In this work, several steps were developed to improve the workflow in the analysis of pesticide residues from cannabis plant material by liquid chromatography tandem mass spectrometry (LC–MS/MS) and gas chromatography (GC)–MS/MS. The goals of the study were to optimize the extraction and analysis process to reduce sample background, improve pesticide recoveries, and allow direct injection of QuEChERS (quick, easy, cheap, effective, rugged, and safe) extracts, without dilution, for high performance liquid chromatography (HPLC) analysis. Reduced background and better pesticide recoveries were achieved by the use of a QuEChERS cleanup with zirconia/C18 functionalized silica, primary secondary amine (PSA), and a low-surface-area carbon blend. HPLC analysis was optimized to provide late elution of coextracted cannabinoids relative to the targeted pesticides, thus making it possible for a dump step to be used to prevent them from entering the MS detector. The HPLC column configuration used improved the peak shapes for the earliest eluted hydrophilic pesticides when injecting extracts in 100% acetonitrile. This then allowed a single set of extracts to be analyzed by both GC–MS/MS and LC–MS/MS.
Consumption of cannabis or cannabis-based products is currently legal in some form in 29 states in the United States plus the District of Columbia. Testing of the plant materials and products is required by many of these states; however, the specific test methods and target compound lists are not mandated in all cases. In October 2016, the state of Oregon took a major step forward by requiring that all laboratories testing cannabis be accredited by the Oregon Environmental Laboratory Accreditation Program (ORLEP) and licensed by the Oregon Liquor Control Commission (OLCC) (1). Consequently, Oregon Administrative Rules (OAR) list specific contaminants to be tested in marijuana samples, along with action levels (2). The pesticides on this list include carbamate, organophosphorus, macrocyclic lactone, neonicotinoid, pyrethroid, and triazole fungicides as well as others. Action levels per OAR vary from 0.2 to 1 µg/g, depending on the specific pesticide. In addition, the state of California, which legalized recreational cannabis in 2016, recently released a proposed list of pesticides that includes all but one of those found on the OAR list, plus eight more (3).
Because of its ease of use and applicability to a wide range of pesticides, the QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach has been adopted by many testing laboratories for use on cannabis. After extraction, incorporation of a cleanup step is important for removing pigments as well as other contaminants. A QuEChERS cleanup using a mixture of primary secondary amine (PSA), C18, and graphitized carbon black (GCB) is often chosen for this purpose. PSA removes acidic interferences, C18 removes hydrophobic interferences, and GCB retains some pigments—specifically the green color imparted by chlorophyll. This mixture of sorbents thus retains a wide range of contaminants; however, it also has potential to reduce recoveries of target pesticides that are susceptible to hydrophobic retention on C18, or planar enough in structure to be strongly retained by GCB. In previous work by Stenerson and colleagues with cannabis, an alternative sorbent mix was evaluated for cleanup in the analysis of various pesticides, and found to offer a better balance than a traditional mixture of PSA/C18/GCB with regards to removal of pigmentation and pesticide recovery (4). This sorbent mix contained PSA, a zirconia-coated C18 functionalized silica, and a specialized carbon. The zirconia retains by Lewis acid-base interactions, and has been found to reduce the background of certain fatty compounds as well as some pigments. The carbon is a low surface area graphitized variety that has been shown to have weaker retention of small, planar molecules such as certain pesticides. In this work, the pesticide list tested previously was expanded to include many of those on the OAR list described above. The alternative sorbent mix was compared directly to a conventional blend of PSA/C18/GCB for cleanup and analysis of spiked replicates of cannabis plant material analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS). For the LC-MS/MS portion of the analysis, the column and mobile phase selection was optimized to improve overall workflow, which is described here.
Dried cannabis was pulverized using an IKA T10 Ultra Turrax mixer. Next, 1.9 g was weighed into a 50-mL centrifuge tube and spiked with pesticides at 50 ng/g. After a 10-min equilibration time, the sample was mixed with 10 mL of deionized water and allowed to sit for 30 min. Then 10 mL of acetonitrile was added, and the sample was shaken at 2500 rpm for 30 min. A mixture of 4 g of magnesium sulfate, 1 g of sodium chloride, 0.5 g of sodium citrate dibasic sesquihydrate, and 1 g of sodium citrate tribasic dihydrate was added, and the sample was shaken for 1 min. The sample was then centrifuged at 5000 rpm for 5 min, and the supernatant was removed for cleanup.
Next, 1 mL of extract was added to a 2-mL tube containing the mixture of cleanup sorbents. Two different sorbent mixtures were used:
- PSA/C18/GCB/MgSO4 (400 mg, 400 mg, 400 mg, and 1200 mg, respectively)
- PSA/zirconia-coated C18 functionalized silica/low-surface-area graphitized carbon (400 mg, 480 mg, and 80 mg, respectively)
Samples were shaken for 1 min, and then centrifuged at 5000 rpm for 3 min. The supernatant was removed for analysis.
- Oregon Health Authority, Oregon Medical Marijuana Program, www.oregon.gov/oha, accessed 6/7/17.
- Exhibit A, Table 3. Pesticide Analytes and their action levels. Oregon Administrative Rules 333-007-0400; Oregon/gov/oha, effective 5/31/2017.
- Chapter 5. Testing Laboratories. Section 5313 Residual Pesticides; Bureau of Marijuana Control Proposed Text of Regulations, CA Code of Regulations, Title 16, Div. 42, pp 23-26.
- K. Stenerson and M. Halpenny, Supelco Reporter 34.1, 17-19 (2016).
- J. Kowalski, J.H. Dahl, A. Rigdon, J. Cochran, D. Laine, and G. Fagras, LCGC North Am. supplement: “Advancing the Analysis of Medical Cannabis” 35(s5), 8-22 (2017).
- B. Kinsella, T.J. Telepchak, D. Mackowsky, D.A. Duncan, and T. Fanning, LCGC North Am. supplement: “Advancing the Analysis of Medical Cannabis” 35(s5), 33-38 (2017).
- Analysis of Bifenazate (sum) by the QuEChERS Method Using LC-MS/MS; EURL-SRM Analytical Method Report, ver. 1, updated 3/17/2017.
- K. Stenerson, MilliporeSigma white paper, document 84901/T416112 (2016).
- F. Hildmann, C. Gottert, T. Frenzel, G. Kempe, and K. Speer, J. Chrom. A 1403, 1-20 (2015).
- B.D. Morris and R.B. Schriner, J. Agric. Food Chem. 63, 5107-5119 (2015).
- J. Han, Y. Sapozhnikova, and J. Matarria, J. Sep. Sci. 39, 4592-4602 (2016).
- L. Han, Y. Sapozhnikova, and S.J. Lehotay, Anal. Chim. Acta 827, 40-46 (2014).
How to Cite This Article
K. Stenerson and G. Oden, Cannabis Science and Technology 1(1), 48-53 (2018).