title: Indacaterol determination in human urine: validation of a liquid-liquid extraction and liquid chromatography-tandem mass spectrometry analytical method.
abstract: 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.
authors.affiliation: 1School of Pharmacy and Medical Sciences, Al-Ahliyya Amman University, Amman, Jordan.
authors.forename: Wesam G
authors.initials: WG
authors.lastname: Ammari
authors.affiliation: 1School of Pharmacy and Medical Sciences, Al-Ahliyya Amman University, Amman, Jordan.
authors.forename: Zainab
authors.initials: Z
authors.lastname: Al-Qadhi
authors.affiliation: 2ACDIMA Centre for Bioequivalence and Pharmaceutical Studies, Amman, Jordan.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Khalil
authors.affiliation: 2ACDIMA Centre for Bioequivalence and Pharmaceutical Studies, Amman, Jordan.
authors.forename: Rabab
authors.initials: R
authors.lastname: Tayyem
authors.affiliation: 1School of Pharmacy and Medical Sciences, Al-Ahliyya Amman University, Amman, Jordan.
authors.forename: Samir
authors.initials: S
authors.lastname: Qammaz
authors.affiliation: 1School of Pharmacy and Medical Sciences, Al-Ahliyya Amman University, Amman, Jordan.
authors.forename: Ghaleb
authors.initials: G
authors.lastname: Oriquat
authors.affiliation: 3School of Pharmacy, Applied Sciences University, Amman, Jordan.
authors.forename: Iman A
authors.initials: IA
authors.lastname: Basheti
authors.affiliation: 4School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom.
authors.forename: Henry
authors.initials: H
authors.lastname: Chrystyn
mesh_terms: D058666:Adrenergic beta-2 Receptor Agonists / Q000652:urine*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D016903:Drug Monitoring / Q000379:methods* / Q000592:standards; D004355:Drug Stability; D006801:Humans; D007189:Indans / Q000652:urine*; D057230:Limit of Detection; D016014:Linear Models; D059625:Liquid-Liquid Extraction / Q000592:standards; D015363:Quinolones / Q000652:urine*; D012015:Reference Standards; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000592:standards
chemical_list: D058666:Adrenergic beta-2 Receptor Agonists; D007189:Indans; D015363:Quinolones; C510790:indacaterol
keywords: LC-MS/MS method; bioanalytical method; indacaterol; liquid-liquid extraction; urine; validation