Abstract

Synthetic cannabinoids sprayed on herbal mixtures have been abused as a new designer drug all over the world since 2004. In 2008, the first compounds, CP 47,497 and JWH-018, were identified as active ingredients in these mixtures. Most of the compounds have been synthesized for research purposes and are potent CB1 and/or CB2 receptor agonists.

To investigate the presence of synthetic cannabinoids in blood samples, a liquid chromatography–tandem mass spectrometry (LC–MS-MS) method was developed using only 100 µL of blood. After the addition of 0.2 mL of trizma buffer, the blood was extracted using liquid–liquid extraction with 1 mL of 1-chlorobutane containing 10% of isopropanol for 5 min on a shaker at 1,500 rpm. After centrifugation at 12,000 rpm for 1 min, the separated solvent layer was transferred to an autosampler vial and evaporated to dryness under N2. The residue was reconstituted in methanol and injected into a Shimadzu 8030 LC–MS-MS system to separate and detect 25 synthetic cannabinoids.

The method has been validated according to international guidelines and was found to be selective for all tested compounds. Calibration was satisfactory from 0.5–100 ng/mL, and from 5.0–500 ng/mL. for HU-210, CP 47,497 and the CP 47,497 C-8 homolog, respectively. The extraction efficiencies ranged from 30–101% and the matrix effects from 67–112%. Accuracy data were within the acceptance interval of ±15% (±20% at the lower limit of quantification) of the nominal values for all drugs.

Introduction

Over the last few years, synthetic cannabinoids sprayed on herbal mixtures have become drugs of abuse (1). This trend commenced in approximately 2004 in Europe and has now spread all over the world. The first identified compounds in products like “Spice,” “Smoke,” “Sense” and “K2” were CP 47,497 and JWH-018 (2–4). Most of these synthetic cannabinoids were synthesized in the 1990s as potent CB1 and CB2 receptor agonists (5, 6) for their possible use in the treatment of pain and for other medical uses (7–9). Users of the so-called “herbal incense” or “legal highs” have described cannabis-like effects after smoking these mixtures (10).

In many European countries, the United States and recently in Australia, some of the most commonly abused compounds were banned (11). Since then, slightly modified compounds appeared in “Spice-products” (12) with more than 20 structurally different synthetic cannabinoids being identified in herbal mixtures to date (2, 3, 10, 11, 13–27).

Emergency centers worldwide have reported the hospitalization of patients with symptoms including short-term loss of consciousness, nausea, tremor and headaches after the abuse of “herbal highs” (15, 28). The number, amount and combination of the compounds included in the smoking mixtures vary amongst the sold products. This diversity explains the variation of their duration of action.

Commonly used laboratory and road side immunoassay-based screening drug tests are not able detect the wide range of synthetic cannabinoids; therefore, there is a need for multi-analyte procedures for the detection of a wide range of synthetic cannabinoids. Although numerous studies for the detection of metabolites of synthetic cannabinoids in urine have been published (4, 13, 18, 29–31), so far only two multi-analyte screenings in blood and serum have been published. Dresen et al. were able to detect 10 different compounds in serum (32). The recently published method by Kacinko et al. included four synthetic cannabinoids analyzed in whole blood (33).

The aim of this study was the detection and quantification of a wide variety of synthetic cannabinoids in human whole blood using liquid chromatography–tandem mass spectrometry (LC–MS-MS).

Materials and Methods

Chemicals and reagents

The analytical standards of AM-694, AM-1241, WIN 55212-2 mesylate, RCS-4-C-4 homolog, JWH-015, JWH-302, JWH-073, JWH-250, JWH-203, JWH-081, JWH-019, JWH-210, JWH-018, JWH-007, JWH-398, CP 47,497, CP 47,497 C-8 homolog, HU-210 and JWH-251 were purchased from Sapphire Bioscience (Waterloo, NSW Australia). WIN 48.098, JWH-030, RCS-8, RCS-4, (3-methoxyphenyl)-(1-pentyl-1H-indol-3-yl)methanone (RCS-4 3-methoxy homolog) and (2-methoxyphenyl)-(1-pentyl-1H-indol-3-yl)methanone (RCS-4 2-methoxy homolog) were purchased from PM separations (Capalaba, Australia). The internal standards (IS) JWH-200-d5, JWH-073-d7, JWH-018-d9, JWH-007-d9 and CP 47,497-d11 were obtained from Sapphire Bioscience (Waterloo, NSW Australia). 1-Chlorobutane, isopropanol and high performance liquid chromatography (HPLC) grade methanol were purchased from Merck (Darmstadt, Germany). Ammonium formate and hydrochloric acid were provided by Ajax Finechem (Taren Point, NSW, Australia) and trizma base was obtained from Sigma–Aldrich (Castle Hill, NSW, Australia). Water was purified using a Milli-Q Ultrapure Water Sytem (Waters, Rydalmere, NSW, Australia).

Trizma buffer was prepared by dissolving 242 g trizma base in 1 L deionized water and pH adjusted to 9.2 with hydrochloric acid.

Specimens

Preserved blank blood samples (containing 1% sodium fluoride–potassium oxalate) for calibration purposes and validation experiments were obtained from a local blood bank. All blood samples were stored at –20°C before analysis.

Apparatus

The LC–MS-MS system consisted of a Shimadzu LCMS-8030 quadrupole mass spectrometer (Melbourne, VIC, Australia) operated in the electrospray ionization (ESI) mode, and a Shimadzu Nexera HPLC system (Melbourne, VIC, Australia) which consisted of a degasser, two eluent pumps, a column oven and an autosampler.

HPLC conditions

Gradient eluent was performed on an Agilent Eclipse XDB C-18 (2.1 × 150 mm, 5 µm) column coupled with an Eclipse XDB C-18 (4.6 × 12.5 mm, 5 µm) guard column. The mobile phase consisted of 5 mmol/L aqueous ammonium formate pH 6.4 (eluent A) and methanol (eluent B). During use, the mobile phase was degassed by the integrated Shimadzu Nexera degasser. Before starting the analysis, the HPLC system was equilibrated for 10 min with a mixture of 80% eluent A and 20% eluent B. The flow rate was set at 0.5 mL/min, the gradient was programmed as follows: 0.01–20.00 min, 20 % eluent B increasing to 100%; 20.01–24.00 min, 100 % eluent B; 24.01–28.00 min, starting conditions (20% eluent B) to re-equilibrate the column.

The column oven was set at 60°C. The autosampler was operated at 4°C and the autosampler needle was rinsed before and after aspiration of the sample using methanol.

MS-MS conditions

The mass spectral data were acquired with the following ESI inlet conditions: nebulizing gas and drying gas were nitrogen at a flow rate of 3.0 and 15.0 L/min, respectively; the interface voltage was set to 4.5 kV; desolvation line (DL) temperature was 280°C and the heat block temperature was 450°C. The mass spectrometer was operated in multiple reaction monitoring mode (MRM) with argon as the collision induced dissociation gas (CID) at a pressure of 230 kPa; the detector voltage was set to 1.72 kV. All other settings were analyte-specific and were auto-optimized by flow injection of 1 µL of a 1 mg/mL solution in methanol containing one analyte. The results of the auto-optimizations are summarized in Table I. The detection window for each compound was set at 0.8 min based on the retention time of the compound. The chemical structures of all included analytes are summarized in Figure 1.

Figure 1.

Chemical structures of the synthetic cannabinoids included in the method.

Figure 1.

Chemical structures of the synthetic cannabinoids included in the method.

Table I

Analyte, retention times (RT) [min], multiple reaction monitoring (MRM) transitions, Dwell times [msec], Q1 Pre Bias [V], collision cell energy (CE)[V], and Q3 Pre Bias [V] used in LC-ESI–MS-MS

Name RT [min] Precursor Ion m/z Product Ion m/z Dwell Time [msec] Q1 Pre Bias [V] CE [V] Q3 Pre Bias [V] 
WIN 48.098 12.99 379.10 135.05 100 -18 -20 -14 
379.10 77.10 100 -18 -64 -14 
379.10 107.00 100 -18 -62 -42 
AM-1241 14.10 504.20 98.15 100 -12 -24 -18 
504.20 112.10 100 -12 -32 -22 
AM-694 14.14 436.00 230.90 100 -20 -24 -26 
436.00 76.00 100 -20 -70 -28 
436.00 202.95 100 -20 -60 -34 
WIN-55212-2 14.64 427.30 155.00 100 -11 -28 -16 
427.30 127.00 100 -10 -50 -24 
427.30 100.10 100 -10 -50 -18 
RCS-4 C-4 homolog 15.02 308.10 135.05 100 -14 -22 -14 
308.10 77.15 100 -14 -66 -28 
308.10 92.05 100 -14 -68 -18 
RCS-4 2-methoxy homolog 15.28 322.10 77.00 50 -8 -62 -14 
322.10 135.05 50 -8 -20 -26 
322.10 92.05 50 -8 -60 -36 
JWH-030 15.63 292.10 154.90 50 -13 -20 -16 
292.10 127.00 50 -12 -44 -24 
292.10 77.05 50 -14 -68 -14 
JWH-015 15.68 328.10 154.90 50 -13 -26 -16 
328.10 127.00 50 -15 -48 -24 
328.10 200.10 50 -8 -24 -22 
JWH-302 15.82 336.20 43.10 50 -16 -48 -17 
336.20 121.10 50 -10 -32 -22 
336.20 214.10 50 -10 -24 -29 
RCS-4 16.01 322.10 77.10 30 -8 -56 -14 
322.10 135.10 30 -8 -20 -26 
322.10 92.10 30 -8 -64 -32 
RCS-4 3-methoxy homolog 16.16 322.10 135.05 30 -8 -20 -14 
322.10 77.00 30 -8 -56 -14 
322.10 92.00 30 -8 -66 -32 
JWH-250 16.19 336.10 121.15 50 -16 -22 -12 
336.10 91.10 50 -16 -70 -16 
336.10 200.15 50 -16 -26 -22 
JWH-073 16.25 328.30 127.10 50 -15 -46 -12 
328.10 155.00 50 -16 -26 -16 
328.10 200.00 50 -16 -26 -22 
JWH-251 16.68 320.00 43.20 50 -15 -48 -16 
319.90 105.20 50 -16 -44 -21 
320.00 214.40 50 -15 -32 -10 
JWH-203 16.77 340.00 214.00 50 -10 -28 -23 
340.00 143.90 50 -10 -44 -26 
340.00 125.00 50 -10 -28 -12 
JWH-018 17.07 342.10 127.10 50 -10 -48 -23 
342.10 155.00 50 -30 -28 -30 
342.10 214.10 50 -16 -24 -24 
JWH-081 17.40 372.10 157.00 50 -15 -46 -16 
372.10 185.00 50 -15 -30 -18 
372.10 214.00 50 -15 -26 -14 
JWH-007 17.43 356.10 127.10 50 -17 -48 -23 
356.10 155.00 50 -17 -28 -30 
356.10 228.10 50 -17 -28 -16 
CP 47,497 17.66 316.90 159.00 50 16 35 15 
316.90 245.00 50 16 35 27 
317.20 299.30 50 12 24 16 
JWH-019 17.81 356.10 228.00 50 -10 -26 -15 
356.10 155.00 50 -15 -28 -16 
356.10 127.00 50 -16 -50 -23 
RCS-8 17.95 376.10 91.15 50 -18 -58 -16 
376.10 121.05 50 -18 -20 -12 
376.10 69.15 50 -18 -56 -24 
CP 47,497 C-8 homolog 18.27 331.20 159.25 50 12 70 30 
331.20 259.10 50 12 32 30 
331.20 185.20 50 12 54 36 
JWH-398 18.27 376.00 160.90 50 -18 -50 -30 
376.00 189.00 50 -11 -28 -20 
376.10 214.10 50 -10 -26 -14 
JWH-210 18.28 370.10 214.00 50 -17 -26 -14 
370.10 183.00 50 -17 -28 -19 
370.10 155.00 50 -10 -44 -30 
HU-210 18.37 385.30 367.10 50 11 30 21 
385.30 280.95 50 12 44 34 
JWH-200-d5 13.32 390.00 114.00 100 -19 -35 -15 
390.00 127.00 100 -10 -35 -15 
390.00 155.20 100 -11 -35 -15 
JWH-073-d7 16.25 335.10 127.10 50 -15 -48 -22 
335.10 155.00 50 -10 -28 -16 
335.10 207.20 50 -16 -26 -23 
JWH-018-d9 17.07 351.20 223.10 50 -14 -26 -15 
351.20 155.00 50 -14 -28 -29 
351.20 127.00 50 -14 -50 -24 
JWH-007-d9 17.43 365.20 127.00 50 -17 -50 -23 
365.20 155.00 50 -10 -30 -15 
365.20 237.10 50 -17 -28 -16 
CP 47,497-d11 17.66 328.00 310.10 50 50 24 50 
328.00 256.15 50 50 28 50 
328.00 282.10 50 50 28 50 
Name RT [min] Precursor Ion m/z Product Ion m/z Dwell Time [msec] Q1 Pre Bias [V] CE [V] Q3 Pre Bias [V] 
WIN 48.098 12.99 379.10 135.05 100 -18 -20 -14 
379.10 77.10 100 -18 -64 -14 
379.10 107.00 100 -18 -62 -42 
AM-1241 14.10 504.20 98.15 100 -12 -24 -18 
504.20 112.10 100 -12 -32 -22 
AM-694 14.14 436.00 230.90 100 -20 -24 -26 
436.00 76.00 100 -20 -70 -28 
436.00 202.95 100 -20 -60 -34 
WIN-55212-2 14.64 427.30 155.00 100 -11 -28 -16 
427.30 127.00 100 -10 -50 -24 
427.30 100.10 100 -10 -50 -18 
RCS-4 C-4 homolog 15.02 308.10 135.05 100 -14 -22 -14 
308.10 77.15 100 -14 -66 -28 
308.10 92.05 100 -14 -68 -18 
RCS-4 2-methoxy homolog 15.28 322.10 77.00 50 -8 -62 -14 
322.10 135.05 50 -8 -20 -26 
322.10 92.05 50 -8 -60 -36 
JWH-030 15.63 292.10 154.90 50 -13 -20 -16 
292.10 127.00 50 -12 -44 -24 
292.10 77.05 50 -14 -68 -14 
JWH-015 15.68 328.10 154.90 50 -13 -26 -16 
328.10 127.00 50 -15 -48 -24 
328.10 200.10 50 -8 -24 -22 
JWH-302 15.82 336.20 43.10 50 -16 -48 -17 
336.20 121.10 50 -10 -32 -22 
336.20 214.10 50 -10 -24 -29 
RCS-4 16.01 322.10 77.10 30 -8 -56 -14 
322.10 135.10 30 -8 -20 -26 
322.10 92.10 30 -8 -64 -32 
RCS-4 3-methoxy homolog 16.16 322.10 135.05 30 -8 -20 -14 
322.10 77.00 30 -8 -56 -14 
322.10 92.00 30 -8 -66 -32 
JWH-250 16.19 336.10 121.15 50 -16 -22 -12 
336.10 91.10 50 -16 -70 -16 
336.10 200.15 50 -16 -26 -22 
JWH-073 16.25 328.30 127.10 50 -15 -46 -12 
328.10 155.00 50 -16 -26 -16 
328.10 200.00 50 -16 -26 -22 
JWH-251 16.68 320.00 43.20 50 -15 -48 -16 
319.90 105.20 50 -16 -44 -21 
320.00 214.40 50 -15 -32 -10 
JWH-203 16.77 340.00 214.00 50 -10 -28 -23 
340.00 143.90 50 -10 -44 -26 
340.00 125.00 50 -10 -28 -12 
JWH-018 17.07 342.10 127.10 50 -10 -48 -23 
342.10 155.00 50 -30 -28 -30 
342.10 214.10 50 -16 -24 -24 
JWH-081 17.40 372.10 157.00 50 -15 -46 -16 
372.10 185.00 50 -15 -30 -18 
372.10 214.00 50 -15 -26 -14 
JWH-007 17.43 356.10 127.10 50 -17 -48 -23 
356.10 155.00 50 -17 -28 -30 
356.10 228.10 50 -17 -28 -16 
CP 47,497 17.66 316.90 159.00 50 16 35 15 
316.90 245.00 50 16 35 27 
317.20 299.30 50 12 24 16 
JWH-019 17.81 356.10 228.00 50 -10 -26 -15 
356.10 155.00 50 -15 -28 -16 
356.10 127.00 50 -16 -50 -23 
RCS-8 17.95 376.10 91.15 50 -18 -58 -16 
376.10 121.05 50 -18 -20 -12 
376.10 69.15 50 -18 -56 -24 
CP 47,497 C-8 homolog 18.27 331.20 159.25 50 12 70 30 
331.20 259.10 50 12 32 30 
331.20 185.20 50 12 54 36 
JWH-398 18.27 376.00 160.90 50 -18 -50 -30 
376.00 189.00 50 -11 -28 -20 
376.10 214.10 50 -10 -26 -14 
JWH-210 18.28 370.10 214.00 50 -17 -26 -14 
370.10 183.00 50 -17 -28 -19 
370.10 155.00 50 -10 -44 -30 
HU-210 18.37 385.30 367.10 50 11 30 21 
385.30 280.95 50 12 44 34 
JWH-200-d5 13.32 390.00 114.00 100 -19 -35 -15 
390.00 127.00 100 -10 -35 -15 
390.00 155.20 100 -11 -35 -15 
JWH-073-d7 16.25 335.10 127.10 50 -15 -48 -22 
335.10 155.00 50 -10 -28 -16 
335.10 207.20 50 -16 -26 -23 
JWH-018-d9 17.07 351.20 223.10 50 -14 -26 -15 
351.20 155.00 50 -14 -28 -29 
351.20 127.00 50 -14 -50 -24 
JWH-007-d9 17.43 365.20 127.00 50 -17 -50 -23 
365.20 155.00 50 -10 -30 -15 
365.20 237.10 50 -17 -28 -16 
CP 47,497-d11 17.66 328.00 310.10 50 50 24 50 
328.00 256.15 50 50 28 50 
328.00 282.10 50 50 28 50 

Preparation of stock solutions, calibration standards and control samples

Stock solutions of each analyte were prepared at a concentration of 1 mg/mL using methanol as solvent. For all solid reference standards, solutions were prepared by weighing separately. Commercially available liquid reference standards were diluted with methanol to give a concentration of 1 mg/mL. Working solutions of each analyte were prepared using methanol by independent dilution of each stock solution at the following concentrations: 0.1, 0.01 and 0.001 mg/mL. All solutions were stored at –20°C for a maximum time frame of three months; the stability of the stock solutions past this time-frame has not been evaluated.

The calibration standards were prepared using pooled blank blood and spiking solutions prepared from the working solutions as mixtures of the 25 synthetic cannabinoids at concentrations 10 times higher than the corresponding calibration standards. The quality control samples were prepared using pooled blank blood and independently prepared mixtures of the 25 synthetic cannabinoids at concentrations 100 times higher than the concentrations of the corresponding quality control samples.

The final blood concentrations of calibration standards and quality control samples were as follows: Calibration standards for CP 47,497, CP 47,497 C-8 and HU-210 were 5, 10, 50, 125, 250, 375 and 500 ng/mL; their respective quality control concentrations were 15 ng/mL (low), 225 ng/mL (med) and 450 ng/mL (high).

For all other analytes, calibrations standards were 0.5, 1, 10, 25, 50, 75 and 100 ng/mL; their respective quality control concentrations were 1.5 ng/mL (low), 45 ng/mL (med) and 90 ng/mL (high). All samples were stored at –20°C before analysis.

Extraction procedure

In a 2-mL Eppendorf tube (Eppendorf Australia, North Ryde, NSW), 0.1 mL blood was mixed with 10 µL of the IS (20 ng/mL of JWH-200-d 5, JWH-073-d 7, JWH-018-d 9, JWH-007-d 9 20 and 100 ng/mL of CP 47,497-d 11). To the blood, 0.2 mL of trizma buffer and 1 mL of 1 chlorobutane containing 10% of isopropanol were added and mixed thoroughly. The sample was extracted for 5 min on a VXR basic IKA Vibrax shaker at 1,500 rpm. After a brief centrifugation to separate layers, the solvent layer was transferred to an autosampler vial and evaporated to dryness using a Ratek dry block heater DBH10 operated at room temperature.

The residue was reconstituted in 50 µL of methanol and 10 µL of the final extract were injected into the LC–MS-MS system.

Validation Experiments

Selectivity

Selectivity experiments were carried out using postmortem and antemortem blood samples sent to the authors' laboratory for toxicological analysis. Ten postmortem and 10 antemortem samples were extracted as described previously without the addition of IS (reference). The samples were analyzed to exclude any interference with endogenous peaks. Additionally, two zero samples (blank sample + IS) were analyzed to check for absence of analyte ions in the respective peaks of the IS.

Linearity

Aliquots of blank blood samples were spiked at concentrations extracted as described previously to obtain calibration standards. Replicates (n = 6) at each of the seven concentration levels were analyzed. Daily calibration curves using the same concentrations (single measurements per level) were prepared with each batch of validation and quality control samples.

Accuracy and precision

Quality control (QC) samples, QC low, QC medium, and QC high were prepared at the concentrations described previously. Two samples of each QC concentration were measured over a period of eight consecutive days (n = 16 at each concentration). Daily calibration curves were used to calculate the concentration of the QCs. Accuracy was calculated for each analyte and bias determined by calculating the percent deviation of the mean of all calculated concentration values at a specific level from the respective nominal concentration. Precision data (given as relative standard deviation; RSD) for within-day (repeatability), and time-different intermediate precision (combination of within-day and between-day effects) of the method were calculated according to Beyer et al. (34, 35) using one-way analysis of variance (ANOVA) with the grouping-variable “day”. The acceptance intervals of within-day (repeatability) and intermediate precision were ≤15% RSD (≤20% RSD at QC low) and ±15% for bias (±20% at QC low) of the nominal values (36).

Processed sample stability

For estimation of stability of the processed samples under the conditions of LC–MS-MS analysis, QC low and QC high samples (n = 8 each) were extracted as described previously. The resulting extracts at each concentration level were pooled to eliminate differences in extraction efficiency. Aliquots of these pooled extracts at each concentration level were transferred to autosampler vials, injected into the LC–MS-MS system and analyzed under conditions given previously. The time intervals between the analyses of the QC samples were extended to 2 h by the injection of five blank samples. Stability of the extracted analytes was tested by regression analysis plotting absolute peak areas of each analyte at each concentration versus injection time. The instability of the processed samples was indicated by a negative slope, significantly different from zero (P ≤ 0.05) (37).

Freeze/thaw stability and bench-top stability

Combined freeze/thaw and bench-top stability were evaluated through analysis of QC samples (six replicates at each concentration) before (control samples) and after eight freeze/thaw cycles (stability samples). For each cycle, the samples were kept at 20°C for 21 h. The thawed samples were kept at room temperature for 3 h before the next freeze cycle to incorporate bench-top stability. The experiments were carried out together with the accuracy and precision experiments and the concentrations of the control and stability samples were calculated via daily calibration curves. Stability was tested against an acceptance interval of 90–110% for the ratio of the means (stability samples versus control samples) and an acceptance interval of 80–120% from the control samples' mean for the 90% confidence interval (CI) of stability samples (37).

Long-term stability

The experimental design for the study of long-term stability was similar to the freeze/thaw stability. Analyte stability for long-term storage was evaluated through analysis of QC samples (n = 6 at each concentration) before (control samples) and after storage for six weeks at –20°C (stability samples). Stability was measured against an acceptance interval of 90–110% for the ratio of the means (stability samples versus control samples) and an acceptance interval of 80–120% from the control samples' mean for the 90% CI of stability samples (37).

Lower limits of quantification and detection

The lower limits of quantification (LLOQ) were defined as the lowest point of the calibration curve, as mentioned previously, and fulfilled the requirement of LLOQ signal-to-noise ratio of 10:1 (37, 38). The limit of detection (LOD) was not systematically evaluated.

Extraction efficiencies and matrix effects

According to the approach of Matuszewski et al. (39) the extraction efficiencies, matrix effects and process efficiencies were estimated with a set of three different samples at two concentrations. Set A was a batch of neat standards. The neat samples were prepared with 10 µL IS, 10 µL of the respective spiking solution for the QC low (five samples) and high (five samples) and 30 µL methanol. For the samples of set B, five different blank bloods (100 µL) were extracted as described previously and the residue of the samples was reconstituted in 50 µL methanol containing the analytes and IS. For set C, identical blank blood samples to those used for set B were spiked at described QC low and QC high concentrations and extracted as described previously.

Extraction efficiencies were estimated by comparison the peak area of the samples of set B to those of set C. For the matrix effects, the peak area of the samples of set B was compared to those of set A and for extraction efficiencies set C was compared to set A. All values are reported in percentage. Values over 100% for matrix effects indicate ion enhancement, while values below 100% indicate ion suppression.

Results and Discussion

Preliminary experiments showed that a commonly used liquid–liquid extraction method in the author's laboratory for the detection of common drugs and drugs of abuse could be applied for the detection of these synthetic cannabinoids (data not shown). Table II shows mean values of extraction efficiencies and the corresponding variation over five different blood samples. Datasets in which the variation (SD in percentage) is greater than 20% difference of the mean value (not acceptable) are marked in bold type. Overall, the method showed satisfactory extraction efficiencies for all analytes, with average extraction efficiencies ranging from 30 to 101%. Table II also shows the mean values of matrix effects and the corresponding variation over five different blood samples. As described for the extraction efficiencies, datasets in which the variation (SD in percentage) is greater than 20% difference of the mean value (not acceptable) are marked in bold type. Overall, the matrix effects were not significant using the described extraction and detection procedure for most compounds. With the exception of RCS-4,3 methoxy-homolog, CP 47,497 and CP 47,497 C-8 homolog at a low concentration, variation of matrix effects did not exceed 20%.

Table II

Matrix effects and recoveries in % [range] off all targets and IS. Datasets with variations (SD in %) greater than 20% difference of the mean value (not acceptable) are marked in bold type

Name Matrix effects
 
Recovery
 
LOW HIGH LOW HIGH 
WIN 48.098 95 [87 - 103] 87 [80 - 93] 82 [69 - 91] 91 [84 - 104] 
AM-1241 93 [84 - 102] 83 [76 - 91] 78 [68 - 87] 86 [80 - 100] 
AM-694 92 [81 - 102] 85 [78 - 91] 74 [63 - 88] 79 [72 - 91] 
WIN-55212-2 92 [84 - 101] 87 [78 - 95] 84 [72 - 91] 88 [78 - 105] 
RCS-4 C-4 homolog 95 [94 - 106] 86 [79 - 92] 76 [66 - 86] 83 [76 - 96] 
RCS-4 2 methoxy-homolog 94 [85 - 102] 86 [79 - 92] 73 [63 - 81] 80 [72 - 92] 
JWH-030 85 [76 - 93] 82 [74 - 89] 70 [64 - 80] 73 [66 - 86] 
JWH-015 94 [84 - 103] 85 [78 - 91] 66 [58 - 79] 72 [65 - 84] 
JWH-302 86 [79 - 95] 86 [80 - 92] 62 [55 - 72] 66 [59 - 75] 
RCS-4 93 [81 - 105] 87 [79 - 93] 68 [54 - 87] 101 [62 - 137] 
RCS-4 3 methoxy-homolog 112 [82-191] 81 [75 - 87] 50 [31 - 63] 57 [56 - 71] 
JWH-250 89 [80 - 98] 85 [78 - 91] 58 [53 - 65] 63 [56 - 72] 
JWH-073 91 [82 - 100] 85 [79 - 92] 57 [50 - 68] 61 [54 - 71] 
JWH-251 93 [83 - 100] 89 [79 - 96] 53 [47 - 63] 58 [50 - 69] 
JWH-203 92 [86 - 98] 88 [81 - 93] 58 [53 - 71] 56 [47 - 65] 
JWH-018 95 [84 - 107] 87 [79 - 93] 43 [36 - 54] 49 [42 - 58] 
JWH-081 98 [98 - 107] 86 [82 - 91] 41 [36 - 51] 45 [41 - 52] 
JWH-007 92 [84 - 101] 88 [81 - 93] 44 [37 - 54] 47 [40 - 55] 
CP 47,497 89 [63 - 118] 82 [85 - 94] 80 [43 - 138] 70 [56 - 90] 
JWH-019 76 [63 - 85] 73 [67 - 77] 37 [29 - 50] 38 [32 - 45] 
RCS-8 75 [63 - 85] 74 [67 - 81] 41 [32 - 52] 40 [34 - 48] 
CP 47,497 C-8 homolog 84 [58 - 113] 73 [63 - 81] 56 [38 - 68] 62 [50 - 78] 
JWH-398 67 [50 - 74] 73 [68 - 83] 32 [19 - 47] 36 [29 - 41] 
JWH-210 70 [51 - 79] 72 [66 - 79] 30 [17 - 46] 32 [27 - 38] 
HU-210 79 [63 - 92] 74 [60 - 83] 66 [45 - 101] 61 [47 - 78] 
Name Matrix effects
 
Recovery
 
LOW HIGH LOW HIGH 
WIN 48.098 95 [87 - 103] 87 [80 - 93] 82 [69 - 91] 91 [84 - 104] 
AM-1241 93 [84 - 102] 83 [76 - 91] 78 [68 - 87] 86 [80 - 100] 
AM-694 92 [81 - 102] 85 [78 - 91] 74 [63 - 88] 79 [72 - 91] 
WIN-55212-2 92 [84 - 101] 87 [78 - 95] 84 [72 - 91] 88 [78 - 105] 
RCS-4 C-4 homolog 95 [94 - 106] 86 [79 - 92] 76 [66 - 86] 83 [76 - 96] 
RCS-4 2 methoxy-homolog 94 [85 - 102] 86 [79 - 92] 73 [63 - 81] 80 [72 - 92] 
JWH-030 85 [76 - 93] 82 [74 - 89] 70 [64 - 80] 73 [66 - 86] 
JWH-015 94 [84 - 103] 85 [78 - 91] 66 [58 - 79] 72 [65 - 84] 
JWH-302 86 [79 - 95] 86 [80 - 92] 62 [55 - 72] 66 [59 - 75] 
RCS-4 93 [81 - 105] 87 [79 - 93] 68 [54 - 87] 101 [62 - 137] 
RCS-4 3 methoxy-homolog 112 [82-191] 81 [75 - 87] 50 [31 - 63] 57 [56 - 71] 
JWH-250 89 [80 - 98] 85 [78 - 91] 58 [53 - 65] 63 [56 - 72] 
JWH-073 91 [82 - 100] 85 [79 - 92] 57 [50 - 68] 61 [54 - 71] 
JWH-251 93 [83 - 100] 89 [79 - 96] 53 [47 - 63] 58 [50 - 69] 
JWH-203 92 [86 - 98] 88 [81 - 93] 58 [53 - 71] 56 [47 - 65] 
JWH-018 95 [84 - 107] 87 [79 - 93] 43 [36 - 54] 49 [42 - 58] 
JWH-081 98 [98 - 107] 86 [82 - 91] 41 [36 - 51] 45 [41 - 52] 
JWH-007 92 [84 - 101] 88 [81 - 93] 44 [37 - 54] 47 [40 - 55] 
CP 47,497 89 [63 - 118] 82 [85 - 94] 80 [43 - 138] 70 [56 - 90] 
JWH-019 76 [63 - 85] 73 [67 - 77] 37 [29 - 50] 38 [32 - 45] 
RCS-8 75 [63 - 85] 74 [67 - 81] 41 [32 - 52] 40 [34 - 48] 
CP 47,497 C-8 homolog 84 [58 - 113] 73 [63 - 81] 56 [38 - 68] 62 [50 - 78] 
JWH-398 67 [50 - 74] 73 [68 - 83] 32 [19 - 47] 36 [29 - 41] 
JWH-210 70 [51 - 79] 72 [66 - 79] 30 [17 - 46] 32 [27 - 38] 
HU-210 79 [63 - 92] 74 [60 - 83] 66 [45 - 101] 61 [47 - 78] 

For the detection of the analytes of interest, three MRM transitions were used for each analyte; their use and their respective peak area ratios enabled unambiguous identification of all analytes included in the assay. For two analytes (AM 1241 and HU 210), limited fragmentation did not allow for the use of three MRM transitions. The identification of the substances using two MRM transitions is still possible; however, positive identification of these substances should be reviewed with caution. The MRM ratios were compared to within-batch calibrators and acceptance criteria of the GTFCh Quality Assurance Guidelines (40) were applied.

In ESI mode, 22 synthetic cannabinoids could be ionized in positive mode and three drugs (CP 47,497, CP 47,497 C-8 homolog and HU-210) formed negative ions. To accommodate the formation of negative ions, the mobile phase A was prepared at 5 mM aqueous ammonium formate and adjusted to a pH of 6.3. Additionally, methanol was used as eluent B without the addition of modifiers. This combination of eluents and pH proved to be acceptable for the formation of positive and negative ions.

The MRM settings described in Table I were chosen by the Shimadzu LC–MS software and critically reviewed. The dwell times were optimized depending on the signal response of each individual synthetic cannabinoid.

The chemical structures of the synthetic cannabinoids used as designer drugs are very similar, because only minor changes to the molecule result in a possibly non-scheduled new compound. It is, therefore, common to encounter isobaric compounds with similar fragmentation. In this method, five sets of isobaric compounds were identified in the list of analytes. These isobaric compound groups are: precursor ion m/z 322 (RCS-4 2 methoxy homolog, RCS-4, RCS-4 3 methoxy homolog), precursor ion m/z 328 (JWH-015, JWH-073), precursor ion m/z 336 (JWH-250, JWH-302), precursor ion m/z 356 (JWH-007, JWH-019), and precursor ion m/z 376 (JWH-398, RCS-8). To avoid misidentifications, chromatographic separation of these isobaric compounds needed to be achieved. Preliminary experiments showed that a chromatographic separation could be achieved using gradient elution on a XBD C18 narrow bore column. A typical sample chromatogram showing the chromatographic separation of all analytes acquired in positive mode is given in Figure 2, and the chromatographic separation of all analytes acquired in negative mode is given in Figure 3. Although RCS-4 and RCS-4 3 methoxy homolog have not been baseline separated, the validation experiments showed that the accuracy and precision of the determination of these compounds was satisfactory.

Figure 2.

MRM chromatogram of all transitions recorded in positive mode of an extract of a calibrator at a concentration of 100 ng/mL.

Figure 2.

MRM chromatogram of all transitions recorded in positive mode of an extract of a calibrator at a concentration of 100 ng/mL.

Figure 3.

MRM chromatogram of all transitions recorded in negative mode of an extract of a calibrator at a concentration of 500 ng/mL.

Figure 3.

MRM chromatogram of all transitions recorded in negative mode of an extract of a calibrator at a concentration of 500 ng/mL.

Validation experiments

The described procedure was validated according to internationally accepted recommendations (37, 38, 41). The assay was found to be selective for all tested compounds and no interfering peaks were observed in the extracts of the different postmortem and antemortem blank blood samples.

Calibration curves were linear in the range described previously. All analytes were visually checked for a linear fit, a weighted second order model fit and a quadratic fit. Linear regression (1/x2 weighting) was applied to all studied analytes. The calibration fit showed a coefficient of determination of r2 > 0.99 for all drugs.

When using mobile phase for reconstitution of the final extracts during method development, most drugs appeared to degrade when stored in the autosampler. Further experiments showed that the analytes did not degrade and that the compounds were re-recoverable from the glass wall of the autosampler vial using methanol. Therefore, methanol was used as a reconstitution solvent. After this modification, no analyte showed signs of instability for up to 24 hours at ambient temperature. All drugs also appeared to be stable over a period of 1 month when stored at –20°C. The LLOQs corresponded to the lowest concentrations used for the calibration curves with a signal-to-noise ratio of at least 10.

The assay showed only minor variations in extraction efficiencies and matrix effects for most analytes. A bigger than acceptable variation of matrix effects and extraction efficiencies was observed for CP 47,497 and CP 47,497 C-8 homolog. These substances are detected in negative mode and showed a lower response possibly due to lower ionization efficiency of the ESI source. Because the variations have been observed in samples containing low concentrations, this might be explained by low peak responses. Variations have also been observed in the isobaric pair of RCS-4 and RCS-4 3 methoxy homolog, which might be explained by the close retention of these compounds. Concentrations in unknown samples should be reviewed with caution.

Accuracy data were for all analytes within the acceptance interval of ±15% (±20% at the LLOQ) of the nominal values for all drugs. Within-day (repeatability) and intermediate precision data were within the required limits of 15% RSD (20% RSD at LLOQ) with the exception of low concentrations of HU-210 (Table III). Concentrations below the QC med value for HU-210 should therefore be considered as semi-quantitative.

Table III

Accuracy, precision (time-different intermediate precision) and repeatability (within-day precision) of the LC–MS-MS method for all analytes. IS for quantification is given in parenthesis. Datasets outside required limits are marked in bold type

Name Accuracy
 
Precision
 
Repeatability
 
LOW MED HIGH LOW MED HIGH LOW MED HIGH 
WIN 48.098 (JWH-073-d7) -3.6 15.0 7.2 8.9 8.3 10.5 5.1 4.0 7.4 
AM-1241 (JWH-073-d7) -0.9 7.6 -4.5 7.2 8.0 9.0 4.5 3.3 6.3 
AM-694 (JWH-018-d9) -5.2 14.1 1.6 9.0 7.2 7.6 6.3 3.0 4.3 
WIN-55212-2 (JWH-200-d5) 1.1 6.9 -5.0 9.1 10.9 10.6 4.0 3.1 4.8 
RCS-4 C-4 homolog (JWH-007-d9) 0.3 13.7 3.8 9.6 6.4 10.3 7.1 3.5 6.5 
RCS-4 2-methoxy homolog (JWH-007-d9) -0.2 10.2 0.9 9.7 9.7 9.1 5.9 5.9 5.9 
JWH-030 (JWH-018-d9) 2.7 10.2 5.3 10.6 11.5 8.7 6.9 6.9 7.1 
JWH-015 (JWH-073-d7) 3.2 12.5 4.7 8.3 8.9 8.1 3.6 3.2 5.8 
JWH-302 (JWH-200-d5) 7.1 11.1 1.0 8.9 9.4 7.5 6.7 2.8 3.0 
RCS-4 (JWH-073-d7) 0.4 14.0 2.5 9.2 9.9 8.2 3.0 5.5 6.6 
RCS-4 3-methoxy homolog (JWH-007-d9) 6.5 12.7 4.4 8.6 8.2 8.9 6.9 4.1 6.8 
JWH-250 (JWH-018-d9) 4.0 12.0 5.9 10.6 6.9 6.6 6.6 2.9 6.2 
JWH-073 (JWH-073-d7) -7.9 2.2 -10.2 8.8 6.6 8.1 3.7 4.2 4.9 
JWH-251 (JWH-200-d5) 10.0 11.9 2.7 8.5 7.1 4.8 7.1 2.9 3.8 
JWH-203 (JWH-073-d7) 7.7 12.3 4.2 10.6 7.3 7.0 3.6 3.8 4.6 
JWH-018 (JWH-018-d9) 3.9 11.6 -7.4 10.2 7.4 8.2 4.3 4.1 5.2 
JWH-081 (JWH-018-d9) 9.4 13.3 -0.3 9.7 7.7 6.8 5.5 4.4 5.4 
JWH-007 (JWH-007-d9) 0.3 4.1 -9.2 8.6 6.8 8.5 3.3 3.0 4.6 
CP 47,497 (CP 47,497-d11) 8.96 2.4 8.6 14.3 14.6 14.0 13.8 14.6 12.0 
JWH-019 (JWH-007-d9) 7.9 9.5 -2.4 10.7 7.7 8.2 4.3 4.0 4.3 
RCS-8 (JWH-007-d9) 9.3 11.5 6.4 11.2 10.9 10.0 4.1 3.8 4.6 
CP 47,497 C-8 homolog (CP 47,497-d11) 5.2 8.9 -3.6 14.4 13.7 12.6 14.4 12.5 10.8 
JWH-398 (JWH-007-d9) 10.9 14.5 7.1 8.3 8.8 9.5 4.7 4.9 5.6 
JWH-210 (JWH-007-d9) -3.2 12.3 5.6 7.6 9.2 9.3 5.6 3.6 5.2 
HU-210 (CP 47,497-d11) -2.8 2.7 -10.8 21.2 14.0 12.3 19.7 14.0 11.9 
Name Accuracy
 
Precision
 
Repeatability
 
LOW MED HIGH LOW MED HIGH LOW MED HIGH 
WIN 48.098 (JWH-073-d7) -3.6 15.0 7.2 8.9 8.3 10.5 5.1 4.0 7.4 
AM-1241 (JWH-073-d7) -0.9 7.6 -4.5 7.2 8.0 9.0 4.5 3.3 6.3 
AM-694 (JWH-018-d9) -5.2 14.1 1.6 9.0 7.2 7.6 6.3 3.0 4.3 
WIN-55212-2 (JWH-200-d5) 1.1 6.9 -5.0 9.1 10.9 10.6 4.0 3.1 4.8 
RCS-4 C-4 homolog (JWH-007-d9) 0.3 13.7 3.8 9.6 6.4 10.3 7.1 3.5 6.5 
RCS-4 2-methoxy homolog (JWH-007-d9) -0.2 10.2 0.9 9.7 9.7 9.1 5.9 5.9 5.9 
JWH-030 (JWH-018-d9) 2.7 10.2 5.3 10.6 11.5 8.7 6.9 6.9 7.1 
JWH-015 (JWH-073-d7) 3.2 12.5 4.7 8.3 8.9 8.1 3.6 3.2 5.8 
JWH-302 (JWH-200-d5) 7.1 11.1 1.0 8.9 9.4 7.5 6.7 2.8 3.0 
RCS-4 (JWH-073-d7) 0.4 14.0 2.5 9.2 9.9 8.2 3.0 5.5 6.6 
RCS-4 3-methoxy homolog (JWH-007-d9) 6.5 12.7 4.4 8.6 8.2 8.9 6.9 4.1 6.8 
JWH-250 (JWH-018-d9) 4.0 12.0 5.9 10.6 6.9 6.6 6.6 2.9 6.2 
JWH-073 (JWH-073-d7) -7.9 2.2 -10.2 8.8 6.6 8.1 3.7 4.2 4.9 
JWH-251 (JWH-200-d5) 10.0 11.9 2.7 8.5 7.1 4.8 7.1 2.9 3.8 
JWH-203 (JWH-073-d7) 7.7 12.3 4.2 10.6 7.3 7.0 3.6 3.8 4.6 
JWH-018 (JWH-018-d9) 3.9 11.6 -7.4 10.2 7.4 8.2 4.3 4.1 5.2 
JWH-081 (JWH-018-d9) 9.4 13.3 -0.3 9.7 7.7 6.8 5.5 4.4 5.4 
JWH-007 (JWH-007-d9) 0.3 4.1 -9.2 8.6 6.8 8.5 3.3 3.0 4.6 
CP 47,497 (CP 47,497-d11) 8.96 2.4 8.6 14.3 14.6 14.0 13.8 14.6 12.0 
JWH-019 (JWH-007-d9) 7.9 9.5 -2.4 10.7 7.7 8.2 4.3 4.0 4.3 
RCS-8 (JWH-007-d9) 9.3 11.5 6.4 11.2 10.9 10.0 4.1 3.8 4.6 
CP 47,497 C-8 homolog (CP 47,497-d11) 5.2 8.9 -3.6 14.4 13.7 12.6 14.4 12.5 10.8 
JWH-398 (JWH-007-d9) 10.9 14.5 7.1 8.3 8.8 9.5 4.7 4.9 5.6 
JWH-210 (JWH-007-d9) -3.2 12.3 5.6 7.6 9.2 9.3 5.6 3.6 5.2 
HU-210 (CP 47,497-d11) -2.8 2.7 -10.8 21.2 14.0 12.3 19.7 14.0 11.9 

Conclusions

The presented LC–MS-MS assay is a suitable procedure for separation, detection and quantification of 25 synthetic cannabinoids in blood samples. It has proven to be selective, linear, accurate and precise for all 25 studied drugs in spiked samples. The method will have to be applied to authentic antemortem and postmortem samples to evaluate pharmacodynamic and pharmacokinetic data, which will help to gain knowledge about the toxicological significance of the detection of the cannabinoids in blood.

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