You are on page 1of 33

_______________________________________________________________

_______________________________________________________________

Report Information from ProQuest


September 26 2013 03:44 _______________________________________________________________

26 September 2013

ProQuest

Table of contents
1. Volatile substance abuse--post-mortem diagnosis....................................................................................... 1

26 September 2013

ii

ProQuest

Document 1 of 1

Volatile substance abuse--post-mortem diagnosis


Author: Wille, Sarah M.R; Lambert, Willy E.E Publication info: Forensic Science International (Online) 142.2 (2004): 135-156. ProQuest document link 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. Links: LinkSource, Base URL to Journal Linker: Full text: Hydrocarbons Aliphatic Alcohols Nitrites Propane Butanol Oxygenated compounds Aromatic Ethers Anaesthetics Toluene Halogenated compounds Esters Ketones Other Isoamylacetate Acetone Chloral hydrate Butylacetate Butanone Tetrachloro-ethylene Pentylnitrite Heptane Methylene-chloride Difluoroethane Ethylchloride

tert -Butylether
Desflurane Ethylbenzene Dimethyl ether Sevoflurane Xylene Trichloro-ethylene Enflurane Halothane Bromochlorodifluoromethane

n -Butylnitrite
Butane Propanol Amylnitrite Neopentane Isoflurane Benzene Hexane Pentane

Table 1 - Volatile organic compounds classification Adapted from [2,10,12].

26 September 2013

Page 1 of 31

ProQuest

Reference Matrix

Compound Add./IS s Bromochlo rodifluorom ethane, butane, FC11, FC12, isobutane, propane, dimethylet her

HS time (min)

HS temp. (C)

Inj.

Comments

Inj. vol. (ml)

[20]

Blood, urine, tissue

Tissue: Subtilisin A IS: ethylbenze 15 ne, 1,1,2trichloroeth ane

65

Manual

Warm needle (10min on heating block)

0.10-0.30

[25]

Blood

Isoflurane

IS: 1,4dioxane

30

55

Needle Autosampl temperatur 1.0 er e: 120C

[32]

Urine, gastric content

Methylene chloride, Sodium volatile petroleum contents, (ethanol) chloride 20 (urine), IS: t -butanol 60 Manual 0.250 [40]

Blood

IS: toluene Hydrocarb d8, cold 20 ons water

60

Manual

[42]

Blood

Halothane, enflurane, IS: isoflurane, dichlorome 15 sevofluran thane e

55

Manual

[43]

Blood

Hydrocarb ons, esters, aldehydes, ketones, ethers, (alcohols)

Potassium fluoride, 33 sodium chloride

50

Valve/loop temperatur e: 54C, Aux. gas pressure: Autosampl 130kPa, 1 er vial pressurisat ion time: 15s, carrier gas: He 70ml/min

[58]

Blood, lung, gastric content, urine, bile, brain, liver, kidney, vitreous humor

Methyl-2pentane, methyl-3hexane, methylcycl ohexane, heptane

26 September 2013

Page 2 of 31

ProQuest

20

80

Needle temperatur e: 90C, loop temperatur e: 100C, vial Autosampl pressurisat 0.070 er ion time: 2min, carrier gas: He 10psi, pressurisat ion pressure: 17psi Blood, urine, tissue Sodium Ethylchlori chloride, de IS: 1propanol

[59]

Blood

Desflurane , sevofluran e, isoflurane, enflurane, halothane

Anticoagul ant, IS: 1,4dioxane

Autosampl [63] er

37

0.7

[64]

Blood, brain, lung, liver, spleen, Enflurane kidney, muscle, adipose tissue

Distilled water, IS: 1,4dioxane

30

55

Vial pressurisat ion time: 0.13min, pressurisat ion: 124kPa, transferline and loop temperatur Autosampl e: 100C, 1 er loop fill time: 0.15min, injection time: 0.20min, loop equilibratio n time: 0.15min

[65]

Blood, urine

Toluene

Sodium citrate solution, 20 IS: isobutanol

55

Autosampl 0.250 er

[66]

Blood, urine

26 September 2013

Page 3 of 31

ProQuest

Difluoroeth IS: 1ane propanol

15

37

Manual

0.3

[67]

Blood, Propane brain, lung

Fluoride (blood)

Manual

0.5

[68]

Diethylethe Blood, r, Sodium urine, lung hydrocarbo chloride ns

26

80

Valve/loop temperatur e: 85C, Aux. gas pressure: 130kPa, vial pressurisat Autosampl ion time: [69] er 15s, sample loop V: 1ml, vent loop fill time: 1s, injection time: 2min

Blood

Hydrocarb Cold water, 20 ons IS

60

Manual

[70]

Blood, urine

Toluene

Sodium citrate solution, 20 shaken, IS: 1,4dioxane

60

Valve and Autosampl transferline 1.0 er temperatur e: 140C

[74]

IS: Chlorodiflu Brain, liver, genetron oromethan lung, blood 502, e shaken

60

RT

[75]

Heart, lung, brain, Trichloroflu liver, oromethan blood, e spleen, kidney

IS: dichlorome 30 thane

40

Manual

[76]

Blood, lung

Methylchlo 60 roform

20

[77]

Dichlorome NaCl, IS: Blood, thane, sec brain, heart chloroform butanol

20

55

0.250

Metabolite [28] s

26 September 2013

Page 4 of 31

ProQuest

Blood, tissue

Tetrachloro ethylene, trichloroeth IS: [52] ylene, chloroform trichloroac etic acid

Blood, lung, liver, kidney, stomach content

IS: n Trichloroet butanol, hylene, NaCl, 35 trichloroac water, etic acid vortex, sonication Acetone, 2-propanol, 2butanone, tert butylether, benzene, 28 toluene, ethylbenze ne, propylbenzene, o -, p -, m xylene

80

Loop temperatur HS Autosampl e: 110C, quantificati [44] er transferline on temperatur e 120C

Blood, tissue

Aux. gas pressure: 210kPa, 130 (FET), not more 80 (MHE) than 10mg of compound

HS chemical

[29]

Blood

Toluene, Distilled ethylbenze water ne

3-5

65

HS followed by COT

[35]

Blood, urine

Ethylacetat e, benzene, butan-1-ol, toluene, butylacetat e, isoamylacetate (=thinner component s)

Distilled water, stirring bar, heparin 30 sodium (in blood), IS: ethylbenze ne

90

Manual

[39]

Blood

Distilled o -, m -, p water, IS: Xylenes aniline

30

100

Manual

[60]

Blood, lung

Propane, isobutane, butane, neopentan e, n 10 pentane, bromochlor odifluorom ethane

50

0.1000.250

[61]

Blood

Sevofluran e, Distilled isoflurane, water, IS: enflurane, halothane halothane

15

26 September 2013

Page 5 of 31

ProQuest

55

Manual

10

[62]

Blood

Distilled Chloroform water, IS: , methylene 20 methylene chloride, chloride chloroform

55

Manual

HS followed by [37] Tenax-trap

Blood

nIS: t -butylPropane, methyl 20 isobutane, ether n -butane

60

Table 2 - Analytical data for HS extraction

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. Compound Cond. HS Ad. Desorp. Fibre s time/temp. time/temp. time/temp. time/temp.

Reference Matrix

Add.

[15]

VOC, incl xylene, Blood, butane, 5min/200 lung, brain, halothane, 1.5h/80C C fat toluene, petroleum residues

30min/-

1min/220 100m C PDMS

Distilled water, IS: cyclohexan one

[27]

Blood, urine, tissues

Tetrachloro ethylene, trichloroeth 100m 5min/60C 1min/60C 15s/250C ylene, PDMS trichloroac etic acid

Ultra Turrax, IS: [32] carbontetra chloride

Urine, gastric contents

Methylene chloride, 10min/60 1min/225 5100m volatile 20min/60 C urine, C urine, 10min/260 PDMS, petroleum C 5min/60C 1min/250 C 85m PA products gastric C gastric (ethanol) 0.1% sodium azide, shaken (rpm 85), IS: 1,4dioxane, (iso butanol)

Sodium chloride in [70] urine, IS: t -butanol

Blood, urine

Toluene

20min/60 1min/250 100m 1min/60C C C PDMS

[71]

Blood, urine

26 September 2013

Page 6 of 31

ProQuest

Thinner component s: toluene, benzene, n 1h/250C butylacetat e, n butanol, n isoamylace tate Halothane, isoflurane, chloroform, 1h/250C diethylethe r

15min/80 3min/200 100m 5min/80C C C PDMS

Distilled water, magnetic stirring bar, [72] IS: ethylbenze ne

Blood, liver, kidney, brain, urine, bile, stomach contents

15min/100 15min/100 2min/250 100m C C C PDMS

Ammonium sulphate, sulphuric [73] acid solution

Blood, urine

Chloroform

Distilled water, Blood, urine

75m shaken SPME , 10min/30 20min/30 2min/240 carboxen/P 10s IS: followed by [24] methylene C C C DMS methylene COT chloride chloride, chloroform Hydrolysed 30min/155 1-3h/20n -butylC 23C nitrite 5100m 1min/240 20min/60 PDMS, C C 85m PA IS: n propanol

[34]

Blood

Table 3 - Analytical data for HS-SPME extraction

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. Oven temp. (C) -5 to 5 inception of the column [35]

Reference

Matrix

Compounds

Extr. method

Cooling gas

Comments

[24]

Blood, urine

Hydrolysed n SPME butyl nitrite Liquid CO2

Liquid CO2

[34]

Blood Ethyl acetate, benzene, butan-1-ol, toluene, butyl acetate, isoamylacetate

Hydrocarbons SPME

-40

Blood, urine

HS

Liquid CO2

[37]

Blood

n -Propane, n butane, isopropane

26 September 2013

Page 7 of 31

ProQuest

HS + Tenax

Liquid N2

-80 cooled inlet-liner (Tenax-trap)

Tenax-trap heated to 200C for 6min

[39]

Blood

o -, m -, p Xylenes

HS

Liquid CO2

[60]

Blood

Propane, butane, isobutane

HS

NS

0-10

[61]

Blood

Sevoflurane, isoflurane, enflurane, halothane

HS

NS

-40

[62]

Blood

Chloroform, methylene chloride

HS

Liquid CO2

-30

Table 4 - Analytical data for HS-COT/CF extraction

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. Extraction liquid

Reference

Matrix

Compounds

Add./IS Chloral hydrate, trichloroethano l, trichoroacetic acid

Vortex time

Centrifuge

Solvent extraction

[49]

Blood, liver, kidney, stomach contents

Water, sulphuric acid, IS: 1,3 Diethylether dichloro-2propanol

2min

10min, 2500g [50]

Blood, urine, stomach content

Chloral IS: hydrate, tetrachloroethy Chloroform trichloroethano lene l Trichloroethyle ne, trichloroethano l, trichloroacetic acid Water, sulphuric acid, IS: 1,3 Diethylether dichloro-2propanol

30s

30s, 10000rpm [53]

Blood, liver, kidney, lung, stomach contents

2min

10min, 2500g [54]

Blood, urine

Trichloroethan ol (chloral hydrate, Diethylether trichloroacetic acid) Add./IS Heating time (s)

3min

4min, 2500rpm

Reference

Matrix

Compounds

Ferromag. cond. temp.

Oven temp. (C)

26 September 2013

Page 8 of 31

ProQuest

Needle temp. (C)

Pulse-heated extraction

[42]

Blood

Halothane, enflurane, isoflurane, sevoflurane Compounds Extraction temp. (C)

160

170 Purge time (min)

200

Reference

Matrix

Add./IS Trap temp. (C)

Gas flow (ml/min) Desorption time/temp.

Dry purge time Line/valve (min) temp. (C)

Trap

Sample P (kPa)

Trap P (kPa)

Purge-trap extraction

[38]

Blood

Aceton, isopropanol, 1propanol, 1,1,1-trichloroethane, isoflurane, Antifoam b diethylether, tert -butanol, toluene, isobutylmethyl ketone, ethylacetate

He 35

10

Tenax 1/8in. 12in. Hydrocarbons

-20

4min/225C

140 Tenax TA (60/80 mesh)

40

[40]

Blood

Cold water, IS: N2 500 toluene d8 Antifoam b, NaF, IS: diethylketone -20

40

220C

[57]

Blood

27 VOC's

He 38

15

150

Tenax 1/8in. 12in.

Room temperature

2min/225C

Table 5 - Solvent-, pulse-heated purge and trap extraction

Abbreviations : Add., addition; Ferromag. cond. temp., ferromagnetic conduction time; temp., temperature; IS,
internal standard. Extr. Inj. Refere Comp Matrix metho temp. nce ounds d (C)

Inj. M

Inj. vol.

Colum Gas n flow

Oven

Detect Det. or limit

Quant. Deriva limit t.

26 September 2013

Page 9 of 31

ProQuest

[20]

Bromo chloro difluor ometh ane butane , Blood, FC11, urine, FC12, HS tissue isobut ane, propan e, dimeth ylether (224 comp)

150

40C (6min) 5C/mi n[arro SPBw 1, 60m He Splitle 0.10right]8 FID/E 0.53 8.6cm3 [25] ss 0.30ml 0C; CD mm /min 80C5m 10C/ min[arr ow right]2 00C

Blood

Isoflur ane

HS

100

Split 1:30

1.0ml

35C (1.4mi DB 5, n)30m He 40C/ 0.25 40cm/ min[arr MS mm s ow 0.25 right]8 m 0C (2min)

1.2g/ 4.7g/ [28] ml ml

Tetrac hloroet hylene , Blood, trichlor HS tissue oethan ol, trichlor oaceti c acid

150

ECD: 65C ECD: (2min) WCOT SPSIL N2 10C/ 5CB, 3ml/mi min[arr ECD 25m n ow 0.53 right]1 mm 30C (1.5mi n) DB 1, 30m He 0.53 15ml/ mm min 5m

MS: BP20, 25m 0.25g 0.36g BSTF 0.22 /ml /m1 A mm 0.25 m

MS: 45C10C/ min[arr MS ow right]2 00C

[42]

Haloth ane, enflura ne, Blood isoflur ane, sevofl urane

HS

120

1ml

Isother mal: MS 60C

PH

170

GS-QGC, Isother 0.2g/ 30 mm mal: [43] ml 0.53 160C mm

Blood

26 September 2013

Page 10 of 31

ProQuest

Hydro carbon s, esters, aldehy des, HS ketone s, ethers, (alcoh ols)

60

Split

1ml

40C (4min) 10C/ DB 1, min[arr 30m ow He 0.25 right]2 MS18m1/ mm 00C, ITD min 0.1 200Cm 50C/ min[arr ow right]2 50C

0.03 till 0.74 mol/l

[44]

Aceton e, 2propan ol, 2butano ne, tert butylet her, benze Blood, ne, tissue toluen e, ethylb enzen e, propylbenze ne, o -, p -, m xylene

HS (FET, MHE) 35mg

150

DB 1, 30m MS0.25 ITD mm 1m

0.41nmol polar voc (blood) , 0.03- [52] 0.1nm ol nonpol ar (brain)

Blood, lung, liver, kidney , stoma ch conten t

Trichlo roethyl ene, HS trichlo acetic acid

220

2ml

100% dimeth ylsilico ne, He 30m 51713 0.32 Pa mm 1.0 m

40C (12min ) 20C/ MS min[arr ow right]2 40C

140

DB 1301, He 30m 27580 FID 0.53 Pa mm 1m

[58]

Blood, lung, gastric conten t, urine, bile, brain, liver, kidney , vitreou s humor

Methyl -2pentan e, methyl -3hexan HS e, methyl cycloh exane, heptan e

Splitle 0.070 ss m1

35C (5min) 624CB 15C/ , 30m min[arr MS 0.25 ow mm right]1 50C (5min)

0.0005 g/ml (blood, heptan e)

26 September 2013

Page 11 of 31

ProQuest

[59]

Desflu rane, sevofl urane, isoflur Blood HS ane, enflura ne, haloth ane

100

Split 1:30

35C (3.5mi XTI-5, n)60m 40C/ He 0.25 min[arr 40cm/ MS mm ow s 0.24 right]1 m 20C (0.68m in)

Below 1g/ml 10g/ [63] ml

Blood, urine, tissue

Ethylc HS hloride

250

Innow ax, 15m He Isother 0.7m1 0.25 1.05ml mal: FID mm /min 50C 0.5 m

[64]

Blood, brain, lung, liver, spleen , Enflur kidney HS ane , muscl e, adipos e tissue 35C (5min) 10C/ MS min[arr ow right]8 5C

150

Split 30:1

1ml

FID: DBWax, He 30m 20ml/ 0.53 min mm 1.0 m

Isother mal: FID 35C

200300ng/ 250 0.25g

MS: HPWax, He 30m 1.0ml/ 0.25 min mm 0.25 m

[65]

Blood, Toluen urine e

HS

220

0.250 m1

AT 5, 30m 0.25 mm

Isother mal: MS 130C

[66]

Difluor Blood, oethan HS urine e

200

Split

0.3ml

RTXBAC1, 30m 0.32 mm 1.8 m

He Isother 2ml/mi mal: FID n 65C

[67]

Blood, Propa brain, HS ne lung

40

DB 1, 30m 0.32 mm

Isother mal: FID 10C

[68]

Diethyl Blood, ether, urine, hydroc lung arbons

26 September 2013

Page 12 of 31

ProQuest

HS

120

Split

DB 1, 30m He 0.25 18ml/ mm min 1m

40C (4min) 10C/ MSmin[arr ITD ow right]2 00C

200C50C/ min[arr [69] ow right]2 50C

Hydro Blood carbon HS s

100

2ml

40C DB 1, (4min) 2 He 30m 8C/mi 30m1/ MS 0.53 n[arro min mm w 5m right]2 00C

0.0010.01g [74] /ml

Brain, liver, lung, blood

Chloro difluor HS ometh ane

150

Split 25:1

100% methyl silicon e, 12m 5l 0.25 (blood) mm He , 50l 0.25 5psi (tissue m; ) glass conne ctor, 0.25m m

35C (2min) 20C/ MS min[arr ow right]4 5C

PEG, 15m 0.25 [75] mm 0.25 m

Heart, lung, brain, liver, blood, spleen , kidney

Trichlo rofluor HS ometh ane

150

Split 1:70

0.1ml

HP 5 Ultra 2, 12m He 0.2m 1psi m 0.33 m

Isother mal: MS 30C

[76]

Methyl Blood, chlorof HS lung orm

350

Split 10:1

40C (3min) DB 5, 15m He 20C/ 0.32 1ml/mi min[arr FID mm n ow 1m right]3 00C (5min)

[77]

Dichlor Blood, ometh brain, ane, HS heart chlorof orm

250

0.250 ml

DB 5, 30m

He 4psi

70C (3min) 10C/ MS min[arr ow right]1 80C

[29]

Toluen e, HSBlood ethylb 250 chem. enzen e

2m1

HP5M S, 30m He 0.25 1ml/mi mm n 0.25 m

26 September 2013

Page 13 of 31

ProQuest

70C (2min) 20C/ min[arr FID ow right]2 30C (2min)

BSTF FTIR A

[70]

Blood, Toluen HS250 urine e SPME

Split 30:1

FID: HPInnow ax, 30m 0.25 mm 0.25 m

FID: 60C (6min) 10C/ FID min[arr ow right]1 40C (3min)

0.001 g/ml SPME, 0.01g /ml HS

HS

MS: HP 5ms, 30m 0.25 mm 0.25 m

MS: isother MS mal: 60C

[15]

VOC, incl xylene , butane Blood, , lung, HShaloth 220 brain, SPME ane, fat toluen e, petrole residu es ECD: 60C (1min) SPB 1, N2 10C/ 30m 3m1/m min[arr ECD 0.25 in ow mm right]1 30C (1.54m in)

35C DE (1min) 1701, 30m He 8C/mi Splitle 0.25 1ml/mi n[arro MS ss mm n w 0.25 right]2 m 70C (1min)

[27]

Tetrac hloroet hylene , Blood, trichlor HSurine, 250 oethyl SPME tissues ene, trichlor oaceti c acid

35ng/g

3Methyl -1-ptoyl triazen e in diethyl oxide

MS: 45C10C/ min[arr MS ow right]2 00C

[32]

26 September 2013

Page 14 of 31

ProQuest

Methyl ene chlorid e, Urine, volatile gastric HS225, petrole conten SPME 250 um ts conten ts, (ethan ol)

30C (5min) 5C/mi n[arro w right]1 DB 1, 25C 30m (5min) Splitle 0.25 or MS ss mm 40C 0.25 (5min) m 5C/mi n[arro w right]1 25C (9min) Blood, liver, kidney , brain, urine, bile, stoma ch conten ts

[71]

Thinne r compo nents: toluen e, benze Blood, ne, n - HS200 urine butyla SPME cetate, nbutano l, n isoam ylacet ate

Splitle ss

DBWax, 30m He 0.32 4m1/m mm in 0.25 m

35C (1min) 20C/ FID min[arr ow right]2 30C

0.0022 [72] 0.0048 g/ml

Haloth ane, isoflur ane, HS250 chlorof SPME orm, diethyl ether

HP 5ms, 30m He Splitle 0.25 1.0ml/ ss mm min 0.25 m

40C (2min) 30C[a rrow right]2 80C

MS

0.004 [73] mg/kg

Chloro form, Blood, methyl HS240 urine ene SPME chlorid e

RTX 624, 30m He Splitle 0.32 2ml/mi ss mm n 1.80 m -40C (1min) 30C/ MS min[arr ow right]2 90C

35C (1min) 10C/ FID min[arr ow right]1 05C

0.3g/ ml (blood) , [34] 0.2g/ ml (urine)

Hydro HSBlood carbon SPME 250 s -COT

XTI 5, 30m He Splitle 0.25 2.1ml/ ss mm min 0.25 m

0.01g /g [24] blood

Hydrol ysed n HSBlood, SPME urine butylni -CF trite

26 September 2013

Page 15 of 31

ProQuest

240

50C (5min) HP 1, 30m 10C/ He Splitle 0.53 min[arr 4ml/mi FID ss mm ow n 2.64 right]2 m 60C (5 or 10min)

0.001 [35] g/ml

Ethyta cetate, benze ne, butan1-ol, Blood, HStoluen urine COT e, butyla cetate, isoam ylacet ate

200

Splitle 5ml ss

DB624 , 30m He 0.32 5ml/mi mm n 1.80 m

5C (1min) 20C/ FID min[arr ow right]1 00C

100C 0.0005 (3min)[ arrow [39] 0.005 right]1 g/ml 10C

o -, m -, p - HSBlood Xylene COT s

180

Splitle 2ml ss

ATWAX, 30m 0.32 mm 0.5 m

5C (1min) He 15C/ 2.2ml/ min[arr FID min ow right]1 80C (4min)

0.020 [60] g/ml

Propa ne, isobut ane, butane , neope Blood, ntane, HSlung nCOT pentan e, bromo chloro difluor ometh ane

30

0.1000.250 DB5 m1

Isother mal: betwe MS en 0 and 10C

[61]

Sevofl urane, isoflur ane, HSBlood enflura COT ne, haloth ane

150

Splitle 10ml ss

RTXVotatil He e, 30m 2ml/mi 0.32 n 1.5 m

-40C (1min) 10C/ FID min[arr ow right]7 0C

70C20C/ 0.010- min[arr 0.100 ow [62] g/ml right]2 50C (4min)

Blood

26 September 2013

Page 16 of 31

ProQuest

Chloro form, methyl HSene COT chlorid e

250

Splitle 5ml ss

RTXVolatil e, 30m He 0.32 3ml/mi mm n 1.5 m

-30C (1min) 10C/ FID min[arr ow right]1 00C

100C20C/ 0.004 min[arr [37] g/ml ow right]2 80C

nPropa ne, Blood isobut ane, n butane

HSCF/Te 200 nax

Split (0.7:1, 0.5ml 40:1)

WCOT CP624 , 41m He Isother 0.25 1.0ml/ mal: MS mm min 35C 2.1 m

0.0013 0.0030 1g/ml

0.0055 [38] 0.012 g/ml

Aceton , isopro panol, 1propan ol, 1,1,1trichlor oethane , isoflur ane, PT Blood diethyl Tenax ether, tert butano l, toluen e, isobut y methyl ketone , ethyla cetate

240

Split 1:4

30C (2min) PoraP LOT He 15C/ Q, 1.5ml/ min[arr FID 25m min ow 0.32 right]2 10m 40C (2min)

0.0510g/ ml

FTIR

(low mol alcoho [40] ls: 100g/ ml)

Hydro PT Blood carbon 250 Tenax s

26 September 2013

Page 17 of 31

ProQuest

2ml

DB 5, 15m 0.53 mm 15m

40C4C/mi n[arro MS w right]9 0C

0.0010.01g [57] /g

Blood

27 VOC

PT 250 Tenax

30C (2min) PoraP LOT He 15C/ Splitle Q, 3.8ml/ min[arr ss 25m min ow 0.32 right]2 10m 50C (5min) 80C (1min) 10C/ min[arr ow DB 1, right]1 30m 10C, 0.32 ECD 110C mm (12C) 5m 20C/ min[arr ow right]2 50C Blood, liver, kidney , lung, stoma ch conten ts

FlD

0.0124g/ ml

FTIR

[49]

Blood, liver, kidney , stoma ch conten ts

Chlora l hydrat e, trichlor Solven 150 oethan t ol, trichlor oaceti c acid

Splitle 0.001 ss m1

Diazo metha ne

[50]

Blood, urine, stoma ch conten t

Chlora l hydrat Solven e, 250 t trichlor oethan ol

100% methyl silicon He Isother Splitle e, 15m 1.5ml/ mal: MS ss 0.25 min 50C mm 1.0 m

[53]

Trichlo roethyl ene, trichlor Solven oethan t ol, trichlor oaceti c acid

Table 6 - Analytical data of separation and detection modes

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. 1 26 September 2013 Page 18 of 31 ProQuest

Introduction 1.1 Prevalence Epidemiological studies indicate that a substantial number 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 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 escapades 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 behavioural 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 separation and detection. Inhaled volatile substances fall into several chemical families, notably hydrocarbons, oxygenated compounds and halogenated compounds (Table 1). The hydrocarbons can be subdivided into aliphatic hydrocarbons 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. 26 September 2013 Page 19 of 31 ProQuest

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 compound itself and on the magnitude, duration and the route of exposure. Abused inhalants have different mechanisms for their toxicological effects [11]. The most important toxicological 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 catecholamines following exercise, fright or sexual activity. Stimulation 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 coronary vasospasm has also been hypothesised as a mechanism. 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 system, especially in chronic users. Most volatiles, especially anaesthetics, act as a central nervous system depressant [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 pulmonary tissue. Asphyxiation can be caused by oxygen displacement, 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, dichloromethane, 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 haematological changes while abuse of nitrite inhalants is associated 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 widespread 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 26 September 2013 Page 20 of 31 ProQuest

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 compounds 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, trichloroethylene, xylene and benzene i.e. hippuric acid, trichloroacetic 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 important, 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 homogenisation [16]. A good seal also protects the sample against contamination from environmental and laboratory sources of volatiles [17-19]. Storage, transport and handling of the sample should always occur at approximately -5 till 4C. 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 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, 26 September 2013 Page 21 of 31 ProQuest

efficient, 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 (HSCOT), 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 (HSmultiple 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 chromatographic 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, minimises 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 sample, 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-mortem 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 proteolytic 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 Fourier 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 performed and 10ml of headspace gas was introduced into a chlorine gas containing vial. This was placed in the sun for 10min 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 physicochemical 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-m 26 September 2013 Page 22 of 31 ProQuest

polydimethylsiloxane phase (PDMS) for absorption of apolair compounds such as toluene and halothane. A polyacrylate 85-m 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 (75m) fibre to 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 sensitivity [30]. However, the absorption capacity is more sensitive for highly volatile and low molecular mass compounds. Sometimes 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 0C with use of liquid carbon dioxide or liquid nitrogen. 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 1ml of headspace air could be injected into narrow bore columns in the split mode, injection of 2 till 10ml 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 hydrolysed 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 effective for compounds with moderate to high water solubility and the concentration step, 'the trapping', enhances sensitivity. 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 recovery 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] suggest 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]. Ojanper et al. even applied the technique to the analysis of tert -butylmethyl ether and 1,1,2-trichloroethene 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 26 September 2013 Page 23 of 31 ProQuest

cryogenic trap is used. Morinaga et al. [40] described a purge and trap method with a Tenax TA-trap combined with a dehydratation 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 sampling techniques over the past few years, solvent extraction is used for chloral hydrate, trichloroethylene and their metabolites. Solvent extraction, however, is time consuming, laborious and more difficult to automate. Interferences in the chromatographic run can occur because of co-extraction 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 discussed 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 ferromagnetic coil quickly heated up by electromagnetic induction [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 30m, 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 10m. 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 0.53mm), 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 ionisation 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). 26 September 2013 Page 24 of 31 ProQuest

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-functions: 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 compound 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 standardisation [16]. The interaction of analytes with matrix components can result in binding of the volatile organic compounds thus becoming unavailable for determination in the gas 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, especially 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 temperature 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 benzene, 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 26 September 2013 Page 25 of 31 ProQuest

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 postmortem 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 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 trichloroacetic acid [28]. Trichloroethanol, a urinary metabolite 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 368nm, while reaction products with chloral hydrate and trichloroacetic acid have maxima at 368 and 530nm, 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% methylsilicone 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 candidates 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 environmental, occupational and therapeutic exposure of the deceased be evaluated, also the possible formation of endogenous volatile compounds due to several inborn errors of metabolism 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 chlorobutanol 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 benzoates, added to food as a preservative. The simultaneous detection of several compounds may also lead to determination of the source, for example petroleum consists of aliphatic and aromatic hydrocarbons, naphtalenes, paraffin, alkenes and eventually tetraethyl lead [55]. Trichloroacetic acid can originate from trichloroethylene, 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 contamination of the sample. 26 September 2013 Page 26 of 31 ProQuest

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, differences 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 simple, 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, compound mixes and differences in extraction techniques and detectors used. The large variety of volatiles requires an analytical method that both detects and identifies the compounds. MS, ECD and FID are usually used for quantitation purposes, while MS and FTIR have a screening and confirmation 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 analysed 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 quantitative results. References Esmail A, Meyer L, Pottier A, Wright S: Deaths from volatile substance-abuse in those under 18 years--results from a National Epidemiologic-Study . Arch. Dis. Child. 69 356-360, 1993. Flanagan RJ, Ruprah M, Meredith TJ, Ramsey JD: An introduction to the clinical toxicology of volatile substances . Drug Saf. 5 359-383, 1990. Marelich GP: Volatile substance abuse . Clin. Rev. Allergy Immunol. 15 271-289, 1997. Dinwiddie SH: Abuse of inhalants--a review . Addiction 89 925-939, 1994. Neumark YD, Delva J, Anthony JC: The epidemiology of adolescent inhalant drug involvement . Arch. Pediatr. Adolesc. Med. 152 781-786, 1998. Williams DR, Cole SJ: Ventricular fibrillation following butane gas inhalation . Resuscitation 37 43-45, 1998. Steffee CH, Davis GJ, Nicol KK: A whiff of death: fatal volatile solvent inhalation abuse . South. Med. J. 89 879884, 1996. Kurtzman TL, Otsuka KN, Wahl RA: Inhalant abuse by adolescents . J. Adolesc. Health 28 170-180, 2001. Balster RL: Neural basis of inhalant abuse . Drug Alcohol Depend. 51 207-214, 1998. Ashton CH: Solvent abuse--little progress after 20 years . BMJ 300 135-136, 1990. Musshoff F, Junker H, Madea B: An unusual case of driving under the influence of enflurane . Forensic Sci. Int. 128 187-189, 2002. 26 September 2013 Page 27 of 31 ProQuest

Tranthim-Fryer DJ, Hansson RC, Norman KW: Headspace/solid-phase microextraction/gas chromatographymass spectrometry: a screening technique for the recovery and identification of volatile organic compounds (VOC's) in postmortem blood and viscera samples . J. Forensic Sci. 46 934-946, 2001. Seto Y: Determination of volatile substances in biological samples by headspace gas-chromatography . J. Chromatogr. A 674 25-62, 1994. Cardinali FL, McCraw JM, Ashley DL, Bonin MA: Production of blank water for the analysis of volatile organiccompounds in human blood at the low parts-per-trillion level . J. Chromatogr. Sci. 32 41-45, 1994. Gill R, Hatchett SE, Osselton MD, Wilson HK, Ramsey JD: Sample handling and storage for the quantitativeanalysis of volatile compounds in blood--the determination of toluene by headspace gas-chromatography . J. Anal. Toxicol. 12 141-146, 1988. Streete PJ, Ruprah M, Ramsey JD, Flanagan RJ: Detection and identification of volatile substances by headspace capillary gas-chromatography to aid the diagnosis of acute-poisoning . Analyst 117 1111-1127, 1992. Dyne D, Cocker J, Streete PJ, Flanagan RJ: Toluene, 1-butanol, ethylbenzene and xylene from Sarstedt Monovette serum gel blood collection tubes . Ann. Clin. Biochem. 33 355-356, 1996. Streete PJ, Flanagan RJ: Ethylbenzene and xylene from Sarstedt Monovette serum gel blood-collection tubes . Clin. Chem. 39 1344-1345, 1993. Drozd J, Novak J: Headspace gas-analysis by gas-chromatography . J. Chromatogr. 165 141-165, 1979. Tytgat J, Daenens P: Solvent-free sample preparation by headspace solid-phase microextraction applied to the tracing of n-butyl nitrite abuse . Int. J. Legal Med. 109 150-154, 1996. Yang NC, Hwang KL, Hung DZ, Wuhh HH, Ho WM: Reliable gas chromatographic-mass spectrometric method combined with a headspace autosampler for isoflurane determination in blood . J. Chromatogr. B 742 277-282, 2000. Pihlainen K, Ojanpera I: Analytical toxicology of fluorinated inhalation anaesthetics . Forensic Sci. Int. 97 117133, 1998. Dehon B, Humbert L, Devisme L, Stievenart M, Mathieu D, Houdret N, Lhermitte M: Tetrachloroethylene and trichloroethylene fatality: case report and simple headspace SPME-capillary gas chromatographic determination in tissues . J. Anal. Toxicol. 24 22-26, 2000. Gaillard Y, Billault F, Pepin G: Tetrachloroethylene fatality: case report and simple gas chromatographic determination in blood and tissues . Forensic Sci. Int. 76 161-168, 1995. El-Haj BM, Al-Amri AM, Hassan MH, Bin-Khadem RK, Al-Hadi AA: A GC-MS method for the detection of toluene and ethylbenzene in volatile substance abuse . J. Anal. Toxicol. 24 390-394, 2000. Liu JT, Cheng P, Suzuki O: Solid-phase microextraction (SPME) of drugs and poisons from biological samples . Forensic Sci. Int. 97 93-100, 1998. Theodoridis G, Koster EHM, de Jong GJ: Solid-phase microextraction for the analysis of biological samples . J. Chromatogr. B 745 49-82, 2000. Brewer WE, Galipo RC, Morgan SL, Habben KH: The confirmation of volatiles by solid-phase microextraction and GC-MS in the investigation of two traffic fatalities . J. Anal. Toxicol. 21 286-290, 1997. Poli DBE, Manini P, Andreoli R, Mutti A: Solid-phase microextraction gas chromatographic-mass spectrometric method for the determination of inhalant anesthetics in urine . J. Chromatogr. B 732 115-125, 1999. Liu JT, Hara K, Kashimura S, Kashiwagi M, Hamanaka T, Miyoshi A, Kageura M: Headspace solid-phase microextraction and gas chromatographic-mass spectrometric screening for volatile hydrocarbons in blood . J. Chromatogr. B 748 401-406, 2000. Lee XP, Kumazawa T, Sato K, Watanabe K, Seno H, Suzuki O: Determination of solvent thinner components in human body fluids by capillary gas chromatography with trapping at low oven temperature for headspace samples . Analyst 123 147-150, 1998. 26 September 2013 Page 28 of 31 ProQuest

Watanabe-Suzuki K, Ishii A, Suzuki O: Cryogenic oven-trapping gas chromatography for analysis of volatile organic compounds in body fluids . Anal. Bioanal. Chem. 373 75-80, 2002. Bouche M, Lambert WE, Van Bocxlaer JFP, Piette MH, De Leenheer AP: Quantitative determination of npropane, iso-butane, and n-butane by headspace GC-MS in intoxications by inhalation of lighter fluid . J. Anal. Toxicol. 26 35-42, 2002. Ojanpera I, Hyppola R, Vuori E: Identification of volatile organic compounds in blood by purge and trap PLOTcapillary gas chromatography coupled with Fourier transform infrared spectroscopy . Forensic Sci. Int. 80 201209, 1996. Hattori H, Iwai M, Kurono S, Yamada T, Watanabe-Suzuki K, Ishii A, Seno H, Suzuki O: Sensitive determination of xylenes in whole blood by capillary gas chromatography with cryogenic trapping . J. Chromatogr. B 718 285289, 1998. Morinaga M, Hara K, Kageura M, Hieda Y, Takamoto M, Kashimura S: A simple, rapid and simultaneous analysis of complex volatile hydrocarbon mixtures in blood using gas-chromatography mass-spectrometry with a wide-bore capillary column . J. Legal Med. 103 567-572, 1990. Ohshima T, Kondo T, Sato Y, Takayasu T: Postmortem alcohol analysis of the synovial fluid and its availability in medico-legal practices . Forensic Sci. Int. 90 131-138, 1997. Saito K, Takayasu T, Nishigami J, Kondo T, Ohtsuji M, Lin ZQ, Ohshima T: Determination of the volatile anesthetics halothane, enflurane, isoflurane, and sevoflurane in biological specimens by pulse-heating GC-MS . J. Anal. Toxicol. 19 115-119, 1995. Schuberth J: Joint use of retention index and mass-spectrum in postmortem tests for volatile organics by headspace capillary gas-chromatography with ion-trap detection . J. Chromatogr. A 674 63-71, 1994. Schuberth J: A full evaporation headspace technique with capillary GC and ITD: a means for quantitating volatile organic compounds in biological samples . J. Chromatogr. Sci. 34 314-319, 1996. Astier A: Chromatographic determination of volatile solvents and their metabolites in urine for monitoring occupational exposure . J. Chromatogr. 643 389-398, 1993. Yang YD: Simultaneous determination of creatine, uric acid, creatinine and hippuric acid in urine by high performance liquid chromatography . Biomed. Chromatogr. 12 47-49, 1998. Fujii T, Kawabe S, Horike T, Taguchi T, Ogata M: Simultaneous determination of the urinary metabolites of toluene, xylene and styrene using high-performance capillary electrophoresis--comparison with highperformance liquid chromatography . J. Chromatogr. B 730 41-47, 1999. Raikhlin-Eisenkraft B, Hoffer E, Baum Y, Bentur Y: Determination of urinary hippuric acid in toluene abuse . J. Toxicol. Clin. Toxicol. 39 73-76, 2001. Meyer E, Van Bocxlaer JF, Lambert WE, Piette M, De Leenheer AP: Determination of chloral hydrate and metabolites in a fatal intoxication . J. Anal. Toxicol. 19 124-126, 1995. Heller PF, Goldberger BA, Caplan YH: Chloral hydrate overdose--trichloroethanol detection by gaschromatography mass-spectrometry . Forensic Sci. Int. 52 231-234, 1992. Levine B, Park J, Smith TD, Caplan YH: Chloral hydrate--unusually high-concentrations in a fatal overdose . J. Anal. Toxicol. 9 232-233, 1985. Coopman VAE, Cordonnier J, De Letter EA, Piette MHA: Tissue distribution of trichloroethylene in a case of accidental acute intoxication by inhalation . Forensic Sci. Int. 134 115-119, 2003. De Baere S, Meyer E, Dirinck I, Lambert W, Piette M, Van Peteghem C, De Leenheer A: Tissue distribution of trichloroethylene and its metabolites in a forensic case . J. Anal. Toxicol. 21 223-227, 1997. McBay AJ, Boling VR, Reynolds PC: Spectrophotometric determination of trichloroethanol in chloral hydrate poisoning . J. Anal. Toxicol. 4 99-101, 1980. Cairney S, Maruff P, Burns C, Currie B: The neurobehavioural consequences of petrol (gasoline) sniffing . Neurosci. Biobehav. Rev. 26 81-89, 2002. 26 September 2013 Page 29 of 31 ProQuest

Tanaka T, Kasai K, Kita T, Tanaka N: Distribution of phenol in a fatal poisoning case determined by gas chromatography mass spectrometry . J. Forensic Sci. 43 1086-1088, 1998. Ojanpera I, Pihlainen K, Vuori E: Identification limits for volatile organic compounds in the blood by purge-andtrap GC-FTIR . J. Anal. Toxicol. 22 290-295, 1998. Gaulier JM, Tonnay V, Faict T, Sayer H, Marquet P, Lachatre G: Analytical aspects of volatile substance abuse (VSA) . J. Forensic Sci. 48 880-882, 2003. Yang NC, Hwang KL, Shen CH, Wang HF, Ho WM: Simultaneous determination of fluorinated inhalation anesthetics in blood by gas chromatography-mass spectrometry combined with a headspace autosampler . J. Chromatogr. B 759 307-318, 2001. Kojima T, Ishii A, Watanabe-Suzuki K, Kurihara R, Seno H, Kumazawa T, Suzuki O, Katsumata Y: Sensitive determination of four general anaesthetics in human whole blood by capillary gas chromatography with cryogenic oven trapping . J. Chromatogr. B 762 103-108, 2001. Watanabe K, Seno H, Ishii A, Suzuki O, Kumazawa T: Capillary gas chromatography with cryogenic oven temperature for headspace samples: analysis of chloroform or methylene chloride in whole blood . Anal. Chem. 69 5178-5181, 1997. Broussard LA, Broussard AK, Pittman TS, Lirette DK: Death due to inhalation of ethyl chloride . J. Forensic Sci. 45 223-225, 2000. Ise H, Kudo K, Jitsufuchi N, Imamura T, Ikeda N: Simple and rapid determination of enflurane in human tissues using gas chromatography and gas chromatography mass spectrometry . J. Chromatogr. B 698 97-102, 1997. Park SW, Kim NY, Yang YG, Seo B, Paeng KJ: Toluene distribution of glue sniffers' biological fluid samples in Korea . J. Forensic Sci. 43 888-890, 1998. Broussard LA, Brustowicz T, Pittman T, Atkins KD, Presley L: Two traffic fatalities related to the use of difluoroethane . J. Forensic Sci. 42 1186-1187, 1997. Avis SP, Archibald JT: Asphyxial suicide by propane inhalation and plastic bag suffocation . J. Forensic Sci. 39 253-256, 1994. Schuberth J: Gas residues of engine starting fluid in postmortem sample from an arsonist . J. Forensic Sci. 42 144-147, 1997. Morinaga M, Kashimura S, Hara K, Hieda Y, Kageura M: The utility of volatile hydrocarbon analysis in cases of carbon monoxide poisoning . Int. J. Legal Med. 109 75-79, 1996. Kim NY, Park SW: The comparison of toluene determination between headspace-solid phase microextraction and headspace methods in glue-sniffer's blood and urine samples . J. Forensic Sci. 45 702-707, 2000. Lee XP, Kumazawa T, Sato K: A simple analysis of 5 thinner components in human-body fluids by headspace solid-phase microextraction (SPME) . Int. J. Legal Med. 107 310-313, 1995. Musshoff F, Junker H, Madea B: Rapid analysis of halothane in biological samples using headspace solidphase microextraction and gas chromatography-mass spectrometry--a case of a double homicide . J. Anal. Toxicol. 24 372-376, 2000. Seno H, Ishii A, Watanabe K, Suzuki O, Kumazawa T: Extraction of chloroform and methylene chloride in human whole blood and urine by headspace solid phase microextraction (SPME) . Med. Sci. Law 39 332-336, 1999. Fitzgerald RL, Fishel CE, Bush LLE: Fatality due to recreational use of chlorodifluoromethane and chloropentafluoroethane . J. Forensic Sci. 38 476-482, 1993. Groppi A, Polettini A, Lunetta P, Achille G, Montagna M: A fatal case of trichlorofluoromethane (freon 11) poisoning--tissue distribution study by gas-chromatography mass-spectrometry . J. Forensic Sci. 39 871-876, 1994. Winek CL, Wahba WW, Huston R, Rozin L: Fatal inhalation of 1,1,1-trichloroethane . Forensic Sci. Int. 87 161165, 1997. 26 September 2013 Page 30 of 31 ProQuest

Kim NY, Park SW, Suh JK: Two fatal cases of dichloromethane or chloroform poisoning . J. Forensic Sci. 41 527-529, 1996. Subject: Volatile organic compounds--VOCs; Teenagers; Solvent abuse; Metabolites Identifier / keyword: Volatile substance abuse, Post-mortem, Sample handling, Headspace analysis, Volatile organic compounds Publication title: Forensic Science International (Online) Volume: 142 Issue: 2 Pages: 135-156 Publication year: 2004 Publication date: 2004 Year: 2004 Publisher: Elsevier Limited Place of publication: Amsterdam Country of publication: United States Publication subject: MEDICAL SCIENCES--FORENSIC SCIENCES Source type: Scholarly Journals Language of publication: English Document type: CAL, Journal Article DOI: http://dx.doi.org/10.1016/j.forsciint.2004.02.015 ProQuest document ID: 1033030125 Document URL: http://search.proquest.com/docview/1033030125?accountid=28110 Copyright: 2004 Elsevier Ireland Ltd Last updated: 2012-08-11 Database: ProQuest Research Library: Health & Medicine

_______________________________________________________________
Contact ProQuest

Copyright 2012 ProQuest LLC. All rights reserved. - Terms and Conditions

26 September 2013

Page 31 of 31

ProQuest