Automatically faster SPME

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Ezine

  • Published: Sep 22, 2008
  • Author: Steve Down
  • Channels: Sample Preparation
thumbnail image: Automatically faster SPME

SPME is such a simple concept but when it was introduced by Janus Pawliszyn in 1990, it revolutionised sample extraction. Coat a fibre with an absorbent material and position it in the sample, be it a gas, liquid or solid. Alternatively, place it in the headspace above the sample in a closed container to absorb the emitted volatiles. The analytes are analysed by inserting the fibre into the injection port of a gas or liquid chromatograph and heating.

The simplicity is reinforced by the versatility in being able to sample volatile compounds from different matrices and select the absorbent material to suit the type of analyte. So successful was the technique that it has since spawned many derivatives such as in-tube SPME, solid-phase aroma concentrate extraction (SPACE) and stir bar sorptive extraction (SBSE). Yet, despite its undoubted popularity, SPME suffers from several major drawbacks.

First of all, it is difficult to replicate the exact timings during the extraction and desorption steps and to reproduce the position of the fibre during extraction. In addition, it is essentially a low-throughput technique. These shortcomings would benefit from automation and this potential solution was addressed by the Pawliszyn group in 2007 when they reported on preliminary attempts at automation.

They concluded that multifibre SPME using hollow-membrane polydimethylsiloxane (PDMS) coatings combined with orbital shaking was the best route to take with the standard 96-well format commonly used in labs. Now, that work has been extended to produce a fully automated SPME-LC/MS/MS system that was optimised for the analysis of drugs in complex matrices such as whole blood. The details were published in Analytical Chemistry by Pawliszyn, Dajana Vuckovic and Erasmus Cudjoe from the University of Waterloo, with Dietmar Hein from PAS Technology, Magdala, Germany.

One of the first problems to overcome was the performance of the PDMS fibre, which had been chosen for its robustness, reproducibility and low cost. In the 96-well-plate system, the inter-fibre reproducibility was satisfactory but the intra-fibre reproducibility was unacceptably high at more than 10%. The kinetics of extraction and desorption were also too slow. The poor reproducibility was attributed to differences in evaporative losses from different wells and changes in temperature due to the long extraction and desorption times required.

To find a replacement, a panel of five types of coated fibres was tested on four common benzodiazepine drugs. The best performance came from a commercial fibre based on RP-amide C16-coated silica particles. It gave the best intra-fibre reproducibility, lowering the value from 9-19% for PDMS to 3-11%, and the fibres showed no loss of coating on repeated use.

The team constructed a 96-fibre SPME device which was attached to one arm of a commercial three-arm robotic autosampler. The second arm held a nitrogen feed for aiding solvent evaporation and analyte concentration. The final arm was fitted with a microsyringe for dispensing solvents, wash solutions and internal standards and for injecting the sample into the HPLC port. The samples were in a 96-well plate positioned on an orbital agitator.

This set up allows the whole operation to be automated. However, in the current application to drugs in blood, the desorption volume of 800-1000 µL was too large for the autosampler so it was dispensed manually or by an automated well microplate reagent dispenser. The accuracy and precision of the autodispenser was equivalent to manual pipetting but allowed time and labour savings.

The system was fully optimised, down to selection of the calibration method (internal standard) and the performance of the agitator. It led to an absolute recovery of 30% for diazepam, which is regarded as excellent for an SPME method where values below 10% are common. Equilibration times were low at 30 minutes, compared to 20 hours with PDMS fibres, and the low carryover of 1-2% was completely eliminated by introducing a preconditioning step.

For diazepam, lorazepam, nordiazepam and oxazepam in whole blood, the preparation of 96 samples was complete within 100 minutes, which is the highest published throughput for any SPME technique. It permits the analysis of about 1200 samples/day. Combined with LC/MS/MS quantification, the method met US FDA validation requirements, achieving limits of quantification of 4 ng/mL.

The speed of the new system is about the same as those of automated SPE and liquid-liquid extraction but it requires no sample pretreatment and is cheaper. It will be applicable to other complex matrices such as tissue homogenates.

Pawliszyn declared that the group are now seeking to improve the system by developing thinner coatings which do not require preconditioning and by exploiting thin film microextraction to give higher recoveries.


The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

J_Pawliszyn
Janus Pawliszyn 

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