Influence of thermal hair straightening on cannabis and cocaine content in hair

Accepted Manuscript Title: Influence of thermal hair straightening on cannabis and cocaine content in hair Author: Jana Ettlinger Michel Yegles PII: DOI: Reference:

S0379-0738(16)00008-6 http://dx.doi.org/doi:10.1016/j.forsciint.2016.01.002 FSI 8268

To appear in:

FSI

Received date: Revised date: Accepted date:

9-10-2015 24-12-2015 2-1-2016

Please cite this article as: J. Ettlinger, M. Yegles, Influence of thermal hair straightening on cannabis and cocaine content in hair, Forensic Science International (2016), http://dx.doi.org/10.1016/j.forsciint.2016.01.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Highlights  Thermal hair straightening has a strong impact on THC and cocaine contents in hair  Heat treatment of hair at 200°C converts cocaine into BZE and THC into CBN

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 Thermal straightening should be considered for hair results interpretation

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Influence of thermal hair straightening on cannabis and cocaine content in hair Jana Ettlinger, Michel Yegles Laboratoire National de Santé, Forensic Toxicology,

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1, rue Louis Rech, L-3555 Dudelange (Luxembourg)

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Corresponding Author Michel Yegles

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Laboratoire National de Santé

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Service de Toxicologie 1, rue Louis Rech

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L-3555 Dudelange

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Phone : +352 28100 577 / Fax: +352 28100 562

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E-mail : [email protected]

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Influence of thermal hair straightening on Cannabis und Cocaine content in hair

Abstract It has been shown that cosmetic treatment like bleaching and perming may lead to an important decrease of drugs of abuse content in hair. Currently, hair straightening has become a regular hair treatment especially for women. The aim of this preliminary study was to

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investigate the effect of in vitro treatment of hair with heat straightener on cannabis and cocaine concentrations in hair.

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17 positive cannabis and 7 positive cocaine hair samples were treated in vitro with a hair straightener. During this treatment hair was put sequentially 30 times in contact with heated

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iron plates at 200°C during 2 s corresponding to a total time of contact of 1 min. THC and Cannabinol (CBN) were analysed in cannabis positive hair and cocaine, benzoylecgonin (BZE) methods using GC/MS in electron impact mode.

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and cocaethylen were analysed in cocaine positive hair. Analyses were performed with routine Regarding cannabis results a decrease of THC concentrations was found in 11 of 17 hair

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samples after thermal treatment, whereas in 6 cases an increase was shown. In all the hair samples CBN concentrations was explicitly higher after the in vitro treatment. Regarding

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cocaine results cocaine and cocaethylene concentrations decreased after treatment in all seven hair samples; in contrast, higher concentrations of BZE were determined.

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The strong increase of CBN and BZE content in hair after thermal treatments may be due to the fact that THC is converted by heat into CBN and cocaine into BZE, thus changing the respective

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ratios of the analysed substances. In conclusion, thermal straightening should be considered as other cosmetic hair treatments for a correct interpretation of hair results. Keywords: pitfalls in hair analysis, thermal straightening, cocaine, cannabis

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1. Introduction It is known that some cosmetic treatments like bleaching, dyeing and perming have to be taken into consideration for a correct interpretation of hair results, as a decrease of drugs of abuse concentration was shown after these treatments [1]. It has been found that bleaching and dyeing lead to a decrease of 34.0 % and 12.3 % respectively of THC concentrations [2]. After dyeing cocaine concentrations decreased between 20.0 and 43.4 % [2-3], whereas bleaching

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caused a decrease of cocaine between 24.6 to 80.0 % [2, 3, 4, 5]. Especially for women hair straightening represents nowadays an often daily used hair

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treatment. During this hair treatment hair strands are put in contact with an excessive heat.

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Iron plates are usually heated between 150 and 250 degrees Celsius. During this treatment hydrogen and disulphide bonds were broken in the hair matrix allowing keratin chains to move around slightly and assume a position resulting in straightened hair. After cooling, the hydrogen

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and disulphide bonds between the keratin chains are reformed. Hair stays now in a straightened shape, because the keratin molecules are in different positions. As exposure to

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moisture causes the hair to revert back to its original shape, this method of straightening hair is not permanent.

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The effect of thermal straightening of hair has already been investigated [6]. Radial and axial cracking along with scale edge fusion were revealed by scanning electron microscopic proprieties of hair fibres.

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examination of treated fibres. Repeated curling showed slight increase in tensile mechanical

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In general, during one heat treatment of hair the strands are passed through heated iron plates 2 to 3 times. As during one pass hair strands are in contact with the curling plates during about 2 s, the total contact time for the hair strands with the heated plates lasts 4 to 6 s. Thus, when extrapolating, in case of daily use during one month, the total contact time for hair with heated iron plates corresponds to 2 to 3 min. A study showing the influence of hair straightening on EtG content in hair has already been published [7]. A decrease or an increase of EtG concentrations were observed depending on hair colour: an increase in hair was found when treating dark coloured hair, whereas a decrease was detected after treatment of light coloured hair. The role of melanin, hair thickness and hair density was proposed to explain this different impact depending on hair colour.

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To our knowledge no studies exist so far about the effect of thermal hair straightening on cannabis and cocaine contents in hair. Thus, the aim of this preliminary study was to investigate the influence of this cosmetic treatment on THC and CBN contents in hair as well as for cocaine and its metabolites benzoylecgonine and cocaethylene contents in hair.

2. Materials and Methods

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2.1 Chemicals and reagents

Reference standards were purchased from LCG Standards (Molsheim, France). Solvents were supplied

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by Biosolve (Valkenswaard, the Netherlands). All chemicals and reagents were of analytical purity grade. A ball mill of type MM2 (Retsch, Haan, Germany) was used for the pulverization of the hair samples.

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Extraction was performed with an ELMA TI-H-15 ultrasonication bath (Elma Hans Schmidbauer GmbH & Co. KG, Singen, Germany) and centrifugation with a Sigma centrifuge (Osterode am Harz, Germany). A

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Supelco VisiprepTM SPE Vacuum Manifold (Bellefonte, CA, USA) with 24 ports was employed to load the hair samples and to dry the cartridges. Solvent evaporation was achieved with a Pierce Reacti-Therm III Heating Module (Rockford, IL, USA).

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2.2 Instrumentation

The study was performed with a gas chromatograph 7890A coupled to a mass selective detector 5975C

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inert XL (Agilent, Waldbronn, Germany). The gas chromatograph was equipped with a VF-5MS (5 % Phenyl Methyl Silox, 12 m x 200 µm x 0.33 µm) column. 2 different methods were used: for cocaine and

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metabolites, temperature was programmed from 60 ˚C (2 min hold), to 170 °C at 40 ˚C/min, to 234°C at 8 ˚C/min and to 300°C at 50 ˚C/min (2 min hold); for THC and CBN, temperature was programmed from

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100°C (2 min hold) to 250 °C at 30 °C/min, to 260 °C at 5°C/min and to 300°C at 30°C/min (2 min hold). 2.3 Hair Specimens

17 positive cannabis and 7 positive cocaine hair samples were used for this study. Specimens were from granting, maintaining or re-granting of driving license cases due to former drug abuse. Colour of hair was documented (table 2 and 3). 3 cm proximal hair segments (if available) were analysed. 2.4 In vitro thermal straightening The samples were split into two strands. One strand was treated in vitro with a curling iron heated at 200°C. Hair was sequentially put 30 times in contact with the heated iron plates during 2 s corresponding to a total time of contact of 1 min. When extrapolating, this treatment would correspond approximately to a daily treatment during one month. The other strand was not treated. Both hair strands were then cleaned as described below and analysed using the method described below. All the samples were analysed in triplicates.

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2.5 Cannabis determination in hair Cannabis positive hair samples were cleaned with water and acetone, then pulverised using a ball mill and incubated with NaOH (1 M) for 30 min at 90°C. A solid-phase extraction was performed with Chromabond C18ec Columns (Machery Nagel). After silylation with Silyl 991, the concentrations were determined by GC/MS in electron impact mode: 386 and 371 m/z for THC and 389 and 374 m/z for THC-D3 (used as internal standard = IS); and 367 and 368 m/z for

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CBN. Validation data for THC and CBN are shown in table 1. 2.6 Determination of cocainics in hair

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Cocaine positive hair samples were washed with water and acetone, pulverised and incubated in phosphate buffer (0.1 M, pH 6.0) for 2 hours in an ultrasonic bath. After solid-phase

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extraction with Clean Screen Columns (UCT), derivatization was performed using pentafluoropropionic anhydride and pentafluoropropanol. Analyses were done by GC/MS in

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electron impact mode: 386 and 303 m/z for cocaine, 389 m/z for cocaine D3 (IS), 300 and 421 m/z for BZE, 303 m/z for BZE D3 (IS), 196 and 317 m/z for cocaethylen and 199 m/z for cocaine, BZE and

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cocaethylen D3 (IS). In table 1 validation data were resumed for

3. Results and Discussion

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cocaethylene.

3.1 Effect of thermal straightening on cannabis content in hair

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The results of the thermal straightening of 17 cannabis positive hair specimen show a decrease or an increase of THC content in hair. In all the samples a median decrease of 10.1 % was

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found. For the majority of hair specimens (11 of 17 hair samples) a decrease of THC content was found. This decrease ranged from 3.0 % to 100 % (median 34.7 %). In only 6 cases an increase was shown ranging from 4.6 % to 67.2 % (median 12.6 %) (Table 2). In all the samples (15 samples, as 2 were negative for CBN) CBN concentrations were explicitly higher after thermal straightening. The increase ranges from 72 % to 751 % (median 138 %) (Table 2). The effect of thermal straightening was also studied on the ratio THC/CBN. The results obtained showed a median decrease from 7.5 in not treated hair to 2.4 in treated hair (figure 1). Although the number of samples analysed in this preliminary study is rather low, a ratio THC/CBN below 4 may be proposed as a hint that the analysed hair may be heat straightened before collection.

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The decrease of THC and the increase of CBN after thermal straightening may be due to chemical transformation of THC into CBN by heat as was already shown by Repka et al [8]. Variation of THC results seems to be dependent on hair colour as higher concentrations of THC are only found in dark hair (figure 2). Similar findings were found in a previous study evaluating the influence of thermal straightening on EtG in hair [7]. Parameters like melanin, thickness or density of hair were proposed to explain this difference depending on hair colour. The decrease of THC may be explained by the fact that light coloured hair is normally thinner resulting in a

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better in vitro conversion of THC into CBN by heat compared to dark coloured hair which is normally thicker. The increase of THC is more difficult to explain. One hypothesis may be that

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THC sequestered in dark coloured hair, thus in a thicker hair matrix, may be less sensitive to heat and thus will be less converted by heat into CBN. Moreover, as hair matrix may be

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degraded after thermal straightening, this may facilitate a better leaching out of the hair matrix of the incorporated substances during hair extraction compared to not treated hair. However,

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to confirm this hypothesis further studies are needed as for example scanning electron microscope examination studies in order to evaluate the effect of thermal treatment on THC

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content relative to hair damage and hair thickness.

3.2 Effect of thermal straightening on cocainics’ content in hair

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Our data show that thermal straightening has a strong impact on cocaine content in hair (table 3). In all the 7 hair samples a median decrease of 75.4 % of cocaine concentrations was shown

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(range: 44.6 % to a total loss). On the other hand, higher concentrations of BZE were determined in all the hair specimens after treatment ranging from 3.3 to 48.2 % (median

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20.5%). Cocaethylene was shown to decrease with a median of (52.1 %) in 4 hair samples (3 were negative of cocaethylene); the decrease ranges from 17.6 to a total loss. The effect of thermal straightening was also studied on the ratio BZE / cocaine as this ratio BZE/cocaine above 0.05 should be an indication to confirm cocaine consumption9. Our data show that after treatment this ratio increased in every specimen (figure 3). The median BZE/cocaine ratio of 0.6 in not treated hair increased to 1.6 in treated hair. Although the number of samples analysed in this preliminary study is rather low, a ratio BZE/cocaine higher than 1 may be proposed as an indication that the analysed hair was heat straightened before collection. The decrease of cocaine and the increase of BZE may be explained by a chemical conversion of cocaine into BZE. As, to the best of our knowledge, no scientific literature describes cocaine conversion into BZE by heat, a further experiment has been performed: 200 ng of cocaine was put in 2 different vials, one with 100 µL water, the other one without water. The 2 vials were Page 7 of 16

closed, then placed during 1 min in an oven at 200°C (the time period of our in vitro heat treatment of hair) and then rapidly cooled down. Cocaine and BZE concentrations were then determined in both vials. In the vial with water it was shown that the cocaine concentration decreased from 200 to 57 ng/vial (decrease of 71.5 %). Furthermore in this vial BZE could be determined with at a concentration of 65 ng/vial. In the vial without water a decrease of the cocaine concentration from 200 to 36 ng/vial (decrease of 82 %) was found with a production of BZE at a concentration of 2 ng/vial. These results confirm that cocaine may be converted by

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heat into BZE. As more BZE was converted in presence of water, the conversion cocaine into

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BZE may be mainly due to hydrolysis. 4. Conclusions

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This preliminary study indicates that thermal straightening has a strong impact on cannabis and cocaine concentration in hair.

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Regarding cannabis results after the performed in vitro heat treatment of hair a decrease or an increase of THC content in hair was found depending on hair colour, whereas CBN content was

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strongly increased in all the hair specimens. Regarding cocaine results, this treatment led to an important decrease of cocaine and cocaethylen concentrations and an increase of BZE concentrations in all the hair specimens.

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The strong increase of CBN and BZE content after thermal hair treatment may be due to the fact that THC and cocaine are converted by heat into CBN and BZE respectively.

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Although the number of analysed hair samples is relatively low, a ratio THC/CBN below 4 and / or a ratio BZE/cocaine higher than 1 may be an indication that the analysed hair may be treated

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by heat before collection. If one if these proposed criteria are fulfilled, results in hair should be interpreted with caution. However, further studies should be done to verify these proposed ratios.

To confirm these preliminary in vitro results, in vivo studies over several weeks should be performed as moisturizing of hair (washing or sweating) may interfere with thermal treatment of hair. Moreover, the studies of the effect of thermal straightening should be extended to other substances like CBD and THC-COOH for cannabis and norcocaïne and anhydroecgonine méthylester for cocainics. Finally, regarding our in vitro results, hair straightening should as well be documented as other cosmetic treatments prior to hair analysis implying also that persons should be informed not do use thermal straightener before hair collection. Page 8 of 16

Legends of figures Figure 1 : Ratio of THC vs CBN in not-treated and treated hair Figure 2: Percentage of variation of cocaine concentrations in not-treated hair compared to concentrations in treated hair related to hair colour

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Figure 3: Ratio of BZE vs cocaine in not-treated and treated hair

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Table 1: Validation Data for the determination of THC, CBN, BZE, cocaine and Cocaethylene in hair Accuracy (% bias) Within-day

Precision (% bias)

Between-day

Within-day

Between-day

4.2

4.0

Recovery (%) 89.0

4.0

9.2

98.3

19.0

9.4

85.9

10.9

0.7

89.6

40.7

5.3

87.6

20.0

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0.7 2.6 3.1 9.4 THC 5.6 4.3 6.9 BZE 12.4 4.1 5.1 Cocaine 5.2 4.3 3.1 Cocaethylene Validation performed at a concentration of 500 pg/mg hair CBN

LLOQ (pg/mg) 29.6

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Substance

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Table 2. Effect of thermal treatment on THC and CBN content in hair THC

CBN

Specimen Hair Colour Not - treated Treated Difference

Not - treated Treated Difference

[ng/mg]

[%]

[ng/mg]

[ng/mg]

[%]

Light brown

5.1

4.6

-10.1

0.18

1.13

543.9

2

Black

2.3

2.7

16.7

0.46

1.40

205.0

3

Blond

1.5

1.2

-18.3

0.27

0.65

137.9

4

Light Brown

1.5

0.69

-53.3

0.19

1.61

751.7

5

Light Brown

1.3

0.71

-45.2

0.40

0.68

72.1

6

Dark brown

1.2

1.6

25.4

0.26

0.62

135.3

7

Blond

0.93

0.81

-13.0

0.04

0.17

339.1

8

Light brown

0.75

0.49

-34.7

9

Black

0.66

1.1

67.2

10

Dark blond

0.43

0.26

-40.4

11

Dark brown

0.23

0.24

4.6

12

Black

0.20

0.21

8.5

13

Dark blond

0.20

0.19

14

Light brown

0.17

15

Blond

0.15

16

Black

0.09

17

Dyed red

0.04

0.10

178.7

0.37

0.84

128.1

0.05

0.08

80.3

0.05

0.12

137.7

0.06

0.12

107.5

-3.0

0.04

0.09

151.3


-100.0

< LLOQ

< LLOQ

-

0.14

-6.3

0.03

0.05

81.8

0.09

7.3

0.03

0.11

296.3

-100

< LLOQ

< LLOQ

-

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0.03

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1

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[ng/mg]

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5 6 7

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29.6

8.7

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4

3.49

1.1

1.26

18.6

0.26

0.32

20.6

0.43

0.44

0.37

0.55

0.56

0.59

2.7

Difference [%]

Treated [ng/mg]

Difference [%]

2.1

-75.4

1.8

0.67

-62.9

2.6

0.97

-62.7

0.08

0.07

-17.6

0.51

0.16

-69.2

0.11

0.06

-41.2

3.3

0.72


-100.0

0.19


-100.0

48.6

0.60

0.33

-44.6



-

4.3

0.47


-100.0



-

0.34

0.40

16.7

0.43


-100.0



-

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3

Treated [ng/mg]

d

2

Dark brown Dark brown Dark brown Dark brown Dark brown Dark brown Dark brown

Difference [%]

Treated [ng/mg]

Cocaethylen Nottreated [ng/mg]

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Hair colour

Nottreated [ng/mg]

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Specimen

Cocaine Nottreated [ng/mg]

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BZE

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Table 2: Effect of thermal straightening on cocaine, BZE and cocaethylen in hair

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Ratio THC/CBN 35 30

not treated

25

treated

20

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15

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10

0 2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

Specimen

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5

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5. References

1

M. Yegles, Pitfalls in hair analysis: cosmetic treatment, Annales de Toxicologie Analytique. 17 (2005), 275-278. 2

Jurado c et al., Influence of the cosmetic treatment of hair on drug testing. Int. J. Legal Med. 110 (1997), 159-163

3

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Welch MJ, Sniegoski LT, Allgood CC, Habram M. Hair analysis for drugs of abuse: evaluation of analytical methods, environmental issues, and development of reference materials. J Anal Toxicol 1993;17:389-398.

4

Cirimele, V., P. Kintz, and P. Mangin, Drug concentrations in human hair after bleaching. J Anal Toxicol, 1995. 19(5): 331-332. 5

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Yegles, M., Y. Marson, and R. Wennig, Influence of bleaching on stability of benzodiazepines in hair. Forensic Sci Int, 2000. 107(1-3): . 87-92. 6 S.B. Ruetsch, Y.K. Kamath. Effects of thermal treatments with a curling iron on hair fiber. Int. J. Cosmetic Sci. 2004, 26, 217. 7

Ettlinger J1, Kirchen L, Yegles M. Influence of thermal hair straightening on ethyl glucuronide content in hair. Drug Test Anal. 2014 J6 Suppl 1:74-77.

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Repka MA, Munjal A, ElSohly MA, Ross SA Temperature Stability and Bioadhesive Properties of Δ9Tetrahydrocannabinol Incorporated Hydroxypropylcellulose Polymer Matrix Systems Drug Dev Ind Pharm. January; 2006 32(1): 21–32. 9

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Society of Hair Testing. Recommendations for Hair Testing in Forensic Cases. Society of Hair Testing. Forensic Science International 84 (1997) 3-6

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