Seeds for one sample were grown in Poland, the remaining seeds used to
press the oils were grown North America. Five Canadian-sourced samples
were grown in provinces the manufacturer did not disclose. Of the
remaining, one was grown in Alberta, three in Manitoba, and two were
grown in Saskatchewan. Samples grown in the United States include three
from seeds grown in Indiana, two from Kentucky, one from Montana, three
from North Dakota, three from New York, and five from Virginia.
The researcher did not investigate cannabinoid variances based on
growing locale since most suppliers reported only a single cultivar
grown in a single location. Therefore, analysis by growing locale would
not provide any new or significantly meaningful information given the
available data.
Suppliers identified the cultivar for twenty-six of the samples, which
included a mix of eleven varietals. The number of different cultivars
ranged from one to four varietals per supplier. Of the four samples of
an unknown varietal, one supplier did not respond to follow-up requests
to identify the cultivar(s), and one sample came from a US manufacturer
that sourced seeds from Poland without the varietal data being
available.
Nineteen of the samples were classified as “conventional” by the
supplier, and seven were classified as “organic.” The researcher did
not attempt to verify compliance with any official organic
certification. The conventional or organic status of four samples was
not provided.
Analytical Procedures and Sample
Analysis
The researcher contracted with a third-party ISO 17205 accredited
laboratory that is also DEA, FDA, and USDA licensed to develop a
validated GLP method to assay sixteen (16) cannabinoids in CPHSO with a
lower LOQ of 50 ppb (0.05 ppm) using UHPLC-MS/MS. The FDA-CVM reviewed
the method before the laboratory conducted the GLP-compliant analysis.
All analytical measurements were conducted using a Shimadzu 8050
LC-MS/MS instrument and are reported in ppm (except density).
The development of a low-LOQ analytical method was required to quantify
cannabinoids that naturally occur in minimal quantities that commercial
laboratories could not assay. Also, there is no commonly-available
method or approved AOAC or AOCS method for testing cannabinoids in CPHSO
at very low LOQs, and the AOCS draft method did not provide
sufficiently-low LOQ for the study requirements. Finally, this research
is part of a series of safety and efficacy investigations relating to
hemp in animal feeds. Existing research indicates some animals have a
lower tolerance to cannabinoids than humans, with CBDA and CBD
hepatotoxicity documented in multiple species, so these cannabinoids are
of particular interest.
The third-party laboratory assayed the acceptable samples using the
validated method and provided the results to this researcher. Each
sample’s oil density and cannabinoid analytes were assayed multiple
times per the GLP requirements. The mean values of density and each
assayed cannabinoid content were reported in ppm and entered into a
spreadsheet for further analysis. The analytical assay found no
detectable levels of Δ8-THC, so this analyte was not included in the
statistical analysis or reported herein. The analyte values (in ppm)
were also summed for each sample.
Data were analyzed using Microsoft Office
Professional® 2016 Excel®spreadsheets and IBM SPSS® version 26 statistical
analysis software. The final documentation was constructed with
Microsoft Office Professional® 2016
Word® and Adobe Acrobat
Professional® 2017.
Data preparation before statistical
analysis
For the phase 1 investigation, a value of 95% of the LOQ was
substituted for cannabinoid analytes detected below the lower LOQ. This
substitution value was chosen because the investigator determined that
this value may represent the true data better than zero (0.0) or
treatment as missing values for the analysis. In many cases, only a few
values are substituted for any given analyte. Notable exceptions are CBN
(20% <LOQ replaced) and THCVA (46.7% <LOQ
replaced). The <LOQ substituted values are highlighted with
light-gray in Table 7.
Descriptive statistics were computed for all values, including a 95%
confidence interval and the inter-quartile lower and upper range limits.
Table 8 shows the descriptive statistics for the trimmed data. Light-red
backgrounds and red text indicate values that exceed the upper limit of
the 95% confidence interval in Table 7.
The untrimmed data was copied to a second spreadsheet for phase 2, and all
substituted <LOQ values and values higher than the upper limit
of the initial 95% confidence interval were deleted. The descriptive
statistics were recomputed for all values. Table 12 (a-b) shows the
trimmed data with recomputed descriptive statistics.