Application of new technology : MEPS and LC-MS/MS for determination of therapeutic drugs

Bioanalysis most often requires an extraction procedure to isolate the target compounds from a complex matrix. The aim of this work was to evaluate microextraction in packed syringe (MEPS) performance as a sample preparation technique by developing new analytical methods for different categories of compounds utilizing MEPS in combination with LC-MS/MS. The overall goal was to provide high throughput methods that can offer automation, on-line coupling to mass spectrometry and short sample preparation time.

MEPS is a new sample preparation method representing a miniaturization of solid phase extraction (SPE) technique that can be fully automated. Sample preparation is performed on the packed bed. Less sorbent material is used with MEPS; about 1 mg of sorbent is inserted in to a syringe needle (100-250 μL) as a plug and thus less solvents are needed. Different categories of compounds have been used to evaluate the performance of MEPS on-line with LC and tandem mass spectrometry (MS/MS), cytostatic, local anaesthetics, immunosuppressive drugs and one anaesthetic.

In study I, the first on-line quantification method for cyclophosphamide was developed and in study II it was the first quantification method for cyclophosphamide from whole blood. This method was applied to test tail vein sampling method from mice by studying the cyclophosphamide pharmacokinetic in mice. In study III, local anaesthetics were quantified from whole blood for the first time, using only 25 μL sample volume. In study IV an on-line method for immunosuppressive drugs was developed which can be implemented in clinical laboratories. Remifentanil in study V was quantified from whole blood utilizing only 20 μL sample volume which might facilitate pharmacokinetic studies on pediatric patients.

Full method validation has been performed according to the FDA guidelines. Sensitive methods have been obtained with small sample volume. The accuracy and precision results were in agreement with the acceptable ranges. Matrix effect was investigated and found to be reduced comparing to other sample preparation methods. Method comparison was done with other well established sample preparation techniques.

Further work on MEPS is recommended regarding applying this technique to more drugs and their metabolites, exploring another applications area such as environmental and food analysis, and modifying the device in a way that can reduce clogging and speed up the extraction process.

List of scientific papers

I. Said Rana, Hassan Zuzan, Hassan Moustapha, Abdel-Rehim, Mohamed (2008). "Rapid and Sensitive Method for Determination of Cyclophosphamide in Patients Plasma Samples Utilizing Microextraction by Packed Sorbent Online with Liquid Chromatography-Tandem Mass Spectrometry (MEPS-LC-MS/MS)" Journal of Liquid Chromatography & Related Technologies 31: 683 - 694

II. Mohamed Kamel, Rana Said, Aziza Elbeqqali, Fatma Bassyouni, Mohamed Abdel-Rehim (2009). "On-Line Determination of Cyclophosphamide in Blood Samples Utilizing Microextraction by Packed Sorbent and Liquid Chromatography Tandem Mass Spectrometry (MEPS-LC-MS/MS)." The Open Spectroscopy Journal 3: 26-30

III. Said Rana, Kamel Mohamed, El-Beqqali Aziza, Abdel-Rehim Mohamed (2010). "Microextraction by packed sorbent for LCMS/MS determination of drugs in whole blood samples. " Bioanalysis 2: 197-205

IV. Determination of remifentanil in human plasma by liquid chromatography and tandem spectrometry utilizing micro extraction in packed syringe (MEPS) as sample preparation (2010). "Determination of four immunosuppressive drugs in whole blood using MEPS and LC-MS/MS allowing automated sample work-up and analysis." (Submitted)

V. Rana Said, Anton Pohanka, Olof Beck, Maria Andersson, Mohamed Abdel-Rehim (2010). "Determination of remifentanil in human plasma by liquid chromatography and tandem spectrometry utilizing micro extraction in packed syringe (MEPS) as sample preparation" (Manuscript)