EBF Hatching Brussels 12-13 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)

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.

Adsorption of analyte onto the coated

fibre Sampling

Desorption of analyte from

the solid phase

Extraction

Detection and

quantification of analyte

LC/MS/MS

Why are GSK Interested in SPME?

Dried Blood Spots

Microsampling

DMPD SPME WP-CM

Advantages for ...

! 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

! 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

Pre-Clinical / Clinical The Analyst

Current SPME Approach

SPME Mode

Direct in vivo SPME

In–Tube SPME

SPME fibre at Sampling site

SPME fibre at Analytical site

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.

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

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).

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 1-1000ng/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.

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.

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.

GSK - Validating The Technique

In Vitro

In Vivo

Regulatory Perspective

Automation

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

References

! Pawliszyn J. SPME commercial devices and fiber coatings. In: Handbook of SPME. Chemical Industry Press, Beijing, China, 86–115 (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, 3367–3375 (2011).

! Musteata FM, Musteata ML, Pawliszyn J. Fast in vivo microextraction: a new tool for clinical analysis,

Clin. Chem. 52(4), 708–715 (2006).

! Vuckovic D, Zhang X, Cudjoe E, Pawliszyn J, Solid-phase microextraction in bioanalysis: New devices and directions, Journal of Chromatography A 1217, 4041-4060 (2010).

! Lord H, Grant R, Walles M, Incledon B, Fahie B, Pawliszyn J. Development and evaluation of a solid-phase microextraction probe for in vivo pharmacokinetic studies. J. Anal. Chem. 75, 5103–5115 (2003).

Questions