Many of the methods currently in use focus primarily on the four major cannabinoids, Δ9-THC, Δ9-THCA, CBD, CBDA, and CBN, to satisfy testing and labeling requirements for cannabis [4] and to meet the regulatory guidelines for hemp [5]. However, there are many other cannabinoids known to be present in cannabis and hemp for which commercial reference standards are available, including cannabigerol (CBG), cannabigerolic acid (CBGA), cannabinolic acid (CBNA), cannabichromene (CBC), cannabichromenic acid (CBCA), tetrahydrocannabivarin (THCV), tetrahydrocannabivarinic acid (THCVA), cannabidivarin (CBDV), cannabidivarinic acid (CBDVA), cannabicyclol (CBL), cannabicyclolic acid (CBLA), and Δ8-tetrahydrocannabinol (Δ8-THC) (Fig. 1). Recent reviews of analytical methods for cannabinoids have discussed analytical techniques for the major and minor cannabinoids, revealing few methods providing adequate quantitative analysis of many of the minor cannabinoids [6, 7]. The methods include high-performance liquid chromatography with ultraviolet detection (HPLC-UV) [8,9,10,11,12,13], liquid chromatography tandem mass spectrometry (LC-MS/MS) [13,14,15,16,17,18], and nuclear magnetic resonance (1H-NMR) spectroscopy [19]. Many of these techniques are limited in sensitivity and specificity, and GC is limited by its inability to directly quantitate the acidic cannabinoids without derivatization [20, 21]. Many methods are quantitative for the major cannabinoids (THC, THCA, CBD, and CBDA) and some of the minor cannabinoids; however, many of the minor neutral and acidic cannabinoids are not quantified due to sensitivity and specificity limitations.
While the large range of cannabinoid concentrations observed in cannabis, and to a lesser extent hemp, is difficult to cover in a single analysis, one approach is to use a wide-range calibration curve coupled with appropriate dilutions of the extracts [14, 18]. Use of a single sample dilution limits the quantifiable range, usually with a sacrifice at the lower concentration ranges. HPLC-UV is commonly used due to the low cost of laboratory set-up and operation [11, 12, 22, 23]. This technique, while providing adequate quantitative results for the major cannabinoids at higher concentration levels, lacks sensitivity and specificity for cannabinoids at lower concentrations [11, 12, 22, 23], limiting the achievable lower limit of quantitation (LLOQ) in matrix. While complete separation is possible for subsets of cannabinoids up to 8 or 12, LC-UV is generally not capable of resolving larger suites of cannabinoids. In some cases, the resolution of challenging cannabinoid pairs relies of precise pH control [11], but this can be problematic and limit the robustness of a method [3]. A fast, 5-min HPLC-DAD method [8] has been reported; however, the LOQ is high at 10 μg/mL, CBDA and most of the minor cannabinoids were not evaluated, and there is no evidence to indicate that many of the cannabinoids do not co-elute.
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LC-MS/MS is a sensitive and specific technique which allows the analysis of both major and minor cannabinoids at low LLOQs in the same method [14, 18]. While not yet commonplace for the routine analysis of cannabinoids in plant material, it is the method of choice for the analysis of cannabinoids and metabolites in other complex matrices such as urine, blood, plasma, and oral fluid [24,25,26,27]. Application of the LC-MS/MS method presented here achieves low LLOQs for 17 cannabinoids with a range of 0.002 to 200 mg/g in matrix by employing a large calibration range with appropriate sample extraction and dilution. The method has been validated according to AOAC [28] and ASTM [29] guidelines in both cannabis and hemp matrices.
CBD was purchased from Toronto Research Chemicals and certified for purity at NRC using quantitative NMR (qNMR) [30] with NIST SRM 350b benzoic acid as internal standard [31]. Reference standards of the other cannabinoids and isotopically labeled cannabinoids were purchased from Cerilliant (Round Rock, Texas, USA). The neutral cannabinoids included Δ9-THC, Δ8-THC, CBG, CBN, CBC, THCV, CBDV, and CBL and were provided at 1.0 mg/mL in methanol (CBL provided at 1.0 mg/mL in acetonitrile). The acidic cannabinoids included Δ9-THCA, CBDA, CBGA, CBNA, CBCA, THCVA, CBDVA and CBLA and were provided at 1.0 mg/mL in acetonitrile (CBLA provided at 0.5 mg/mL in acetonitrile). The isotopically labeled cannabinoids included Δ9-THC-d3, CBD-d3, and CBN-d3, which were provided at 0.1 mg/mL in methanol. Dried cannabis and hemp samples were obtained from licensed producers in Canada via the Ontario Cannabis Store. A candidate NRC certified reference material for cannabis was used for validation and quality control purposes. This material has been rigorously tested to be homogeneous and stable with respect to the 14 cannabinoids it contains, with value assignment for cannabinoids based on a combination of results from a validated LC-UV method [3, 11] and a more targeted version of the LC-MS/MS method reported here that employs narrow calibration ranges that bracket the cannabinoid levels.
Calibration standards were used to generate calibration curve regressions while QC sample concentrations were derived from the regressions to verify accuracy and precision of the method. The LLOQ (lower limit of quantitation) and ULOQ (upper limit of quantitation) were set to 10 and 10,000 ng/mL respectively for all cannabinoids with the 1000-fold calibration range resulting in a higher likelihood that diluted matrix extract concentrations fell within the calibration curve limits. Matrix sample results were derived from the regressions followed by calculations to account for matrix sample mass, extraction solvent volume, and dilutions with final results reported in mg/g. Calibration curve regressions, QC sample results, and matrix sample results were generated using Xcalibur software, Version 4.0.27.10 (Thermo Scientific, San Jose, CA, USA).
Due to the high concentration of cannabinoids in matrix and the lack of adequate blank matrices, it is not practical or affordable to spike cannabinoids or internal standards directly into matrix prior to extraction. A common method of quantitation is to prepare calibration curves in solvent and dilute matrix extracts into the calibration curve range. Internal standards may then be added to calibration standards, QC samples, and diluted matrix extracts as the last preparation step. We opted for this approach with calibration curves and cannabis extracts diluted in methanol, and believe it is a reasonable compromise between cost and performance. While only three isotopic derivatives were available commercially at the time of this work, it is recommended that additional internal standards be incorporated into the method as they become available.
Ion suppression of 14 cannabinoids present in the candidate NRC reference material and 3 internal standards was evaluated by spiking cannabinoids into diluted matrix extracts to yield double the incurred concentration of the extract. The pure cannabinoid solutions at 1 incurred matrix concentration (Sample1) and 2 incurred concentration (Sample4) as well as the un-spiked diluted extract (Sample2) and spiked diluted extract (Sample3) were then analyzed. Sample1 and Sample4 were used to determine cannabinoid response factor (RF) in pure solution: \( \mathrmRF=2\times \Big(\frac\mathrmpeak\ \mathrmarea\ \mathrmSample1\mathrmpeak\ \mathrmarea\ \mathrmSample4 \)).
Seven cannabis samples were selected to evaluate the method with samples representing a range of THC and CBD concentrations. Two samples with label claims for medium, balanced THC/CBD concentrations, two with high THC/low CBD concentrations, two with low THC/high CBD concentrations, and one with unknown concentrations, were extracted and analyzed in triplicate. While three dilution levels (1/100, 1/10, and neat) were analyzed for each cannabis sample, only the average of the 1/100 and/or 1/10 dilutions (triplicate samples) was reported if a valid result was obtained. A valid result was defined as a result within the calibration curve range. The neat extract sample results were reported only if a valid result was not obtained for the 1/100 and/or 1/10 diluted samples. Including all three dilution levels (1/100, 1/10, and neat), the quantifiable range was from 0.002 to 200 mg/g in matrix, representing a 100,000-fold range.
Sample results (average of triplicate analysis on three separate days) were compared to label claims for THC, total THC, CBD, and total CBD. Label claims did not specify THCA or CBDA as individual results. Total cannabinoid concentrations in mg/g were determined as neutral equivalent as follows:
The rapid increase in demand for the analysis of cannabinoids in cannabis and hemp has resulted in a similar demand for analytical methods that are able to meet the current regulatory requirements and be adaptable to future requirements such as cannabinoid analysis in edibles. HPLC-UV has been and continues to be a heavily used technique to meet this demand; however, it has limitations with respect to sensitivity, specificity, and peak separation requirements. LC-MS/MS has become a common analytical technique in many laboratories due to its superior sensitivity, specificity, and less stringent peak separation requirements. Additional cannabinoids, as standards become available, can be added more easily to LC-MS/MS methods than to HPLC-UV methods which require complete chromatographic separation of all cannabinoids. The low limits of quantitation and wide range of quantitation in matrix reported here for 17 cannabinoids are made possible by the advantages provided by LC-MS/MS. We expect that this method will be easily adapted to more challenging matrices such as oils and edibles [18, 36] providing the opportunity to utilize a consistent technique for all matrices, making it a logical choice for cannabinoid analysis.
Three-dimensional (3D) optical imaging of whole biological organs with microscopic resolution has remained a challenge. Most versions of such imaging techniques require special preparation of the tissue specimen. Here we demonstrate microtomy-assisted photoacoustic microscopy (mPAM) of mouse brains and other organs, which automatically acquires serial distortion-free and registration-free images with endogenous absorption contrasts. Without tissue staining or clearing, mPAM generates micrometer-resolution 3D images of paraffin- or agarose-embedded whole organs with high fidelity, achieved by label-free simultaneous sensing of DNA/RNA, hemoglobins, and lipids. mPAM provides histology-like imaging of cell nuclei, blood vessels, axons, and other anatomical structures, enabling the application of histopathological interpretation at the organelle level to analyze a whole organ. Its deep tissue imaging capability leads to less sectioning, resulting in negligible sectioning artifact. mPAM offers a new way to better understand complex biological organs. 2ff7e9595c
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