Cannabis Pesticides Testing and Quantification Using Reference Standards: How Does it Work?

In the absence of federal pesticides regulations, states with legalized cannabis have enacted their own pesticides use and testing guidelines.

These guidelines are based largely on those used for the agricultural and food safety industries, although the range and levels of accepted pesticides that require testing vary from state to state.

Oregon was the first state to establish comprehensive pesticides guidelines, setting limits for 59 pesticide residues in cannabis. California penned more stringent action limits for 66 pesticides, all but one of those found on the Oregon list and 8 more. As the industry has grown, the types and allowable limits for pesticides have changed in step with the adoption of new policies.

The changing landscape of cannabis pesticides regulations

The dynamic, state-based system for pesticides testing has proved a challenge for testing labs and cultivators alike. Staying current with the latest requirements while operating proficient and accredited testing practices is a necessity if testing labs are to remain successful.

These challenges will remain at least until federal regulations are drafted. Until then, access to high-quality analytical materials and resources can ensure testing laboratories operate at the highest levels of accuracy and precision — supporting transparent, clean, and safe cannabis products.

Methods for cannabis pesticides analysis

Third party testing laboratories are tasked with ensuring cultivators and grow operations generate compliant cannabis crops according to state regulations. Testing may include initial validation of grow operations as well as periodic mandatory pesticides screening of crops.

Liquid Chromatography Mass Spectrometry (LC-MS) and Gas Chromatography Mass Spectrometry (GC-MS) are the gold standard instrument platforms for pesticides analysis. LC-MS typically handles the workload of soluble polar compounds, whereas GC-MS is suitable for more organic volatile compounds.

Custom methods can and have been developed to handle and extended range of pesticides with varying compound chemistries. For instance, atmospheric pressure chemical ionization (APCI) techniques have been developed which allow analysis of pesticides, typically analyzed on a GC-MS instrument, on a central LC-MS instrument. This ability circumvents the need for two separate instruments and the methods, reagents, logistics associated with the use of both. Many other examples exist of hybrid and novel methods and instruments. Again, a constant challenge involves changes in testing criteria, and methods and instruments have a need to be flexible in this respect.

Pesticides detection and quantification

A very basic breakdown of analysis includes two steps: 1) detection of pesticides in the cannabis background, followed by 2) quantitative analysis of the detected pesticides.

The cannabis background or matrix is complex and compounds of interest may be enshrouded with overlapping peaks and excessive noise in the data. To address this, upstream fractionation, extraction, and enrichment techniques, as well as custom instrument components and settings, assist in boosting the detection sensitivity and range of pesticide identification. Custom techniques may also suit specific cannabis source material — such as flower, trim, or whole plant extracts for example — which may vary according to testing criteria and location.

“If you can’t see it, you can’t quantify it”. Once methods are refined to permit the necessary detection of pesticides, quantification methods are developed to enable accurate and precise pesticides measurements. Such methods typically make use of quantitation standards — either internal standards or reference standards.

Internal Standards versus Reference Standards

An internal standard is a compound or set of compounds that are “spiked” into the cannabis testing material prior to analysis. This can involve an analog or a close relative of the compound of interest. Alternatively, this can be a near duplicate of the compound of interest, such as a stable isotope labeled or deuterated form of the compound. In theory, the closer the internal standard is to the compound to be measured, the more accurate (and precise) the measurement will be. Of course, accurate knowledge of the internal standard concentration is required for analysis of target compound concentration in the sample.

The major advantage of internal standards? They are added to the sample matrix upstream of the analysis. Any fractionation, enrichment, or influences of the instrumentation will affect both the target compound and the internal standard just the same. Thereby recovery will be the same and measurement accuracy should be of high integrity.

A major disadvantage with the use of internal standards is the need for a custom compound which behaves near identically to the target compound, with minute but detectable differences. The mass difference (or delta mass) of a stable isotope or dueterated labeled standard must be within the resolution of the mass spectrometer in order for accurate detection  The greater the internal standard diverges chemically from the target compound, the larger the potential loss measurement accuracy.

Alternatively, and a more common approach for pesticides analysis, includes a series of dilutions which can be used to form a linear range or standard curve. In this “external standard” approach, the dilution series are made in a background matrix similar in complexity or content as the experimental matrix, prior to analysis. This may include a cannabis extract produced through the same process as the test material, but devoid of the target compounds, i.e. a sample blank. This may be easier said than done, and can work better for potency testing than for pesticides testing, where pesticides may be present in both blank and experimental samples. Other methods for linear range construction include “surrogate” blanks, which in the case of cannabis may be alternative plants extracts, such as those used in the food testing industries. Others, such as hops or Humulus lupulus, a close relative of cannabis in the Cannabaceae family, have been recently used with success.

Reference standards are often mandated as certified reference materials or CRMs, which are ISO/IEC 17925 certified according to ISO Guide 34 international guidelines. CRMs are tested using validated analytical methods on qualified instrumentation to ensure traceability to SI units of measurement, i.e. the purity, concentration, and stability are very well defined.

Reference Materials for pesticides are readily available, as they have been used for the agricultural and food safety industries for some time. The same cannot be said for some cannabinoids, terpenes, and other cannabis compounds which have more slowly appeared, due in part to challenges with purification, synthesis, or other factors. As is the case with the cannabis industry overall, this is changing rapidly as more compounds are studied and methods for production, purification, and analysis have rapidly evolved.


Measurement of peak area versus that of the internal standard(s) or comparison with the plot of linear range provides the basis for concentration determination. These calculations are typically performed online by instrument software. This is the same general concept no matter the testing instrument, HPLC, GC, LC-MS, GC-MS.

What other species may be hiding within that peak, thereby compromising the accuracy of concentration measurement? This is a persistent challenge, particularly for rich samples with complex chemical backgrounds. The range, the sensitivity, and the accuracy required for pesticides testing have cultivated ongoing efforts towards fractionation, including multi-dimensional LC, GC, and MS/MS method development. These approaches and techniques are topics for subsequent posts, inlcuding the following:

New Cannabinoid Purification and Testing Solutions

Cannabis Consumables and Critical Advancements in Testing Standardization

New Cannabis Potency Testing Instruments and Equipment

Mass Spectrometry Pesticides Detection in Modern Food Safety and Testing

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Updated October 2021