Cannabis sativa has been cultivated around the world for centuries [1,2]. Originally, cultivation may have been predominantly for fiber, but historical records indicate contemporaneous medicinal use for ailments like rheumatic pain, constipation and malaria [3,4,5,6]. Evidence suggests that psychotropic use of Cannabis began in the early 1600s in North America with medicinal use following in the 1800s for ailments such as tetanus, epilepsy and rabies and as a muscle relaxant [7]. In the United States, the Controlled Substance Act of 1970 created five schedules of controlled substances primarily based on accepted medical use and the potential for abuse [8]. Schedule I drugs are defined as having no currently accepted medical use and a high potential for abuse. Schedule II drugs have accepted medical use, but with a high potential for abuse, and Schedule V drugs have accepted medical use and a much lower potential for abuse. When the US law was enacted, there was no accepted medical use and fears about abuse, so both marijuana (Cannabis, marihuana) and the tetrahydrocannabinols (THC, delta-8 THC, delta-9 THC, dronabinol and others) were put on Schedule I, which controls the possession of both the plant and the chemical. Recent documentation of numerous medical uses such as for nausea/vomiting, pain, muscle spasticity, mood disorders, etc., combined with limited potential for abuse, suggest similarity to Schedule III drugs [9].

The Agriculture Improvement Act of 2018, otherwise known as the Farm Bill [10], legalized the nationwide cultivation of Cannabis sativa containing ≤0.3% total delta-9 THC on a dry weight basis, designated as hemp, while maintaining Cannabis sativa > 0.3% total delta-9 THC, designated as marijuana, as still illegal in the United States, as it is in many countries globally [11,12]. Following legalization in the US and other countries, it has been estimated that around 147 million people consume the plant globally, mainly for its THC-derived effect [13]. In the US Farm Bill, hemp growers are required to have plant material collected up to 30 days before harvest, and test results must show compliance with the 0.3% regulatory threshold. Crops with THC levels exceeding the threshold are to be destroyed. Notably, the law specifically states that the method of analysis for compliance testing must include the postdecarboxylation products. This stipulation requires the primary plant product of tetrahydrocannabinolic acid (THC-A) to be added to the Delta-9 tetrahydrocannabinol for a total Delta-9 THC concentration. Gas chromatography utilizes hot injection (250 °C), which decarboxylates THC-A in situ and produces one test result. Liquid chromatography utilizes a lower temperature injection, producing a result for THC and THC-A separately, which are combined by formula to produce one test result designated as total THC. The ratio of THC to THC-A is irrelevant for compliance purposes relative to the Farm Bill. While the Farm Bill removed hemp from the Schedule I list, it did not at the time specify what may be done with the harvested commodity [10]. Those consumer products must still meet regulatory requirements issued by the United States Food and Drug Administration (FDA) and/or Drug Enforcement Administration (DEA) to be moved between states and may have additional requirements within states [10].

The biosynthesis of THC and CBD is related by shared pathways involving the activities of a cannabidiolic acid synthase, a cannabichromenic acid synthase and a tetrahydrocannabinolic acid synthase. Respectively, these enzymes convert a common precursor, cannabigerolic acid, into the acidic forms of cannabidiol, cannabichromene and tetrahydrocannabinol [14,15]. With respect to these cannabinoids, the difference between hemp and marijuana is the genetic coding for enzymes that produce either THC-A or cannabidiolic acid (CBD-A) from the precursor molecule cannabigerolic acid, and the only reliable method to distinguish the two analytes is with laboratory testing. Cannabis sativa cultivars are sometimes described in terms of chemotypes: chemotype I is predominantly THC (marijuana), chemotype II is predominantly CBD with moderate THC and chemotype III is predominantly CBD with low THC (hemp) [16,17]. The previous illegal status of the plant has meant that the limited knowledge about breeding, varietal differences, growth conditions, etc., has restricted many types of scientific study until recently [18,19]. Furthermore, though it remains federally illegal in the United States, marijuana has been legalized in many states for medicinal and/or adult use. In 2012, Colorado and Washington became the first states to legalize Cannabis. By 2023, 38 states had some form of legalized medical cannabis, which included 24 states with fully legalized adult use [20]. With increases in indoor and outdoor cultivation, and as a wind-pollinated crop, the development of hemp seed may be complicated by both feral and cultivated marijuana.

As required by the Farm Bill, the United States Department of Agriculture (USDA) Agricultural Marketing Service (AMS) published a final rule, effective on 22 March 2021, that established a national regulatory framework for hemp production in the United States [21]. The final rule requires test samples for each field lot to be collected within 30 days of harvest for compliance testing. A lot is defined as a contiguous growing area containing the same cultivar of cannabis throughout and could be a field or greenhouse location. Sampling agents are directed to cut 5–8 inches from the terminus of branches to represent a lot, and the USDA standard sampling protocol states “The standard sampling protocol ensures, at a confidence of 95 percent, that no more than one percent of the plants in each lot would exceed the acceptable hemp THC level and ensures that a collected sample represents a homogeneous composition of the lot” [22]. The document contains formulas to calculate the number of primary plants to sample for field lots larger than ten acres, and for lots smaller than ten acres, an “example 2” is provided that specifies a minimum sample size of one plant per acre. In the US, the State of Connecticut (CT) had 109 licensed producers of hemp in 2019, which increased to 140 in 2020 but then decreased to 98 in 2021 and 78 in 2022. The average lot size has ranged from 1.44 to 0.56 acres, so for most lots in CT, the final rule allows a lot to be represented by only one or two plants [23].

As an ISO/IEC 17025:2017 state testing laboratory, the Analytical Chemistry laboratory of the Connecticut Agricultural Experiment Station (CAES) is suited to address how field sampling affects both THC and CBD test results. The laboratory has access to both an experimental farm where hemp may be grown and collected and regulatory testing facilities. Testing laboratories routinely comminute and homogenize samples and can measure the uncertainty related to those processes. Sample agents develop protocols to properly collect samples in the field or greenhouse that are representative of a lot. A laboratory is usually unable to assess how test items relate back to the decision unit or the lot that the sample is meant to represent. Moreover, while the number of plants that compose a sample is estimated based on the variability between the plants, the actual variability between plants is usually unknown, making the development of the sampling protocol difficult. Therefore, while test portions should be reflective of the item delivered to the laboratory, the effect of the field collection procedure on the final test result, a potentially significant parameter, is rarely studied. Furthermore, while the AMS guidance allows a minimum sample size of one plant per lot of one acre, it provides little guidance on how to ensure that no more than one percent of the plants would exceed the acceptable hemp THC level, or how many plants are required to represent a homogeneous composition of the lot [10].

Previous studies have investigated the levels of THC and CBD in hemp cultivars over time. However, plant-to-plant differences within the cultivar have not been assessed [24,25,26]. Overall, the current study aimed at providing evidence for the occurrence of plant-to-plant differences in THC and CBD production as a function of time to support regulatory efforts. Understanding plant-to-plant variations in THC production within the same cultivar will help in the selection of hemp varieties by growers to ensure that varieties of preference are suited for maintaining the regulatory limit. Moreover, it could provide useful feedback to hemp breeders regarding the fidelity of their germplasms. To this end, gas chromatography with flame ionization detection was used to evaluate hemp plant material for their THC and CBD contents with the specific objective of examining the rate of increase in THC and CBD in different cultivars during the growing season and assessing plant-to-plant variability within the same cultivar. It is anticipated that this study will provide information that is useful to regulatory bodies ensuring compliance with federal regulations. While CBD has no bearing on the legal status of the crop in the US, understanding the relative rate of increase for THC and CBD is critical to yield calculations of compliant hemp, the creation of sampling strategies that meet the AMS guidance and ultimately the economic viability of hemp as a crop.