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Fluticasone furoate (FF) and vilanterol trifenatate (VT) is a widely prescribed combination in the management of asthma and chronic obstructive pulmonary disease. In the present study, two quantitative methods based on HPLC-UV and spectrofluorimetric analysis had been developed and validated for simultaneous estimation of FF and VT in rabbit plasma using baclomethasone as internal standard (ISTD). Analytes and ISTD were separated from plasma using simple step of protein precipitation with acetonitrile. Chromatographic separation was achieved on Spherisorb S5 ODS2 (250 mm × 4.6 mm, 5.0 µm) column using mobile phase that constitute acetonitrile-0.01% glacial acetic acid in water (70:30, v/v) and then detected on a UV detector at 235 nm wavelength. Spectrofluorimetric detection was performed using absorption/emission wavelength (λ abs/em ) of 286/352 nm and 362/407 nm for FF and VT, respectively. For both analytes, linearity ranged from 4-200 ng/mL to 10-200 ng/mL using HPLC-UV and spectrofluorimetric method, respectively. Methods were validated as per FDA recommendations. Statistical analysis revealed that these detection methods are statistically insignificant difference and can be used interchangeably without any bias. Further, these methods were applied in pharmacokinetic study for simultaneous estimation of FF and VT in rabbit plasma., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This paper reports an LC/MS/MS method for analysis of salemeterol and fluticasone propionate in human plasma based on combined SPE-based extraction and separate LC/MS/MS conditions. Previously reported interaction between analytes was confirmed and eliminated by their separation in the sample preparation step to ensure no negative impact on their quantitation. The method was validated per FDA guidelines in the range of 2.5-500 pg/mL for salmeterol and 5-500 pg/mL for fluticasone propionate. The method is suitable for plasma analysis of combined salmeterol/fluticasone formulation without adverse effects of inter-analyte interactions on quantitation.

A new and very sensitive analytical method has been developed and validated to jointly determine the anti-inflammatory drug ciclesonide (CIC), its active principle metabolite M1 (CIC-M1) and fluticasone propionate (FP) in human serum, in the low concentration range from 10 to 1000 pg/mL. This was accomplished by high-performance liquid chromatography and tandem mass spectrometry using atmospheric pressure photo ionisation (HPLC-MS/MS with APPI) using 0.5 mL of serum. Serum was mixed with the internal standards (IS) D11-CIC and D11-CIC-M1 and extracted with diisopropylether. A gradient with acetonitrile (containing 10 mM of acetic acid and 10% of acetone) was used. HPLC-MS/MS of the acetic acid adducts of the analytes was performed in negative mode. The novel aspect of this method is that instead of the dopant being introduced directly into the source by means of an external HPLC pump, it was added to the mobile phase. This provided significantly better sensitivity than the usual method of in-source addition of the dopant, and with no loss in HPLC performance. Sensitivity for the analytes was about four times greater than with either APCI or ESI. Validation was performed in three batches. The inter-batch precision (CV) of the quality control samples in human serum ranged from 4.08% to 6.78% for CIC, from 2.57% to 7.74% for CIC-M1, and from 2.38% to 9.61% for FP. The inter-batch accuracy (with reference to the mean value) of the quality control samples in human serum ranged from 99.3% to 110.0% for CIC, from 101.8% to 104.7% for CIC-M1, and from 100.4% to 101.8% for FP. Calibration data and LLOQ data are also presented in this paper. The analytes were stable in human serum over three freeze/thaw cycles, or for 4h at room temperature, or for at least 18 months when stored at below -20 degrees C. This method was used for quantifying the analytes after inhalation of low-mug amounts of the drugs by patients.

The manufacture of micro-HPLC columns with combined stationary phases, a body of 3.5-microm XTerra-C18 particles, and poly(styrene-divinylbenzene) (PS-DVB) frits is described in detail. The efficiency of the columns was assessed by rapid separation of neutral and acid compound mixtures. Direct analysis of some pharmaceuticals in plasma resulted in lower limits of detection (LOD) for salmeterol xinofate of 12.5 nanograms on-column.

A highly sensitive and selective liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry assay was developed and validated for simultaneous determination of epimeric budesonide (BUD) and fluticasone propionate (FP) in plasma. The drugs were isolated from human plasma using C18 solid-phase extraction cartridges, and epimeric BUD was acetylated with a mixture of 12.5% acetic anhydride and 12.5% triethylamine in acetonitrile to form the 21-acetyl derivatives following the solid-phase extraction. Deuterium-labelled BUD acetate with an isotopic purity >99% was synthesized and used as the internal standard. The assay was linear over the ranges 0.05-10.0 ng/ml for epimeric BUD, and 0.02-4.0 ng/ml for FP. The inter- and intra-day relative standard deviations were <14.3% in the assay concentration range.

A sensitive and selective high-performance liquid chromatographic method for the determination of 6-methylen-androsta-1,4-diene-3,17-dione (exemestane) and its 17-dihydro metabolite in human plasma has been developed. The analytes and internal standard (Norgestrel) were extracted from plasma samples with a methylene chloride-iso-octane mixture; the organic phase was dried and the residue was reconstituted with an acetonitrile-water mixture, then analyzed by reversed-phase liquid chromatography. Quantification was achieved by ultraviolet detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the constituents of human blank plasma was observed. The lower limit of quantification was 10 ng/ml plasma. The suitability of the method for in vivo samples was checked by analysis of plasma samples drawn from healthy male volunteers who had received a 200-mg single oral dose of the test compound.

An highly sensitive and selective high-performance liquid chromatographic method was developed for the determination of epristeride [17 beta-(N-tert.-butyl carboxamido)-androst-3,5-diene-3-carboxylic acid, SK&F 105657], a potent inhibitor of the prostatic steroid 5 alpha-reductase enzyme, in human plasma samples. Epristeride is currently in development for the treatment of benign prostatic hyperplasia. The analytical method involves isolation of epristeride and the internal standard [17 beta-(N,N-diisopropyl carboxamido) estra-1,3,5 (10)-triene-3-carboxylic acid, SK&F 105419] from plasma by solid-phase extraction prior to chromatographic separation on an aminopropyl silica column, using hexane-methylene chloride-2-propanol-acetic acid as the mobile phase, with subsequent ultraviolet absorption detection. The absolute recovery of epristeride from plasma was 90.2 +/- 2.96. The limit of quantification for epristeride was 2.5 ng/ml. Linear response was observed for concentrations of epristeride ranging from 1 to 500 ng/ml plasma. The assay was sufficiently sensitive, accurate and precise to support pharmacokinetic studies in human subjects.