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ANALYSIS OF INHALED GLUCOCORTICOSTEROIDS - EPIMERIC BUDESONIDE AND
FLUTICASONE PROPIONATE IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY
ATMOSPHERIC PRESSURE CHEMICAL IONIZATION TANDEM MASS SPECTROMETRY
YUAN N (Benny) Lia ,BRUCE TATTAM a, KENNETH. F. BROWNa AND JOHN. P. SEALE b
a. Department of Pharmacy, University of Sydney, NSW 2006 Australia
b. Department of Pharmacology, University of Sydney, NSW 2006 Australia
Budesonide and Fluticasone Propionate are two of the leading drugs used in the treatment of asthma andrhinitis, both are topically applied (eg as nasal spray or as an inhalation), with the percentage of thepopulation experiencing asthma or asthma like symptoms these two drugs are excellent aids in theirtherapy's. The clinical data on the patient samples and the method development will be discussed.
API mass spectrometry is the method of choice for glucocorticosteroids, in particular AtmosphericPressure Chemical Ionization (APCI) has high sensitivity for this class of compounds, with this we havedeveloped a highly sensitive and selective quantitative assay to determine plasma concentrations fromhealthy subjects after inhalation of epimeric budesonide (BUD) or fluticasone propionate (FP),respectively. Previous analysis of BUD in thermospray1 or APCI/LCMS2 have been used but had minorproblems. Plasma concentrations were quantified in 144 samples using liquid chromatographyatmospheric pressure chemical ionization tandem mass spectrometry(LC/APCI/MS/MS). The drugs were isolated from human plasma using a C18 solid phase extractioncartridges BUD was acylated with a mixture of 12.5% acetic anhydride and 12.5% triethylamine inacetonitrile to form the 21- derivatives following the solid -phase extraction. The FP did not have to bederivatised and did not undergo that part of the protocol. Deuterium-labelled budesonide acetate wassynthesised and used as the internal standard for both compounds.
The LC method used is able to separate the 22R & 22S epimers of BUD, using 2.1mm ODS column and asimple isocratic elution the sample analysis time is <15min.
Other assays such as radioimmunoassay (RAI) proved to be good but had suffered poor precision( up to 25% CV). RAI for FP is also subject to nonspecific interference and varying cross -reactivity withdrug metabolites and other unrelated compounds, although the antibodies used in the RIA were claimedto be highly specific3.
The assay was linear over the ranges 0.05 -10 ng/ml for BUD and 0.02 - 10 ng/ml for FP, respectively.
The interassay and intraassay relative standard deviations were <14.3% in the assay concentration range.
The structures of BUD and FP are shown below.
1. Lindberg, C., Blomqvist , A. and Paulson, J. Biol. Mass Spectrom., 21 (1992) 5252. Li, Y.N., Tattam, B. N., Brown, K.F., Seale, J.P. J.Chromatography. B 683 (1996) 259-268.
3. Makie, A.E., Ventresca, G.P., Fuller, R.W., Bye, A. Br. J. Clin. Pharmacol. 41 (1996) 539 - 542
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