High-Resolution Ion Mobility Spectrometry for Rapid Cannabis

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High-resolution ion mobility spectrometry for rapid cannabis potency testing Marianne Hädener, Michael Z. Kamrath, Wolfgang Weinmann, and Michael Groessl Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02180 • Publication Date (Web): 26 Jun 2018 Downloaded from http://pubs.acs.org on June 27, 2018

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Analytical Chemistry

High-resolution ion mobility spectrometry for rapid cannabis potency testing

Marianne Hädener1, Michael Z. Kamrath2, Wolfgang Weinmann1, Michael Groessl3

1

Affiliation and Address:

Institute of Forensic Medicine University of Bern Bühlstrasse 20 3012 Bern Switzerland

2


Tofwerk AG Uttigenstrasse 22 3600 Thun Switzerland

3


Department of Nephrology and Hypertension and Department of BioMedical Research, Inselspital, Bern University Hospital University of Bern 3010 Bern Switzerland E-Mail: [email protected] Tel.: +41 31 63 29477

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ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract We report initial results from an ion mobility spectrometry (IMS) based analysis of natural cannabis samples and explore the possibility of using this technique to distinguish medical marijuana from illegal forms of the drug as defined by Swiss legislation. We analyzed cannabis extracts by electrospray ionization IMS-MS and found that high-resolution drift-tube IMS (R > 150) can effectively isolate and quantify the controlled substance, Δ9-tetrahydrocannabinol (THC), even in the presence of other non-controlled cannabinoid isomers including cannabidiol (CBD). We used this information to determine whether the THC content of a given sample surpassed the legal limit, which is 1% by weight in Switzerland. Our IMS-MS methodology produced equivalent quantification results to standard HPLC-based methods and offers the additional advantage of significantly shorter time requirements for the analysis. In addition, IMS-based analysis offers flexibility over HPLC in that it can be performed on portable devices. As such, these findings may have implications for cannabis testing in police laboratories.

Introduction Δ9-Tetrahydrocannabinol (THC) and cannabidiol (CBD) are two isomeric cannabinoids present in cannabis1. CBD lacks the psychoactive effects of THC but possesses pharmacological activity that is being explored for therapeutic applications 1-3. In Switzerland, only plant material with a THC content of 1% or higher is considered illegal by the narcotics legislation and high-CBD/low-THC marijuana products can be legally sold. However, legal CBD- and illegal THC-cannabis cannot be reliably distinguished by appearance or smell and require forensic chemical analysis of organic extracts thereof. Additionally, with the legalization of cannabis in several US states (either for medicinal or recreational use), potency testing becomes an important part in the quality control of these consumer products4. Potency testing of cannabis is usually performed by GC- or LC-based techniques. Due to the structural similarity of isomeric cannabinoids, long run times (> 15 min) are required to achieve baseline separation by HPLC5. Whereas GC analysis results in shorter runtimes, quantitation of 2


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the cannabinoid acids Δ9-tetrahydrocannabinolic acid-A (THCA) and cannabidiolic acid-A (CBDA) is rendered impossible by the high temperature used in this method, which leads to decarboxylation of the analytes6,7. Yet, as THCA and CBDA are converted to THC and CBD, respectively, during consumption of cannabis, the sum of both THC and THCA is relevant for potency testing. Within this paper, we investigate the suitability of direct analysis of cannabis extracts by ion mobility spectrometry coupled to mass spectrometry (IMS-MS) as a rapid alternative for the analysis of these cannabinoids.

Experimental Section Cannabinoid Standards and Cannabis Samples THC, THCA and THC-d3 reference standards were purchased from Cerilliant Corporation (Round Rock, TX, USA). CBD and CBDA were obtained from Lipomed (Arlesheim, Switzerland) and THCAd3 was generously provided by the Institute of Forensic Medicine Freiburg, Germany. Calibration solutions were prepared by appropriate dilution of the reference standards in methanol. Concentration levels for THC and CBD were 20, 50, 100, 200, 400, and 1000 ng/mL for THC and CBD, and 5, 20, 50, 100, 250, and 500 ng/mL for THCA and CBDA. Calibration was performed by linear regression analysis using 1/x2 weighting and analytes were quantified by normalization with their deuterated internal standards. All solvents used were of analytical or HPLC grade. Cannabis material (flowers, leaves) used in this study had been seized by the police and had been submitted to the Institute of Forensic Medicine Bern, Switzerland for routine determination of the cannabinoid content by HPLC-DAD5. Extraction of the plant material was carried out as described for the HPLC-DAD analysis. Briefly, plant material was dried at 40 °C in an oven and then homogeneously ground. Approximately 500 mg of the resulting powder were extracted with 10 mL of a mixture of methanol/hexane 9:1 (v/v) by ultrasonication for 20 min. For IMS-MS analysis, the cannabis extracts were diluted with methanol and THC-d3 and THCA-d3 were added at final concentrations of 400 ng/mL and 80 ng/mL, respectively.

HPLC 3



HPLC-DAD analysis was performed using a Waters Alliance 2695 HPLC system equipped with a Waters photodiode array detector 2996 (Waters, Switzerland), a Merck LiChrospher 60 RPSelect B (5 μm) precolumn and a Merck LiChrospher 60 RP-select B (5 µm) LiChroCART, 125 x 4 mm analytical column (all Merck, Switzerland). Mobile phase A was a 25 mM aqueous solution of triethylammonium phosphate and mobile phase B was acetonitrile. Chromatographic separation was achieved under isocratic conditions with a 1.0 mL/min flow of 64% B (run time: 18 min). The injection volume was 10 µL and the detection wavelength was set at 210 nm. Further details of the method and exemplary chromatograms can be found in the work of Ambach et al.5

Ion Mobility Spectrometry – Mass Spectrometry All measurements were carried out on a high-resolution low field, drift tube IMS-MS instrument from Tofwerk (Thun, Switzerland), comprising a nanoESI source and a pressure- and temperature-controlled IMS cell coupled to a high-speed TOF mass spectrometer (Tofwerk HTOF TOFMS). The IMS cell contains a 10 cm desolvation tube and an 20 cm drift tube, both made from resistive glass8, along which a uniform electric field of ca. 400 V/cm (reduced electric field strength of ca. 2 Td) is applied. The IMS cell was maintained at 1000 mbar and 60°C, with high-purity nitrogen as buffer gas. The instrument uses a Hadamard-type multiplexed gating scheme to pulse ions into the drift region9, thus achieving 50% IMS duty cycle and improving signal intensity, sensitivity, signal-to-noise ratio, and effective resolution. Measurements were carried out in both positive and negative ion modes at an applied ESI potential of ca. 2 kV. Mass spectra were acquired from m/z 100 to 500. Sample aliquots of 0.8 µL were infused into the nanoESI source with a 1 μL/min flow of 0.1% formic acid in methanol (positive ion mode) and methanol (negative ion mode), respectively, using a WPS-3000TRS thermostatted plate autosampler and an HPG-3200RS binary pump (Dionex, Olten, Switzerland). After 1.5 min, the flow rate was increased to 10 μL/min for 1.7 min to clean the sampling system. Then, the flow rate was returned to its initial value for system re-equilibration, resulting in a total run time of 4 min. Reducing sample-to-sample time to 2 min yielded comparable results to the 4 min method. 4


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Raw IMS-MS data was post-processed using Tofware version 2.5.10 (Tofwerk, Switzerland) running in the Igor Pro (WaveMetrics, OR, USA) environment. For quantification, the ion signal of the protonated cannabinoids (m/z 315) and the deprotonated acids (m/z 357), respectively, was averaged over 0.5 min once the relevant ion current signal had appeared and stabilized. The signal was then distributed to the individual analytes according to the ratio measured with IMS. Inter-assay reproducibility and IMS drift time stability were evaluated by analyzing two cannabis samples in duplicate in both positive and negative ion modes on five consecutive days (n = 10). Accuracy was assessed with nettle extracts spiked with cannabinoids standards at concentrations of 60, 250 and 800 ng/mL for the neutral cannabinoids and 15, 125 and 400 ng/mL for the acids (n = 3 per concentration level). Nettle (Urtica dioica), which belongs to the same order as cannabis sativa L., was extracted as described for cannabis samples. Carry-over was investigated by injecting a methanol blank directly after a cannabis sample (n = 5). For determination of reduced mobilities K0 and CCS, IMS measurements were carried out at five different IMS drift voltages, ranging from 8 to 12 kV. K0 and CCS were automatically calculated by Tofware by normalizing the corrected drift time to drift length, voltage, pressure and temperature and using a modified zero-field equation which includes field-dependent corrections for both collisional momentum transfer and collision frequency10,11. The IMS-MS device was calibrated externally using a mixture of tetraalkylammonium salts12.

Results and Discussion Initially, the performance of the IMS-MS instrument to separate the isomeric pairs THC/CBD and THCA/CBDA was investigated. The instrument was operated with factory settings (IMS drift cell at 60°C and 1000 mbar) and no special tuning was performed. In contrast to other commercial IMS-MS instruments which are operated at low pressures (in the range of 3 mbar)13, performing IMS at atmospheric pressure allows to reach high resolving power using relatively short IMS drift cells8. Figure 1 shows the structures and the separation of cannabinoid standard mixtures at different relative concentrations in comparison to an authentic cannabis sample. As can be seen by looking at the chemical structures, THC and THCA have an ether-bridged closed-ring 5



structure whereas in CBD/CBDA the ring is cleaved, resulting in an alkene and hydroxyl-group. This leads to a change in their collision cross sections of approximately 1 %, which consequently requires an IMS resolving power of > 150 for near-baseline separation of the isomeric pairs.

Figure 1. (a) Structures and (b) representative ion mobility spectra of cannabis samples in comparison with different mixtures of reference standards.

Subsequently, validation of the method for quantitative analysis by assessing the following analytical parameters were performed: limit of detection (LOD), lower and upper limit of quantitation (LLOQ and ULOQ, respectively), linear range, inter-assay precision, accuracy and carry-over. The results of the validation are summarized in Table 1. LODs of approximately a factor 20 lower were achieved for the cannabinoid acids compared to their neutral counterparts which is due to the efficient ionization via deprotonation of organic acids during ESI. Otherwise, all analytes showed comparable analytical performance with a linear range of approximately two orders of magnitude, inter-assay precision of < 10% and an accuracy within 20% of the target value at 3 different concentrations. Also, carry-over was negligible with a background signal of < 20% of the LLOQ. 6


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Table 1. Method validation parameters for the IMS-MS assay.

Analyte

a

LOD

LLOQ

a

ULOQ

a

R

2b

Accuracy (%)

Inter-assay precision

c

d

Carry

Level 1

Level 2

Level 3

over

THC

6.6

20

1000

0.998

3.8

112.8

119.6

100.7

< 10

CBD

7.7

20

1000

0.986

9.9

112.1

107.9

113.7

< 10

THCA

0.3

20

1000

0.999

5.7

83.6

112.6

91.0

< 20

CBDA

0.5

20

1000

0.999

6.4

94.0

118.0

99.9

< 20

a

LOD: limit of detection; LLOQ: lower limit of quantification; ULOQ: upper limit of quantification; all in ng /ml

b

coefficient of determination of the linear calibration curve

c

in % RSD (n= 10)

d

determined in nettle extracts spiked with analyte standard. For THC and CBD: Level 1/2/3 = 60/250/800 ng/mL for THC and

CBD, 15/125/400 ng/ml for THCA and CBDA. n = 3 per level. e

in ng/ml

Additionally, the IMS drift time stability was investigated and both ion-neutral collision cross sections (DTCCSN2) and reduced mobility constants (K0) calculated for use as future reference values. Due to the small difference in IMS drift time between the isomeric pairs, IMS drift time stability is crucial for confident analyte identification. The relative standard deviation of the reproducibility was well below 0.1 % for all analytes, showing that reliable compound identification based on IMS drift time is possible. The IMS parameters are summarized in Table 2.

Table 2. IMS drift time, inter-assay reproducibility (n=20), collision cross section and reduced mobility constant for each cannabinoid. CCSN2 (Å )

2

K0 (cm V s )

0.02

187.4 ± 0.1

1.066 ± 0.001

39.72 ± 0.05

0.03

185.3 ± 0.1

1.078 ± 0.001

44.01 ± 0.06

0.01

203.1 ± 0.2

0.979 ± 0.001

Analyte

IMS drift time (ms)

Reproducibility (% RSD)

THC

40.22 ± 0.04

CBD THCA

DT

7


2 -1 -1

e


CBDA

43.32 ± 0.02

0.05

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199.8 ± 0.1

0.996 ± 0.001

Next, we were interested to see how the IMS-MS assays performs in comparison to a commonly used HPLC method for the analysis of cannabis samples; the corresponding Bland-Altman plots are shown in Figure 2. These plots show the agreement between the HPLC and IMS-MS methods 14

; the difference between the HPLC and the IMS-MS measurements is plotted on the y-axis and

the average measured value by HPLC and IMS-MS on the x-axis. As can be seen on the y-axis, the IMS-MS method shows a negative bias in all cases (between 5 and 20%; blue line), with confidence intervals of approximately 20% for all analytes (red lines). A reason for the negative bias might lie in the higher selectivity of MS detection compared to the diode-array detector used for HPLC (co-eluting substances might give rise to an unspecific UV signal).

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Figure 2. Bland-Altman plots for comparing results obtained by HPLC to IMS-MS for seized cannabis samples. Blue lines indicate bias of the IMS-MS method, red lines the confidence intervals (2 standard deviations).

From a practical point of view, it would be highly desirable to have a portable instrument for evaluating the potency of cannabis in the field. Even though the IMS-MS instrument used in this study is designed for robustness and ease of transportability, only a less complex and simple-touse instrument would be suited for use in daily routine by police. We therefore investigated if identification of the cannabinoids is also feasible by IMS only. For this purpose, the total ion current of the MS detector was used as signal (thus essentially simulating a Faraday cup detector as commonly used for IMS) in contrast to IMS-MS where only the selected ion current of the analyte of interest is analyzed. Furthermore, as short analysis times are crucial for field use, we evaluated if an integration time of as short as 3 seconds is also sufficient to distinguish the isomeric cannabinoids (Figure 3). Signal quality deteriorates in IMS only mode due to higher background (especially at 3 second integration time) but the isomers are still clearly detectable and the isomeric ratio determined by IMS only shows less than 5 % deviation form ratio determined by IMS-MS. Even though more detailed investigations on the use of IMS with simple ion current detection for cannabis potency testing are required, the construction and use of a portable device for this purpose seems feasible. In this regard, a sampling interface which employs an ambient ionization source also suitable for the direct analysis of solids (e.g. low temperature plasma 15) would be required for field use.

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Figure 3. Comparison of IMS-MS and IMS-only analysis of a cannabis sample in positive ion mode for THC/CBD (left) and negative ion mode for THCA/CBDA (right).

Conflict of Interest The authors declare no competing financial interest.

References

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(4) Smart, R.; Caulkins, J. P.; Kilmer, B.; Davenport, S.; Midgette, G. Addiction 2017. (5) Ambach, L.; Penitschka, F.; Broillet, A.; Konig, S.; Weinmann, W.; Bernhard, W. Forensic science international 2014, 243, 107-111. (6) Cochran, J. Chromatography Techniques 2014, 8-9. (7) Gambaro, V.; Dell’Acqua, L.; Farè, F.; Froldi, R.; Saligari, E.; Tassoni, G. Analytica Chimica Acta 2002, 468, 245-254. (8) Kaplan, K.; Graf, S.; Tanner, C.; Gonin, M.; Fuhrer, K.; Knochenmuss, R.; Dwivedi, P.; Hill, H. H., Jr. Analytical chemistry 2010, 82, 9336-9343. (9) Zhang, X.; Knochenmuss, R.; Siems, W. F.; Liu, W.; Graf, S.; Hill, H. H., Jr. Analytical chemistry 2014, 86, 1661-1670. (10) Groessl, M.; Graf, S.; Knochenmuss, R. The Analyst 2015, 140, 6904-6911. (11) Siems, W. F.; Viehland, L. A.; Hill, H. H., Jr. Analytical chemistry 2012, 84, 9782-9791. (12) Fernández-Maestre, R.; Harden, C. S.; Ewing, R. G.; Crawford, C. L.; Hill, H. H. The Analyst 2010, 135, 1433-1442. (13) Lanucara, F.; Holman, S. W.; Gray, C. J.; Eyers, C. E. Nature chemistry 2014, 6, 281. (14) Bland, J. M.; Altman, D. G. Statistical Methods in Medical Research 1999, 8, 135-160. (15) Huang, M.-Z.; Yuan, C.-H.; Cheng, S.-C.; Cho, Y.-T.; Shiea, J. Annual review of analytical chemistry 2010, 3, 43-65.

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High-Resolution Ion Mobility Spectrometry for Rapid Cannabis

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