source: [20] Fig. 2

(p. 1114).


technique is used for the extraction of trichloroacetic acid in

combination with its mother compound, as well as for the

extraction of tetrachloroethylene and its metabolite tri-

chloroacetic acid [28]. Trichloroethanol, a urinary meta-

bolite of chloral hydrate, is extracted by solvent extraction

[49,50,54].

4.3. Separation and detection

4.3.1. Spectrophotometric detection

Heating trichloroethanol in combination with pyridine

and sodium hydroxide results in a reaction product with a

spectral maximum at 368 nm, while reaction products with

Fig. 4. (A) Mass spectra of butane and 2-methylpropane. Source: Mass Spectral and GC Data (second ed., ISBN 3-527-26989-4) p. 57. (B)

Gram-Schmidt reconstruction chromatogram obtained from 0.5 ml of case 1 blood and the vapour phase FTIR spectrum of ethyl acetate (7)

superimposed with the corresponding library spectra. And the mass spectrum of ethylacetate. Source: [38] Fig. 1 a, h (p. 205), Mass Spectral

and GC Data (second ed., ISBN 3-527-26989-4) p. 278.


chloral hydrate and trichloroacetic acid have maxima at 368

and 530 nm, respectively [54].

4.3.2. Gas chromatographic determination

Gas chromatography is used in combination with ECD,

FID, MS or FTIR. Polar metabolites have a lower volatility

and therefore derivatisation could be necessary for a better

peak shape and thus higher sensitivity. BSTFA is used for

silylation of trichloroacetic acid [28], while methylation of

this acid also occurred after use of diazomethane [49,53].

Column phases vary from apolair such as 100% methylsi-

licone to polair phases such as a cyanopropyl phenyl phase.

5. Precautions in interpretation of results

When interpreting qualitative results attention should be

paid to wrong conclusions due to the fact that compounds of

related character generate very similar mass spectra such as

butane and 2-methylpropane, thus resulting in several can-

didates as possible cause of death [43]. Detection of volatile

organic compounds does not always indicate death by

solvent abuse. Not only must the possibility of environmen-

tal, occupational and therapeutic exposure of the deceased

be evaluated, also the possible formation of endogenous

volatile compounds due to several inborn errors of metabo-

lism must be taken in account [2,14,20]. Ketotic patients

have a high concentration of acetone in their blood, while

acetone and butanone are seen in large quantities in children

with acetoacetyl CoA thiolase deficiency. Volatiles such as

halothane are used for therapeutic purposes, while chloro-

butanol is a sedative and a bactericide present in some

heparin preparations [20].

Volatiles may also occur in vivo after metabolism of

specific compounds such as acetone after metabolism of

isopropanol. Compounds such as alkylnitrites are unstable

and degrade rapidly in vivo into the corresponding alcohol

[19]. Hippuric acid can originate from ingestion of benzo-

ates, added to food as a preservative.

The simultaneous detection of several compounds may

also lead to determination of the source, for example pet-

roleum consists of aliphatic and aromatic hydrocarbons,

naphtalenes, paraffin, alkenes and eventually tetra-ethyl lead

[55]. Trichloroacetic acid can originate from trichloroethy-

lene, chloralhydrate, triclofos and dichloral phenazone [19],

while a thermal decomposition of trichloroacetic acid can

lead to chloroform. Most abused products are commonly

used in the lab and therefore are an easy source of con-

tamination of the sample. Xylene, toluene, butanol and

ethylbenzene can be detected after storage of blood in a

Sarstedt Monovette Serum gel blood collection tube,

whereas 1-butanol and 2-methyl-2-propanol are detected

in EDTA-coated tubes [19].

The main reason why scepticism of quantitative results is

necessary is the occurrence of losses of analytes during

sampling, sample handling and storage, which results in

‘false’ quantitation, especially in the case of very volatile

analytes such as propane, butane and the halon aerosol

propellants [19]. Due to the volatile nature of the compounds

and individual differences in susceptibility such as rates of

absorption, delivery to target tissue compartments, rates of

metabolism and elimination, and protective response, differ-

ences in blood concentrations may occur, thus leading to a

lack of a strong correlation between blood levels and clinical

features [2,14].

6. Summary

The extraction methods for volatile compounds are sim-

ple, inexpensive and solvent-free in most cases. Injection of

large volumes or preconcentration of the sample is applied in

view of the low levels of the target compounds in the sample.

By HS-COT, HS-CF, SPME, sample loads are higher and

thus sensitivity is enhanced. A variety of capillary columns

can be used for the separation of volatile organic compounds

due to differences in polarity of these compounds, com-

pound mixes and differences in extraction techniques and

detectors used. The large variety of volatiles requires an

analytical method that both detects and identifies the com-

pounds. MS, ECD and FID are usually used for quantitation

purposes, while MS and FTIR have a screening and con-

firmation purpose. Quantification can be based on addition

of an internal standard or by matrix-negating procedures

such as MHE, FET and SAM.

For occupational and environmental exposure and in

monitoring of abuse of volatile organic compounds, analysis

of metabolites in urine can be interesting because of the

extended detection window and ease of sampling. In post-

mortem cases, metabolites seem of less importance,

although trichloroethanol and trichloroacetic acid are ana-

lysed in cases of chloral hydrate and trichloroethylene

(ab)use. These analytes were determined after solvent- or

HS-extraction through spectrophotometric detection or GC

detection after derivatization.

Of great importance are sample handling due to the

volatile nature of the compounds and caution remains

necessary by the interpretation of the qualitative and quan-

titative results.

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volatile substance abuse—post-mortem diagnosis

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