Cannabis Lab 101: Validation

I originally posted the article on Medium

When it comes to analytical testing — validation is key. In order to be a licensed and accredited testing facility, a laboratory needs to fully validate all methodologies that will be used to quantify and report any value to the public. From Cannabis testing labs to Pharmaceutical facilities, validation of methods is a critical component that must be performed, maintained, amended, and inspected routinely.

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“Method validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Results from method validation can be used to judge the quality, reliability and consistency of analytical results; it is an integral part of any good analytical practice.” — Dr. Ludwig Huber (source)

So, how does one go about performing a validation? I will quickly outline the steps that most cannabis labs must include in the validation of each testing assay. For the sake of this article, I will be showing the steps needed (on average) for a Potency Determination Method.

Step 1: Write up a validation protocol outline

Before embarking on the daunting task of validation, it is critical to assess all of the guidelines for method validation supplied by the accrediting licensing bodies for your lab (ISO 17025:2017, USP, FDA CGMP, EPA, ICH) as well as the state’s cannabis regulations of which your lab abides by. This is where a Quality Control/Quality Assurance Specialist shines as it is their job to ensure your lab is meeting all of the requirements needed. Start-up labs tend to hire professional QC/QA consultants whereas larger facilities end up creating entire departments dedicated to QC and QA. Hiring third-party consultants to perform compliance audits is a great way to prepare for large audits and obtain a new perspective on your laboratory procedures.

Once you have competency over the required regulations you should write up a method of approach for each of the validation topics covered in this article from here on out:

  • Pre-Validation
  • Selectivity
  • Calibration/Linearity
  • Accuracy/Precision/Intermediate Precision
  • Limit of detection/Limit of quantification/Sensitivity/Dynamic Range
  • Robustness
  • Proficiency Testing — Earn your badge

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Step 2: Pre-Validation

In the cannabis industry, labs oftentimes are not attempting to reproduce already validated analytical methodologies but rather opt to create their own unique methods. When new methodologies are being created, a pre-validation becomes needed as an exploratory process with the aim of establishing operational limits and performance characteristics of the method being considered.

It is in this step where the lab determines a separations method for the analytes of interest to ensure they can appropriately identify (not quantify) the analytes of interest (see my article on Cannabinoid Separation Methodologies). They also work on their extraction/processing methods for the cannabis matrices that will pass through the lab and also determine what dilution factors should be employed to not oversaturate the instrument detector being utilized (See my article on Potency Determination Overview).

Once the lab has determined their extraction, processing, and separations procedures show promise — it is time to attempt validation of the method to test if it is ready for production.

Step 3: Selectivity

This is where we establish that our test method is quantifying only the analytes that we intend to quantify. Showing that your method has a consistent and well-resolved separations method alongside spectral verification is one step and showing that the method is free from interferences is another step.

Showing your separations method has an acceptable resolution of peaks (at low, mid, and high concentrations), predictable retention times, and the ability to extract a spectrum from each cannabinoid is more than enough for most accreditation bodies.

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I, on the other hand, suggest labs take it a step further and also run a terpenes mixture at a concentration that is slightly higher than the samples that will be run. This will determine if your cannabinoids method is selective against terpenes (which are in abundance in cannabis products).

Step 4: Calibration/Linearity

Now that you are ready to start validating your method, you must first calibrate your instrumentation with Certified Reference Materials — standards/controls that have a verified and known concentration.

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With the certified reference materials (CRM’s), you can create a range of concentrations from high to low and run these on your instrumentation to develop a calibration curve. A linear fit calibration curve with greater than five points is preferred for cannabinoid testing with an R² value of greater than 0.9950 (closer to 1.0000 the better). Calibration with CRM’s is one step that helps a lab be confident in their results.

Calibration-curve-of-9-tetrahydrocannabinol

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It is at this point that you can determine your Linearity as well. Test the concentration values ran and determine whether or not the points are holding true to a linear fit and not showing signs of a logistic curve (seen below). The linear range is the range at which your instrument response has a linear relationship versus concentration.

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Step 5: Accuracy/Precision

With your calibration complete, you are ready to obtain your accuracy and precision data.

Accuracy is the closeness of the test results versus the true value.

Precision is the degree of agreement among individual test results when an analytical method is used repeatedly

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Accuracy can be determined in many ways but one commonly used method is via spike-and-recovery. A common method is taking a sample with a known concentration — using a blank matrix is most common in the cannabis industry — and spiking into it, known concentrations of cannabinoids that quantify at the low, mid, and high points within your calibration curve. Ensure the spiked sample is treated exactly as a normal sample would be (if possible — due to the limits of cannabinoid CRM concentrations this can be difficult). Collecting a minimum of three measurements at each level on each instrument to be used is good practice.

Due to the overwhelming amount of matrices in the cannabis industry — it is suggested to perform a spike-and-recovery study for multiple matrices, i.e. solvent, flower, oil, gummy, chocolate, lotion, salve, drink, etc. This helps provide evidence that your method is not prone to matrix interference. Be sure to perform accuracy in pure solvent to have a baseline recovery.

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From these measurements, you can find the accuracy by determining how close your method is quantifying to the true value expected.

Precision: Use the data collected in the Accuracy portion to determine the precision by finding the percent relative standard deviation between results.

Intermediate precision: The precision reproducibility over a series of changes that address method variabilities such as — injection-to-injection, preparation-to-preparation, day-to-day, instrument-to-instrument, and lab-to-lab precision. What you decide to collect is up to your lab but I tend to perform the following:

Injection-to-Injection/Instrument-to-Instrument Precision: The statistical variance introduced by the instruments themselves — tested by generating low, mid, and high concentration samples, splitting them into multiple vials (one vial per instrument) and running the samples multiple times to find the relative percent standard deviation for each instrument at each level. This test lets you calculate the precision and uncertainty of each instrument to be used.

Day-to-Day Intermediate Precision: Perform the same test above but on the next day and to determine day-to-day precision. This shows that the method is reproducible after instruments have been shut down.

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Take it even further challenge — Person-to-Person Precision/Accuracy: Truly test your labs’ method by having different chemists and lab-techs perform the accuracy and precision tests to include the uncertainty of pipetting error and prep/extraction error.

Step 6: Limit of Detection / Limit of Quantification / Sensitivity / and Dynamic Range

Sensitivity: Method sensitivity via HPLC-DAD/UV-Vis is mainly dependent on the analytical wavelength chosen for the detection of the analytes of interest (220-230nm, 270–280nm, and 300–306nm for cannabinoids ). It is also dependent on the flow cell, pump mixer volumes, injection volume, detector response, bandwidth, detector slit width, column width, and overall performance of the chromatographic method and instrumentation.

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Limit of Detection: The limit of detection is the point at which your method can confidently identify a compound of interest is present but not quantify said compound.

Limit of Quantification: The point at which your method can confidently quantify a compound of interest.

Surprisingly, to this day, there are a plethora of methodologies for the determination of the LOD (see this article for an in-depth study on LOD determinization). If you are lucky, some state regulations will give you specific direction on how they wish you to determine the LOD for your chromatographic methodologies.

My personal preference for determining the LOD is to prepare a solution consisting of all analytes at the concentration of my lowest calibration point (typically, around 0.25µg/mL to 1.0µg/mL for cannabinoids) and run it 3 to 5 times. I then use the following equations based on ICH guidelines.

The detection limits may be expressed as below:

LOD = 3.3 σ/Slope

LOQ = 10 σ/Slope

Where: σ = the standard deviation of the response at low concentrations

Slope = the slope of the calibration curve.

Another method that is often used in methodologies with easily calculated signal-to-noise values(mass-spectrometry and FID based methodologies) is seen below:

The detection limits may also be expressed as below:

LOD is where the Signal to Noise Ratio = 3:1

LOQ is where the Signal to Noise Ratio = 10:1

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Dynamic Range: The range in which your method has been demonstrated to confidently quantify with an appropriate level of accuracy and precision.

Step 7: Robustness

Robustness: The capacity of a method to remain unaffected by small, yet deliberate variations in the method parameters. This is a measure of how reliable and resilient the method is to change.

Testing for robustness is quite fun if you have the time, yet sadly it is oftentimes overlooked in the cannabis industry. If it is required, most labs will do the absolute bare minimum required.

I on the other hand like to perform the following robustness tests for Potency:

Step 8: Test your method with a third party proficiency test — EARN YOUR BADGE!

If you think your method is ready for the final test visit Emerald Scientific and pick up a few practice proficiency test samples or try for the official Emerald Scientific proficiency badge!

“An Emerald Badge™ will be awarded to labs that perform within a specific tolerance in each proficiency test category, with criteria established by the ISO accredited PT manufacturer with input from the Emerald Test™ Advisory Panel. Labs that receive a Badge are not only recognized for their capabilities in comparison to their peers within a given series, the Emerald Badge™ affirms the labs’ ability to meet regulatory requirements for licensure. In addition, Emerald Test™ ILC/PT participation satisfies ISO/IEC 17043 accreditation requirements for external proficiency testing.” — Emerald Scientific

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Emerald Proficiency Testing Badge

Another method you can try also is to go to the local dispensary and pick up some samples tested by competitor labs and see how your lab fairs against the product’s certificate of analysis. Use the other labs as guidelines but don’t rely on other labs’ values being a target — rely on your own team and trust the science.

Step 9: Send your validation to the licensing agency and get validated!

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Contact your licensing bodies and have them look over your validation protocol. If there is something missing that they would like to see then they will let you know what it is and you can hop on it. Sending over a copy of your pre-validation outline to get reviewed is a good way to ensure you will have everything your licensing agency and your state regulating bodies mandate.

And that is it for today — I hope you learned something new! Thanks for the read.

Obviously this is a very quick overview of the steps needed to validate an analytical method but I hope it helps you get a better understanding of what it takes to actually validate an analytical procedure in a lab. There are many other valid ways to perform the steps I spoke about today. I am always happy to hear new methodologies, suggestions, and ideas.

Some good sources for more information:

Waters Guide to Analytical Validation

Guidelines for the validation and verification of quantitative and qualitative test methods — NATA

Limit of Detection and Limit of Quantification Determination in Gas Chromatography

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