Tobacco acids reacted in syringe

One of the recently developed microextraction techniques that have been designed to replace large-volume solvent extractions is single drop microextraction (SDME). By definition, it uses one drop of solvent to accomplish the extraction of volatile compounds, by holding the drop in the headspace above the sample, or by immersing the drop in the liquid sample. In the latter case, the drop and the sample should be immiscible liquids, or the drop will be lost.

The drop is generally held at the end of a microsyringe during extraction, then withdrawn into the syringe when extraction is completed. SDME has the advantage of requiring very small volumes of organic solvent, reducing the environmental impact compared with liquid-liquid extractions, and cutting the cost of the procedure.

Since the compounds extracted from the headspace are volatile, one of the commonly used analytical techniques is GC/MS. With judicial selection of the solvent, the drop can be injected directly from the syringe into the inlet. However, not all volatile compounds are suitable for immediate analysis by GC/MS, due to their polar nature, so a derivatisation step may be required.

One solution devised by a group of Chinese scientists is in-drop derivatisation, in which the derivatising agent is dissolved in the extraction solvent before SDME begins. As soon as the compounds are adsorbed into the drop, their derivatisation begins, removing the need for a separate derivatisation step. Once reaction is deemed to be complete, the contents can be injected into the GC/MS system.

The same team began to study the content of volatile organic acids in cigarette tobacco, using SDME for extraction. These straight-chain and branched acids, such as formic, acetic and butyric acid up to heptanoic acid, would need to be derivatised for subsequent GC/MS analysis. The researchers chose the standard, highly efficient agent, bis(trimethylsilyl)trifluoroacetamide (BSTFA), but it is too volatile to remain in the drop for in-drop derivatisation SDME.

So, they adopted a recently developed technique called in-syringe derivatisation in which the reagents could be held in a controlled space. Following SDME, the drop is withdrawn into the microsyringe before a small volume of BSTFA is also drawn in. The plunger is moved gently up and down to encourage mixing and reaction within the syringe.

Optimisation of the method using standard solutions of the acids was described in the Journal of Separation Science by Da Wu, Yunfei Sha and Yichun Zhang from the Shanghai Tobacco (Group) Corporation Technical Center and Jiaoran Meng and Chunhui Deng from Fudan University, Shanghai. The acids were analysed by GC/MS in selected ion monitoring mode.

Of the three preferred solvents for SDME, decane was selected because dodecane and heptadecane interfered with the organic acid derivatives. Using an extraction volume of 1 µL, equilibrium was achieved within 30 s. The optimal conditions for in-syringe derivatisation were a BSTFA volume of 0.8 µL and a reaction time of 3 min at room temperature.

The recoveries of 10 organic acids were 92.2-108.1% and the detection limits ranged from 0.10-3.37 µg/g. Linear calibration curves were obtained and the repeatabilities were less than 15.2%.

The optimised method was applied to the organic acids in cigarette tobacco removed from 7 commercial brands, using hexanoic acid-d11 as an internal standard. All 10 acids were found: formic, acetic, propionic, butyric, isobutyric, isovaleric, valeric, isocaproic, caproic and heptanoic acid.

The proportions of the acids differed from brand to brand but the two most abundant in all cases were formic and acetic acid. The third most abundant acid varied between propionic, butyric, isobutyric and isovaleric acid. The C2-C7 organic acids are important flavour components of tobacco, so this data is important in assessing the odour and flavour of the individual brands.

SDME with in-syringe derivatisation proved to be a simple, rapid and low-cost method using minimal solvent for the extraction and derivatisation of volatile organic acids for subsequent GC/MS analysis. It provides an alternative to in-drop derivatisation for reagents such as BSTFA that are too volatile.

Related links:

* Journal of Separation Science 2010, 33, 212-217: "Determination of volatile organic acids in tobacco by single-drop microextraction with in-syringe derivatization followed by GC-MS"