Tips on the analysis of pesticides and mycotoxins in cannabis products: Matrix matters! (Part I)

The growing variety of cannabis products that are currently available in the market speaks volumes about the creativity of entrepreneurs within the industry. Vapes, lotions, balms, cookies, hard candy, gummies, infused drinks, chocolate, fudge, soaps, and tinctures are only a handful of the product types that are currently being commercialized in this ever-growing market. Such explosion of marketing creativity in launching innovative cannabis goods to satisfy the needs of different consumers, demands resourcefulness from analytical chemists in finding smart and reliable solutions to comply with regulations in different countries/states. For instance, in the majority of states where cannabis has been legalized, potency testing (analysis of cannabinoids) is required in plant material, concentrates, and in all sorts of final products to ensure that the label claims truly match the product composition. Given the high concentration of cannabinoids in any cannabis commodity, HPLC-UV is the instrumental technique of choice; however, special attention should be paid to the sample preparation process to ensure close to exhaustive recoveries from any matrix. Testing pesticides and mycotoxins is a completely different story. Analysis of these compounds is normally required at part per billion (ppb) levels, which demands the use of more sophisticated equipment, namely liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography – tandem mass spectrometry (GC-MS/MS). In the majority of the states and in Canada, analysis of pesticides and mycotoxins is obligatory only in plant material. However, the state of California, which is known for having some of the most rigorous cannabis regulations, demands testing for these contaminants in any cannabis good. To support California cannabis testing, we recently published a technical article on the analysis of pesticides and mycotoxins in brownies. In that article, we described the different steps we followed to optimize the extraction of the California list of pesticides and mycotoxins using brownies as a model matrix.  Based on this work, on my recent experience working with dark chocolate, and after talking to several analysts from Cannabis testing labs that still have concerns about how to properly quantitate pesticides and mycotoxins, I decided to write this series of blogs to highlight some important points, critical for performing quantitation of these contaminants in any matrix. The first point I would like to discuss is matrix effects.

1. Matrix effects

To extract all the target pesticides and mycotoxins from any cannabis matrix, solvents like acetonitrile are commonly used. However, such extraction conditions will also favor the extraction of multiple matrix components, which will vary depending on the sample type. Co-extracted matrix components can have an effect on the MS/MS response of target analytes. That effect is what is collectively known as matrix effects. In the case of LC-MS, both electrospray (ESI) and atmospheric pressure chemical ionization (APCI) are susceptible to matrix effects (ESI is more prone to matrix effects than APCI). So, how do you know if your methodology is being impacted by matrix effects? One of the best ways to evaluate matrix effects is by following the approach proposed by Matuszewski et al. [1,2]. In this approach, you compare the responses of your analytes post-spiked in blank matrix extracts versus their response in neat solvent by using the following equation:

 

Matrix effects = Analyte response (post-spiked in blank extract)/Analyte response (spiked in neat solvent))*100

 

In an ideal world, your result should be 100. Values below 100 indicate that you have signal suppression, and values above 100 show signal enhancement. To illustrate the importance of performing this test in each type of matrix that you have to analyze, we can refer to Figure 1.

Figure 1. Matrix effects corresponding to daminozide in brownies, dark chocolate and cannabis biomass extracts. Brownies samples were prepared as described in our technical article. Dark chocolate samples (0.5 g) were extracted by using isopropyl alcohol (0.5 mL) and acetonitrile acidified with acetic acid at 1% (2.5 mL); subsequently, 2 mL of the extract were passed through a Restek Resprep C18 cartridge (Restek Cat.#26030). Cannabis biomass was prepared by adding 6 mL of acidified acetonitrile to 1g of sample; the final extract was passed by a Restek Resprep C18 cartridge (Restek Cat.#26031) (cannabis biomass experiments were conducted at Convergence Lab facilities in Santa Rosa, CA). In all cases, extracts were mixed with water in a ratio 3:1 (extract:water), and 2 µL were injected in the LC-MS/MS system.

 

As can be seen, the same pesticide, daminozide, analyzed under the same chromatographic conditions displays different levels of ionization enhancement in brownies (209%) and chocolate (175%) while, a certain level of ionization suppression is observed in cannabis biomass extracts (76%). Since this compound is highly polar and therefore poorly retained under reversed phase conditions (retention time = 0.7 min), it co-elutes with multiple co-extracted polar interferences that can affect its ionization in matrix extracts. Unfortunately, improving chromatographic retention in the same method for the analysis of all the California regulated pesticides and mycotoxins is difficult as most of the compounds are well analyzed via reversed phase chromatography. Typically, when you are performing multiresidue pesticides testing, compromises need to be made for certain analytes. Now, you may wonder what are the best conditions to assess matrix effects once you have a potential analytical workflow ready?  Here are some tips.

1. First, check your matrix blanks. The surrogate matrix that you choose to develop your method may contain some of your target analytes. Ensuring that your blanks are true blanks is important not only for the assessment of matrix effects, but also for method development in general. If a matrix blank is not easily available, in some cases, subtracting the area of the blank from the post-spiked blank extract could help to assess matrix effects.
2. Check your MS/MS transitions in different samples (matrix blanks vs. post-spiked extracts vs. standards). Although this step is not directly related to evaluating ionization effects, it is always important to make sure that you don’t have interferences sharing the same transitions of your target analytes and eluting at the same retention time. If this is the case, picking another transition with higher selectivity for the detection of your target analyte, even if it isn’t the one with the highest intensity, is the best choice (you may want to check the blog written by my colleague Dan Li).
3. Evaluate matrix effects at different concentration levels. If you pick a concentration that is too high to investigate matrix effects, your results may indicate that your method is free of ionization suppression/enhancement. However, evaluation at levels close to the LOQ or even at the requested action level, ionization effects may become apparent. In our brownies method, we spiked extracts at 15 ng/mL to assess matrix effects. Why? If we take into account that we used was 0.5 g of sample, 3 mL of solvent for the extraction, and that the lowest action level in the California list of pesticides is 100 ng/g, in the case of having close to exhaustive recoveries we were expecting analytes to be at a final concentration of 16.7 ng/mL in sample extracts. Based on this, we considered it appropriate to choose a concentration close to 16.7 ng/mL. Nonetheless, it is important to highlight that the evaluation of matrix effects at levels close to the LOQ is always recommended.
4. Use the average response of at least three replicates. Technical replicates are critical to ensure that you have a representative value with an estimated error.
5. Make sure that your neat solvent composition matches your final extract composition. In our case, we used acidified acetonitrile to prepare the neat solvent samples to assess matrix effects in brownies extracts (you can check our technical article to see why we used acified acetonitrile).

 

So, what if I have matrix effects? Matrix effects are very common, and that is why we recommend the use of a matrix matched calibration to account for variations in the ionization conditions during the entire chromatographic run. In cannabis testing it can be difficult to perfectly match the sample that is being analyzed due to the broad variety of products. However, we recommend trying to find a surrogate matrix very close to what you are analyzing. It is worth emphasizing that the use of neat solvent calibration solutions can lead to biased results. For instance, in the case of daminozide, quantitation in brownies and chocolate (Figure 1) using calibration solutions in solvent will cause an over-estimation of the analyte concentration. In addition to matrix-matched calibration, the use of isotopically labeled analogues as internal standards is essential. Although we understand that this represents an extra cost that many people would prefer to avoid, internal standards are necessary to quantitate your target analytes in the great majority of cases, and especially when dealing with complex matrices. Selecting a handful of representative deuterated analogues that elute at different retention times in the chromatographic run is the best way to go when dealing with multiple analytes. In the case of daminozide, we had to introduce its deuterated analogue to account for ionization effects at the right retention time as well as to account for low absolute recoveries (this story will be part of our next blog). If your matrix effects are significantly affecting the detection of your analyte of interest at the required levels, you may need to play with your chromatographic method to separate your target analytes from interferences, or you many need to adjust your sample clean-up conditions.

What about GC-MS/MS analysis? Are there any matrix effects?

Indeed, co-extracted interferences can also affect analytes’ responses in GC-MS/MS analysis. Co-injected and non-volatile matrix components can interact with active sites in the GC column or injection port, and this causes an alteration in the response of analytes injected in matrix extracts in comparison to neat solvent standards. This effect is known as “matrix-induced chromatographic response enhancement” and it is particularly problematic for compounds that are polar and/or thermally sensitive [3,4]. The non-volatile matrix components that are co-injected help to block active sites in the GC system, hence preventing adsorption and thermal degradation of analytes. This leads to an enhancement of the analytes’ response in comparison to neat solvent injections where broader peaks with lower responses can be observed. Although this may sound as if having co-extracted matrix components could be beneficial, having appropriate sample clean-up is crucial to avoid matrix built-up in the GC system after multiple sample injections [3]. Running extracts that are too dirty can lead to losses in analyte response, peak tailing, and eventually MS system contamination.

The best way to prevent “matrix-induced chromatographic response” effects from leading you to biased quantitative data is by using matrix-matched calibration. For this reason, in our brownies method we used the same extract for both LC and GC analysis (for GC injections we did an extra clean-up step). Alternatively, the use of certain compounds capable of blocking GC active sites, also known as analyte protectants, has been proposed. Although we haven’t tested this strategy yet for the analysis of cannabis products, it is definitely in our to-do list!

I hope this blog was helpful for your cannabis method development work! Please stay tuned for part II. If you want to do some extra reading on this topic, here are some of the cited references:

 

 

[1]         B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng, Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS., Anal. Chem. 75 (2003) 3019–30. http://www.ncbi.nlm.nih.gov/pubmed/12964746.

[2]         P. Panuwet, R.E. Hunter, P.E. D’Souza, X. Chen, S.A. Radford, J.R. Cohen, M.E. Marder, K. Kartavenka, P.B. Ryan, D.B. Barr, Biological Matrix Effects in Quantitative Tandem Mass Spectrometry-Based Analytical Methods: Advancing Biomonitoring, Crit. Rev. Anal. Chem. 46 (2016) 93–105. doi:10.1080/10408347.2014.980775.

[3]         J. Hajšlová, J. Zrostlíková, Matrix effects in (ultra)trace analysis of pesticide residues in food and biotic matrices, J. Chromatogr. A. 1000 (2003) 181–197. doi:10.1016/S0021-9673(03)00539-9.

[4]         D.R. Erney, A.M. Gillespie, D.M. Gilvydis, C.F. Poole, Explanation of the matrix-induced chromatographic response enhancement of organophosphorus pesticides during open tubular column gas chromatography with splitless or hot on-column injection and flame photometric detection, 1993.