Indacaterol determination in human urine: validation of a liquid-liquid extraction and liquid chromatography-tandem mass spectrometry analytical method.

Background: Indacaterol is a novel once-a-day inhaled ultra-long-acting β2-agonist. Quantitative bioanalysis supports pharmacokinetic and clinical research. The aim of the current work was to validate an in-house developed high performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) analytical method for indacaterol determination in human urine samples.

Methods: A liquid-liquid extraction method has been developed to extract indacaterol from human urine samples using ethyl acetate. Indacaterol dry extract was reconstituted with 200 μL of the mobile phase (acidified water:methanol (30:70, v/v)) of which 5 μL was needed for the HPLC-MS/MS analysis. Indacaterol was eluted on a reversed C18 stationary phase with an isocratic mobile phase at a flow of 1 mL/min. Formoterol was the internal standard (IS). The MS/MS detection was employed with a turbo-ion spray ionization in the positive ion mode. A consensus of the international Guidelines for Bioanalytical Method Validation was followed.

Results: Indacaterol was detected at a mass to charge ratio (m/z) of 393.3 and its MS/MS daughter at 173.2. The retention times of indacaterol and IS were 1.60 and 1.20 min, respectively. Validated calibration curves were linear over a range of 0.075-100 ng/mL with correlation coefficients (r)≥0.990. The curves' regression weighting factor was 1/x. Method specificity was established in six different human urine batches. No matrix interference was observed. The intra- and inter-batch precision and accuracy within±20% (at lower limit) and±15% (other quality control (QC) levels) were confirmed. The indacaterol mean recovery (precision) percentages at Low, Mid, and High QC levels were 93.5 (3.84), 89.8 (2.15), and 92.2 (2.17), respectively. Short-term, long-term, freeze-thaw, and auto-sampler stability results were accepted.

Conclusions: A specific, accurate and precise HPLC-MS/MS method has been validated for indacaterol quantification in human urine. This simple method is reproducible and robust to support future, indacaterol-related pharmacokinetic, bioequivalence and clinical studies.