Why Spike Recovery Fails for Cannabis Flower: Using Sequential Extractions to Validate Cannabinoid Extraction Efficiency

For cannabis flower, cannabinoids occur at concentrations far too high for traditional spike recovery experiments to meaningfully assess extraction efficiency. Even large spike additions contribute only a small fraction of the total analyte mass, making recovery results insensitive to extraction performance. This article explains why extraction efficiency for cannabinoid methods must instead be established through matrix-exhaustive, sequential extraction studies. The approach employs repeated extractions of the same test portion until additional extraction steps no longer yield measurable cannabinoids, providing direct evidence of extraction completeness. Practical guidance is provided for designing sequential extraction studies. This strategy allows laboratories to confidently demonstrate that their methods fully extract cannabinoids from the complex botanical matrix of cannabis flower.

Introduction

This article explains why traditional spike recovery methods fail for high-cannabinoid content matrices like cannabis flower, describes how sequential extraction studies work, and provides practical guidance for implementing these studies as part of a defensible method development and method validation strategy. By incorporating matrix-exhaustive extraction assessments, laboratories can significantly improve the reliability of their cannabinoids data and better satisfy accreditation and regulatory expectations. Most importantly, these practices allow labs to maintain complete confidence when defending “potency” results to clients who anticipate higher values.

Accurate measurement of cannabinoids begins prior to sample introduction into chromatographic systems. The extraction process, which facilitates the release of cannabinoids from the resinous, trichome rich cannabis flower, plays a pivotal role in ensuring that analytical methods reflect actual concentrations. However, many labs validate cannabinoid analysis methods with tools ill-suited for the flower and typical concentration levels. In some cases, regulatory requirements mandate spike recovery experiments that ultimately provide limited valuable insight. The primary concern lies in reliance on spike recovery procedures to evaluate extraction efficiency. Using spike recovery as a standard for cannabinoid extraction is misguided, especially since related fields like botanical testing employ alternative and often more appropriate validation strategies. (1) Accurate characterization of cannabinoid extraction is essential both from a scientific perspective and to ensure the validity of results, especially given external pressures to overstate “potency” values.

Spike recovery is a long-established and useful tool in analytical validation for many commodities and analytes. Unfortunately, it is not suitable for cannabis flower when testing cannabinoids. Cannabinoids are endogenous and some present at such high concentrations that a spike adds only a tiny fraction to the total analyte mass. As a result, spike recovery is essentially blind to extraction inefficiencies and cannot provide meaningful evidence of method performance.

To work around the lack of in-matrix reference materials and the mismatch between high native cannabinoid levels and low-concentration chemical standards, some laboratories spike samples after the initial extraction, once the extract has been diluted for analysis. However, this approach is even less informative, because it evaluates only instrumental performance and does nothing to characterize the extraction step itself.

To accurately evaluate extraction efficiency for cannabis flower, laboratories should turn to alternative approaches. I prefer using matrix-exhaustive sequential extraction, ideally combined with comparisons of different solvents or extraction conditions to assess extraction completeness and robustness. This article will focus on the concept of sequential extraction or re-extraction.

Why Spike Recovery Experiments Cannot Measure Extraction Efficiency in Cannabis Flower

Spike recovery experiments are a common tool in analytical method validation, where a known amount of analyte is added to a sample and its recovery is measured to assess the loss, if any, of the analyte due to sample preparation (extraction and clean-up). While this approach works well for matrices with low or absent endogenous analytes, it is fundamentally unsuitable for cannabis flower.

Cannabis flower contains high concentrations of cannabinoids, typically 15–30% by mass. For a 250 mg test portion, this corresponds to 37,500–75,000 µg of native cannabinoids. In contrast, the largest feasible spike addition—limited by standard availability, expense and practical preparation—rarely exceeds 1,000 µg, representing less than 3% of the total analyte. Because the spike constitutes such a small fraction of the total cannabinoid amount, the additional analytical signal it produces can be negligible relative to the method measurement uncertainty. This means the change caused by the spike is statistically indistinguishable from the unspiked sample, making the spike effectively indiscernible in the analytical data.

This masking effect can create a false sense of method performance. Even if the extraction fails to recover a significant portion of the native cannabinoids, the spike recovery can still appear near 95–105%. The instrument accurately quantifies the spike, but this measurement provides no information about the extraction efficiency of the native analytes. Consequently, problems such as incomplete trichome disruption, insufficient solvent penetration, inadequate extraction time, or poor mixing go undetected. Spike recovery experiments, in this context, cannot reliably demonstrate method effectiveness and can give laboratories a false sense of confidence in their results.

In short, the combination of extremely high native cannabinoid levels, the small relative size of the spike, and the limits of method measurement uncertainty makes spike recovery experiments unsuitable for evaluating extraction performance in cannabis flower. Alternative approaches, such as matrix-exhaustive sequential extraction studies, are required to directly assess whether a method effectively extracts cannabinoids from the plant matrix. This approach allows laboratories to detect poor-extraction, assess extraction completeness, and establish reliable extraction efficiency, providing a scientifically defensible foundation for method validation.

Sequential Extraction/Re-extraction: A Scientifically Defensible Alternative

Sequential extraction or re-extraction is valuable not only for validation but also during method development. It can be used to optimize extraction parameters, such as solvent composition, solvent-to-sample ratio, extraction time, agitation technique, and temperature, by providing direct feedback on how these variables influence extraction completeness. Once optimal conditions are established, sequential extraction studies can then be incorporated into formal validation to demonstrate that the selected parameters achieve a thorough, exhaustive extraction of cannabinoids.

Additionally, sequential extraction can reveal matrix-specific challenges, such as solvent penetration limitations, analyte retention in plant tissue, or ineffective comminution that might otherwise go undetected with spike recovery experiments.

How Sequential Extraction Works

In this study design, a homogenized test portion of cannabis flower is subjected to the laboratory’s proposed extraction method. After the initial extraction (Extraction 1), the extract is collected and analyzed. The same solid flower test portion is exposed to fresh solvent and extracted again. The extract from this Extraction 2 is collected and analyzed.

A sample from each extraction round is analyzed separately, and the results can be used to construct a cumulative recovery curve. As extraction rounds progress, the amount of cannabinoids recovered should rapidly decrease. Once additional extractions contribute minimally to the cumulative total, extraction can be considered complete. Under appropriate parameters, the first extraction would be considered complete, and negligible amounts of analytes would be detected.

Designing a Sequential Extraction Study

1. Extraction Relevant Parameters

Sequential extraction must use the laboratory’s proposed method parameters, including:

• Particle size (comminution process)
• Solvent composition
• Solvent volume
• Extraction time
• Temperature
• Agitation method
• Test portion size

2. Number of Extractions

Most studies require 3–4 extraction rounds, depending on solvent strength and method performance. More extraction rounds can be added if measurable analytes remain.In many cases, Extraction 1 can be expected to recovery 85–~100% of cannabinoids, Extraction 2 recovers ~0–5% and Extraction 3 recovers 0–2%. The final extraction mass should be near or below the method’s detection limit, demonstrating that essentially all cannabinoids have
been extracted.

3. Data Collection and Calculations

For each extraction round:

1. Record the mass of cannabinoids recovered.
2. Calculate the cumulative mass extracted across all extraction rounds.
3. Express each extraction round as a percentage of the total cumulative mass by calculating extraction efficiency.

For example, here is the equation for calculating the extraction efficiency of the method used in Extraction 1.

The key is not only the absolute percentage recovered, but also a clear demonstration that extraction is complete.

4. Helpful Considerations

During a sequential extraction study, pay attention to two often overlooked details: adjusting for any leftover solvent and adjusting testing for extracts with low concentrations.

During sequential extraction, decanting the solvent from the first extraction inevitably leaves a small amount of liquid in the tube and wetting the flower. This remaining solvent contains extracted cannabinoids, and if not accounted for, it can inflate the analyte amount recovered in subsequent extractions while slightly underrepresenting the analyte from the first extraction. To correct for this, the remaining solvent can be weighed and its volume determined using solvent density. The known concentration of cannabinoids in the initial extract can then be used to calculate the amount of analyte in the remaining solvent. This quantity is added back to the first extraction and subtracted from the second, ensuring that the data appropriately reflects the true distribution of cannabinoids across extraction steps.

To use remaining solvent correction:

1. Decant sample extraction solvent as much as possible.
2. Weigh extraction tube with wetted flower.
3. Leave to dry in the fume hood until completely dry.
4. Reweigh the extraction tube.
5. Calculate the evaporated solvent weight by the difference in weigh (wet weight – dry weight)
6. Convert the evaporated solvent weight to volume (mL) using the solvent density (e.g. 0.7913 g/mL for methanol).
7. Calculate the amount of analyte remaining in the evaporated solvent using the volume (mL)
   and the concentration of the previous extraction.
8. This amount of analyte can be added to the previous extraction and subtracted from the subsequent extraction.
   

Another important consideration arises from the low concentration of cannabinoids in the second and subsequent extractions. Typically, the first extraction removes the majority of cannabinoids, often around 90% or greater, leaving only a small amount in the flower. For instance, a flower sample with 20% THCA may contain only ~ 2% THCA after the initial extraction. Many analytical methods include a post-extraction dilution to bring typical flower samples within the method calibration range. For the second and subsequent extractions, this dilution can be reduced or eliminated to ensure that the lower concentrations remain detectable. I typically eliminate the post-extraction dilution step when testing the second and subsequent extractions. Without this adjustment, the analyte in later extractions may fall below the limit of quantitation, making the assessment of extraction efficiency less exact and potentially giving the incorrect impression that the extraction is complete after the first extraction.

Careful planning of sequential extraction studies—including proper sample homogenization, consistent extraction parameters, correction for residual solvent, and appropriate handling of low-concentration extractions—is critical for generating reliable, defensible data on cannabinoid extraction efficiency. By addressing these details, laboratories can ensure that sequential extraction studies are a valuable tool that can correctly reflect method performance.

Conclusion

Cannabinoid testing in cannabis flower presents unique analytical challenges due to the exceptionally high concentration of endogenous cannabinoids. Spike recovery experiments, though widely used in other analytical situations, are incapable of measuring extraction efficiency under these conditions.
The spike simply represents too small a portion of the total analyte mass to reveal whether the extraction step is complete.

Matrix-exhaustive sequential extraction studies provide a scientifically sound and practical alternative. By repeatedly extracting the same test portion until additional extraction yields become negligible, laboratories can directly quantify extraction completeness and accurately determine extraction efficiency.

As cannabis regulations evolve and the market matures, the industry’s expectations for analytical quality should continue to rise. Implementing robust extraction efficiency studies is a critical step toward producing cannabinoid data that laboratories, regulators, producers, and consumers can trust. Perhaps most importantly, this enables laboratories to confidently defend their "potency" results when clients expect higher numbers and when regulators raise concerns about inflated values.

References

1. Appendix K: Guidelines for Dietary Supplements and Botanicals, Official Methods of Analysis of AOAC INTERNATIONAL (2016) 20th Ed., AOAC INTERNATIONAL, Rockville, MD, USA (http://www.eoma.aoac.org/app_k.pdf). Also at: J. AOAC Int. 95, 268(2012); DOI: 10.5740/jaoacint.11-447

About the Columnist

Julie Kowalski is a technical consultant primarily serving the cannabis and hemp testing market. She earned her graduate degree in Analytical Chemistry. Her professional experience includes troubleshooting, method development and validation for GC, GC-MS, LC, and LC-MS/MS in addition to extensive pesticide residue analysis experience and chromatography method development. With over ten years at a technology provider, she has served as Scientific Director and Chief Scientific Officer for a cannabis testing lab. She has served as the President of the North American Chemical Residue Workshop, served on AOAC Expert Review Panels, the Cannabis Scientific Task Force for Washington State, chaired the AOAC CASP Chemical Contaminants Working Group and is currently chairing the AOAC CASP Pesticide Think Tank. Julie is a part of Saturn Scientific, LLC, an interdisciplinary team of experienced scientists that leverage their collective expertise to perform technical investigations for the cannabis and hemp industries.

How to Cite this Article

Kowalski, J. Why Spike Recovery Fails for Cannabis Flower: Using Sequential Extractions to Validate Cannabinoid Extraction Efficiency, Cannabis Science and Technology, 2025, 8(6), 14-17.