Development and validation of an LC‐MS/MS method for ...

Research article Received: 5 October 2012

Revised: 5 December 2012

Accepted: 6 December 2012

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jms.3152

Development and validation of an LC-MS/MS method for quantification of Δ9-tetrahydrocannabinolic acid A (THCA-A), THC, CBN and CBD in hair Nadine Roth,a,b Bjoern Moosmanna,b and Volker Auwärtera* For analysis of hair samples derived from a pilot study (‘in vivo’ contamination of hair by sidestream marijuana smoke), an LC-MS/MS method was developed and validated for the simultaneous quantification of Δ9-tetrahydrocannabinolic acid A (THCA-A), Δ9-tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD). Hair samples were extracted in methanol for 4 h under occasional shaking at room temperature, after adding THC-D3, CBN-D3, CBD-D3 and THCA-A-D3 as an in-house synthesized internal standard. The analytes were separated by gradient elution on a Luna C18 column using 0.1% HCOOH and ACN + 0.1% HCOOH. Data acquisition was performed on a QTrap 4000 in electrospray ionization-multi reaction monitoring mode. Validation was carried out according to the guidelines of the German Society of Toxicological and Forensic Chemistry (GTFCh). Limit of detection and lower limit of quantification were 2.5 pg/mg for THCA-A and 20 pg/mg for THC, CBN and CBD. A linear calibration model was applicable for all analytes over a range of 2.5 pg/mg or 20 pg/mg to 1000 pg/mg, using a weighting factor 1/x. Selectivity was shown for 12 blank hair samples from different sources. Accuracy and precision data were within the required limits for all analytes (bias between 0.2% and 6.4%, RSD between 3.7% and 11.5%). The dried hair extracts were stable over a time period of one to five days in the dark at room temperature. Processed sample stability (maximum decrease of analyte peak area below 25%) was considerably enhanced by adding 0.25% lecithin (w/v) in ACN + 0.1% HCOOH for reconstitution. Extraction efficiency for CBD was generally very low using methanol extraction. Hence, for effective extraction of CBD alkaline hydrolysis is recommended. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: cannabinoids; hair analysis; LC-MS/MS; Δ9-tetrahydrocannabinolic acid A; validation

Introduction

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* Correspondence to: Volker Auwärter, Institute of Forensic Medicine, Forensic Toxicology, Albertstraße 9, 79104 Freiburg, Germany. E-mail: volker.auwaerter@ uniklinik-freiburg.de a Institute of Forensic Medicine, Forensic Toxicology, Albertstraße 9, 79104 Freiburg, Germany b Hermann Staudinger Graduate School, University of Freiburg, Hebelstraße 27, 79104 Freiburg, Germany

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Differentiation between an active cannabis consumption and passive drug exposure, e.g. through side stream marijuana smoke, is a known problem in hair analysis.[1–4] The Society of Hair Testing (SoHT) published recommendations for the analysis of hair for drugs of abuse proposing several washing steps (with both organic solvent and aqueous washes), the use of cut-offs (recommended LOQ for Δ9-tetrahydrocannabinol (THC) ≤ 50 pg/mg) and the identification of metabolites like 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH, recommended LOQ ≤ 0.2 pg/mg) to prove the consumption of cannabis products and to exclude false positives.[5,6] Despite such requirements and the use of standard procedures for hair analysis, the exclusion of an external contamination of hair keeps being an important issue, not only in the case of marijuana but also for other common drugs of abuse.[1,7,8] So far, the identification of THC-COOH alongside the psychoactive THC was used to differentiate between an active consumption and a passive drug exposure.[3,9–12] Since THC-COOH is generally present in the low pg/mg range in hair of cannabis users[9,13] and sometimes not detectable at all despite using extremely sensitive analytical methods, the missing detection of this oxidative metabolite does not prove an external contamination. Furthermore, many forensic laboratories do not possess the adequate technical equipment to detect low concentrations of THC-COOH.

A specific marker for an external contamination of hair could give useful additional information. Δ9-tetrahydrocannabinolic acid A (THCA-A) seems to be a promising candidate for this purpose. This compound is the non-psychoactive precursor of THC and the main cannabinoid in fresh plant material. When heated or smoked, THCA-A is de-carboxylized (Fig. 1), but not completely.[14,15] The incorporation of THCA-A into hair has already been studied in a pilot study 2010 and revealed no evidence for an incorporation of THCA-A into hair after oral intake of relatively high doses of THCA-A for 30 days.[16] This led to the assumption that THCA-A found in hair may arise from external contamination[16] brought by, for example, sidestream marijuana smoke or contaminated hands after being in contact with cannabis material. For the analysis of the pilot study samples from 2010, a semi quantitative GC-MS-SIM method was used.[16] However, for a follow-up study focusing on the external contamination of hair by

N. Roth, B. Moosmann and V. Auwärter

Figure 1. Decarboxylation of THCA-A leading to the psychoactive THC and CO2.

sidestream marijuana smoke, exact quantification of THCA-A was required. Therefore, a fully validated and more sensitive analytical method, applying a deuterated internal standard (IS) for each analyte was needed. In previous studies, THC-COOH-D3 was used as an IS for THCA-A,[17,18] because deuterated THCA-A is not commercially available. However, this was not an ideal solution, particularly with regard to matrix effects.

Material and methods Chemicals, reagents and blank hair

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THCA-A was purchased from Lipomed (Arlesheim, Switzerland) and dissolved in methanol to obtain a stock solution of 0.1 mg/mL. THC, cannabinol (CBN) and cannabidiol (CBD) (1 mg/mL each) were obtained from Cerilliant (Round Rock, TX, USA). THCA-A-D3 was synthesized by an in-house procedure[19] yielding a mixture of THCA-A-D3 (> 90%), THC-D3 (approx. 9%) and THCA-A (< 1%). THC-D3 (0.1 mg/mL) was obtained from Cerilliant (Round Rock, TX, USA), CBN-D3 (0.1 mg/mL) from Lipomed (Arlesheim, Switzerland) and CBD-D3 (1 mg/mL) from THC-Pharm (Frankfurt, Germany). Acetone (p.a., ACS) and ethyl acetate (p.a., ACS, ≥ 99.5% GC) were obtained from Sigma Aldrich (Steinheim, Germany). Methanol and acetonitrile were obtained from J.T.Baker (Deventer, The Netherlands), formic acid (ROTIPURANW ≥98%, p.a.) and petroleum ether (ROTIPURANW 40–60  C) from Carl Roth (Karlsruhe, Germany). Deionized water was prepared using a cartridge deionizer from Memtech (Moorenweis, Germany). Different additives were evaluated to improve processed sample stability of THCA-A: Lecithin (egg phosphatidylcholine) was obtained from Lipoid (Ludwigshafen, Germany), polyethylene glycol 400 (ROTIPURANW Ph. Eur), octoxynol-9 (TritonW X 100, reinst) and polysorbate 20 (TweenW 20) from Carl Roth (Karlsruhe, Germany). Polyoxyethylen (23) laurylether (BrijW 35), albumin from bovine serum (lyophilized powder ≥ 96%), polysorbate 80 (TweenW 80) and sodium dodecyl sulphate (> 99% GC) were obtained from Sigma Aldrich (Steinheim, Germany). Poloxamer 188 (LutrolW F 68) was obtained from BASF (Ludwigshafen, Germany). Sodium hydroxide pellets (for preparation of 1 M NaOH solution) and n-hexane (EMSURE ACS, Reag. Ph Eur) were supplied by Merck (Darmstadt, Germany). Blank hair was provided by three volunteers, pooled and tested for the four cannabinoids prior to use by LC-MS/MS analysis. Positive hair for determination of extraction efficiency was prepared as follows: the donor person ran his fingers through the hair after handling marijuana flowers. After one night of ‘incubation’ and a hair wash with shampoo in the morning, several hair samples were obtained from the donor. Four hair strands were taken the next morning. The hair was washed according to the protocol (see sample preparation) and cut into pieces of 1–2 mm length.

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Sample preparation First, an IS solution in methanol, comprising 0.15 mg/mL THCA-A-D3 as well as 1 mg/mL THC-D3, CBN-D3 and CBD-D3, was prepared. Furthermore, three concentrations of working solutions (1 mg/mL, 0.1 mg/mL and 0.01 mg/mL of THCA-A, THC, CBN and CBD) for spiking blank hair with the appropriate analyte concentration were prepared. Blank hair was washed before use with three different solvents (deionized water, acetone and petroleum ether), in each case by shaking hair in 4 mL washing agent for 4 min. After the hair dried overnight in a pleated filter, it was cut into small pieces (1–2 mm length). For sample preparation 50 mg washed hair was used. 2 mL methanol was added together with 20 mL IS and the appropriate amount of working solution. After 4 h of incubation under occasional shaking at room temperature, 1.5 mL of the organic solvent was evaporated to dryness under a stream of nitrogen at 40  C. The dried extracts were reconstituted in 100 mL acetonitrile + 0.1% HCOOH + 0.25% lecithin (w/v). Finally, 20 mL sample volume was injected into the LC-MS/MS system. LC-MS/MS analysis Instrumentation The chromatographic system (Shimadzu HPLC system, Prominence series 20A, Duisburg, Germany) consisted of two pumps (LC-20AD SP), an autosampler (SIL-20AC), a degasser (DGU20A3), a column oven (CTO-20AC) and a communication bus module (CBM-20A). Detection was carried out by a QTrap 4000 triple quadrupole linear ion trap mass spectrometer from AB Sciex (Darmstadt, Germany). Analytes were separated on a Luna C18 column (2) (150 mm  2 mm, 5 mm) from Phenomenex (Aschaffenburg, Germany) with a corresponding guard column (Luna C18 4 mm  2.0 mm). The LC-MS/MS system was equipped with a TurboIonSpray interface and AnalystW software version 1.5.1. LC-MS/MS conditions and optimization As a starting point for the optimization of the LC-MS/MS conditions, an already published method for quantification of THCA-A in serum[20] was used. Chromatographic separation was realized using gradient elution (Fig. 2) with 0.1% HCOOH in water (mobile phase A) and ACN + 0.1% HCOOH (mobile phase B). The flow rate was increased after 9.5 min from 0.6 mL to 0.8 mL per min to speed up the run. The column oven was heated to 50  C. The autosampler was heated as well (30  C) to prevent precipitation of hair matrix constituents in the glass vials. Overall run time was 15.5 min. Data acquisition was performed in multi reaction monitoring mode (MRM) using negative electrospray ionization (ESI) for THCA-A, and positive ESI for THC, CBN and CBD. The ion source potentials and collision energies were optimized for each MRM transition and are listed in Table 1.

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LC-MS/MS method for cannabinoids in hair Furthermore, two hair samples were analyzed to test for incomplete deuteration in the IS solution. For this purpose, blank hair was spiked with 20 mL IS solution and incubated in MeOH as described above. Limits of detection and quantification (LOD/LLOQ) The analytical limits were studied using spiked samples in the expected concentration range (2.5, 10, 20, 40 and 100 pg/mg THCA-A, THC, CBN and CBD). A signal-to-noise ratio of at least 3:1 or higher, for the target as well as for the qualifier ion was set to assess limit of detection (LOD). Lower limit of quantification (LLOQ) was evaluated by measuring the lowest concentration level (2.5 pg/mg for THCA-A and 20 pg/mg for THC, CBN and CBD) five times, and assessing accuracy and precision, which should lie in the required ranges (bias  20% and RSD ≤ 20%).

Figure 2. Liquid chromatography gradient and flow rate.

Linearity Table 1. MRM transitions of deuterated and non-deuterated THCA-A, THC, CBN and CBD: (T: Target = Quantifier, Qu: Qualifier, DP: Declustering Potential, EP: Entrance Potential, CE: Collision Energy, CXP: Collision cell Exit Potential) Analyte

THCA-A

ion Q1 mass Q3 mass dwell time DP

T Qu THCA-A-D3 T THC T Qu THC-D3 T CBN T Qu CBN-D3 T CBD T Qu CBD-D3 T

EP

CE CXP

amu

amu

ms

V

V

V

V

357.2 357.2 360.3 315.2 315.2 318.2 311.2 311.2 314.3 315.2 315.2 318.2

313.2 245.2 316.4 193.2 259.3 196.2 323.2 241.2 223.2 193.2 259.3 196.2

20 10 20 20 10 20 20 10 20 20 10 20

45 45 95 60 60 60 60 60 60 60 60 60

10 10 10 10 10 10 10 10 10 10 10 10

34 43 34 34 28 34 30 28 30 31 31 34

7 5 7 3 5 3 4 6 4 14 7 3

Nitrogen was used as curtain gas (35 psi) and collision gas (6 psi). Ion source gas 1 (nebulizer gas, 40 psi) and ion source gas 2 (turbo gas, 70 psi) were also nitrogen. Ion source temperature was 600  C, and ion source voltage switched between 4250 V and + 4500 V with a cycle time of 1.66 s. Validation Validation was carried out according to the guidelines of the German Society of Toxicological and Forensic Chemistry (GTFCh) for the quality assurance of forensic toxicological analysis (attachment B).[21] Further recommendations for hair analysis, which can be found in attachment C,[22] were also considered. Valistat (version 2.00.1; Arvecon GmbH 2010) was used for calculations. Selectivity

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Determination of accuracy and precision Since it is not feasible to prepare a homogenous hair pool with a defined incorporated analyte concentration for each analyte from drug positive hair, quality controls had to be prepared similar to calibrators. Duplicates of spiked blank hair were tested on five different days in the low and high concentration range (THCA-A: 8 pg/mg and 800 pg/mg; THC, CBN and CBD: 50 pg/mg and 800 pg/mg). The acceptance criteria were bias  15% of the nominal value (near the LLOQ  20%) and RSD ≤ 15% (near the LLOQ ≤ 20%). Matrix effects and recovery Matrix effects and recovery were evaluated using the Matuszewski protocol.[24] The analyte area ratio of samples spiked after extraction (Matuszewski set 2) and spiked control samples in mobile phase without matrix (Matuszewski set 1) provided information about ion enhancement or suppression caused by hair matrix. Recovery was calculated from the analyte area ratio of samples spiked before the extraction (Matuszewski set 3) in relation to samples spiked after extraction (Matuszewski set 2). For each set, hair from five different sources were prepared in the low and high concentration range (THCA-A: 8 pg/mg and 800 pg/mg; THC, CBN and CBD: 50 pg/mg and 800 pg/mg). Matrix effects should lie between 75% and 125%, and recovery of each analyte should be ≥ 50%, both with a standard deviation (SD) ≤ 25%. Long-term stability Stability of the dried extract was tested over a time period of one to five days in the dark at room temperature. For this purpose, three blank hair samples were spiked with each analyte in the

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Twelve different hair specimens, including child, dyed and grey hair, were screened for interfering signals. 2 mL MeOH was added to 50 mg of blank hair. After 4 h incubation under occasional shaking at room temperature, 1.5 mL solution was transferred into an empty LC-vial and evaporated to dryness followed by reconstitution in 100 mL mobile phase B + 0.25% lecithin (w/v).

Calibrators covered the whole calibration range (THCA-A: 2.5, 10, 20, 40, 100, 200, 500, 1000 pg/mg; THC, CBN and CBD: 20, 40, 100, 200, 500, 1000 pg/mg) and were prepared using three different working solutions as specified in chapter material and methods/ sample preparation. The analysis of each calibrator concentration was repeated five times as required for the analysis of hair samples.[22] A linear calibration model was accepted if accuracy was sufficient ( 20%) for each concentration with the calculated regression equation.[23] Additionally, the chosen calibration model was tested with Grubbs test for stragglers (≥ 95% significance) and Cochran test for variance of homogeneity (99% significance).

N. Roth, B. Moosmann and V. Auwärter low as well as in the high concentration range (THCA-A: 8 pg/mg and 800 pg/mg; THC, CBN and CBD: 50 pg/mg and 800 pg/mg). The dried extracts were reconstituted and analyzed after one and five days of storage in reference to three freshly spiked control samples. Stability criteria were fulfilled when the mean value of the three stability samples lay within the 90–110% interval of the control samples and the 90% confidence interval lay within the 80–120% interval of the mean value of the control samples.

Processed sample stability Processed sample stability was tested in the low and high concentration range (THCA-A: 8 pg/mg and 800 pg/mg; THC, CBN and CBD: 50 pg/mg and 800 pg/mg). Six blank hair samples were used for each concentration level and spiked with the appropriate analyte concentrations. After incubation for 4 h in 2 mL MeOH, 1.5 mL of the solution was evaporated, and the residue was reconstituted in 100 mL mobile phase B + 0.25% lecithin (w/v).

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Figure 3. Comparison of the relative extractive performance of different extracting agents after 4 h incubation (medium of threefold analysis, highest medium extraction yield was set 100%; bars indicate the range of the measured values; CBD not shown due to generally low extraction yields).

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LC-MS/MS method for cannabinoids in hair Finally, all six reconstituted samples of one concentration level were combined in a glass vial, mixed and portioned again in five 100 mL aliquots. Samples were analyzed at intervals of 4 h, with the last sample after 16 h.

Results and discussion Sample preparation First, a suitable solvent for the simultaneous extraction of THCA-A, THC, CBN and CBD from hair matrix had to be found. In the literature alkaline hydrolysis is applied for the extraction of cannabinoids from hair.[25–29] Unfortunately, this extraction procedure leads to a decarboxylation of the base-labile THCA-A[16] and was therefore not applicable. The use of an ultrasonic bath for enhanced liberation of analytes from the hair matrix was also not taken into consideration, since this procedure also leads to thermal degradation of THCA-A.[30] Therefore, different organic solvents and mixtures of solvents were tested to find the optimal extracting agent for all analytes. Extractive performance of different organic solvents For determination of the extraction efficiency of different solvents, large amounts of positive hair were required. Since such a large quantity of positive hair was not available, ‘contaminated’ hair was prepared by an in-house procedure. Subsequently, the hair was washed according to the protocol and cut into pieces of 1–2 mm length (see chapter material and methods/ sample preparation). For evaluation of extraction efficiency, three hair aliquots were used for each extracting agent (post-extraction addition of the deuterated IS) and the medium concentration in the extract after 4 h incubation was plotted for each analyte versus the extracting agent (Fig. 3).

Since sensitive detection of THCA-A was the main focus, methanol was chosen as a good compromise for the extraction of all four analytes. It should be mentioned that the extracted CBD from authentic hair samples was generally very low with quantitative values close to the LLOQ/LOD (CBD = 20 pg/mg). This means that in hair samples with low concentrations of CBD, the extractable amount can drop under the LLOQ/LOD and CBD would no longer be detectable. Therefore, alkaline hydrolysis followed by liquidliquid extraction seems to be more suitable for analyzing CBD and should be applied if this particular analyte is of interest. In addition, the influence of extraction time was evaluated. For this purpose, three different hair aliquots were extracted with methanol for 1, 2, 4 and 6 h. After 4 h, no further increase of the concentrations could be observed. Therefore, 4 h was chosen as the extraction time. LC-MS/MS THCA-A showed a retention time of 9.0 min, THC of 8.68 min, CBN of 8.41 min and CBD of 7.97 min. In contrast to the LC-MS/MS method for the quantification of THCA-A in serum,[20] where an autosampler temperature of 5  C is used, the temperature was increased to 30  C for the analysis of hair samples. This was necessary, since components of the hair matrix precipitated in the glass vials while standing in the cooled autosampler, leading to a pronounced loss of sensitivity. Validation results and discussion Selectivity experiments showed no interfering signals for THCA-A, THC, CBN and CBD. Traces of non-deuterated THCA-A, originating from the in-house synthesized THCA-A-D3 (THCA-A < 1%) in the IS solution did not interfere with the analysis.

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Figure 4. MRM chromatograms for calibrators of 2.5 pg/mg (LLOQ for THCA-A) and 20 pg/mg (LLOQ for THC, CBN and CBD).

N. Roth, B. Moosmann and V. Auwärter Linearity was shown in the range of 2.5 pg/mg to 1000 pg/mg for THCA-A and 20 pg/mg to 1000 pg/mg for THC, CBN and CBD using a weighted calibration model (1/x). Weighting was applied for compensation of heteroscedasticity since the range of calibration covers about two orders of magnitude. The correlation coefficients were r ≥ 0.9977 for THCA-A, r ≥ 0.9988 for THC, r ≥ 0.9981 for CBN and r ≥ 0.9968 for CBD. The chosen calibration model passed all statistical tests (no stragglers with a significance level of 99%, homogeneity of variances with a significance level of 99%). LOD and LLOQ were 2.5 pg/mg for THCA-A and 20 pg/mg for THC, CBN and CBD. MRM chromatograms of calibrators at the appropriate LLOQ levels are shown for each analyte in Fig. 4. Accuracy (bias  20%) and precision (RSD ≤ 20%) data for determination of LLOQ lay in the required ranges (THCA-A: RSD 15.5%, bias 10.7%, THC: RSD 10.8%, bias 5.0%, CBN: RSD 4.4%, bias 3.0% and CBD: RSD 7.5%, bias 10.2%). Quality controls in the low and high concentration range were prepared on five different days; the obtained accuracy and precision data are summarized in Table 2. Dried hair extracts were stable over a time period of one to five days in the dark at room temperature. After five days of storage values for low and high concentrations lay within the 90–110%

interval of the freshly spiked control samples. Likewise, the 90% confidence interval lay within the 80–120% interval of the mean value of the control samples. Recovery for all analytes in the low and high concentration range was higher than 50% with a SD lower than 23% (see Table 2). It should be mentioned that recovery strongly depends on the extraction efficiency of the chosen extraction method (extracting agent) when analyzing authentic hair samples. Since methanol shows only very low extraction efficiency for CBD in authentic hair samples, analysis after alkaline hydrolysis and liquid-liquid extraction should be taken into consideration if quantification of CBD is of concern. Matrix effects were also determined using Matuszewski protocol.[24] For THCA-A and CBN, we obtained matrix effects above, for CBD below the acceptance interval of 75% to 125% (Table 2). However, SD of the matrix effects were ≤ 30% leading to acceptable accuracy and precision (Table 2). Therefore, the use of an isotopically labeled IS is strongly recommended for each analyte. Evaluation of processed sample stability revealed another problem caused by hair matrix. The analyte peak area of THCA-A in the high concentration range decreased about 25% during the first 4 h in the autosampler. Silanized glass inlets (30  60 mm, tip 15 mm, A-Z Analytik-Zubehör GmbH, Langen,

Table 2. Summary of accuracy, precision, recovery and matrix effects for low and high concentration levels Analyte

Mean value

Accuracy (bias  15/20%)

Precision (RSD ≤ 15/20%)

Recovery

Matrix effects

THCA-A (low: 8 pg/mg) THCA-A (high: 800 pg/mg) THC (low: 50 pg/mg) THC (high: 800 pg/mg) CBN (low: 50 pg/mg) CBN (high: 800 pg/mg) CBD (low: 50 pg/mg) CBD (high: 800 pg/mg)

8.0 pg/mg 851.4 pg/mg 50.1 pg/mg 810.5 pg/mg 51.0 pg/mg 798.6 pg/mg 51.2 pg/mg 799.3 pg/mg

0.5% 6.4% 0.2% 1.3% 1.9% 0.2% 2.4% 0.1%

11.5% 3.7% 4.1% 4.4% 6.3% 4.2% 4.6% 3.7%

60% (SD 10%) 70% (SD 19%) 64% (SD 22%) 63% (SD 16%) 59% (SD 23%) 62% (SD 13%) 56% (SD 23%) 64% (SD 15%)

180% (SD 15%) 163% (SD 20%) 94% (SD 21%) 91% (SD 12%) 133% (SD 30%) 121% (SD 28%) 69% (SD 20%) 57% (SD 13%)

232

Figure 5. Overlayed ion chromatograms of an authentic study sample (50 mg hair + 20 mL IS) from posterior vertex (proximal segment, THCA-A = 35 pg/mg, THC = 950 pg/mg, CBN = 330 pg/mg and CBD = 0 pg/mg). T: Target (quantifier), Qu: Qualifier.

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LC-MS/MS method for cannabinoids in hair Germany) were tested without success to prevent adsorption effects. Furthermore, nine different solubilizers (lecithin, polyethylene glycol 400, octoxynol-9, polysorbate 20 and 80, poloxamer 188, bovine serum albumin, polyoxyethylen (23) laurylether and sodium dodecyl sulphate) were evaluated to improve the solubility of THCA-A. The addition of sodium dodecyl sulphate led to a considerable loss of THCA-A signal. Bovine serum albumin, polyoxyethylen (23) laurylether, octoxynol-9, polysorbate 20, polysorbate 80 and poloxamer 188 led to ion suppression especially for THC, CBN and CBD. Lecithin and polyethylene glycol 400 showed acceptable ion intensities, but solely the addition of 0.25% lecithin to mobile phase B for reconstitution led to an enhancement of processed sample stability for THCA-A. Using this solubilizer for sample preparation, all analytes were stable in low and high concentrations over 16 h in processed samples in the autosampler (see chapter material and methods/validation). There was no negative slope when plotting peak areas against time of injection and the maximal decrease of peak area was 18% for THCA-A at the high concentration level after 8 h (required < 25% when using deuterated IS).

[10]

[11]

[12] [13] [14]

[15]

[16]

[17]

Performance in practice The presented method was successfully applied for analyzing hair samples from a study dealing with the external contamination of hair by sidestream marijuana smoke. The results of the study will be published separately. In brief, THCA-A could be quantified in a range of 2.5 to 35 pg/mg in hair samples after daily exposure to the smoke of a joint over a three week period (Fig. 5). Concentrations differed depending on hair length and head region of sampling. THC and CBN were found in higher concentrations than THCA-A. Additionally, this method already proved to be useful for the analysis of hair samples contaminated with THCA-A by handling of cannabis material.

[18]

[19]

[20]

[21]

Acknowledgements The authors would like to thank Dr. Ariane Wohlfarth, Jessica Traber and Dr. Martin Holzer for their help. The project was funded by the ‘Deutsche Forschungsgemeinschaft’ (DFG-AU: 324/3-1).

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