Determination of drug plasma protein binding by solid phase microextraction

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Abstract

The plasma protein binding of drugs has been shown to have significant effects on the quantitative relationship between clinical pharmacokinetics and pharmacodynamics. In many clinical situations, measurement of the total drug concentration does not provide the needed information concerning the unbound fraction of drug in plasma, which is available for pharmacodynamic action. Therefore, the accurate determination of unbound plasma drug concentrations is important in understanding drug action. Many methodologies exist for determining the extent of plasma protein binding, but different methods produce a rather wide range of results for the same compound at the same concentration level. The solid phase microextraction (SPME) method reported in the present study attempts to eliminate many experimental variables that could lead to the lack of reproducibility, such as the variable content of organic solvent or ionic strength in plasma, pH shifts, and volume shifts. Five well-known drugs were chosen to study plasma protein binding: ibuprofen, warfarin, verapamil, propranolol, and caffeine, with high, intermediate and low binding properties. Dilution of plasma with isotonic PBS or incubation with 10% CO2 in the atmosphere was found to compensate for changes in pH during incubation. The data obtained using these pH-controlled methods correlate well with the average values of plasma protein binding found in the literature. SPME, which uses an extraction phase that dissolves or adsorbs the drug of interest and rejects proteins, overcomes several limitations of currently available techniques and is a thermodynamically sound method, since the measurements are always performed at equilibrium. Compared to other methods, SPME offers several advantages: small sample size, short analysis time, possibility to automate, and ability to directly study complex samples.

Section snippets

INTRODUCTION

Determining the amount of drug binding to plasma proteins is an essential step in both drug discovery and in clinical phases of drug development. Binding of drugs to plasma proteins is important in understanding the pharmacokinetics and pharmacodynamic relationship of a drug.1., 2., 3. Therefore, plasma protein binding (PPB) is normally recognized as an important factor in assessing drug disposition, efficacy, and safety.4 In the early drug development stage, the knowledge of drug protein

Chemicals and Reagents

Verapamil (99%), propranolol (99%), warfarin (98%), ibuprofen (99%), lithium perchlorate (95%), pyrrole (98%), PBS pouches, and acetic acid were purchased from Sigma (Mississauga, ON, Canada); ammonium acetate, acetone, and nitric acid were obtained from BDH, Inc. (Toronto, ON, Canada); caffeine (99%) was obtained from Aldrich (Mississauga, ON, Canada); diazepam standard (1 mg/mL in methanol, 98%) was purchased from Cerilliant (Austin, TX); HPLC grade acetonitrile and methanol were purchased

EXPERIMENTAL RESULTS AND DISCUSSION

Experiments were carried out in 2 mL vials with polypropylene inserts. For increased throughput, the inserts can be arranged in standard 96-well format. After the inserts were filled with sample or desorption solution, the SPME fibers were introduced through the vial septum and the whole assembly was incubated and/or shaken.

Five well-studied drugs (ibuprofen, warfarin, verapamil, propranolol, and caffeine) with a wide range of physicochemical properties were selected for evaluating the

CONCLUSIONS

Due to the important clinical implications of plasma protein binding data and its role in characterizing a drug's behavior and proper dosing, there is an increasing need to make this measurement as early as possible in the discovery process in order to understand drug disposition and to optimize individual drug therapy.

This study details a new method for determining the value of PPB based on the partitioning of a drug between an SPME fiber and plasma proteins. The suitability of this method was

Acknowledgements

Authors gratefully acknowledge the financial support of Millennium Pharmaceuticals (Cambridge, MA) and the Natural Sciences and Engineering Research Council of Canada.

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