A reliable and validated LC-MS/MS method for the simultaneous quantification of 4 cannabinoids in 40 consumer products

<div><p>In the past 50 years, <i>Cannabis sativa</i> (<i>C</i>. <i>sativa</i>) has gone from a substance essentially prohibited worldwide to one that is gaining acceptance both culturally and legally in many countries for medicinal and recreational use. As additional jurisdictions legalize <i>Cannabis</i> products and the variety and complexity of these products surpass the classical dried plant material, appropriate methods for measuring the biologically active constituents is paramount to ensure safety and regulatory compliance. While there are numerous active compounds in <i>C</i>. <i>sativa</i> the primary cannabinoids of regulatory and safety concern are (-)-Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD), and their respective acidic forms THCA-A and CBDA. Using the US Food and Drug Administration (FDA) bioanalytical method validation guidelines we developed a sensitive, selective, and accurate method for the simultaneous analysis CBD, CBDA, THC, and THCA-A in oils and THC & CBD in more complex matrices. This HPLC-MS/MS method was simple and reliable using standard sample dilution and homogenization, an isocratic chromatographic separation, and a triple quadrupole mass spectrometer. The lower limit of quantification (LLOQ) for analytes was 0.195 ng/mL over a 0.195–50.0 ng/mL range of quantification with a coefficient of correlation of >0.99. Average intra-day and inter-day accuracies were 94.2–112.7% and 97.2–110.9%, respectively. This method was used to quantify CBD, CBDA, THC, and THCA-A in 40 commercial hemp products representing a variety of matrices including oils, plant materials, and creams/cosmetics. All products tested met the federal regulatory restrictions on THC content in Canada (<10 μg/g) except two, with concentrations of 337 and 10.01 μg/g. With respect to CBD, the majority of analyzed products contained low CBD levels and a CBD: CBDA ratio of <1.0. In contrast, one product contained 8,410 μg/g CBD and a CBD: CBDA ratio of >1,000 (an oil-based product). Overall, the method proved amenable to the analysis of various commercial products including oils, creams, and plant material and may be diagnostically indicative of adulteration with non-hemp <i>C</i>. <i>sativa</i>, specialized hemp cultivars, or unique manufacturing methods.</p></div

Precision and accuracy of the determination of cannabinoids in plant material (Intra-day n = 5).

<p>Precision and accuracy of the determination of cannabinoids in plant material (Intra-day n = 5).</p

Extraction recovery, matrix effect of 4 cannabinoids (n = 6) and internal standards (n = 24) in plant material and cream matrices (mean +/- SEM).

<p>Extraction recovery, matrix effect of 4 cannabinoids (n = 6) and internal standards (n = 24) in plant material and cream matrices (mean +/- SEM).</p

Precision and accuracy of the determination of cannabinoids in cream (Intra-day n = 5).

<p>Precision and accuracy of the determination of cannabinoids in cream (Intra-day n = 5).</p

Stability of cannabinoids (n = 5), (mean +/- SEM).

<p>Stability of cannabinoids (n = 5), (mean +/- SEM).</p

Comparative concentrations of cannabinoids in 40 commercial hemp finished products.

<p>CBD, THC, CBDA and THCA-A were extracted from test articles and quantified using LC-MS/MS. Measured levels, reported in % w/w of (A) CBD, (B) THC, (C) CBDA, and (D) THCA-A.</p

Precision and accuracy of the determination of cannabinoids in extra virgin olive oil (Inter-day n = 5x3; Intra-day n = 5).

<p>Precision and accuracy of the determination of cannabinoids in extra virgin olive oil (Inter-day n = 5x3; Intra-day n = 5).</p

LC-MS/MS profiles of cannabinoids in matrix.

<p>Chromatograms of blank matrix (olive oil) detected in (A) positive mode, (B) negative mode, blank matrix spiked with 2 ng/mL CBD, THC, CBDA and THCA-A and 10 ng/mL CBD-d3, THC-d3 and THCCOOH-d3 detected in (C) positive mode and (D) negative mode and test article # 32 analyzed in (E) positive mode and (F) negative mode. Asterisk denotes a small peak (CBDA*) with the same MRM pair of THCA-A due to concurrent fragmentation (D and F).</p

Extraction recovery, matrix effect of 4 cannabinoids (n = 6) and 3 internal standards (n = 24) in Extra Virgin Olive Oil (mean +/- SEM).

<p>Extraction recovery, matrix effect of 4 cannabinoids (n = 6) and 3 internal standards (n = 24) in Extra Virgin Olive Oil (mean +/- SEM).</p

Comparative pharmacokinetics and safety assessment of transdermal berberine and dihydroberberine

<div><p>The natural alkaloid berberine has been ascribed numerous health benefits including lipid and cholesterol reduction and improved insulin sensitivity in diabetics. However, oral (PO) administration of berberine is hindered by poor bioavailability and increasing dose often elicits gastro-intestinal side effects. To overcome the caveats associated with oral berberine, we developed transdermal (TD) formulations of berberine (BBR) and the berberine precursor dihydroberberine (DHB). These formulations were compared to oral BBR using pharmacokinetics, metabolism, and general safety studies <i>in vivo</i>. To complete this work, a sensitive quantitative LC-MS/MS method was developed and validated according the FDA guidelines for bioanalytical methods to simultaneously measure berberine, simvastatin, and simvastatin hydroxy acid with relative quantification used for the berberine metabolite demethylene berberine glucuronide (DBG). Acute pharmacokinetics in Sprague-Dawley rats demonstrated a statistically relevant ranking for berberine bioavailability based upon AUC_0-8 as DHB TD > BBR TD >> BBR PO with similar ranking for the metabolite DBG, indicating that transdermal administration achieves BBR levels well above oral administration. Similarly, chronic administration (14 days) resulted in significantly higher levels of circulating BBR and DBG in DHB TD treated animals. Chronically treated rats were given a single dose of simvastatin with no observed change in the drugs bioavailability compared with control, suggesting the increased presence of BBR had no effect on simvastatin metabolism. This observation was further supported by consistent CYP3A4 expression across all treatment groups. Moreover, no changes in kidney and liver biomarkers, including alanine aminotransferase and alkaline phosphatase, were observed between treatment formats, and confirming previous reports that BBR has no effect on HMG-CoA expression. This study supports the safe use of transdermal compositions that improve on the poor bioavailability of oral berberine and have the potential to be more efficacious in the treatment of dyslipidemia or hypercholesterolemia.</p></div