Microdose clinical trial: Quantitative determination of fexofenadine in human plasma using liquid chromatography/electrospray ionization tandem mass spectrometry

https://doi.org/10.1016/j.jchromb.2007.08.011Get rights and content

Abstract

A sample treatment procedure and high-sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for quantitative determination of fexofenadine in human plasma was developed for a microdose clinical trial with a cold drug, i.e., a non-radioisotope-labeled drug. Fexofenadine and terfenadine, as internal standard, were extracted from plasma samples using a 96-well solid-phase extraction plate (Oasis HLB). Quantitation was performed on an ACQUITY UPLC system and an API 5000 mass spectrometer by multiple reaction monitoring. Chromatographic separation was achieved on an XBridge C18 column (100 mm × 2.1 mm i.d., particle size 3.5 μm) using acetonitrile/2 mM ammonium acetate (91:9, v/v) as the mobile phase at a flow rate of 0.6 ml/min. The analytical method was validated in accordance with the FDA guideline for validation of bioanalytical methods. The calibration curve was linear in the range of 10–1000 pg/ml using 200 μl of plasma. Analytical method validation for the clinical dose, for which the calibration curve was linear in the range of 1–500 ng/ml using 20 μl of plasma, was also conducted. Each method was successfully applied for making determinations in plasma using LC/ESI-MS/MS after administration of a microdose (100 μg solution) and a clinical dose (60 mg dose) in eight healthy volunteers.

Introduction

The development of new drugs has been stagnated in the last decade, the cause of which has been neither prohibitive costs nor lack of industriousness. Research and development budgets in pharmaceutical companies are increasing beyond their cost-cutting efforts. A major problem is how misleading extrapolation of absorption, distribution, metabolism and excretion (ADME) data from animals to dosing in humans can be. In fact, of the new drugs under development, up to 40% are dropped at Phase I even though promising ADME data were obtained in animals [1]. Pharmacokinetic (PK) data in humans prior to a traditional first-in-human study (Phase I study) is highly advantageous in selecting candidate drugs. These factors gave rise to the concept of a microdosing study. The aim of the microdosing study is to obtain human PK data for a single or multiple drug candidates prior to Phase I studies with the intention of reducing the dropout rates in Phase I development. A position paper released in 2003 by the European Agency for the Evaluation of Medicinal Products defined the amounts for microdosing in human to be less than 1/100th of the therapeutic dose predicted from animal and in vitro models, while also not exceeding 100 μg [2]. Thus, analysis of drug concentration in human plasma requires the use of ultra-sensitive instruments such as that used in accelerator mass spectrometry (AMS) [3]. Among the merits of AMS is its ability to obtain sensitivity in the range of 10−21 to 10−18 mol [4], thus enabling the instrument to directly detect 14C atoms which are 10−12 less existent on earth than 12C. Subsequently, 0.5–100 μg amounts are sufficient for dosing in human and the volume of blood collection can be reduced from the beginning. On the other hand, there are several disadvantages. The dosing of 14C-labeled drugs, or “hot drugs”, is absolutely essential and the syntheses involved are both costly and time consuming. It puts financial burden on the pharmaceutical company and can cause temporary suspension at development stages. Additionally, the process for sample treatment takes extra man-hours and a spacious facility is needed to install AMS equipment. The AMS equipment itself is expensive and its analysis is also costly compared with that of analysis using liquid chromatography–tandem mass spectrometry (LC/MS/MS) [4].

In contrast to this is LC/electrospray ionization (ESI)-MS/MS, which has been used most frequently in the analysis of drugs in human matrices. ESI makes it possible to ionize almost all polar compounds [5]. The most remarkable aspect is that determination of concentration in plasma can be done after administration of a non-labeled drug (cold drug). We have already reported on actual examples of determination of drugs at a picogram per milliliter level in human plasma using LC/ESI-MS/MS [6].

In this report, we show that we have developed a sample treatment procedure and analytical methods for a microdose clinical trial using LC/ESI-MS/MS after administration of a cold drug, having been fexofenadine in this case, and studied how the determination of drug concentration using LC/ESI-MS/MS is useful and effective in a microdose study. As a subject of comparison, a clinical dose study was also conducted.

Section snippets

Materials and reagents

Fexofenadine hydrochloride and terfenadine hydrochloride were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). HPLC grade acetonitrile and methanol, and analytical grade ammonium acetate and acetic acid were purchased from Wako Pure Chemical Industries, Ltd. (Kyoto, Japan). The solid-phase extraction (SPE) plate, Oasis HLB (10 mg/well) was supplied by Waters Corporation (Milford, MA, USA).

Preparation of stock and standard solutions

Fexofenadine stock solution (100 μg/ml) and terfenadine stock solution (100 μg/ml) for the internal

LC/ESI-MS/MS analysis

We selected an ACQUITY UPLC system and an XBridge C18 analytical column with the aim of greater high-through-put analysis because these have been designed to tolerate high pressure [8]. We attempted to obtain a sharper peak and shorter retention time for fexofenadine on MRM chromatograms. This was accomplished by increasing the organic solvent content and flow rate of the mobile phase by use of a column which is packed with particles synthesized by ethylene-bridged hybrids technology [9]. This

Conclusions

The sample treatment procedure and high-sensitive LC/ESI-MS/MS method we developed for quantitative determination of fexofenadine in human plasma were applied to the analysis of fexofenadine in human plasma for a microdose clinical study. We concluded that the quantitative determination of drug at a picogram per milliliter order in human plasma using LC/ESI-MS/MS was useful and effective for a microdosing study. There is the potential that analysis using LC/ESI-MS/MS could be advanced to being

References (13)

  • J.B. Fenn

    J. Am. Soc. Mass Spectrom.

    (1993)
  • M.I. Churchwell et al.

    J. Chromatogr. B

    (2005)
  • I. Fu et al.

    J. Pharm. BioMed. Anal.

    (2004)
  • J.A. Dimasi

    Clin. Pharmacol. Ther.

    (2001)
  • European Agency for the Evaluation of Medicines for Human Use (EMEA), Position Paper on the Nonclinical Safety Studies...
  • K.W. Turteltaub et al.

    Curr. Pharm. Des.

    (2000)
There are more references available in the full text version of this article.

Cited by (0)

View full text