The Application of SPME; A Multipurpose Micro-Technique for Bioanalysis

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1 EBF Hatching Brussels June 2012 The Application of SPME; A Multipurpose Micro-Technique for Bioanalysis Sheelan Ahmad Bioanalytical Science and Toxicokinetics, PTS DMPK, GlaxoSmithKline, Ware, UK (Sheelan.2.Ahmad@gsk.com)

2 What is SPME?! Solid Phase Micro-Extraction is a sample preparation technique based on an equilibrium process in which the analyte partitions between the SPME coating and the sample matrix.! The amount of analyte extracted by SPME is directly proportional to the unbound concentration of the drug present in a given system. Sampling Adsorption of analyte onto the coated fibre Extraction Desorption of analyte from the solid phase LC/ MS/MS Detection and quantification of analyte

3 Why are GSK Interested in SPME? Dried Blood Spots Microsampling DMPD SPME WP-CM

4 Advantages for... Pre-Clinical / Clinical! No blood withdrawal (Direct immersion SPME approach)! Ethical benefits - maintains the benefits of the 3Rs in animal use Refinement! Elimination / reduction of rodent warming Reduction! Reduction / removal of satellite rodents! Data quality Serial vs composite TK from central study animals! Enables juvenile studies! Enables paediatric studies! Costs Animal numbers Procedures The Analyst! SPME combines Sampling, Sample Preparation and Extraction in one step! Reduced overall number of sample processing steps No sub-aliquoting No centrifugation No sample freezing/thawing! Increased speed and improved efficiency! Simplified sample preparation, reduced blood handling by analytical personnel! Allows effective sample clean up achieving high sensitivity

5 Current SPME Approach SPME Mode Direct in vivo SPME SPME fibre at Sampling site In Tube SPME SPME fibre at Analytical site

6 Direct in vivo SPME! On site sampling! No blood withdrawal! The extraction phase comes into concentration equilibrium with the chemicals in the surrounding sample matrix.

7 Sampling Devices! Metal wire or blade coated with biocompatible polymer Miniaturization of device to minimise tissue damage Biocompatibility of SPME coating to prevent adverse and toxic reactions! Needle-like device housing biocompatible SPME fibre with a fused silica! Non-invasive sampling tools for breath and skin analysis Headspace: SPME fibre protected by inert tubing with a small opening to expose breath to the fibre Skin sampling performed by placing skin sampling patches onto the skin

8 In-Tube (Offline) SPME Small blood sample volumes aliquoted into Micronics. SPME fibre dipped into sample tube for extraction. Same fibre can be used for multiple extractions (>50).

9 Application to Pharmacokinetics! The first in vivo study on the determination of drug concentrations in dog was reported in 2003 (Lord H et al, 2003).! SPME used for in vivo monitoring of circulating blood concentrations of benzodiazepines. The method was used to monitor the pharmacokinetic profiles of diazepam and its metabolites in dogs.! Extraction of drug molecules directly from a peripheral vein eliminating the need to draw blood.! Subsequent quantification by LC/MS/MS.! Method range ng/mL.! SPME probe based on polypyrrole was prepared and evaluated for extraction characteristics.! Drug concentrations in blood evaluated by both direct SPME probe and conventional blood draws for cross-validation.! The results compared favourably with profiles determined by conventional methods.

10 Diazepam pharmacokinetic profile, from three studies on three dogs (n = 9). Blood : in vivo SPME from whole blood, Plasma : conventional analysis, SOF PPY : in vivo on the fibre with PPY probes, SOF PEG : in vivo standard on the fibre with PEG probes.

11 Conclusion! Diazepam and its metabolites were successfully monitored over the course of a full pharmacokinetic study, repeated three times on three beagles.! All of the data sets demonstrate good correlation of reported drug concentrations between SPME probes and conventional blood draws in that the profiles reported agree within experimental error.! The SPME-based method was faster than conventional approaches, interfered minimally with the investigated system, minimized errors associated with sample preparation and limited personnel exposure to hazardous biological samples.

12 GSK - Validating The Technique In Vivo In Vitro Regulatory Perspective Automation

13 Acknowledgements! GSK, PTS, DMPK, Bioanalytical Science and Toxicokinetics Dr. Neil Spooner Dr. Scott Summerfield (UK)! University of Waterloo/Canada Professor Janusz Pawliszyn! University of Hertfordshire Dr. Ute Gerhard Dr. Darragh Murnane! Sigma/Aldrich - Supelco Alan Farnaby Bob Shirey Craig Aurand Klaus Buckendahl Len Sidisky

14 References! Pawliszyn J. SPME commercial devices and fiber coatings. In: Handbook of SPME. Chemical Industry Press, Beijing, China, (2009).! Vuckovic D, de Lannoy I, Gien B et al. In vivo solid-phase microextraction for single rodent pharmacokinetics studies of carbamazepine and carbamazepine-10,11- epoxide in mice. J. Chromatogr. A 1218, (2011).! Musteata FM, Musteata ML, Pawliszyn J. Fast in vivo microextraction: a new tool for clinical analysis, Clin. Chem. 52(4), (2006).! Vuckovic D, Zhang X, Cudjoe E, Pawliszyn J, Solid-phase microextraction in bioanalysis: New devices and directions, Journal of Chromatography A 1217, (2010).! Lord H, Grant R, Walles M, Incledon B, Fahie B, Pawliszyn J. Development and evaluation of a solidphase microextraction probe for in vivo pharmacokinetic studies. J. Anal. Chem. 75, (2003).

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