Microextractions doubling up: Two techniques combined for drugs in biological fluids

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  • Published: Jun 13, 2011
  • Author: Steve Down
  • Channels: Sample Preparation
thumbnail image: Microextractions doubling up: Two techniques combined for drugs in biological fluids

Microextraction combination

Microextraction techniques were devised largely to minimise or eliminate the use of solvents, providing less expensive and greener procedures, and to combine extraction and preconcentration and, in some cases, sample introduction to the analytical instrumentation. Since the introduction of the first recognised microextraction method, solid-phase microextraction, many more different types have been conceived, each with their own little twist.

A team of Iranian scientists has now provided a further novel technique which combines the advantages of two separate microextraction methods. Homeira Ebrahimzadeh and colleagues from Shahid Beheshti University and Payame Noor University, both in Tehran, developed the novel combination for the extraction of drugs from biological solutions.

The first of the two techniques was ultrasound-assisted emulsification microextraction (USAEME), in which a small volume of organic solvent is added to the sample solution. Ultrasonication is applied to aid formation of an emulsion to enhance mass transfer of the analytes and the solvent is recovered by centrifugation which separates the two phases.

The principal disadvantage of USAEME is the use of toxic solvents such as carbon tetrachloride, chloroform and chlorobenzene.

The second technique was liquid-phase microextraction with solidification of a floating organic droplet (LPME-SFO). Here, a droplet of the extractant liquid is suspended in the sample solution or floated on the surface, with stirring to maximise contact with the droplet. Then, the sample holder is transferred to an ice bath to freeze the droplet which is easily removed and melted for analysis.

The main drawback with this method, say the researchers, is the extraction time, which can be on the long side.

The combination of the two techniques, which was named ultrasound-assisted emulsification microextraction based on solidification of floating organic droplet, was described in the Journal of Separation Science.

Fine tuning

The process was optimised for the extraction of the three antidepressant drugs citalopram (CIT), fluoxetine (FLX) and venlafaxine (VLF) from standard solutions in water, before application to human urine and plasma. Drug concentrations in the extracts were determined by HPLC with UV detection at 225 nm.

In the first instance, the extracting solvent was selected from a panel of six chosen for their low aqueous solubility, the ability to form an emulsion after ultrasonication, and their melting points, which were in the range 10-30°C. The best one was 1-undecanol, which outperformed hexadecane, cyclohexanol, undecane, heptadecane and 1-dodecanol by at least 100%. Based on a sample size of 5 mL, the optimum volume of 1-undecanol was 30 µL.

The extractant was added to the sample and ultrasonicated, producing a turbid solution. Following centrifugation, the droplets of 1-undecanol collected on the surface of the aqueous solution and were solidified using an ice bath. They were removed, melted and mixed with HPLC mobile phase for analysis.

The optimum ultrasound extraction time was 20 minutes and the other optimised parameters included the ionic strength, which was adjusted by adding sodium chloride and the pH, adjusted with potassium hydroxide.

Under these conditions, the preconcentration factors for CIT, FLX and VLF in water were 316, 174 and 201, respectively, leading to HPLC detection limits of 3 µg/L.

Getting more from plasma and urine

The procedure was applied to the extraction of the three drugs from plasma and urine samples obtained from a local clinic for patients undergoing treatment for depression.

The recoveries from plasma were >91.4, >92.4 and >95.5% for CIT, FLX and VLF, respectively and the corresponding values for urine were 105, 109 and 93.6%. The interday and intraday relative standard deviations were also good, all values being less than 10.4% for CIT and VLF and less than 14% for FLX.

The procedure had wider linear calibration ranges and greater preconcentration factors than four alternative published procedures for the drugs, including SPME-GC/MS, SPE-LC/MS/MS and liquid-liquid extraction followed by GC-NPD.

So, the combination of USAEME with LPME-SFO allows the extraction and preconcentration of drugs from human biological fluids without the use of toxic solvents and in a relatively fast time. It is simple and inexpensive, providing good analytical figures of merit, and could provide a novel option for analytical labs.

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

Antidepressant drugs in plasma and urine have been successfully extracted by a combination of two microextraction techniques involving ultrasound-assisted emulsification and solidification of a floating organic droplet, to give high preconcentration factors 

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