To our knowledge, this study presents the first validated method capable of quantifying THC at 0.10 ng/mL using 50 µL of whole blood, while simultaneously quantifying CBN, CBG, CBD, 11-OH-THC, and THC-COOH. Previously published methods have required ≥100 µL of blood [22-24], relied on the cleaner matrix of plasma to increase sensitivity [25-27], employed SPE [28-30], or a combination of these strategies to reach sub-ng/mL sensitivity. Some approaches also use separate acidic and basic extractions to optimize recovery of both THC and THC-COOH [31-33]. Our method incorporates both NaOH and formic acid in a single extraction, providing a flexible point of adjustment. Increasing basicity enhances extraction efficiency for THC, which is particularly relevant when testing for impairment. Conversely, more acidic conditions can improve recovery of THC-COOH, which can be more informative for assessing cannabis use history. In this study, the method was optimized for THC, reflecting its envisioned application in roadside microsampling, where timely collection provides more accurate estimates of THC levels at the time of collision or other driving incident.

While the use of SPE is well established for improving sensitivity through reduced analyte loss and enhanced matrix clean-up [34], our LLE method achieves superior LLOQs for THC without the need for SPE. This is particularly advantageous for high throughput applications, as the high cost of solid phase cartridges can be a practical barrier. Although our LLOQs for THC metabolites THC-COOH and 11-OH-THC are higher than those reported in some methods [24,27], they are consistent with several recent studies [22,23,29] and remain suitable for quantitation. Including 11-OH-THC is particularly important, as it is psychoactive and often reaches higher blood concentrations after oral cannabis consumption compared to inhalation [35]. Further, our method also validates the quantitation of additional cannabinoids, CBN, CBG, and CBD, which are often excluded from published methods. Although these compounds are non-psychoactive and less studied, they may provide valuable contextual information.

CBG, known as the “mother cannabinoid” for its role as the precursor of other cannabinoids in the cannabis plant, undergoes rapid hepatic metabolism [36] and may therefore indicate recent consumption. CBN, which forms from the oxidative degradation of THC in the cannabis plant, could provide insight into the type or age of product consumed [37]. CBD measurement is also increasingly relevant, given the widespread availability of CBD-rich cannabis products and edibles, as well as its growing use for medical purposes [38,39]. Together, inclusion of these cannabinoids extends interpretive power beyond THC alone and may strengthen forensic assessments of cannabis use.

The present method not only offers a robust alternative to existing liquid blood assays but also provides a practical counterpart to dried blood sampling approaches. By using only 50 µL of whole blood, it preserves the advantages of microsampling while avoiding key limitations associated with DBS and VAMS®, such as hematocrit-related variability and higher consumable costs. In our experience, obtaining 50 µL of blood via a fingerstick is feasible with appropriate lancets, and collection into microtubes such as Sarstedt Microvette® devices can provide a simple, low-cost option for storage. Moreover, we demonstrate that a 1:1 dilution can be performed without compromising accuracy, supporting the feasibility of using as little as 25 µL of liquid blood when smaller volumes are collected.

Another important distinction lies in sample preparation. DBS and VAMS® methods rely on protein precipitation with methanol or acetonitrile [14-16], taking advantage of the partial retention of cells and proteins within dried matrices to reduce matrix complexity and achieve low LLOQs. In contrast, liquid blood methods generally incorporate an additional organic separation step, which helps remove endogenous material. However, phospholipids, particularly lysophosphatidylcholines and lysophosphatidylethanolamines, can co-elute with THC in LC-MS workflows and have been shown to cause pronounced ion suppression of cannabinoids in acetonitrile extracts of whole blood [40,41]. The long-term impact of residual phospholipid co-extraction from DBS and VAMS® extracts on instrument performance has not yet been characterized. Future studies of dried blood sampling should therefore include assessments of instrument robustness, including evaluations of sensitivity and column backpressure over extended injection series (>1000 injections) in comparison with conventional organic extraction workflows.

A major advantage of dried blood sampling is the ability to store samples at room temperature, since water loss during drying halts most enzymatic degradation reactions. Recent studies have shown that THC, 11-OH-THC, and THC-COOH remain stable on DBS cards for up to three months at room temperature [14], while VAMS® devices maintain stability for at least 30 days [15]. For liquid blood (≥2 mL), however, the available stability data are much older, with reports from the 1980s indicating that cannabinoids remain stable for approximately one month in glass tubes at room temperature [42], whereas storage in plastic containers leads to markedly greater analyte loss [43]. Given advances in collection devices designed for very low blood volumes [44], updated stability studies are needed to confirm storage conditions suitable for microsampling applications.

Practical limitations to consider when utilizing our method include the challenge of working with low blood volumes. When only °50 µL of blood is available, accurately aliquoting the entire amount be difficult during forward pipetting using positive-displacement pipettes, as bubbles may form. However, our results demonstrate that sample dilution is possible without compromising accuracy or precision. It should also be emphasized that although our extraction procedure has been highly optimized, the results reported here were achieved using a state-of-the-art Agilent 6495 liquid chromatography-triple quadrupole mass spectrometer, which has an instrument detection limit of 0.30 fg of reserpine when injected onto the column [45]. Comparable results may therefore depend on access to instrumentation of similar sensitivity.