This high-throughput method achieves the lowest published LLOQ's for fluticasone propinate (0.1 pg/mL) and salmeterol (0.05 pg/mL).
Fluticasone and Salmeterol are two inhaled compounds that are often co-administered in the treatment of asthma and COPD. Fluticasone is a synthetic trifluorinated glucocorticoid receptor agonist with antiallergic, anti-inflammatory and antipruritic effects. Salmeterol is a highly selective, long-acting beta-2 adrenergic agonist with bronchodilatory activity. Both compounds were designed to act on the lungs and airways with limited to zero systemic exposure. As a result, their circulating levels are extremely low with peak concentrations of sub pg/mL, thus requiring a very high sensitivity assay is to detect these compounds. Currently published methods are able to quantify fluticasone at 0.2 pg/mL. In this poster, we present an optimized method which allows for quantification of fluticasone propionate as low as 0.1 pg/mL.
Human plasma is spiked to generate a calibration curve and quality control samples from 0.1-10 pg/mL and 0.05-5 pg/mL for Fluticasone propionate and Salmeterol respectively. 400 uL of each point on the calibration curve, QC's and samples is transferred to a 2 mL 96-well plate and pre-treated with equal volumes of an organo-aqueous solution containing Zinc sulphate and ammonium hydroxide. Analytes of interest are then extracted using Oasis HLB u-Elution plates. Analytical separation is achieved using a UPLC system and a C18 column in less than 5 minutes and detected using MRM mode on a tandem quadrupole mass spectrometer using transitions 501.3 > 293.3 and 416.4 > 232.2 for fluticasone propionate and salmeterol respectively.
Preliminary Data or Plenary Speakers Abstract
Use of Oasis HLB u-Elution plates for sample processing followed by carefully fine-tuned wash and elution steps allows for rapid extraction of analytes of interest with low matrix interference and increased signal to noise. Use of UPLC system with BEH C18 columns with small particle size and optimized chromatography results in excellent separation of the analytes of interest achieving peak widths of < 6 seconds in under 5 minutes providing high throuput.
Using the method described above, we can accurately quantify fluticasone propionate and salmeterol from human plasma at LLOQ's of 0.1 and 0.05 pg/mL respectively. The calibration curve is linear for fluticasone propionate from 0.1-10 pg/mL and for salmeterol from 0.05-5 pg/mL, with r2 > 0.99 for both analytes. The lowest point on the calibration curve for both analytes has inter and intra day accuracy and precision of <20% across multiple days. For all other points on the calibration curve and QC's the inter and intra day accuracy and precision is <15%. This assay performance meets the FDA bionalytical method validation guidelines and is suitable for use in any high-throughput regulated/non-regulated laboratory.