58 February / March 2018
Chromatography Today Help Desk The Use of Derivatising Reagents
The GC analysis of cannabinoids is routinely performed using derivatising reagents that look to silylate the polar functional groups on the acidic form of the cannabinoid, thereby rendering the molecule more stable in the injector. This ensures both the acidic and neutral forms of the cannabis can be accurately quantified. This process also has the advantage that a non-polar column can be used in the analysis, making the chromatography more robust. Two commonly used reagents for derivatisation are N-Methyltrimethylsilyltrifluoroacetamide (MSTFA) and Bis(trimethylsilyl)trifluoroacetamide (BSTFA), however there are a vast array of derivatising agents used in both LC and GC.
The derivatising reagent can be used to alter the molecule into a form that is more amenable to the mode of chromatography being utilised, or to make the analyte more sensitive to the detector employed. The reaction undertaken by the derivatising reagent should be selective to a certain functional moiety and should also reach as close to 100% conversion to the final product as possible, with little or no side reactions occurring. It is also critical that the reagent as well as the final derivatised molecule be stable to the environment that they will be exposed to, whether that be the solvents, pressure or temperature of the resulting chromatographic system employed.
Derivatisation reactions used for gas chromatography (GC) fall into three general reaction types, namely:
• Alkylation, which is actually esterification and is the predominant mechanism employed
• Acylation • Silylation
Each of these three processes will make the analyte either more volatile, more stable at elevated temperatures, or more detectable and hence more amenable to the analysis by GC. A classic example of this can be viewed through the derivatisation of the hydroxyl groups within the cannabinoid functionality the more thermally stable tri methyl silylated form of the molecule, thus stopping the conversion of the acid into the neutral form in the injector. The derivatisation to the tri methyl silylated form also results in a more
hydrophobic molecule which delivers superior chromatography on a neutral stationary phase such as a 5% biphenyl.
In addition to choosing the correct derivatising reagent, it is also essential to consider other factors which will affect the overall assay performance. Since the derivatisation process in this example selectively targets the hydroxyl group, it is evident that if the matrix has an abundance of hydroxyl groups present then the efficiency of the derivatisation process will be reduced. This could happen for example if the cannabis being measured is in the presence of sugars, a potentially popular scenario would be a cannabis laden chocolate brownie. If GC was the only approach to the analysis of cannabinoids then this could help reduce the detection, however other orthogonal approaches such as LC-MS ensure that this is not an issue. The sample preparation approach also plays an important role here as it will determine the amount of possible interferants that are left in the sample prior to the derivatisation process.
Caution must be exercised to prevent any water from entering the samples as this will lead to hydrolysis of the derivatisation reagent and could suppress of the targeted analytes from undergoing derivatisation. Thus, when a sample pre-treatment such as SPE is utilised to clean up a a complex matrix such as blood or plasma it is essential that the final elution solvent is completely removed if water is present or an organic final elution solvent is used. In general, evaporating the final SPE eluant down to dryness before the addition of the derivatising reagent will ensure that the water content from the sample is kept to a minimum. While the use of a closed vial during the derivatisation process will keep the water level to a minimum, it is also important that the derivatising reagent is kept dry. To address this concern, the use of fresh reagents will ensure that the quality of the assay is maintained.
For the analysis of cannabis samples, the silylation reaction is driven by a good leaving group, in this case a group with a low basicity: namely chlorine. The reagents that are used to silylate will have the ability to stabilise a negative charge in the transitional state, with little or no back bonding between the leaving group and silicon atom. The mechanism involves the replacement of the active hydrogens (in the case of cannabinoids the hydrogen on the
Figure 1: The general mechanism of silyation using BSTFA.
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