ISO is an abbreviation for the International Organization for Standardization. The ISO organization in collaboration with the International Electrotechnical Commission (IEC) develops joint ISO/IEC documents that specify performance standards that are used to accredit various types of organizations throughout the world. 

The ISO/IEC 17025 standard applies only to testing and calibration laboratories. Any testing or calibration laboratory that seeks ISO/IEC 17025 accreditation must contact an accreditation body such as Perry Johnson Laboratory Accreditation (PJLA), the American Association for Laboratory Accreditation (A2LA), American National Standards Institute (ANSI), and others. The accrediting bodies like PJLA accredit laboratories that fulfill all the requirements for accreditation specified in the ISO/IEC 17025 documents. Gaining accreditation by a testing laboratory is recognition that the laboratory has documented procedures in place that assure that the standards specified by ISO/IEC 17025 are being met on a continuing basis. 

An laboratory accredited to the standards of ISO/IEC 17025 is permitted to report its test results on a certificate of analysis bearing the ISO/IEC 17025 logo.

The DEA is the Drug Enforcement Administration, an arm of the US Department of Justice. Any person or entity that handles controlled substances is required to register with the DEA and to maintain records that account for the disposition of each controlled substance that they obtain. A hemp or cannabis laboratory is usually registered with the DEA because it may need to purchase reference standards of controlled substances for use in developing methods or calibrating instruments. In such cases, DEA registration and recordkeeping are required. However, DEA registration never means that the hemp or cannabis laboratory shares its results with the DEA. 

A laboratory that is registered with the DEA doesn’t have necessarily test samples better than a laboratory that is not registered with the DEA. However, the registered laboratory may have greater access to standards of controlled substances than the laboratory that is not registered. Access to these standards will enable the registered laboratory to test for more substances than those that are not registered.

The test results produced by KCA Labs are provided only to the company or agency that submitted the samples for testing.

Delta-8-THC (D8-THC or (-)-Δ⁸–trans-THC) is a psychoactive substance that differs chemically from Delta-9-THC ( D9-THC or (-)-Δ⁹–trans-THC) only in the location of one double bond in the THC molecule. Delta-8-THC and Delta-9-THC have the same numbers of carbon, hydrogen, and oxygen atoms, the same molecular weight, the same stereochemistry, and nearly the same three-dimensional shape. Consequently, Delta-8-THC and Delta-9-THC are nearly identical cannabinoids that share many chemical, physical, and pharmacologic properties. 

Figure 2. Structures of Delta-8-THC and Delta-9-THC compared

The close chemical and physical properties of Delta-8-THC and Delta-9-THC make them a challenge to separate by high performance liquid chromatographic (HPLC) methods when reversed-phase separations are used. If the HPLC instrument is connected to a UV-visible light detector, Delta-8-THC and Delta-9-THC produce nearly identical absorption spectra so they cannot be differentiated based on differences in their UV spectra so the only way to identify them by HPLC methods with UV-visible detection is to separate them chromatographically. Modifications of mobile phases and use of more efficient chromatographic separations can produce baseline separation of Delta-8-THC from Delta-9-THC thereby allowing them to be differentiated based on differences in characteristic retention times.

Although Delta-8-THC can arise in plant materials from acid-catalyzed isomerization of Delta-9-THC, the rate of this reaction in plant material is very slow so the amount of Delta-8-THC in natural plant material is so low that it is usually undetectable. Therefore, plant material is not a useful commercial source of Delta-8-THC, so it must be obtained from other sources. The Delta-8-THC that has been commercially available for several years is made by the acid-catalyzed isomerization of cannabidiol (see Figure 3). Delta-8-THC that is made by this method typically contains variable amounts of Delta-9-THC plus numerous side products. Two of these side products are Delta-8-iso-THC and Delta-4(8)-iso-THC (see side products in Figure 3). 

Figure 3. Conversion of cannabidiol (CBD) to Delta-9-THC and Delta-8-THC as well as side-products Delta-8-iso-THC and Delta-4(8)-iso-THC

Although these side-products have names that suggest a close relationship to Delta-8-THC, their shapes differ substantially from Delta-8-THC, and they probably do not possess any of the psychoactivity of Delta-8-THC or Delta-9-THC. If they have other pharmacologic effects, they have not been reported.

The physicochemical properties of these side products are so similar to those of Delta-8-THC and Delta-9-THC that they are not separated from them by reversed-phase HPLC methods. Delta-4(8)-iso-THC is not adequately separated from Delta-9-THC and Delta-8-iso-THC is not adequately separated from Delta-8-THC when the HPLC method that is widely used to determine cannabinoids in plant materials is used. 

The chromatogram in Figure 4 was obtained from the analysis of a Delta-8-THC product that contains Delta-9-THC as well as these side-products that are not adequately resolved. Consequently, concentrations of Delta-9-THC may be overestimated in Delta-8-THC materials because the D4(8)-iso-THC is not resolved from the Delta-9-THC. Alternatively, the Delta-4(8)-iso-THC, which appears slightly later than Delta-9-THC, overlaps the peak for Delta-9-THC and masks it so that it is not reported.

Figure 4. HPLC reversed-phase separation of a Delta-8-THC sample that contains Delta-9-THC, Delta-4(8)-iso-THC, and Delta-8-iso-THC

KCA Labs recognized and addressed this analytical challenge several years ago and developed a GC-MS method that completely resolves Delta-4(8)-iso-THC, Delta-8-iso-THC, Delta-8-THC, and Delta-9-THC chromatographically so that each substance can be quantified in a mixture containing these four substances plus other cannabinoids. The consequence of our use of this alternative and superior methodology is that we accurately quantify Delta-9-THC in Delta-8-THC samples that have been analyzed by HPLC because the presence of Delta-4(8)-iso-THC in these samples has masked the presence of Delta-9-THC when analyzed by HPLC. Since the GC-MS method separates Delta-4(8)-iso-THC from Delta-9-THC (see Figure 5) and is highly sensitive, samples of Delta-8-THC containing Delta-9-THC near the regulatory threshold can be determined more accurately and a clear distinction can be made between samples that contain more Delta-9-THC than is permitted. Consequently, a Delta-8-THC sample that is reported to contain no detectable Delta-9-THC when analyzed by HPLC may be found to contain  Delta-9-THC above 0.3% Delta-9-THC when analyzed by GC-MS. 

Figure 5. GCMS chromatogram illustrating separation between Delta-4(8)-iso-THC, Delta-8-iso-THC, Delta-8-THC, and Delta-9-THC

HPLC is high performance liquid chromatography whereas GCMS is gas chromatography coupled to a mass spectrometric detector. The HPLC instrument is typically connected to a UV-visible absorption detector for analysis of cannabinoids, but other detectors are sometimes used for this and other applications. When a mass spectrometer is connected to a high-performance liquid chromatograph, the resulting instrument is typically abbreviated LCMS.

The liquid chromatograph provides separation of the components of a mixture by forcing a liquid through a narrow stainless-steel tube containing an absorbent that usually has hydrocarbon chains attached to the surface of small particles that have been packed into the tube. Substances dissolved in the mobile phase interact with the immobilized hydrocarbon chains if they have an affinity for them. The rate of their passage through the column is slowed by these interactions because they are not moving while they are attached to the hydrocarbon chains. Substances that do not interact with the hydrocarbon chains pass through the column rapidly and enter the detector where the molecules are measured. These substances are characterized by short retention times (i.e., the total time that they are in the chromatographic column). Two different substances that interact identically with the hydrocarbon chains will be characterized by the same retention time and will not be differentiated unless their absorption spectra differ. In the case of Delta-8-THC, Delta-9-THC, Delta-4(8)-iso-THC, and Delta-8-iso-THC, the absorption spectra are identical so they cannot be differentiated from each other unless their retention times differ. As noted earlier, they are not adequately resolved by reversed-phase chromatography so a different stationary phase or a different chromatographic method is required to resolve them.

Gas chromatography, in contrast to liquid chromatography, is an orthogonal technique to HPLC because the mode of chromatographic separation is completely different. In gas chromatography, a gas (known as a carrier gas) flows through a narrow but very long (15-30 meters) column that has been coated on its inner surface with a thin film of a stationary phase that contains immobilized functional groups with which molecules may interact. Those that  interact weakly are characterized by short retention times whereas those that interact extensively are characterized by longer retention times. Substances are carried by the gas from the end of the column into the mass spectrometer where they are bombarded with a stream of electrons that cause the molecules to ionize and break into characteristic fragments that are measured by the mass spectrometer and reported as a mass spectrum. 

Analysis of the cannabinoids Delta-8-THC, Delta-9-THC, Delta-4(8)-iso-THC, and Delta-8-iso-THC by gas chromatography on an appropriate column affords resolution of these substances and detection by mass spectrometry assures identification because the mass spectra are different although the molecular masses are identical.

Samples are introduced into the GCMS through a heated injector that volatilizes the components of the mixture. Any substance that is unstable at the temperature of the injector or the chromatographic column is likely to degrade. Those substances are known or are identified during method development and method validation studies and measures may be taken to mitigate or eliminate degradation during analysis.

In some methods (such as the GC determination of total THC), the acid form of THC is intentionally decarboxylated during GC analysis by raising the injector temperature to cause complete decarboxylation. On the other hand, the acids can be analyzed by GC methods if the acids are stabilized by converting them to derivatives before analysis. We routinely analyze acids of cannabinoids like CBDA or THCA by GCMS after derivatization without any evidence of decarboxylation.

A recent publication reported the results of an investigation of the degradation of cannabidiol to Delta-9-THC during GCMS analysis and identified several factors that contributed to this analytical problem. These factors included the type of glass used in te injector liner, the design of the liner, the temperature of the liner, the presence of packing materials in the injection liner, the temperature of injection, whether the injection volume was split or was splitless, the condition of the mass spectrometer detector (i.e., cleanliness), whether the sample was derivatized.

It is not uncommon for identified substances to account for only 80-90% of the total mass of a distillate material leading to questions about the identities of the substances that make up the rest of the weight of the material. There are several possible explanations for this apparent discrepancy.

GCMS analysis of distillate materials is typically performed in a targeted mode in which the instrument is instructed to search for specific cannabinoids and ignore all other substances. In this mode of analysis, no substances other than the targeted substances will be detected so it is obvious that substances that are not targeted may be present and account for some or all of the difference. This scenario is unlikely in most cases because we have reanalyzed many distillate products in full-scan mode in which any substance that is ionizable under the conditions of analysis can be detected. The mass spectral data for additional substances are subjected to automated searching in large libraries of mass spectral data to obtain preliminary identifications of substances that produce a mass spectral signal, but these analyses rarely result in our detecting peaks that could be expected to account for the differences in weight. The analysis of distillate materials is performed by GCMS analysis at the higher temperatures required to detect and identify all the cannabinoids, but these temperatures are typically higher than those used for analysis of volatile substances like monoterpenes, sesquiterpenes, and related substances, so they would not be detected even when present. If we lower the initial temperature of the GCMS instrument to analyze these mixtures, we may see peaks for various terpenes and terpenoids plus some fatty acids and tocopherols.

When GCMS analysis is used for analysis of distillate products, any substance that is not volatile at the temperatures used for analysis (e.g., dimers and polymers) or substances that are not ionizable will not be detected. Therefore, these substances may account for some of the unknown materials.

There are several possible reasons for the observations that you have made. A common explanation for observations such as this is that the materials that were used to prepare the formulation were analyzed by one laboratory and the formulated product was analyzed by another laboratory using a method with different specificity and sensitivity from the original laboratory. For example, analysis of Delta-8-THC by reversed-phase HPLC overestimates the Delta-8-THC and may not detect the Delta-9-THC because the HPLC method cannot resolve Delta-8-THC and Delta-9-THC from side-products of the acid-catalyzed isomerization of CBD that was used to make the Delta-8-THC. If the product is formulated based on the results reported in the COA for the Delta-8-THC, the formulated product will likely contain less Delta-8-THC than intended and it may contain detectable or even non-compliant concentrations of Delta-9-THC if a more specific method like GCMS is used to analyze the product. For these reasons, it is prudent to analyze the starting material before formulating it by submitting it to the same laboratory that will analyze the formulated product. For best results, you should select a laboratory that can differentiate Delta-8-THC and Delta-9-THC from the side-products that are present in Delta-8-THC.

Another possible explanation is that one or more of the cannabinoids degraded or has been chemically transformed to another cannabinoid during storage. For example, Delta-9-THC is oxidized to CBN, THCA is decarboxylated to Delta-9-THC, CBD is isomerized to both Delta-9-THC and Delta-8-THC, Delta-9-THC is isomerized to Delta-8-THC, and CBD is oxidized to CBDQ (HU-331), a cannabidiolquinone, and hydroxy-cannabielsoin. Most of these degradation products can be measured by the laboratory so material balance calculations may explain changed ratios of cannabinoids in the formulated product.

Conversion of acids to neutrals?

Decarboxylation of THCA to Delta-9-THC occurs slowly at room temperature and at a much higher rate at high temperatures. If your original sample contained THCA and no detectable Delta-9-THC, decarboxylation of the THCA will occur and eventually the amount of Delta-9-THC in the sample will become detectable. Since the detection limit varies between laboratories and methods, a sample may contain detectable D-THC when tested in one laboratory or by one method but not in another laboratory or another method.

Acidic condition converting CBD to Delta-9-THC?

Weak acids catalyze the isomerization of CBD to Delta-9-THC whereas stronger acids catalyze the isomerization of CBD to Delta-8-THC. Therefore, the presence of acids in the formulated CBD product can catalyze the isomerization of CBD to Delta-9-THC. Weak acids such as ascorbic acid and citric acid that are often found in food products are sufficiently acidic to produce Delta-9-THC from CBD in formulated products.

Misidentification of Delta-9-THC peak

The acid catalyzed isomerization of CBD to Delta-8-THC that is commonly used to prepare it also produces varying amounts of side products depending on reaction conditions and the specific acid catalyst that was used. One of these side-products, D4(8)-iso-THC is often present in Delta-8-THC. When analyzed by reversed-phase HPLC, Delta-9-THC and D4(8)-iso-THC are not chromatographically resolved and they have the same absorption spectra so a sample containing no Delta-9-THC could be reported to contain Delta-9-THC if the sample contains D4(8)-iso-THC that is misidentified as Delta-9-THC. GCMS analysis of these samples resolves Delta-8-THC, Delta-9-THC, and the side products so that all can be determined without interference or misidentification.

Delta-9-THC is produced by Cannabis sativa L from cannabigerolic acid (CBGA) along a biosynthetic pathway that is separate from the pathway that produces cannabidiolic acid from CBGA. However, Delta-9-THC can be produced by the acid-catalyzed isomerization of cannabidiol (CBD). Therefore, various CBD products that contain acids like carbonic acids, citric acid, phosphoric acid, and tartaric acid may contain increasing amounts of Delta-9-THC because of the effects of the acids on the CBD. Therefore, a CBD product containing no detectable Delta-9-THC when it was manufactured may contain increasing amounts of Delta-9-THC during storage if the formulation contains an acid that can catalyze the isomerization of CBD to Delta-9-THC.

A chromatogram is the graph that shows the data that we obtain when we analyze a sample. We use the data shown in the graph (see Figure 6) to determine which cannabinoids are present and estimate the concentration of each. We label the cannabinoids present in the sample, and we analyze the peak sizes, shapes, and space between them because they indicate useful information. In the chromatogram in Figure 6, the peaks are numbered, and a table lists the identities of the numbered peaks. For example, peaks number 11 and 12 are due to Delta-9-THC and Delta-8-THC, respectively, in this chromatogram:

HPLC chromatogram of a mixture of sixteen neutral and acidic cannabinoids

The vertical axis of the chromatogram shows the magnitude of the detector response whereas the horizontal axis shows the time (usually in minutes) from the start of the analysis to the end of the analysis for one test sample. The time at which a peak is observed in the chromatogram is the retention time for that substance and is characteristic of it. For example, Delta-8-THC has a characteristic retention time when analyzed by HPLC according to our standard operating procedure. Every time that we analyze a sample containing Delta-8-THC under those conditions, we expect to find it at that characteristic retention time. If the retention time changes more than the normal run to run variability, then we would know to stop the analysis and identify the reason for the changed retention time.

The retention time and the area under each peak are determined automatically by the software that comes with the analytical instrument. The area under the peak increases as the amount of the substance increases so the peak areas are necessary for determining the amount or concentration of each substance that is identified. The peak area for any identified substance (e.g., Delta-8-THC) in a test sample can be compared to the peak areas for calibrators prepared with that substance from reference standards at different concentrations. When the relationship between the peak areas and the concentrations in calibrators have been established, the concentration of the substance in the test sample can be calculated. Since different substances often produce different responses for the same amount of material, each substance needs to be quantified using calibrators prepared from reference standards of that substance. Therefore, if a substance is an unknown substance or one for which reference standards are not available, it cannot be quantified.

Accuracy – Accuracy is a measure of the ability of a measuring instrument (e.g., HPLC with UV-visible detector) to give responses close to the true value of the amount or concentration of a substance being measured. 

LOD – The limit of detection is abbreviated LOD. It is the lowest concentration of an analyte that can be reliably detected with an analytical procedure.

LOQ – The limit of quantification is abbreviated LOQ. It is the lowest concentration of an analyte that can be reliably quantified with an analytical procedure. Since analytes can be detected at concentrations below the LOQ, the LOD is always lower (approximately three-fold) than the LOQ.

Precision – Precision is a measure of the spread of values obtained with repeated measurements of the concentration or amount of a substance being measured. 

Selectivity – The selectivity of an analytical procedure is the extent to which a particular method can be used to determine an analyte under given conditions in the presence of other components of similar behavior. The selectivity of a method is assessed during method validation studies that are performed by the laboratory before implementing the method. The selectivity of the GCMS method for determining Delta-9-THC in the presence of Delta-4(8)-iso-THC that arises from the acid catalyzed isomerization of CBD to Delta-8-THC has been referenced whereas the selectivity of HPLC methods for determining Delta-9-THC in materials containing Delta-8-THC from the conversion of CBD has not been demonstrated.

Sensitivity – Different and conflicting definitions of the sensitivity of an analytical procedure are in use. However, when we refer to the sensitivity of an analytical procedure, we are using the definition of sensitivity that is like the LOD. An analytical procedure for an analyte is more sensitive than another if the LOD is lower.