# Sampling method development and optimization in view of human hand odor analysis by thermal desorption coupled with gas chromatography and mass spectrometry

Vincent Cuzuel, Eglantine Portas, Guillaume Cognon, Isabelle Rivals, François Heulard, Didier Thiébaut, Jérôme Vial

► To cite this version:

Vincent Cuzuel, Eglantine Portas, Guillaume Cognon, Isabelle Rivals, François Heulard, et al. Sampling method development and optimization in view of human hand odor analysis by thermal desorption coupled with gas chromatography and mass spectrometry. Analytical and Bioanalytical Chemistry, 2017, 409 (21), pp.5113-5124. 10.1007/s00216-017-0458-8. hal-04018375

HAL Id: hal-04018375

https://hal.science/hal-04018375

Submitted on 10 Mar 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1 Sampling method development and optimization in view of

2 human hand odor analysis by thermal desorption coupled

3 with gas chromatography and mass spectrometry.

4 Authors: Vincent Cuzuel a,b, Eglantine Portas b, Guillaume Cognon b, Isabelle Rivals c, François

5 Heulard b, Didier Thiébaut a, Jérôme Vial a

6 Corresponding author: Vincent Cuzuel, vincent.cuzuel@espci.fr, +33140794644

7 a Laboratoire Sciences Analytiques Bioanalytiques et Miniaturisation, ESPCI Paris, PSL

8 Research University, UMR CBI 8231, CNRS, 10 rue Vauquelin, PARIS, France

9 b Institut de Recherche Criminelle de la Gendarmerie Nationale, Caserne Lange, 5 Boulevard

10 de l'Hautil, BP 20312 PONTOISE, 95037 CERGY PONTOISE CEDEX, France

11 c Equipe de Statistique Appliquée, ESPCI Paris, PSL Research University, INSERM, UMRS 1158

12 Neurophysiologie Respiratoire Expérimentale et Clinique, 10 rue Vauquelin, PARIS, France

13 Keywords:

14 Hand sampling; thermodesorption; forensics; odor; gas chromatography

15
16

Abstract:

17

Forensic profiling of human odor is challenging and would be useful to support information

18

provided by dogs in courts of justice. Analyses of volatile compounds constitutive of human

19

odor are commonly performed with gas chromatography coupled with mass spectrometry.

20

All developed methods and sampling prototypes have to be easy-to-use in the field by crime

21

scene investigators. This paper will focus on techniques for human hand odor sampling prior

22

to analysis by a thermodesorption device coupled with gas chromatography and mass

23

spectrometry. Thermodesorption and gas chromatography methods were developed using a

24

sorbent phase spiked with a mixture of 80 compounds representative of human hand odor.

25

Then the crucial sampling step was performed indirectly with a home-made device based on

26

air suction and trapping on a sorbent. This indirect sampling device was evaluated with the

27

same synthetic mixture for optimization. An innovative polymer sorbent called Sorb-Star®

28

was compared to classic Tenax TA® packed tubes. Sorb-Star® provided similar recovery to

29

Tenax TA® packed tubes and a smaller pooled coefficient of variation (6% vs 13%). Thus, it

30

appeared to be fully suited to the indirect sampling of human hand odor. The developed

31

methods were successfully applied to real samples, the ultimate aim being the comparison

32

of a suspect's sample to a sample collected from a crime scene.

33
34

# 1. Introduction

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

Forensic profiling of human hands odor is challenging and requires the development of an
entire strategy, from sampling to statistical data processing. Several studies have been
carried out focusing on the analysis of hand odor (1–4) and the state of the art in this field is
described in details in a review (5). Hands are the body part the most susceptible to be in
contact with an element at the crime scene. It has been shown they generate volatile
profiles which provide sufficient stability through time, and variations from one subject to
another for differentiation (2,6). However, enough information has to be collected to allow a
proper discrimination between individuals: thus, sampling step is critical for efficient and
reliable collection of human hands odor for forensic purpose. There is no standardized
protocol for the collection of odor for analytical purpose yet and two kinds of samplings,
with or without contact, can be implemented. As for chromatographic separation and
detection, gas chromatography coupled with mass spectrometry (GC-MS) appears to be one
the most appropriate techniques and have a direct incidence on the sample preparation and
injection.

Direct sampling procedure relies on the contact between a sorbent and the subject or at
least an object left with its odor. Sorbent choice remains critical and has an influence on the
trapping and release of compounds (7). Contact sampling with cotton material has been
shown to be the collection method that yields the greatest number of volatile compounds
and the highest scent mass amounts (7–10). Cotton swabs were also successfully used for
the molecular cartography of the human skin surface (11). But a wide diversity of material
can be used:glass beads were said to allow the preferential concentration of “oily” residues
while minimizing collection of aqueous perspiration (3,12), Sorptive tape extraction (13,14)
57 which uses polymer "patches" made of a thin layer of flexible polymer put on a solid matrix,
58 hydrogels (15) (16). Many parameters (water content, temperature, composition, purity)
59 (17) are influential and make their use complicated. On the contrary, the use of commercial
60 sorbent, already conditioned, and ready-to-use could consequently facilitate the sampling
61 step, especially in the field. For instance, polydimethylsiloxane (PDMS) coated stir bars used
62 for stir bar sorptive extraction (SBSE) (18-20) eliminate the need for pads or cotton gauzes
63 which are not analytically clean and could lead to overloaded blanks. After conditioning, the
64 stir bar can be used directly on skin, or possibly stored and eventually analyzed.

65 However, contact sampling techniques are useless if the suspect was cautious, did not touch
66 anything at the crime scene and just left an odoriferous trail. In this case, the
67 implementation of non-contact/indirect sampling would be useful too, especially to sample
68 someone's odor in a room or ambient air at a crime scene. "Scent Transfer Unit" is the most
69 commonly used in the USA for dog identification (21-23). This device is made of a vacuum
70 pump connected to a gauze, allowing the collection of volatile organic compounds. For such
71 an approach, many parameters need optimization and a validated protocol should be set up.

72 For instance, flow rate and time of collection have to be carefully chosen not to lose
73 compounds which can break through the collection material (24). Molecular backbone of the
74 sorbent, its chemical composition as well as the weave of the material used, play a part in
75 the trapping and release of compounds (9,25) and were studied in depth (26). Dormont et al.
76 evaluated several analytical preconcentration techniques such as dynamic head-space
77 sorbent tube sampling (DHS), head-space SPME, contact SPME-opposed to head-space
78 SPME (1,27). According to the authors, contact SPME sampling appeared to be faster and
79 more convenient for field experiments. However, only DHS allowed trapping more volatile
80 compounds and isolating trace-levels compounds that were not detected using SPME. The
81 "flow sampling chamber" is also a promising indirect sampling technique. Subject's hand is inserted into a hermetic device coupled with a pump ($N_2$ flow). Compounds carried by the flow are trapped with a Solid Phase Micro Extraction (SPME) fiber, which is then thermodesorbed in GC-MS (28).

85 Whatever the sampling method, with or without contact, desorbing directly samples into the GC-MS reduces steps of sample preparation, compared with solvent based elution techniques, and avoids losing compounds. Different materials and sorbent phases can be thermally desorbed (20) and are suitable for human hands odor analysis. Thermodesorption is then a relevant approach (29) and is a promising alternative to SPME. The latter was used by Colon-Crespo et al. in a recent paper but the extraction time was set to 15 hours (30) which would barely be suitable for routine analysis.

92 The present paper aims at developing and optimizing two methods for both direct and indirect sampling involving a thermodesorption device. It is essential to keep in mind that profiling human odors use for forensic applications will not be possible as long as no standardized sampling procedures are clearly defined and validated. In real cases, sampling steps won't be performed at the bench but directly at a crime scene. This is one of the reasons why all developed methods and prototypes have to be easily transferrable in the field. For now, sampling methods are validated in the lab while testing in real conditions is carried out in partnership with dog handlers.

100 In this paper, thermodesorption parameters for both direct and indirect sampling were optimized. An original and easy-to-use polymeric sorbent phase called Sorb-Star® was evaluated and compared to Tenax TA® packed tubes. Specifically for indirect sampling, a prototype was developed with an industrial partner. This paper gives the results of the
preliminary study of the prototype device at our disposal. At this stage of the project, both
thermodesorption device and indirect sampling methods were developed using
experimental design and a synthetic mixture of 80 standards representative of the
compounds likely to be present in human hand odor (5,31–33). Both GC-MS and
comprehensive 2D gas chromatography coupled with mass spectrometry (GC×GC-MS) were
used in this study. While GC-MS was sufficient enough to perform preliminary experiments,
real samples are likely to be more complex and the use of GC×GC-MS will enhance the
separation.

## 2. Material and methods

### 1) Chemicals and reagents

Standards were purchased from Sigma-Aldrich (Saint Louis, USA). The detailed composition
of this mixture is reported in supplementary information (SI). Methanol was purchased from
Carlo Erba Reagents (Cornadero, Italy). Standards were not purchased in solution. Ultra-pure
water was produced using a Direct-Q UV 3 system (18.2 MΩ/cm) from Millipore (Darmstadt,
Germany).

### 2) Thermodesorption and separation devices

The purge and trap system Versatile Sample Preparator (VSP4000) was purchased from
Innovative Messtechnik GmbH (Vohenstrauß, Germany). Volatile substances are purged
from the sample matrix by the carrier gas of the GC. This concentration step is done with
adsorption on a suitable adsorbent in the system trap by freezing out at low temperatures.
After completion of the purging process the concentrated substances are transferred by fast
126 thermal desorption from the trap onto a transfer line and then separated by gas

127 chromatography.

128 The thermodesorption device was coupled with a GC-MS Q2010Plus purchased from

129 Shimadzu (Kyoto, Japan). A ZB-1MS column (30 m x 0.25 mm, 0.25 µm) (Phenomenex,

130 Torrance, USA) was used to conduct the chromatographic separation. Initial temperature

131 was 40 °C, held 1 min, then raised to 250 °C at 2.5 °C/min, held 1 min. Mass spectrometer

132 was used with the electronic ionization source (70 eV) heated at 200°C. The acquisition was

133 made with scan mode. The scan range was 29-250 m/z. Data were acquired with GC Real

134 Time Analysis and processed with GC PostRun Analysis 4.20 (Shimadzu softwares).

135 For GCxGC-MS analyses, a ZB-1MS column (30 m x 0.25 mm, 0.25 µm) (Phenomenex,

136 Torrance, USA) coupled with a ZB-1701 column (1.5 m x 0.1 mm, 0.1 µm) (Phenomenex,

137 Torrance, USA) were used. Modulation was performed with a N₂ cooled Zoex ZX1 thermal

138 modulator (Zoex, Houston, USA). Modulation time was 8 seconds. The same gradient and

139 mass spectrometry conditions as the GC-MS method were used. Data were acquired with GC

140 Real Time Analysis 4.20 and processed with GC Image 2.5 (Shimadzu and Zoex softwares)

141 and ChromSquare 2.2 (Shimadzu and Chromaleont softwares).

142 All statistical comparisons and significance tests were carried out by ANOVA with a type I

143 error risk α set at 5% (JMP® software, SAS Institute).

144

145 3) Analytical standards for optimization

146 To optimize analytical methods, a mixture of 80 standards was prepared in heptane at a

147 concentration of 10 mg/kg (6.84 mg/L) each. The detailed composition of this mixture is

148 reported in supplementary information (SI). The selected standards are compounds likely to
149

be found in human hand odor (5). A particular attention was given to covering a wide range

150

of compounds with different chemical properties.

151

152

## 4) Sorbent phase for compounds trapping

153

Sorb-Star® were purchased from Action Europe (Sausheim, France). This sorbent is a

154

patented silicon-based polymeric phase and is subject to specific conditioning processes to

155

avoid contaminations as far as possible. The Sorb-Star® has a density of 1.12 g / cm³ and is

156

compliant with FDA 177.2600. It is physiologically safe and even suitable for applications in

157

the food industry. It is meant to be used while direct sampling. People will be asked to rub it

158

in their hands during a defined period of time.

159

Sorb-Star® packaging, before and after sampling, could impact the final results by

160

introducing interfering compounds. The Sorb-Star® was conditioned in a 2mL vial in

161

controlled atmosphere. 6 different packaging were evaluated: 1 - amber glass vial with blue

162

polypropylene (PP) cap, aluminium foil seal; 2 - amber glass vial with blue PP cap, silicone

163

and PTFE seal; 3 - amber glass vial with blue PP cap, butyl and red PTFE seal; 4 - amber glass

164

vial, silicone and PTFE seal; 5 - PP vial with red polyamide (PA) cap, transparent silicone and

165

PTFE seal. 6 - amber cartridge - silver silicone aluminium cap

166

To optimize analytical methods, Sorb-Star® were spiked with 5 µL of the mixture of 80

167

standards prepared in heptane (10 mg/kg) to mimic a direct sampling. Spiked Sorb-Star® was

168

then placed into a stainless steel vial for analysis with VSP4000-GC-MS.

169

Optimization of desorption parameters was performed by monitoring 9 compounds – out of

170

80 - using only GC-MS separation (cf. table 1). The compounds were selected to cover the

171

whole chromatogram with a wide range of boiling point and different chemical families.
172

173

## 5) Indirect sampling device

174

A lab sampling prototype (cf. fig 1) was provided by Action Europe (Sausheim, France).

175

This is the first step to the development of a field user-friendly sampling device. Despite the

176

fact that this device is not fully adapted to the use in the field yet, it provided crucial

177

preliminary results to methods development. It consisted of a glass chamber with one inlet

178

and one outlet: a gas inlet valve connected to a flowmeter and an outlet to connect solid

179

sorbent tubes and collect compounds. The whole device is connected to a portable pump

180

which can work at flowrates from 10 to 80 mL/min. Two different sorbents were used: Tenax

181

TA® and Sorb-Star®. Both Tenax TA® packed tubes and Sorb-Star® cartridges were purchased

182

from Action Europe (Sausheim, France). A plastic hand covered with a nitrile glove and 5 µL

183

of the mixture of 80 standards prepared in heptane (10 mg/kg) were put into the chamber.

184

The use of a plastic hand allowed to mimic real sampling conditions.

185

Glass chamber was washed carefully with perfume-free soap, rinsed with milliQ water and

186

dried at 150°C during one hour between analyses. The seal of the glass chamber were

187

disposable and changed before any experiment (cf. fig 1). Analyses were performed at

188

ambient temperature between 22 and 23 °C.

189

Optimization of the indirect sampling device was performed by monitoring 16 compounds –

190

out of 80 – using GC×GC-MS separation (cf. table 2). These compounds were selected

191

according to their retention time so that they are scattered all over the 2D chromatogram

192

(cf. fig 2).
193 With Tenax TA packed tubes, a vacuum sampling pump (Sigma Aldrich, St Louis, USA) was
194 used at a flowrate of 70 mL/min during 15 minutes. Higher values can increase breakthrough
195 phenomena and are hence not well suited (34).

196 With Sorb-Star® cartridges, a specific prototype was developed by Action Europe (Sausheim,
197 France) to drill the cartridges and control the flowrate. A flowrate of 70 mL/min was used
198 during 15 minutes. For now, flowrates cannot be higher than 70mL/min because of the
199 prototype limitations.

200

201 3. Results and discussion

202 Prior to method development, the impact of the use of different vials seals for the
203 conservation of samples –sorbent- was evaluated using GC×GC-MS. Then, the desorption
204 conditions were optimized by TD-GC-MS using spiked Sorb-Star® in order to mimic a direct
205 sampling. Once the desorption method was developed, the indirect sampling procedure was
206 optimized by TD-GC×GC-MS.

207 The whole mixture was run and then a subset of peaks was selected to reduce data
208 processing, for both GC-MS and GC×GC-MS (cf. tables 1 and 2). They were selected so they
209 are scattered all over the chromatogram. Both GC-MS and GC×GC-MS were used in this
210 study. Because of technical issues, GC×GC-MS was not available to carry out every
211 experiment but it did not have an impact on the results as only data obtained with the same
212 technique were compared. The desorption conditions were optimized with 1D separation
213 while the indirect sampling procedure was developed using comprehensive 2D gas
214 chromatography. As sampling and desorption steps are totally independent of the
chromatographic separation, using 1D or 2D GC cannot modify the results and conclusions. 215

All comparisons and significance tests were carried out by ANOVA with a type I error risk α 216

set at 5%. 217

218

1) Packaging and reduction of external contamination of samples 219

Six different vial seals (cf. fig 3-2 to 3-7) were tested to study and minimize their possible 220 effect on the contamination of Sorb-Star®. The analyses were performed on blank Sorb Star 221 with GC×GC-MS to compare the different packagings qualitatively, using the color plots 222 which were all drawn with the same intensity scale. The differences between the 223 chromatograms are significant and this figure highlights the importance of packaging 224 selection. It appeared essential to reduce matrix effects and seal number 2 (cf. fig 3-2) was 225 selected because it minimized the contamination (peaks of silica derivatives). Its 226 chromatogram was the closest to a “fresh” Sorb-Star® (cf. fig 3-1) – analysed directly after 227 conditioning process. Thus, the same seal was selected for Sorb-Star® stored in vials for 228 direct sampling and in cartridges for indirect sampling. Moreover, the impact of the use of 229 nitrile gloves during sample preparation was evaluated. A blank Sorb-Star® was put during 230 15 minutes inside a nitrile glove. No additional contaminants were noticed (cf. fig 3-8). 231

232

233 2) Optimisation of desorption

The study was carried out by GC-MS coupled to the thermodesorption device. To optimize 234 analytical methods, Sorb-Star® were spiked with 5 µL of the mixture of 80 standards 235 prepared in heptane (10 mg/kg). Spiked Sorb-Star® was then placed into a stainless steel vial 236 for analysis with TD-GC-MS. 237
238 Eight parameters had to be optimized: sample temperature, valve temperature, purge time,
239 purge flow, trap temperature, trap desorption temperature, desorption time and split flow.
240 According to constructor recommendation, desorption time was set to 10 min. A shorter
time would not be enough to desorb the heaviest compounds whereas a longer one could
241 reduce the lifetime of the trap. Many parameters had to be taken into account so that, prior
242 to the experimental design, repeatability analysis and some preliminary tests were carried
243 out to determine the temperature conditions to be applied to the valve, the trap and for
244 desorption. Then, a design of experiment was conducted to optimize the sample
245 temperature, the purge time, the purge flow and the split flow with a reduced number of
246 experiment.
247

248 2.1 Repeatability evaluation

249 Analyses were performed with GC-MS nine times to assess the repeatability of the peak
250 areas. The coefficients of variation (CV) of the compounds (10 mg/kg in heptane) were
251 calculated (cf. table 3). Thermodesorption induces many sources of variability which are not
252 easy to control, and CV under 10% are quite acceptable. The pooled CV is 7.6%.
253

253

254 2.2 Preliminary tests

255 The valve, trap and desorption temperatures were determined with preliminary tests. Their
256 interactions with other parameters to be studied using the DOE were considered
257 unimportant. To evaluate the influence of these parameters, each sample was analyzed in
258 triplicate and ANOVA (35) was performed.
259

### 2.2.1 Valve temperature

260

The valve temperature can theoretically be set from 50°C to 280°C. In practice, temperatures

261

higher than 210°C prevent the trap from being cooled at -35°C. Thus, 150°C, 180°C, 210°C

262

were tested to determine if this parameter had an influence, but no significant difference

263

was observed (cf. table 3). Thus, the valve temperature was set to 210°C to avoid any cold

264

point in the system.

265

266

### 2.2.2 Trap temperature

267

The trap temperature can theoretically be set from -40°C to -10°C. In practice, when the

268

valve temperature was set to 210°C, it prevented the trap from being cooled at -40°C. Thus,

269

-30°C and -35°C were tested to determine if this parameter needed further optimization.

270

There was no significant difference between these two temperatures except for octanoic

271

acid, methyl ester (cf. table 3). The trap temperature was set to -30°C.

272

273

### 2.2.3 Desorption temperature

274

The trap desorption temperature can be theoretically set from 100°C to 400°C. According to

275

the constructor recommendations, temperatures higher than 240°C could damage the trap

276

and reduce its lifetime. Yet, Tenax TA was successfully used in other studies at temperature

277

higher than 240°C (36). Two temperatures were tested: 240°C and 260°C. There was no

278

significant difference between these two temperatures except for acetophenone (cf. table

279

3). Moreover, for both temperatures, a second analysis of the tubes showed that all
280

compounds were desorbed during the first analysis. Thus, the desorption temperature was

281

set to the recommended value of 240°C.

282

283

## 2.3 Design of experiment

284

A 2⁴ full factorial design was conducted in order to check whether sample temperature,

285

purge flow, purge time and split flow had a significant effect on the analytical process. The

286

peak areas were used as response in the experiments. The “+1” level was set as follows:

287

sample temperature = 220°C, purge flow = 30mL/min, purge time = 30 min, split =

288

50mL/min. The “-1” level was set as follows: sample temperature = 160°C, purge flow =

289

10mL/min, purge time = 10 min, split = 10mL/min. Four repeated measurements were

290

performed at the center of the domain (sample temperature = 190°C, purge flow =

291

20mL/min, purge time = 20 min, split = 30mL/min). This whole design was repeated twice to

292

increase the number of degrees of freedom. The lack of fit test was also performed to check

293

whether the proposed model was correct.

294

The split had a major effect on all compounds intensity. This effect is expected as only a

295

fraction of the injected sample goes into the column when a split is used. Unexpectedly, in

296

the tested conditions, the effects sample temperature, purge time and purge flow were not

297

significant. This thermodesorption method appears to be robust with respect to the other

298

experimental parameters (cf. fig 4). For the further analyses, the sample temperature, purge

299

time and purge flow were set to the “0” level – respectively 190°C, 20 minutes and 20

300

mL/min - while the split was decreased to the “-1” level - 10 mL/min.
301

In conclusion of this section, the direct sampling can be performed using Sorb-Star®: the
desorption parameters of this sorbent phase are now optimized and will be used for the
analysis of real samples collected with direct sampling procedures.

302

303

304

### 3) Indirect sampling

305

306

307

308

309

310

311

312

313

314

### 3.1 Sorb-Star® cartridges

315

#### 3.1.1 Repeatability tests

316

317

318

319

320

321

322

323

First of all, the repeatability of every part of the sampling device was checked. To evaluate
separately the different parts of the device, the glass chamber was replaced by a 20 mL glass
vial. The pump – without the glass chamber – provided quite good results at 70 mL/min
during 15 minutes since the coefficient of variation of target compounds did not exceed 6%
except for nonane (which is close to the solvent tail) and eicosane (which is close to its limit
of detection). However using the complete device - with the glass chamber - was the pooled
coefficient of variation rose up to 30%. Different hypotheses can be made to explain this
increase. First, the flow rate of 70 mL/min was not suited (too low) and needed a proper
optimization. To solve this problem, a new pump prototype is being designed and should
reach flow rates up to 200 mL/min. Also, the design of the glass chamber may not be
appropriate and could be improved. To this end, a new glass chamber is being designed to
create turbulences inside the chamber and to enhance repeatability.

328 **3.1.2 Preliminary tests for indirect sampling optimization**

The first tests showed that the complete device - glass chamber and pump – was not
optimized. For all preliminary tests – except for the fan - the glass chamber (cf. fig 1) was
replaced by a 20 mL glass vial to reduce sources of variability and get a better evaluation of
the tested effect. Effectively, as the geometry of the glass chamber is independent of the
cartridge optimization, it is possible to decouple the problems.

334

335 **Use of a fan**

Considering the bad repeatability of the first results, the use of a fan was evaluated during
sampling. Whereas agitation was expected to create turbulences which would enhance the
repeatability of the process, it was shown not to be decisive. Pooled coefficient of variations
reached 31% without the fan (CV ranged from 2 to 56%) and decreased to 28% with it (CV
ranged from 12 to 44%). These CV (cf. table 4) are high but are not dramatic and are close to
classic headspace CV (37,38). In the meantime, further testing was done to design the
cartridges containing the Sorb-Star®.

343
344

## Use of a centering piece in the cartridge

345

346

347

348

349

350

351

352

353

354

To maximize the contact between the Sorb-Star® and the flow, the use of a centering piece inside the cartridge was evaluated. It consisted in an aluminum cross which maintained the Sorb-Star® at the center of the cartridges and avoided contact with the cartridge wall, thus maximizing the available surface. Surprisingly, whereas it was expected to collect more compounds as the available surface increased, centering or not the sorbent made no difference. Moreover, the variability increased despite the fact that the position of the sorbent in the cartridge was better controlled with the centering piece (cf. table 4): the pooled coefficient of variations was 10% without the centering piece (CV ranged from 4 to 20%) and it rose up to 17% with the centering piece (CV ranged from 6 to 32%).

Thus, this centering piece will not be used for further experiments.

355

356

## Pulsed mode

357

358

359

360

361

362

363

364

365

Unlike Tenax, with Sorb-Star® cartridges, the polymer did not fill the whole cartridge. It is important that compounds adsorb on the sorbent phases and do not go through the cartridge. The use of a pulsed mode of the pump was evaluated to create turbulences and enhance the trapping of compounds. The pump is on during a predefined time, then turned off during a predefined time, this cycle being repeated during all the sampling procedure.

The pulses aimed at creating some turbulences to stir in the cartridge and maximize the contact between the Sorb-Star® and the compounds. 2mL cartridges were used with a maximal flow rate of 80 mL/min. Pulses cannot be lower than 500 ms (device limitation) and should not exceed 1500 ms (homogenous agitation).
Pulses of 500 ms and 1000 ms were evaluated. There were no significant differences with or without pulse. The use of a pulsed mode increased the variability (cf. table 4), preventing us from considering this mode of sampling. The pooled coefficient of variation was 10% without the centering piece (CV ranged from 4 to 20%) and it rose up to 12% and 13% with the short-pulsed mode (CV ranged from 0.1 to 34%) and the with the long-pulsed mode respectively (CV ranged from 1 to 38%).

372

373 Use of restricted cartridges

374

Another attempt was to reduce the volume of the cartridges to trap more compounds on the sorbent phase. Since the volume of the cartridge was reduced, the loss of compounds going through the cartridge was expected to be limited. There were significant differences between experiments with or without restriction. Surprisingly, more compounds were collected without using the restricted cartridge.

As for the variability, pooled coefficient of variation (cf. table 4) was 10% without the restricted cartridge (CV ranged from 4 to 20%) and it rose up to 17% with the restricted cartridge (CV ranged from 2 to 42%).

383

384 3.2 Tenax TA phase

First of all, the Tenax TA packed tubes repeatability was assessed (n=3) by measuring the intensities of the 16 targeted compounds. The results were quite promising as there was no significant difference between the three tubes.
388 Tenax TA packed tubes and Sorb-Star® cartridges performance used in indirect sampling
389 mode were compared to direct thermodesorption of Sorb-Star® spiked with the synthetic
390 mixture of 80 compounds. The pooled coefficient of variations was 19% for direct sampling
391 (CV ranged from 0.1 to 39%).

392 This experiment gave interesting results (cf. fig 5). First, the variability of recovered
393 compounds is more important with Tenax phase - pooled coefficient of variation was 13%
394 (CV ranged from 1 to 30%) – than with Sorb-Star® cartridges where the pooled coefficient of
395 variation was 6% (CV ranged from 1 to 16%).

396 As expected, for most compounds, there was a lower recovery between direct spiked Sorb-
397 Star® and indirect sampling process on the two phases (Tenax and Sorb-Star® cartridges).

398 Tenax and Sorb-Star® provided quite similar results for recovering our compounds of
399 interest. However, the developed methods have to be easily transferable in the field. The
400 use of a disposable, cheap and easy-to-use sampling phase such as Sorb-Star® will be
401 preferred by police forces. A Sorb-Star® costs around 3 euros while a Tenax packed tube is
402 much more expensive and costs around 100 euros. And even if it can be reused after a
403 proper conditioning procedure, Tenax can only be used for indirect sampling. Nevertheless,
404 the use of reconditioned sampling phases is not viable as it would weaken the probative
405 value of evidence brought in courts of justice. These are the reasons why we selected Sorb-
406 Star® for both direct and indirect sampling and gave up tests with Tenax phase.

3.3 Application to real samples

408 The developed methods were applied to real samples. Several hundreds of peaks are detected
409 (cf. figure 6) showing that the developed protocol is relevant and efficient to collect volatiles
410 from human hands. The use of a bidimensional separation technique is also relevant
411 considering the number of compounds involved in human odor. GC×GC-MS provided an
412 increased peak capacity, thereby allowing the separation of compounds which would not have
413 been possible using classical GC-MS. A selection of 40 compounds identified in this
414 chromatogram using NIST library is given in the table 5. All of them were described at least
415 once as constitutive compounds of human hands odor (5). It should be noted that a large
416 diversity of chemical families is present, validating the choice of the 80 standards for previous
417 optimization.

418 For real samples, putting a hand in the confined space of the sampling chamber raises
419 substantially the temperature during sampling. It also increases the humidity as compared to
420 tests carried out with the synthetic mixture. Further experiments will be carried out to see to
421 what extent this parameter has an influence on the sampling procedure.

422

423 4. Conclusion

424 Forensic profiling of human hand odor is complex and needs the development of a validated
425 sampling procedure. Desorption optimisation and indirect sampling method using a thermal
426 desorption device coupled with GC×GC-MS were developed for human hand odor, from the
427 selection of the packaging seal to the optimization of indirect sampling and
428 thermodesorption conditions. To mimic human hand odor and perform proper comparison
429 between techniques, a synthetic mixture of 80 compounds representative of human hand
430 odor was used. The methods developed constitute a solid background to perform further
431 testing, to carry out analyses of real samples and to assess the transfer of the different
432 methods to crime scene investigators. Sorb-Star® was selected for both direct and indirect
433 sampling while tests with Tenax phase given up. Unlike Tenax, Sorb-Star® can be used for
434 both direct and indirect sampling. The use of a disposable, cheap, easy-to-use and unique
435 sampling phase will be preferred by police forces. While the pumping mode and the
436 cartridge were successfully optimized, a new prototype of glass chamber is being developed
437 and will be evaluated using the preliminary results obtained in this study. Moreover,
438 different tests would be performed with dogs to check if enough information was collected
439 with Sorb-Star® to individualize people. Whatever the sampling method – direct or indirect –
440 dogs should be able to trace back a person just by sniffing out a Sorb-Star®.

441 All the developed methods will be easily transferable in the field. This first study was
442 restricted to 80 compounds and GC-MS could be used in this preliminary approach. The
443 developed protocols were successfully applied to real samples. Several hundreds of
444 compounds were detected, making the use of GC×GC-MS a true asset at this stage. The
445 combination of increased selectivity, peak capacity, as well as sensitivity due to the
446 cryomodulation will enhance the separation and enable to collect more information. The
447 application to real samples will need a proper statistical processing to validate the relevance
448 of the collected information, to assess its repeatability given the variations of human odor,
449 and finally to perform proper individualization.

450

451 Acknowledgements

452 We would like to thank Olivier Hoffarth (Action Europe, Sausheim, France) for his help and
453 the development of prototypes.

454 We would like to thank Shimadzu for the trial license of ChromSquare 2.2

455
456 *Conflicts of interest*

457 The authors declare having no conflict of interest of any kind.

458

459

460
483

7. Hudson DT. Variables Affecting the Collection and Preservation of Human Scent Components through Instrumental and Biological Evaluations. FIU Electronic Theses and Dissertations. Florida International University; 2009.

484

485

486

8. Prada P a, Curran AM, Furton KG. The evaluation of human hand odor volatiles on various textiles: a comparison between contact and noncontact sampling methods. J Forensic Sci. 2011 Jul;56(4):866-81.

487

488

489

9. Hudson-holness DT, Furton KG. Comparison between Human Scent Compounds Collected on Cotton and Cotton Blend Materials for SPME-GC/MS Analysis. J Forensic Res [Internet]. 2010 [cited 2014 Oct 2];1(1):1-6. Available from: http://www.omicsonline.org/2157-7145/2157-7145-1-101.digital/2157-7145-1-101.html

490

491

492

493

494

10. Hudson DT, Curran AM, Furton KG. The stability of collected human scent under various environmental conditions. J Forensic Sci. 2009 Nov;54(6):1270-7.

495

496

11. Bouslimani A, Porto C, Rath CM, Wang M, Guo Y, Gonzalez A, et al. Molecular cartography of the human skin surface in 3D. Proc Natl Acad Sci U S A [Internet]. 2015;112(17):E2120-9. Available from: http://www.ncbi.nlm.nih.gov/pubmed/25825778%5Cnhttp://www.pnas.org/content/112/17/E2120.full.pdf

497

498

499

500

501

12. Verhulst NO, Beijleveld H, Qiu YT, Maliepaard C, Verduyn W, Haasnoot GW, et al. Relation between HLA genes, human skin volatiles and attractiveness of humans to malaria mosquitoes. Infect Genet Evol. Elsevier B.V.; 2013 Aug;18:87-93.

502

503

504

13. Bicchi C, Cordero C, Liberto E, Rubiolo P, Sgorbini B, Sandra P. Sorptive tape extraction in the analysis of the volatile fraction emitted from biological solid matrices. J Chromatogr A. 2007 May 4;1148(2):137-44.

505

506
507

14.

Sisalli S, Adao A, Lebel M, Fur I Le, Sandra P. Sorptive Tape Extraction - A Novel
Sampling Method for the in vivo Study of Skin. LCGC Eur [Internet]. Advanstar
Communications Inc.; 2006 Jan 1 [cited 2014 Oct 20]; Available from:
http://www.chromatographyonline.com/lcgc/article/articleDetail.jsp?id=284456&pageI
D=1&sk=&date=

508

509

510

511

512

15.

Rodrigues LADS, Figueiras A, Veiga F, de Freitas RM, Nunes LCC, da Silva Filho EC, et al. The systems containing clays and clay minerals from modified drug release: a review. Colloids Surf B Biointerfaces. Elsevier B.V.; 2013 Mar 1;103:642-51.

513

514

515

16.

Dutkiewicz EP, Lin J, Tseng T, Wang Y, Urban PL. Hydrogel Micropatches for
Sampling and Profiling Skin Metabolites. Anal Chem. 2014;

516

517

17.

Gámiz E, Martín-García JM, Fernández-González MV, Delgado G, Delgado R.
Influence of water type and maturation time on the properties of kaolinite–saponite
peloids. Appl Clay Sci [Internet]. Elsevier B.V.; 2009 Sep [cited 2014 Oct
27];46(1):117-23.
Available
from:
http://linkinghub.elsevier.com/retrieve/pii/S0169131709001756

518

519

520

521

522

18.

Penn DJ, Oberzaucher E, Grammer K, Fischer G, Soini H a, Wiesler D, et al. Individual and gender fingerprints in human body odour. J R Soc Interface. 2007 Apr 22;4(13):331-40.

523

524

525

19.

Hasegawa Y, Yabuki M, Matsukane M. Identification of new odoriferous compounds in human axillary sweat. Chem Biodivers. 2004 Dec;1(12):2042-50.

526

527

20.

David F, Sandra P. Possibilities and limitations of recent developments in micro sample preparation techniques. In: SBSE Technical meeting. 2015.

528

529

21.

Curran AM, Prada PA, Furton KG. Canine human scent identifications with post-blast debris collected from improvised explosive devices. Forensic Sci Int. 2010 Jun

530
531 15;199(1-3):103-8.

532 22. DeGreeff LE, Furton KG. Collection and identification of human remains volatiles by
533 non-contact, dynamic airflow sampling and SPME-GC/MS using various sorbent
534 materials. Anal Bioanal Chem. 2011 Sep;401(4):1295-307.

535 23. Stockham RA, Slavin DL, Kift W. Specialized Use of Human Scent in Criminal
536 Investigations. Forensic Sci Commun Res an Technol. 2004;6(3):1-10.

537 24. Eckenrode BA, Ramsey SA, Stockham RA, Van Berkel GJ, Asano KG, Wolf DA.
538 Performance evaluation of the Scent Transfer Unit (STU-100) for organic compound
539 collection and release. J Forensic Sci. 2006 Jul;51(4):780-9.

540 25. DeGreeff LE. Development of a dynamic headspace concentration technique for the non-
541 contact sampling of human odor samples and the creation of canine training aids
542 [Internet]. Florida International University; 2010 [cited 2014 Nov 14]. Available from:
543 http://search.proquest.com/docview/858862059

544 26. DeGreeff LE, Curran AM, Furton KG. Evaluation of selected sorbent materials for the
collection of volatile organic compounds related to human scent using non-contact
545 sampling mode. Forensic Sci Int. 2011 Jun 15;209(1-3):133-42.
546

547 27. Dormont L, Bessière J-M, McKey D, Cohuet A. New methods for field collection of
548 human skin volatiles and perspectives for their application in the chemical ecology of
549 human-pathogen-vector interactions. J Exp Biol. 2013 Aug 1;216(Pt 15):2783-8.

550 28. Kabir A, Holness H, Furton KG, Almirall JR. Recent advances in micro-sample
551 preparation with forensic applications. TrAC Trends Anal Chem [Internet]. 2013 Apr
552 [cited 2014 Oct 2];45:264-79. Available from:
553 http://linkinghub.elsevier.com/retrieve/pii/S0165993613000241
554

29. Woolfenden E. Gas Chromatography [Internet]. Gas Chromatography. Elsevier; 2012

555

[cited 2015 Jun 11]. 235-289 p. Available from:

556

http://www.sciencedirect.com/science/article/pii/B9780123855404000109

557

30. Colón-Crespo LJ, Herrera-Hernández D, Holness H, Furton KG. Determination of VOC
marker combinations for the classification of individuals by gender and race/ethnicity.

558

559

Forensic Sci Int [Internet]. Elsevier Ireland Ltd; 2016; Available from:
http://linkinghub.elsevier.com/retrieve/pii/S0379073816304170

560

561

31. de Lacy Costello B, Amann A, Al-Kateb H, Flynn C, Filipiak W, Khalid T, et al. A
review of the volatiles from the healthy human body. J Breath Res. 2014;8(1).

562

563

32. Dormont L, Bessière J-M, Cohuet A. Human skin volatiles: a review. J Chem Ecol. 2013
May;39(5):569-78.

564

565

33. Curran AM, Rabin SI, Prada P a., Furton KG. On the Definition and Measurement of Human Scent: Response by Curran et al. J Chem Ecol [Internet]. 2006 Aug 2 [cited 2014 Jun 20];32(8):1617-23. Available from: http://link.springer.com/10.1007/s10886-006-9096-x

566

567

568

569

34. Cuccia L, Bourdon R, Dugay J, Bontemps D, Carrette P-L, Vial J. Novel approach for the quantitative analysis of monoethanolamine degradation products present in gas effluent of CO2 capture process by Thermal Desorption-Gas Chromatography-Mass Spectrometry: development and validation. Int J Greenh Gas Control.

570

571

572

573

35. Jardy A, Vial J. L'apport des méthodes statistiques dans la maîtrise de la qualité des analyses. Analusis. 1999;27(6):511-519.

574

575

36. Chahen L, Huard T, Cuccia L, Cuzuel V, Dugay J, Pichon V, et al. Comprehensive monitoring of MEA degradation in a post-combustion CO2 capture pilot plant with identification of novel degradation products in gaseous effluents. Int J Greenh Gas

576

577
578

Control [Internet]. Elsevier Ltd; 2016;51:305-16. Available from:

579

http://linkinghub.elsevier.com/retrieve/pii/S1750583616302638

580

37. Sghaier L, Vial J, Sassiat P, Thiebaut D, Watiez M, Breton S, et al. An overview of recent

581

developments in volatile compounds analysis from edible oils: Technique-oriented

582

perspectives. Eur J Lipid Sci Technol. 2016;1-27.

583

38. Marsili RT. Comparison of solid-phase microextraction and dynamic headspace methods

584

for the gas chromatographic-mass spectrometric analysis of light-induced lipid oxidation

585

products in milk. J Chromatogr Sci. 1999;37(January):17-23.

586