Analysis of Veterinary Hemp-Based Oils for Product Integrity by LC/MS

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Cannabis Science and Technology, May/June 2019, Volume 2, Issue 3

Some hemp-based products are mislabeled regarding their level of CBD. The authors explain why and how that happens.

Pet owners continue to seek alternative therapies for pain relief in their favorite companion animals. Instead of routine nonsteroidal anti-inflammatory drug (NSAID) treatment, there is a trend toward trying hemp-based products rich in cannabinoids. There are increasing numbers of companies producing hemp-based oils enriched with various mixtures of cannabinoids for pain and other maladies including seizures, cancer and anxiety. These products post labels attesting to the composition in the product, usually labeling for cannabidiol (CBD) concentrations in their products. But in the absence of regulatory control we questioned how accurate the information is on the label. The chemical composition of 13 commercially available oils intended for veterinary or a crossover of human and veterinary use was determined by selected ion monitoring liquid chromatography/mass spectrometry (SIM LC/MS). It was found that many of the labels were inaccurate regarding the cannabidiol concentrations and/or the presence of other cannabinoids. In general, the labels on most of the medicinal veterinary samples indicated higher levels of CBD than found in these studies by 20% or more. The precision and accuracy of SIM LC/MS analysis of the samples fell within the acceptable limits of regulated bioanalysis guidelines of +/- 15%.

Many pet owners consider their animals beloved members of the family. When their ‘companion’ is not behaving normally or suffering from pain, they will seek professional help from their favorite veterinarian, and if that is not successful they will sometimes take it upon themselves to treat their ‘best friend’ with other remedies. Although tempting anecdotal reports on the benefits of cannabis- and hemp-based products continue to appear, most veterinarians remain reluctant to recommend cannabis to their clients for several reasons (1). Unfortunately, without proper research, the effects of medications containing CBD on animals remain unknown (2). Foremost is the fact that marijuana and hemp are still considered controlled substances by the U. S. Drug Enforcement Administration (DEA). The American Veterinary Medical Association considers them illegal depending on the state laws, and discourages veterinarians from prescribing them for pets.

Although not currently considered pharmaceutical agents per se, there is clearly a lot of interest in the role that marijuana and hemp and their derived products may have in veterinary healthcare. Increasingly, pet owners have already experimented with advertised medicinal cannabis products. These include oils reportedly containing therapeutic levels of CBD, in addition to other ingredients with product claims of beneficial effects. In states where recreational cannabis is legal, veterinarians are seeing increasing incidences of toxic cannabis cases in dogs. These popular companion animals have indiscriminate eating habits and often eat discarded cannabis products. In addition, veterinarians have had to respond to the pet owners’ queries regarding the pros and cons associated with the proper use of medicinal cannabis. Since there is very little research to-date on these topics, veterinarians are often limited on their ability to wisely respond to these queries.

Medicinal marijuana, or medicinal cannabis, is a treatment regimen that has attracted considerable national attention recently (3). Controversy continues surrounding the legal, ethical, and societal implications associated with its use. Issues include safe administration, adverse health consequences and reported deaths attributed to marijuana intoxication. Therapeutic indications are based on limited clinical data and represent some of the complexities associated with this treatment. Cannabis indica or Cannabis sativa are considered marijuana or hemp. Regardless of what it is called, cannabis is currently considered by the DEA Comprehensive Drug Abuse Prevention and Control Act (Controlled Substances Act) of 1970 as a Schedule I controlled substance. This definition suggests cannabis has a high potential for abuse, no currently accepted medicinal use in treatment in the United States, and a lack of accepted safety data for use of the treatment under medical supervision (4). There is a dearth of research in support of its safety or therapeutic value in clinical settings.

Marijuana and hemp have been used medicinally worldwide for thousands of years (5). In the early 1990s, the discovery of cannabinoid receptors in the central and peripheral nervous systems created interest in other potential therapeutic values of marijuana (6). Since then, marijuana has been used by patients experiencing chemotherapy-induced anorexia, nausea and vomiting, pain, and forms of spasticity. Use among patients with glaucoma and human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS) has also been widely reported (7). It should not be surprising that veterinarians may be interested in the potential for medicinal cannabis to relieve pain and other clinical issues associated with their animal patients (8,9).

The historically restrictive legal guidelines for cannabis have significantly limited research on its safety and efficacy in both humans and animals (10). However, a recent study by researchers at the Cornell University College of Veterinary Medicine reported on the potential beneficial effects of CBD oils administered orally to osteoarthritic (OA) dogs (11). A representative commercial veterinary medicinal CBD oil was purchased and administered orally in the food of known OA canine patients. The publication described the pharmacokinetics, safety and clinical efficacy of cannabidiol treatment in osteoarthritic dogs, which suggested that a 2 mg/kg of CBD twice daily can help increase comfort and activity in dogs with this condition.

These findings spurred us to examine whether the selected 13 commercial oils were similar in chemical composition. In this report, we describe the chemical analysis of some representative commercial veterinary oils by SIM LC/MS to determine the concentration of the constituents reported on the labels of those products.

This analytical approach is one of several available for such an application as described in a recent review (12). We also monitored common other cannabinoid constituents which were not listed on some of the product labels. There are an ever-increasing number of suppliers of such oils and related products which are sold in the absence of any regulatory control.

The goal of this study was to compare our analysis results on the cannabinoid composition with the indicated concentrations of selected compounds on the product label. A recent report on phytocannabinoids common to the cannabis cultivars present in medicinal oils for human use was reported, where high-performance liquid chromatography/ultraviolet detection (HPLC/UV) analyses were described (13). The report described herein expands upon hemp oil analysis with a focus on commercially available oils intended for pet consumption using the more sensitive and selective analytical capabilities of SIM LC/MS.



Instrumental and Chromatographic Conditions

The mass spectrometer employed in this report was the Expression L-model single quadrupole system (Advion, Ithaca, NY). The mass spectrometer was operated in the SIM mode using electrospray ionization coupled with LC/MS while monitoring the characteristic ions of cannabidiolic acid (CBDA) (m/z 341.3), cannabigerol (CBG) (m/z 317.2), CBD (m/z 315.3), cannbinol (CBN) (m/z 313.3), THC (m/z 315.3) and tetrahydrocannabinolic acid (THCA) (m/z 341.3). The positive ion mode was employed with a capillary temperature of 250 °C, capillary voltage of 180 V, a source span of 0, a source offset of 25 V and a source gas temperature of 250 °C. The chromatographic conditions used employed an Avant binary HPLC system (Advion, Ithaca, NY) equipped with a Restek Raptor ARC 18 (3.0 mm x 100 mm) column packed with 1.8-micron particles. The binary gradient mobile phase consisted of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The initial HPLC conditions commenced with 75% B programmed to 95% B over two min which was then held isocratic at 95% B for a total run time of 3.5 min. After this gradient program, the mobile phase was recycled over 2 min to the initial 75% B conditions. The injection volume was 20 microliters, the flow rate was 0.6 mL/min and the column temperature was maintained at 30 °C.

Hemp Oil Sample Preparation

Ten µL of each oil sample were added to 490 µL of isopropyl alcohol and mixed on a vortex mixer to dissolve into a uniform solution for each oil sample. Fifty µL of this initial diluted oil sample in isopropyl alcohol were then further diluted into 450 µL of 50% aqueous methanol. These secondary dilutions were then filtered through 0.2 µm syringe filter into the respective HPLC vials for SIM LC/MS analysis (total dilution factor was 1000-fold for the determination of CBD and 500-fold for the quantitative determination of the other, lower concentration cannabinoids). In each experiment, the protonated molecules for each of the six cannabinoids were monitored along with the corresponding protonated molecules for the available stable isotope internal standard cannabinoids which included CBD-d3 at m/z 318.3, THC-d3 at m/z 318.3, and CBN-d3 at m/z 314.3. The isobaric cannabinoids were measured with the added selectivity of the combined chromatographic separation and the measured protonated molecules for each cannabinoid. The selected deuterium-labelled cannabinoids co-eluted with their unlabeled analogs which provided added selectivity and confidence for these quantitative measurements. In addition, the co-elution of the stable isotope labelled internal standards can mitigate common matrix suppression or enhancement issues which can occur when other endogenous chemicals may co-elute. These internal standards should be used whenever they are available. The samples had such high concentration levels of the target compounds that they were substantially diluted to produce measurable concentrations using the very sensitive and selective analytical capabilities of SIM LC/MS. Thus for the targeted cannabinoids for which we did not have stable isotope internal standards, we speculate that matrix effects and/or chemical interferences will be minimal.


Standard Curves

One milliliter of coconut oil was dissolved in 49 mL of isopropyl alcohol. The coconut oil was used as a representative negative control matrix relative to the veterinary oils. Five mL of this isopropyl solution of coconut oil was further diluted with 45 mL of 50% aqueous methanol. This diluted coconut oil was used as the solvent matrix for preparation of calibration curves for the six targeted cannabinoids. The latter contained CBDA, CBG, CBD, CBN, THC and THCA which produced a calibration curve with concentrations of 1, 5, 10, 50, 100, 500, 1000, 5000, 10,000, and 20,000 ng/mL for each cannabinoid compound. Ten µL of the internal standards (CBD-d3, CBN-d3 and THC-d3 at 1000 ng/mL) were added into 200 µL of calibrators containing each of the six targeted cannabinoids. The internal standard final concentrations were 47.6 ng/mL. Quality control (QC) samples were prepared in the same manner independently at concentrations of 10, 100, 1000, and 10,000 ng/mL fortified with the same stable isotope internal standards as calibrators. The QC samples were analyzed by SIM LC/MS in six replicates within the day of analysis to produce the intra-day precision and accuracy values shown in Table I. It is noted in Table I that the accuracy for the targeted cannabinoids are in general quite acceptable pursuant to regulated bioanalysis guidelines, with the exception of THCA. These results are in agreement with another report which employed LC/MS/MS techniques (14). This may result from the absence of a stable isotope internal standard for this compound or it may suggest that negative ion detection may be a better choice for this application. Additional experimentation may be required to better understand this unexpected discrepancy.


Results and Discussion

Our objective in this work was to analyze a representative set of commercial veterinary CBD oils to determine whether the labeled contents were accurate.  Also, in the absence of any regulatory control, we were interested to learn whether the THC content in these samples was within legal limits of, for example, the Canadian 10 parts per million (ppm) guidelines.

There is a long list of the naturally occurring chemicals occurring in cannabis plants (15). Cannabis is very complex in its chemistry due to the vast number of its constituents and their possible interaction with one another.

These compounds represent many of the chemical classes-for example, mono- and sesquiterpenes, sugars, hydrocarbons, steroids, flavonoids, nitrogenous compounds and amino acids, among others. However, hemp oil extracts usually contain relatively high (several percent) concentrations of CBD and the major carboxylic acids along with much lower levels of other cannabinoids along with a wide range of many other chemicals (16).

The final composition of commercial hemp oil will depend upon the type of extraction used and any subsequent sample extract purification or treatment. The relative amounts of these chemicals will also depend upon the hemp cultivar selected (6).

Figure 1 shows the chemical structures for six common cannabinoids present in hemp oil that were measured in this study (rows 1 and 3). Also shown in Figure 1 are the structures for three of the stable isotope incorporated internal standards used for quantitative analysis (row 2). These include THC-d3, CBD-d3, and CBN-d3. These three internal standards were added to the diluted hemp oil samples at concentrations in the lower quartile of the calibration curve. 

The LC/MS analysis of these samples produced LC/MS chromatograms with these stable isotope labelled internal standards co-eluting with their non-deuterated analogs while displaying the same chemical behavior through the chemical analysis procedure.

They may be easily differentiated from their non-labelled analogs via their mass differences which are 3Da higher in each case.

Shown in Table II is a summary of the concentrations determined by SIM LC/MS of the six targeted cannabinoids shown in Figure 1 measured in veterinary CBD oils of this study which are labelled Vet CBD Oil samples 1-13. As a representative example, the chromatogram shown in Figure 2 was obtained from the SIM LC/MS bioanalysis of medicinal veterinary oil sample number 8 (Table II). Employing the experimental conditions described (vide supra) the analysis of each sample was completed within three min and clearly shows the six targeted cannabinoids in addition to a few additional chemical constituents in the oil sample. The simplicity of this chromatogram is due in part to the additional selectivity provided by a mass spectrometer detector operated in the SIM LC/MS acquisition mode. It is interesting to note in Figure 2 the relatively high level of THC in this sample (when there was no reference on the product label that THC was present in the sample) observed at a retention time of 2.33 min in contrast to the expected high concentration of CBD shown with its retention time of 1.68 min. With reference to Table II, the corresponding concentrations for these compounds were 0.348 mg/mL for THC and 15.5 mg/mL for CBD, respectively.

The cannabinoid concentrations observed in the medicinal veterinary oil samples ranged from not detectable (N/D) to very low concentrations for CBDA, CBN and CBG. This perhaps is not too surprising as it is generally known that some of these cannabinoids are present at relatively low levels in cannabis and hemp (7). An exception is sample number 12 which had a concentration of 11.8 mg/mL for CBDA. This sample also had elevated levels of some other cannabinoids. It is unclear why sample number 12 would have such elevated levels for some of the cannabinoids based upon the available information, but it still falls under the 2018 Farm Bill’s limits of less than 0.3% THC as a hemp product. Since these medicinal veterinary oils were marketed as CBD oils, it was anticipated that the CBD content would be elevated in the samples. CBD has received considerable “press” as a potentially useful drug for a variety of ailments (4). Recently the U.S. Food and Drug Administration (FDA) approved Epidiolex, which is CBD derived from its extraction of the marijuana plant (17). The CBD column in Table II shows the CBD concentrations across the 13 samples ranging from 0.88 mg/mL for sample number 6 up to 27.5 mg/mL for sample number 12.

Looking at the concentration of CBD in the rest of 13 medicinal veterinary oils listed in Table II, it is interesting to note the range of concentrations observed. To help see this more graphically one may look at the bar graph shown in Figure 3. This figure shows a comparison of the CBD concentrations in all 13 medicinal oil samples (Table II) determined by SIM LC/MS compared with the indicated levels of CBD on the product label. The black bars in Figure 3 graphically show the vendor-reported concentration in mg/mL (the concentration units often shown on the product labels) for CBD in each of the 13 medicinal oil samples. Adjacent to each black bar is a solid grey bar showing the SIM LC/MS experimentally derived concentration for CBD in each sample. In samples numbered 3, 4, 7, 9, 10, and 12, the concentrations for CBD were reported to be substantially higher (black bars) on the label than the actual measured concentration measured by SIM LC/MS in this study (grey bars). Thus, the experimental results show the amount of CBD in the oil is often lower (grey bars in Figure 3) than the amounts reported on the label (black bars in Figure 3) except for oils 5 and 13. In general, the manufacturers for these products are labeling CBD concentrations higher than the content actually present in the product. 

Some of these products did not provide any indication on the label of the CBD concentration in their products. However, it is encouraging to note that some vendors provide accurate indications of the CBD contents as noted for the measured CBD concentrations in samples 5, 9 and 11 which were close to the label indications. It is sobering to note that some vendors label their product contents such that the buyer is unable to know the total cannabinoid or individual cannabinoid concentrations. For example, for samples 1, 2, 6 and 8 the indicated concentration on the bottle was “mg of hemp extract per milliliter.” This claim provides the buyer no idea of the actual levels of cannabinoids in the product. Clearly, a systematic format for indicating the concentration content would be helpful for the buyer. Table III shows the variety in the CBD label nomenclature used for the 13 samples used in this study.

Since THC is the psychoactive component in cannabis and hemp that has been a concern in the recent past, it is important to know that the concentration of THC in medicinal oils and products is below government action levels. In Canada, the level of THC in such samples is limited to 10 ppm (10 microgram/gram), while the U.S. 2018 Farm Bill suggests that THC concentrations of hemp products must be less than 0.3%. In the absence of any regulatory authority the customer has no way of knowing whether the vendor abides by the established guidelines. The experimentally determined THC concentrations listed in Table II are plotted in Figure 4 with reference to the units of ppm. A horizontal red dashed line is plotted at the 10 ppm THC level allowed by Canada in such samples. One can see that only sample 4 in this study contains THC below this 10 ppm level. Most of the samples have THC content well above the 100 ppm level, with sample 12 weighing in close to 1300 ppm. It is worth noting the bottle label on sample number 12 as well as the other samples analyzed in this study made no reference to THC being in the product. These results suggest the need for dependable accuracy and consistency in the labelling of veterinary or possibly other medicinal CBD oils and products.

To help the reader gain a better understanding of the ‘analytical view’ of these results that is not so obvious in tabulated data we reference the results shown in Figure 5. This figure shows the SIM LC/MS chromatograms for two of the samples analyzed in this study. It is worth noting these chromatograms reveal relatively few peaks or chromatographic components due in part to the rather high concentrations of the targeted cannabinoids as well as the fact that SIM acquisition experiments measure only those m/z values selected for the experiment. Figure 5a shows the SIM LC/MS chromatogram for medicinal oil sample number 6 which as shown in Figure 4 had a THC concentration just above the 10 ppm allowed level. As is apparent in Figure 5a the THC peak at 2.34 min retention is a minor component along with the other minor cannabinoids with the obvious exception of the large CBD peak at a retention time of 1.68 min retention time. However, in Figure 5b the THC peak in sample 12 is a much more abundant component relative to the other cannabinoids with again the exception being the CBD peak at 1.68 min retention time. Note that the y-scale in both Figure 5a and Figure 5b is the same such that the reader can directly compare the relative quantities of THC in these two samples. The authors suggest the quantitative determination of cannabis constituents in medicinal products can be reliably acquired from results such as those shown in Figure 5.

This report has highlighted label discrepancies for the commercial veterinary medicinal CBD oil products. In contrast, Table IV shows concentration data for a different commercial medicinal cannabis sample where the vendor’s reported concentrations appear to be quite accurate. The vendor provides analytical concentration results for the product cannabinoids from two independent laboratories which agree quite closely. Accordingly, the SIM LC/MS procedure described in this report was also employed with this product and our results concur with those provided by the vendor. The first column in Table IV shows the six cannabinoids reported to be contained in the product. The second column summarizes the results obtained by the authors’ laboratory while columns 3 and 4 in Table IV are the analytical results from two other independent laboratories labelled Laboratory 1 and Laboratory 2. These three sets of bioanalysis results provide confidence to the customer that the product composition is accurate for this particular product.



The potential benefit of medical marijuana or hemp nutraceuticals for veterinary patients is attracting increased attention, but many veterinarians remain relatively uninformed and somewhat skeptical of recommending cannabis-derived products to their customers. Researchers and practitioners would welcome clinical research on the medical utility, safety and efficacy of marijuana-derived products. Instead, many companion animal owners are taking it upon themselves by responding to commercial claims that medicinal oils and products may help alleviate pain and other ailments of their favorite pet. This can lead to the purchase and administration of unproven or unreliable medications or even potentially dangerous treatments that may not help but hurt the animal. Since we are at the early stages of the medicinal marijuana and hemp nutraceutical hype, there is a need for accuracy and consistency in the products used for these purposes. FDA-approved medicines provide patient confidence and safety in the products. In the absence of any regulatory control, there are many companies, both small and large, that are joining in the business of producing medicinal hemp products when there is a dearth of documented clinical benefits from such treatments. The “product” is often an extract of unsubstantiated composition which may or may not be beneficial or safe for the patient, particularly with no standards for cannabinoid content, pesticide contamination or heavy metal limits.

Simple yet major questions for a consumer are: Does the product contain what is advertised; Is it safe?; and Is it effective? A lot of research is still needed to answer these questions, although it is entirely possible to provide accurate chemical analysis of the product composition as this publication has attempted to demonstrate. Just as the last example described in Table IV, a reputable company can provide accurate composition data if they are legitimate. The analytical technology described in this report is entirely capable of providing this information if the vendor has the integrity to employ it.




The authors thank their Advion colleagues, Drs. Daniel Eikel and Changtong Hao, for helpful discussion and contributions to this work, as well as Mr. Seth Richardson of Kingsland Partners Inc. for helpful input on hemp plant biology.


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Ben Nie is a Scientist and Jack Henion, PhD, is Cofounder and Chief Science Officer at Advion Inc. Joe Wakshlag, PhD, is with the College of Veterinary Medicine, University of Florida. Direct correspondence to:;


How to Cite This Article:

Nie B et al., Cannabis Science and Technology 2(3), 36-45 (2019)