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To determine the effectiveness and robustness of the LC–MS-MS method, 10 seized samples and a cannabis sample were analyzed. The samples were present in two forms: tablets and herbal products (incense stick, cigarette, and a cannabis plant). They were finely ground, then aliquots of 5–10 mg of the resulting powder were transferred to 10-mL volumetric flasks and dissolved in 70:20:10 methanol–water–acetonitrile containing 1% formic acid. Following this, the solutions were vortexed for 2 min, sonicated for 10 min, and vortexed again for 3 min. The supernatant was filtered through a 0.45-μm pore polytetrafluoroethylene (PTFE) syringe filter (Phenomenex). Herbal samples required an additional centrifugation step at 3500 rpm for 10 min to avoid mass overloading of the syringe filter. Filtrates were diluted 10–100-fold in 80:20 water–acetonitrile, the initial mobile phase, before injection.
LC–MS-MS Operating Conditions
Data were acquired on an LTQ Orbitrap XL mass spectrometer coupled to an Acella HPLC system (Thermo Scientific). Xcalibur 2.1 and Thermo LTQ Tune Plus 2.5.5 software (Thermo Scientific) were used to control the system and process the data. External mass calibration was used throughout the project. Four analytical columns were initially tested for their chromatographic performance: 100 mm × 2.1 mm, 3.5-μm dp XTerra C18 and 100 mm × 2.1 mm, 1.7-μm dp Acquity BEH C18 columns, both from Waters; a 75 mm × 2.1 mm, 2.6-μm dp Kinetex C18 column from Phenomenex; and a 100 mm × 2.1 mm, 2.6-μm dp Accucore aQ C18 column from Thermo Scientific. Two eluent systems were tested during method development, water–methanol and water–acetonitrile, both containing 0.1% formic acid, under generic gradient conditions (5–95% organic).
Optimized separations were carried out using the Accucore aQ column coupled to a 4 mm × 2.0 mm Phenomenex C18 guard column, both maintained at 40 °C, and the water–acetonitrile gradient. The autosampler temperature was set at 10 °C to avoid sample degradation. Eluents consisted of 0.1% formic acid in water (eluent A) and 0.1% formic acid in acetonitrile (eluent B), and the initial mobile phase contained 20% B. The following gradient elution was applied at a flow rate of 350 μL/min: 20–58% B over 1 min, held at 58% B for 1 min, increased to 85% B over 1 min, then held at 85% B for 2 min. Eluent B was then returned to 20% B over 0.2 min. The system was allowed to reequilibrate for 2.8 min, giving a total cycle time of 8.0 min. The injection volume was 3–5 μL. A needle wash step using 70:20:10 methanol–water–acetonitrile was included in the method. A 5-μL blank, consisting of the initial mobile phase, was injected after each sample to monitor and reduce any potential carryover.
The electrospray interface was operated in positive ion mode. Nitrogen was used as both sheath gas and auxiliary gas while helium was used as collision gas. Using direct infusion, instrumental parameters were adjusted semiautomatically for every analyte using the tune tool in the LTQ Tune Plus software. The parent ions of all analytes showed similar behavior due to their similar structures. After screening every compound individually, the experimental parameters of the full scan event that were found to be suitable for all analytes were set to the following values: sheath and auxiliary gas at flow rates of 44 and 17 (instrument units), respectively; spray voltage, +3500 V; capillary temperature, 310 °C; capillary voltage, 28 V; tube lens, 101. The MS2 and MS3 transitions for every compound were also determined using the tune tool by varying the normalized collision energy. They ranged between 25% and 33% after they were optimized. Therefore, a three-step collision energy function set at 25%, 30%, and 35% was used to perform average fragmentation on every compound. The ion transitions MS2 and MS3 for each standard are shown in Table I, where the MS3 transitions arise from the MS2 value shown in boldface type. Mass spectra were acquired from m/z 50 to 1000 using two scan events: the first was a Fourier transform (FT)-MS full scan for accurate mass detection and the second was a data dependent step with MS-MS acquired only for precursors from the parent mass list with a dynamic exclusion of 10 s. Every standard was then injected onto the column individually to determine its retention time and confirm the parent ion accurate mass, MS2 and MS3. To determine if there were any interactions between the compounds, a mixture of the 24 standards was injected.
Results and Discussion
Method Development and Validation
As seen in Figure 1, many of the 24 compounds have similar structures, which makes the chromatographic separation challenging. Between the mobile phases tested, the acetonitrile gradient gave better selectivity. Between the columns tested, the Accucore aQ gave the best selectivity and retention of early eluted analytes.
Selectivity was further enhanced by coupling the optimized chromatographic method to the high-mass-resolution orbital ion trap mass spectrometer. By individual direct infusion of the standards, the final optimized parameters for full-scan detection (values presented in the LC–MS-MS operating conditions section) were selected to provide appropriate sensitivity for all 24 components in a single analysis. The mass accuracy across the whole study was within 3 ppm.
Figure 2: Typical chromatograms obtained by LC–MS-MS in positive electrospray mode for a mixture containing all 24 analytes. The upper trace of the first panel represents the total ion chromatogram (TIC), followed by the extracted ion chromatograms of all the compounds spread over three panels for ease of presentation (parent ions shown).
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