1 Introduction

With the increase in research done on Cannabis and its unique compounds, various extraction methods from either hemp fibres or Cannabis flowers are commercially used to produce valuable extracts [1]. The global market for Cannabis-derived products, i.e. cannabinoid isolates and extracts, was estimated to be USD 7.1 billion in 2020 with a projected compound annual growth rate (CAGR) of ~ 35% between 2021 and 2027. The market size of these products is projected to exceed USD 108.8 billion by 2027 [2], which necessitates an increase in production or process efficiency to meet the demands of the larger market.

Commercial cannabinoid extraction generally follows the route of primary solid–liquid extraction from plant material with ethanol, followed by a winterisation step to precipitate co-extracted plant waxes [3, 4]. Another effective extraction technique used is supercritical-CO2 (SC-CO2) extraction, which has been demonstrated to effectively extract the cannabinoids from the flowers at high purities [3, 5]. Ethanol can be added as a co-solvent during SC-CO2, which has been reported to extract cannabinoid acids at higher efficiencies [6]. However, the use of SC-CO2 also results in the co-extraction of waxes, and therefore, the winterisation stage for wax removal is also required when using this procedure. On an industrial scale, ethanol extraction is generally implemented [5].

The precipitated waxes are removed from the primary stream through filtration as the presence of lipophilic compounds in the extracts negatively affects the performance of the various downstream distillation stages and the product quality. The amount of wax generated is significant, at 5–10% (w/w) from biomass input is generated [7], and so the utilisation or valorisation of this Cannabis wax may hold significant potential in unlocking additional value from Cannabis processing. However, there remains significant uncertainty in the exact composition of the Cannabis by-product wax [7], which is an obstacle for potential further valorisation of this by-product. The wax differs from, for instance, hemp wax or hemp seed oil, as it is a co-extracted by-product and not a targeted extraction such as described in Noppawan et al. [8] or Montserrat-de la Paz et al. [9], who reported on the composition of Soxhlet extracted waxes from hemp and hemp seed oil, respectively. Due to the different extraction conditions and feed material used, the compositions of these products are significantly different.

Identification and quantification of the major compounds present in these wax by-products would allow for the identification of individual compounds or groups of compounds to target for further recovery. Plant waxes are commonly composed of aliphatic compounds such as long-chain alkanes, fatty alcohols, fatty acids, and wax esters [10,11,12]. Cyclic compounds such as terpenoids and sterols are also present in most plant waxes, including those derived from Cannabis [7, 12, 13]. One might also expect a fraction of this wax to contain cannabinoids, since this wax is a by-product from cannabinoid extraction.

One study, conducted by Leyva-Gutierrez et al. [5], characterised Cannabis by-products, but the focus was aimed more towards the later by-products emerging from the distillation stages, i.e. resins and tars removed from the distillation column, as well as impurities in the final cannabidiol (CBD) isolate. The composition of these further downstream products will most certainly be significantly different from the winterisation wax, a stream which is also significantly larger in volume than those other waste streams. It was further reported that the generation of waste products from commercial Cannabis processing can be up to 58% (w/w relative to total crude extract) from all different processing stages, which due to lack of characterisation remain underutilised [7].

Waste residues and underutilised by-products, such as those generated by Cannabis processing, can effectively be converted through a biorefinery approach into value-adding products and materials, which through integration of process streams and stages can allow for the valorisation of multiple waste streams [14]. This concept is however dependent on knowledge of the composition of the feedstock, necessitating detailed characterisation of the streams before attempted valorisation [15]. A further complication is that the composition of these streams is likely variable, depending on processing route, and the source biomass (in this case a major differentiator might be whether the source Cannabis is from a high THC- or CBD-producing strain), and so a wide number of compositional studies examining various feedstocks will be useful for the bioprocess engineer designing such a biorefinery.

Analysis of plant waxes can be challenging in general due to the relatively high molecular weight of the lipophilic compounds found in the wax, and several problems that could arise during analysis are related to volatility, transport through the analytical columns, and elution from the columns [16]. In cases where compounds have difficulty eluting chemical derivatisation needs to be implemented to produce sufficiently volatile compounds [17]. Different methodologies for gas chromatography (GC) and liquid chromatography (LC) have already been established for the analysis of plant waxes [13, 18,19,20,21,22]; however, in order to create a clearer picture of plant wax compositions some of these methods can be combined or adapted for the analysed substrate.

This study aimed to fill to the gap in knowledge regarding the compositional characterisation of Cannabis processing wax by-products by the implementation of a targeted quantitative analysis to characterise the major compounds present in the wax by-product using gas chromatography–mass spectrometry (GC–MS). Two industrial wax by-products were received from commercial Cannabis processors (labelled wax A and wax B), and were chemically characterised with respect to the major wax components, i.e. n-alkanes, fatty acids, fatty alcohols, sterols, and volatiles (terpenes), and the entrapped cannabinoid fraction. The results are discussed with the focus on identifying fractions with value-adding potential to be recovered from the wax by-product.

2 Materials and methods

2.1 Wax by-products from Cannabis processing

Two wax by-products were acquired from commercial Cannabis processors, labelled wax A and wax B. Wax A was generated from the processing of a CBD-high strain of Cannabis, whereas wax B was generated from the processing of a THC-high strain of Cannabis.

2.2 Standards and reagents

Pure standards for the main classes of compounds, namely long-chain alkanes, fatty acids, fatty alcohols, sterols, and terpenoids, were employed in this study. A long-chain C21–C30 alkane standard was purchased from Sigma-Aldrich (Johannesburg, South Africa) as a 50 mg/L solution in dichloromethane, along with a pure standard (≥ 99%) of nonacosane (C29). Linoleic acid, palmitic acid, 1-hexacosanol, 1-octacosanol, sitosterol, cholesterol, and stigmasterol were purchased from Sigma-Aldrich (Johannesburg, South Africa) as pure compounds, with purities in the range of 90–99%. ß-Caryophyllene was purchased as a 2000 µg/mL solution in methanol along with ursolic acid (≥ 90%) from Sigma-Aldrich (Johannesburg, South Africa). A mixture of terpenoids, namely myrcene, alpha-pinene, camphene, beta-pinene, limonene, alpha-phellandrene, eucalyptol, gamma-terpinene, terpinolene, linalool oxide, linalool, alpha-terpineol, and alpha-bisabolol (purities ranging from 90 to 99%), were provided by the GC department of the Central Analytical Facility (CAF) at Stellenbosch University, the service lab that performed the GC–MS analyses. Further standards for long-chain alkanes and fatty acids were also provided by CAF. Isotopically labelled analytes for use as internal standards were, however, not available from CAF and were not purchased.

2.3 Determination of residual solvent

Both waxes contained residual solvent from the primary extraction that was carried over into the wax separation in the winterisation stage, with wax B containing more solvent than A, as it was not subjected to an evaporation stage after the winterisation stage by the industrial supplier. Thermogravimetric analysis (TGA) was performed with a TGA 5500 (Waters) on wax A and B to determine the fraction of solvent-free wax. A gas flow rate of 70 mL/min was used for this analysis and the samples were equilibrated at 50 °C for 0.16 min after which it was ramped up to 80 °C at 10 °C/min, where it was kept at isothermal conditions for 2 min. This temperate was chosen as it is above the boiling point temperature of the residual solvent, in this case ethanol, which was 78.4 °C. This was followed by a further increase in temperature to 350 °C at 20 °C/min at which it was kept at isothermal conditions for 2 min again. At this point, the cannabinoids would largely have evaporated, and the remaining mass could be attributed to the wax and ash content. For determination of ash content, the temperature was increased to 850 °C and the final weight was measured. The weight of the sample was continuously recorded to generate the TGA curves. The temperature profile was determined by the boiling points of the various fractions, such as the residual solvent, the cannabinoids, and the wax compounds.

2.4 GC–MS analysis

The characterisation of the wax samples was completed by the Central Analytical Facility (CAF) GC–MS laboratory division of Stellenbosch University. Samples were prepared, as described in the upcoming sections, and the analyses were completed by CAF. The resulting characterisation data were processed to generate the characterisation results. All samples were analysed once for each of the four procedures used.

2.4.1 Sample preparation

Fatty alcohol and sterol analysis

Three millilitres of a saponification reagent was added to approximately 100 mg of sample for the analysis of sterol and fatty alcohols. The samples were incubated in an oven at 70 °C for 1 h for completion of the saponification process. The saponified samples were cooled on ice, followed by the addition of 5 mL of distilled H2O and 2 mL of dichloromethane. The mixture was centrifuged for 5 min to facilitate the separation of the layers. After centrifugation, the bottom organic layer was transferred into a clean vial and dried with Na2SO4 to remove any water. One-hundred microliters of the sample was derivatised with 100 µL pyridine and 30 µL of BSTFA followed with incubation in an oven at 100 °C for 1 h [19].

Fatty acid analysis

For the fatty acid analysis, the samples were derivatised to their methyl-ester derivatives. Approximately 100 mg of the sample was taken up into 1.5 mL of 2:1 (v/v) chloroform and methanol mixture and 3 mL of 20% NaCl was added. Samples were sonicated at 55 °C for 30 min and the layers were separated with centrifugation. The bottom chloroform layer containing the fatty acids was extracted and dried with Na2SO4. Two-hundred microliters of dried sample was transferred to a GC-vial insert and dried under a gentle stream of N2. For the wax samples, 100 µL of the dried sample was diluted with 100 µL of chloroform before drying with N2. Dried samples were then derivatised by adding 170 µL of tert-butyl methyl ether(TBME) and 30 µL of trimethylsulfonium hydroxide (TMSH) [20].

Alkane analysis

One millilitre of hexane was added to 10 and 100 mg of wax and oil samples, respectively, for the analysis of alkanes. The solution was then sonicated for 60 min to extract and then 5 mL distilled H2O was added. After the samples were vortexed, it was centrifuged at 2520 RCF for 3 min and 200 µL of the upper hexane layer was transferred into an insert positioned in a 2-mL vial. The wax samples were diluted 1:1 with hexane [21].

Terpene analysis

Terpenes were analysed by solid phase micro-extraction (SPME) of the sample headspace with a polydimethylsiloxane/divinylbenzene (pink) SPME fibre. Five millilitres of 12% (v/v) alcoholic solution (ethanol in water) was added to approximately 100 mg of sample followed by 5 mL of 20% NaCl solution. The samples were vortexed, positioned on the sample tray, and analysed by SPME–GC–MS [22].

2.4.2 Instrumental parameters

The instrumental parameters for each analysis are displayed by Table 1. Generated data were processed using the chemical software Xcalibur (Thermo Fisher Scientific) and compounds were identified by comparison of the relative retention times to those of available standards, analysing the mass fragmentation patterns, and comparison of mass fragmentation patterns with spectra contained in the NIST library.

Table 1 Instrumental parameters and temperature programme for each analysis
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The characterisation was done using externally calibrated linear calibration curves and the values for these calibrations have been given in Table 2. A minimum of 6 calibrators were used for each component’s calibration curve. Values are given as mg/L, as generated by the instrument, and was later for each wax sample converted to mg/g from the sample weights. No recovery experiments were performed on these samples, as it was not available at these facilities.

Table 2 Performance parameters for the characterisation of wax components using GC–MS
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The mass ions monitored during the fatty alcohol and sterol analysis using MS/MS (SRM mode) were given in Table 3. For the fatty alcohol analysis, MRM mode was used with three product ions monitored for each analyte, whereas for the sterol analysis SRM mode was used. For the alkane and terpene analyses scan mode was used with a triple-quadrupole mass spectrometer as the concentrations of these analytes were well above trace levels, and therefore, no issues regarding sensitivity were experienced.

Table 3 SRM transitions of fatty alcohols and sterols
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2.5 Cannabinoid quantification

The fraction of cannabinoids present in both wax by-products was characterised through a combination of gravimetric and HPLC analyses. The analysis was performed by the LC department of CAF at Stellenbosch University, which provides cannabinoid analysis as a service, and therefore had standards for cannabinoids available. These standards were not available to be transferred between departments and were therefore not available to be used for GC analysis by the authors.

A known amount of each wax (5.14 g of wax A and 10.04 g of wax B) were dissolved in 100 mL of ethyl acetate and mixed. The mixture was then cooled and centrifuged at − 5 °C, after which the supernatant was taken off and heated until all solvent was boiled off and only the cannabinoid fraction remained. The cannabinoid profile was characterised by HPLC analysis. One-hundred milligrams of each sample was extracted by 2 mL of a methanol and water mixture with a ratio of 80:20 (v/v) methanol to water. The samples were sonicated with an AC-150H sonicator (MRC) for an hour before being centrifuged, after which the supernatant was drawn off and sent for analysis. The analysis was performed on a Synapt G2 instrument (Manufacturer: Waters) using electrospray ionisation. The column used was BEH C18 (Manufacturer: Waters) with dimensions 2.1 × 100 mm and pore size 1.7 µm. The mobile phase used was a mixture of water and acetonitrile, and the ratios are summarised in Table 4.

Table 4 Mobile phase parameters for the HPLC cannabinoid analysis
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Standards for CBD, ∆9-THC, and THCA were available, but CBDA was quantified relative to THCA. The R-squared values for these calibrations ranged from 0.9903 to 0.9978, provided by CAF. The highest concentration standard was 33.3 ppm and the samples with higher concentrations were extrapolated. Performance parameters such as calibration range, LOD, LOQ, and injection volume were not provided by CAF as it is proprietary knowledge of the LC department.

3 Results and discussions

This work set out to characterise the by-product waxes formed during cannabinoid extraction, using different chromatographic techniques and methods. Two wax by-products were acquired from commercial Cannabis processers, wax A originating from a high-CBD strain of Cannabis sativa and wax B from a high-THC strain. It was expected that there would be significant differences in the cannabinoid composition between the waxes as this profile is strain-specific, but all the main lipophilic molecules were expected to be present in both wax samples [8]. The two by-product waxes were substantially different based on preliminary inspection. Wax A was a dark brown, sticky substance with a distinct herbal smell. Wax B was a yellow-green slurry with a faint herbal smell, and further differed from wax A in that it was not subjected to an ethanol evaporation step beforehand by the industrial supplier. TGA analysis showed that wax A had a solvent content of 34.7% (w/w) and wax B contained 69.9% (w/w) solvent. Figure 1 contains the TGA graphs for the two wax samples that underwent the analyses. All component concentrations reported further in this discussion have been normalised to the dry wax (solvent-free) content and are therefore reported as weight of compound to weight of dry wax (mg/g).

Fig. 1
figure 1

TGA graphs for wax A (top) and B (bottom), showing reduction in mass and increase in temperature over time

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3.1 Characterisation results

Separate analyses for the different lipophilic compounds were necessary owing to co-elution of compounds. Initial analyses attempted to separate the fatty acids, fatty alcohols, and sterols in a single analysis; however, significant overlapping of the fatty acids and alkanes occurred, indicative of co-elution. Therefore, the analysis of fatty acids was done separate from the other compounds. The results of the combined analysis are shown in Table 5 and are reported based on a dry, solvent-free wax basis. The compounds characterised, other than the cannabinoid fraction, made up 42.3% (w/w) of wax A and 18.3% (w/w) of wax B. The fractions for n-alkanes, fatty acids, and fatty alcohols were reported to be the majority of identified lipophilic compounds for both wax samples by the different GC–MS methods. The total quantified mass was 87.7% (w/w) for wax A, but only 54.75% (w/w) of wax B was quantified with the same methodology. It was hypothesised that due to the difference in entrained solvent content between the waxes, the higher levels of ethanol in wax B could have influenced the extraction efficiencies of the target components in their respective sample preparations. However, the reported results regarding the cannabinoid content, determined through gravimetric analysis and HPLC, would not have been influenced by this and significant conclusions could be made regarding the identification of potentially valuable fractions within the wax by-product.

Table 5 Quantitative characterisation of wax by-product A and B, reported based on dry wax content
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The cannabinoid fraction was the most abundant fraction in both waxes, reporting a total fraction of 509.3 mg/g for wax A and 392.6 mg/g for wax B. In wax A, the most abundant cannabinoid was CBDA (314.8 mg/g) followed by the neutral form CBD (119.5 mg/g). For wax B, the most abundant cannabinoid was THCA (266.3 mg/g) and relatively high amount of the neutral form ∆9-THC (114.0 mg/g) was also reported. Cannabinoid products have high market value (CBD distillates sell for USD 3000 per kg CBD and isolates sell for USD 1000 per kg CBD [8]) and combined with their high concentration in the wax by-product makes this fraction very attractive as a potential target fraction for recovery. Suitable pathways for the recovery of the cannabinoids from the wax waste are, however, lacking in the current scientific and patent literature and further research in the development of such a process is required [23]. A previous study also reported high levels of cannabinoids in the wax by-product, at 416.6–463.7 mg/g [7], indicating that significant entrainment of cannabinoids during the removal of the wax by-product is a general concern for commercial Cannabis processing.

The fatty acid profile was significantly different between the two waxes. The chromatogram of this analysis is provided in Fig. 2. Wax A reported a total fraction of 172.2 mg/g and B, 12.02 mg/g. The major fatty acids present in wax A were palmitic acid (C16:0, 23.19 mg/g), oleic acid (C18:1n9c, 18.83 mg/g), linoleic acid (C18:2n6c, 68.46 mg/g), and α-linolenic acid (C18:3n3, 39.53 mg/g). The same fatty acids were also detected in wax B but were quantified in lower amounts. The major fatty acid in wax B was α-linolenic acid (C18:3n3) with a concentration of 3.011 mg/g. α-Linolenic acid is a highly favourable fatty acid used in cosmetics as it cannot be synthesised naturally in animals. It is often used as an emollient where it serves a hydrating purpose [24].

Fig. 2
figure 2

Overlayed chromatograms generated from the fatty acid analysis, with the quantified compounds and respective peaks labelled

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Previous literature reported that the fatty acids were only present in trace amounts [7], but a major difference done in this work was the individual fatty acid analysis, due to high levels of co-elution with other components. As a result, this work was able to quantify the fatty acids at more accurate levels than trace amounts, which is important for subsequent investigations into potential valorisation of this by-product.

The n-alkane fraction was composed of chain lengths C20 up until C33. The chromatogram for this analysis is provided in Fig. 3. As was expected from plant waxes, the odd-numbered chain lengths were dominant with only a few even-numbered chain lengths detected in both samples [25,26,27]. The n-alkane fraction was reported as 135.1 and 54.55 mg/g for wax A and B, respectively. The major alkanes detected for both waxes were C29 (n-nonacosane), followed by C27 (n-heptacosane) and C31 (n-hentriacontane). Wax A contained 52.19 mg/g and wax B 24.47 mg/g of n-nonacosane. The highest chain length quantified in the sample was C33 (n-tritriacontane). Long-chain alkanes are the main compound class in paraffinic waxes [28] and the large fraction in the Cannabis wax can therefore be utilised as a potential natural source of paraffinic wax. The range of chain lengths quantified in this work was comparable to what was reported previously on Cannabis wax. In both cases, the odd-numbered chain lengths were dominant and ranged from C21 to C33 in this work and C25 to C37 for the results previously reported in literature [7]. In both analyses C29 (nonacosane) was the most abundant alkane. The relative total alkane fraction quantified in this work was however much less than reported by literature (290.7–291 mg/g) [7].

Fig. 3
figure 3

Overlayed chromatograms generated from the n-alkane analysis, with the quantified compounds and respective peaks labelled

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The fatty alcohols reported a total fraction of 24.68 mg/g for wax A and 36.10 mg/g for wax B. The chromatogram for this analysis is provided in Fig. 4. The dominant chain length in wax A was C28 (1-octacosanol) which had a concentration of 14.23 mg/g. C26 was dominant in wax B and had a reported concentration of 18.88 mg/g. Fatty alcohols are commonly used in lotions, moisturisers, creams, and other personal care products. Its main contribution to these products is acting as an emulsifier, prohibiting the separation of oil and other liquids [29].

Fig. 4
figure 4

Overlayed chromatograms generated from the fatty alcohol and sterol analysis, with the quantified compounds and respective peaks labelled

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The low levels of fatty alcohols in the wax by-product would, however, not be economically feasible to recover specifically, and therefore, the wax would likely not be a viable source of these components. As with the fatty acids, the fatty alcohol fraction was reported only in trace amounts in a previous study [7], which reported on the free fatty alcohol content. In this work, the fatty alcohol fraction was quantified as both the free fatty alcohols and the released fatty alcohol chains from the transesterification of the wax esters. It is therefore highly likely that the free fatty alcohol concentration in the wax by-product is relatively low in comparison to the fatty alcohols bound in the wax esters.

The sterol fraction was relatively small, with a total of 5.946 mg/g and 0.273 mg/g for wax A and B, respectively. ß-Sitosterol was the major compound in both waxes, a high-value compound that has been found to lower cholesterol and reduce the risk of certain cancers when used in medicine [30]. Stigmasterol was detected in lower amounts than ß-sitosterol and cholesterol was detected in wax A only, but at a very low level of 0.0003 mg/g and could therefore be assumed to be negligible. Although these components have high commercial value, the low levels in which they are present in the Cannabis wax by-product would likely not be commercially worthwhile to recover.

The terpenoid fraction composed largely of monoterpenes with a few terpenoids and sesquiterpenoids also reported in detectable quantities, and the chromatogram for this analysis is provided in Fig. 5. The total fraction was reported as 0.954 µg/g for wax A and 1.519 µg/g for wax B. The major monoterpenoid compounds were α-pinene, ß-pinene, α-phellandrene, and limonene. Myrcene, a common monoterpenoid found in Cannabis, was also detected in lesser amounts. In the terpenoid fraction the major compounds were linalool, α-eudesmol, and eudesm-4(14)-en-11-ol. A concentration of 0.061 µg/g and 0.114 µg/g for wax A and B, respectively, was reported for (-)-trans-caryophyllene, another common terpenoid in Cannabis. There was a significant difference in the concentrations of the terpenoid fraction when compared to a previous study, where it was reported as 241.1–302.2 mg/g [7]. The major terpenoids reported previously were p-cymene (33.2–51.5 mg/g) and (-)-trans-caryophyllene (59.0–72.2 mg/g) [7], both of which were only quantified in trace amounts in this work. The reasons for the differences in terpenoid amount and profile between the current and prior work are not apparent, but may be because the terpenoid profile of Cannabis is strongly affected by the Cannabis strain [31] or due to the different industrial and sample preparation prior to wax analysis.

Fig. 5
figure 5

Overlayed chromatograms generated from the terpene analysis, with the quantified compounds and respective peaks labelled

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Another major compound class that is commonly found in plant waxes are wax esters, products of an esterification reaction between a fatty acid and a fatty alcohol. These compounds are typically very large organic molecules, with carbon chain lengths ranging between C40 and C54, and due to their sizes are difficult to analyse using GC, relating to column elution and limited volatility. These compounds can be accounted for through analysis of both derivatised and underivatised samples from fatty alcohol and fatty acid analysis [32]. A previous study that investigated the composition of a lipophilic extract from hemp reported the wax ester fraction to be up to 11.7% (w/w) of this extract [33], and therefore, it was attempted to account for the wax esters through the fatty acid and fatty alcohol analysis. During the fatty alcohol analysis, saponification of the samples resulted in the transesterification of the wax esters and the release of the fatty alcohol. The free fatty alcohols as present in the wax, along with the fatty alcohols from the wax esters, were then further derivatised and quantified as a total fatty alcohol fraction. During the fatty acid analysis, derivatisation of the fatty acids to their methyl ester forms was also a form of transesterification, resulting in the release and derivatisation of the fatty acid chain from the wax ester. Therefore, the wax esters were indirectly accounted for through the release and quantification of the respective fatty acids and fatty alcohols through.

Whilst a significant portion of these waxes have been positively identified, a fraction remains unidentified. A possible explanation regarding the unidentified mass can be attributed to abundant compounds that fell beyond the methods of analysis. These could include various fatty alcohols and fatty acids for which chemical standards were not acquired and therefore were not quantified. Another compound class that was not investigated was aldehydes, which have been reported as present in substantial amounts in other plant waxes [32,33,34,35]. As the wax by-product was removed from the initial ethanol extract though filtration, plant material from the raw Cannabis entrained in the initial extraction likely also formed part of the removed wax. This insoluble material was assigned to the ash content fraction and was found to be 30.2 mg/g for wax A and 51 mg/g for wax B.

From evaluation of the results generated by this work and literature, it is evident that the composition of the waxes is dependent on upstream processing, specifically the type of primary extraction done and solvent evaporation, as well as the source biomass and strain of C. sativa used. Major differences were reported between the composition of wax A and wax B due to difference in processing and strains used. It is therefore clear that there will be inherent variability in the composition of wax by-products from C. sativa, highlighting the importance of establishing robust analysis methods, and of conducting and publishing this analysis on various samples, so that sensible ranges can be established.

4 Conclusions

The work demonstrates a combination of GC–MS and HPLC methods to determine the composition of two wax by-products from commercial Cannabis processing. The major finding of this study was that a significantly large fraction of cannabinoids was entrained in the wax by-product, determined to be 509.3 mg/g for wax A and 392.6 mg/g for wax B, indicating a prominent loss of value during the initial stages of commercial Cannabis processing. This fraction was also determined to be the most abundant fraction in both these waxes. From both research and economic perspectives, it would be advantageous to recover the cannabinoids from wax and prevent the loss of these valuable components. This would furthermore provide a potential valorisation application for the wax by-products, which is currently not described in literature. The characterisation also identified large amounts of fatty acids and n-alkanes, which have found application in cosmetics where they serve as the base or a binder in numerous products. Refined Cannabis wax can potentially also find application in the cosmetics field based on this compositional result. When compared with current literature, significantly different values were reported for both the fatty acid and terpenoid fractions, potentially due to the improved method used in this work. Only trace amounts were reported for the fatty acid fraction in literature, but these were found to be the second most abundant fraction for wax A in this work.