(M1030-06-37) Strategies and Case Studies to Develop Ultra-sensitive LC-MS/MS Assays for Quantification of Inhalation Drugs in Human Plasma

Purpose: Inhalation dosing often results in low plasma concentrations of drug. Thus, a sub-picogram level (sub-pg) lower limit of quantification (LLOQ) assay is required. Even if the most sensitive mass spectrometer is used, the desired sensitivity is difficult to achieve. A large sample volume is necessary, which results in extensive sample cleanup and significant matrix effect. A simple protein precipitation extraction (PPE) normally would not resolve this. A liquid‑liquid extraction (LLE) or solid-phase extraction (SPE), or a combination of the two, or a third orthogonal extraction may be needed. Additionally, many inhaled drugs are combination formulations, which require a single method to simultaneously quantify two vastly different analytes. Here, we report two challenging assays that we developed: a Tiotropium (0.1 pg/mL LLOQ) assay and a Fluticosone furoate/Indacaterol/Glycopyrrolate (2/4/1 pg/mL LLOQ) assay, and we provide strategies to address issues from sample preparation to liquid chromatography with tandem mass spectrometry (LC‑MS/MS) optimization.

Methods: Two assays for the quantification of inhaled drugs in human plasma are presented. For the Tiotropium (0.1 pg/mL LLOQ) assay, 500 µL of plasma was extracted using Phenomenex Strata-X-CW weak cation mix mode SPE plate. For the Fluticosone furoate/ Indacaterol/ Glycopyrrolate (2/4/1 pg/mL LLOQ) assay, 600 µL of plasma was extracted using a Strata-X-CW weak cation mix mode SPE plate. After a thorough wash, three consecutive elution steps were applied in which each collection plate contained only fluticosone furoate or indacaterol or glycopyrrolate. Samples were analyzed separately on a Sciex Triple Quad 6500+ or Sciex QTrap 5500 LC-MS/MS system equipped with Shimadzu LC-30ADvp series LC system. C₁₈ or biphenyl LC columns were used to separate the analyte from the internal standard. Multiple reaction monitoring (MRM) transitions were measured using positive electrospray ionization MS.

Results: When developing an ultra-sensitive LC-MS/MS method, the following strategies should be considered: (1) Understand the structure of the analytes, including chemical structure, critical functional groups, and equilibrium acid dissociation constant (pKa) value; (2) Increase the amount of the analytes on the LC column; in other words, increase the sample aliquot volume or decrease the reconstitution solvent volume, or increase the injection volume; (3) Optimize the sample extraction method using an orthogonal extraction (PPE+SPE, LLE+SPE); specifically, mixed-mode SPE was useful in optimizing the washing and elution conditions based on the chemical structure of each analyte; (4) Optimize the LC conditions by incorporating a switching valve to back- forward-flush columns and use two-dimensional (2D) chromatography, such as guard column trapping or “heart cut” chromatography, to reduce the background noise, thus increasing the signal-to-noise ratio; (5) Increase MS sensitivity; use the most sensitive in-house MS system and add several MRM transitions to gain sensitivity. At last, ion mobility can be an additional resource, if available. A previously published tiotropium assay at a quantification level of 0.2 pg/mL requires a complicated 2-step LLE and 2D-LC. Not only have we achieved lower LLOQ at 0.1 pg/mL LLOQ, but we have also significantly simplified the method using only 1-step SPE on 96-well plate format and a simple 1D-LC-MS/MS condition with cycle time of 3.5 minutes. No method has been published for the combination assay to simultaneously quantify fluticosone furoate, indacaterol, and glycopyrrolate. Based on the different physical and chemical properties of these three analytes, the sample extraction procedure was designed so that the three analytes were eluted separately to generate the cleanest extract and allow the LC-MS condition to be optimized to the specific analyte. These methods were fully validated and were used to support several bioequivalence studies.

Conclusion: The novel strategies were successfully applied in the quantification of ultra-low inhalation drugs in human plasma by LC-MS/MS.