Research Articles
Stabilization and HPLC Analysis of Betamethasone Sodium Phosphate in Plasma

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Abstract

The analysis of corticosteroid prodrugs in pharmacokinetic (PK) studies poses the risk of overestimation of corticosteroid concentrations due to in vitro hydrolysis of prodrugs after sample collection. This study tests the effectiveness of enzyme inhibitors as stabilizers for betamethasone sodium phosphate (BSP) in pregnant sheep plasma samples collected during PK studies with betamethasone (BET) and provides simultaneous high-performance liquid chromatography analysis of BSP and BET. A rapid, sensitive, and specific ion-paired reversed-phase high-performance liquid chromatography assay for simultaneous measurement of BET and BSP in plasma was developed. This assay was used for analyzing samples from an in vitro prodrug hydrolysis study. Enzyme inhibitors tested were sodium arsenate (Na2HAsO4) and ethylenediaminetetraacetic acid. The BSP was administered intramuscularly to three pregnant sheep to assess in vivo PK. Samples were split with part treated with Na2HAsO4 and part left natural. In vitro hydrolysis of BSP in plasma to BET could be completely inhibited by Na2HAsO4, but not by ethylenediaminetetraacetic acid. The PK study showed lower concentrations of BET in samples with Na2HAsO4 compared with natural samples. This study demonstrates that artifacts in PK profiles of corticosteroids due to in vitro prodrug hydrolysis can be prevented by sample treatment with enzyme inhibitors. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association

Section snippets

INTRODUCTION

Administration of corticosteroids via the parenteral route for therapeutic purposes poses problems because of their limited aqueous solubility. To overcome this problem, corticosteroids have often been formulated as water-soluble hemisuccinate or phosphate ester prodrugs. These prodrugs are converted to the active steroid in vivo and the conversion is thought to occur rapidly and completely. Betamethasone (BET) in the form of its phosphate ester sodium salt is used in perinatal medicine.

Liquid Chromatography

The high-performance liquid chromatography (HPLC) system consists of a Waters model 510 pump, a Waters intelligent sample processor (model 717), and a model 486 Waters fixed wavelength UV detector (Waters Associates, Milford, MA). Analyte peaks are integrated using a Hewlett Packard (Avondale, PA) 3392A integrator. Separations are achieved using an Alltech Adsorbosphere HS (Alltech Associates, Inc., Deerfield, IL) C18 7 μm column (150 × 4.6 mm) attached to an Upchurch Scientific (Oak Harbor, WA)

Assay Characteristics and Validation

Published HPLC methods for measuring corticosteroids and their phosphate esters in plasma samples involve complex extraction procedures and utilize large sample volumes.3,7 Furthermore, determination of the prodrug requires either a second estimation step involving indirect estimation by hydrolysis to the steroid8 or a chromatographic method that makes use of highly acidic conditions to allow extraction and retention of the highly ionized phosphate prodrug.9 To circumvent these problems, an

Acknowledgements

The authors appreciate the provision of fresh blank sheep blood samples from the laboratory of Dr. Daniel Swartz of the University at Buffalo. This work was supported by grant GM24211 from the National Institutes of Health.

REFERENCES (12)

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    Both esters are expected to be hydrolyzed in vivo to the active glucocorticoid BET [3]. Several analytical techniques have been published for the analysis of BET in different matrices, e.g., high performance liquid chromatography (HPLC) [3–5], gas chromatography with mass detection (GC–MS) [6,7] or liquid chromatography with mass detection (LC–MS/MS) [8–19]. The mentioned methods showed low sensitivity, e.g., 10, 50 and 300 ng/ml [3–5], implicated derivatization [6], were not fully validated or were not applicable to clinical or pharmacokinetic (PK) studies in human [12–19].

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    Potassium fluoride was added and the plasma was frozen until analysis. The azide and fluoride were added to prevent the Beta-PO4 and Beta-Ac prodrugs from enzymatic cleavage to free Beta.14,15 Analyses for Beta, Beta-PO4, and Beta-Ac were performed by mass spectroscopic techniques by Analpharm, Quebec, Canada.

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    Although the different preparations of Beta will have effects while the drug is in the circulation, this corticosteroid binds tightly to receptors and can have prolonged effects that extend well beyond plasma availability. A further complication for the interpretation of plasma levels of Beta is the presence of prodrug (Beta-PO4 or Beta-Ac) in plasma that may be hydrolyzed before assay, which will elevate plasma Beta levels artificially.20 The link between plasma corticosteroid levels and lung maturational responses in the fetus remains poorly characterized.

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