Determination of Phosphodiesterase Inhibitors Tadalafil, Roflumilast and Roflumilast N-Oxide Using LC–MS in Guinea Pig Plasma

Abstract

Phosphodiesterases are known as a super-family of 11 isoenzymes, which can exert various functions based on their organ distribution. In this work, a rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method was developed for quantification of tadalafil (phosphodiesterase five inhibitor), roflumilast (RF) (phosphodiesterase four inhibitor) and its active metabolite, RF N-oxide in guinea pig plasma. Chromatographic separation was carried out on UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) at a flow rate 0.5 mL/min, using 0.2% formic acid in acetonitrile and 0.2% formic acid in water as mobile phases within 4 min. Detection was performed using a triple quadrupole mass spectrometer employing electrospray ionization operated in positive mode using multiple reaction monitoring mode. The method utilized deuterium labeled internal standards, and was validated according to European Medicines Agency guidelines. It showed excellent linearity in the range of 0.5-500.0 ng/mL for all analytes with coefficient of determination >0.99. The intra- and inter-day precisions (relative standard deviation %) were within 6.7%, and the recoveries were greater than 73.4%. Using this method, plasma samples from experiments of phosphodiesterase four, and five inhibitors in a model of ovalbumin-induced allergic inflammation in guinea pigs were analyzed.

Simultaneous quantification and pharmacokinetic evaluation of roflumilast and its N‐oxide in cynomolgus monkey plasma by LC–MS/MS method

“…For LC–MS/MS analysis, an appropriate IS is usually required to eliminate interference from the sample matrix and guarantee consistent extraction efficiency. In previous studies, the internal standard that was used for guinea pig plasma was tritium‐labelled isotope markers while it was chlorzoxazone for rat plasma (Kertys et al, 2018; Moussa, El‐Zaher, El‐Ashrey, & Fouad, 2019). An isotopic‐labelled IS is the optimal choice as it has the same retention behaviours and fragmentation schemes (Kertys et al, 2018); however, it is unavailable to most laboratories.…”

“…The precursor/product ion pairs for quantification were monitored at m/z 403.0 → 186.9 for ROF, m/z 419.0 → 186.9 for RNO and 276.2 → 102.9 for IS. Other iron fragments for identification are listed in Table 1, and have been proved to be correct (Kertys et al, 2018; Li et al, 2017; Thevis, Krug, & Schänzer, 2013). MS parameters of ROF, RNO and IS are also shown in Table 1.…”

“…The concentration of the oral dosing solution was determined by the LC–MS/MS method developed. After oral administration of the ROF suspension at a dose of 1 mg/kg, referring to previous studies and the rules for calculating equivalent doses between animals (Ansari et al, 2019; Kertys et al, 2018; Thappali et al, 2012; Xu & Bian, 2002), aliquots of 0.50 ml blood samples were collected in K 2 EDTA polyethylene tubes, at different time points (0.25, 0.50, 1.00, 2.00, 4.00, 6.00, 8.00, 12.0, 24.0 and 48.0 h). The blood collected was immediately placed on ice and centrifuged within 30 min.…”

“…Notably, several liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods have been developed for the simultaneous quantification of ROF and RNO in biological samples obtained from rats (Thappali, Varanasi, Veeraraghavan, & Vakkalanka, 2012), guinea pigs (Kertys, Urbanova, & Mokry, 2018) and humans (Cui et al, 2016; Huang et al, 2018; Knebel, Herzog, Reutter, & Zech, 2012; Li, Wang, Liu, Ma, & Ding, 2017). These LC–MS/MS methods have been validated for detecting ROF and its active metabolite RNO in different biological samples.…”

A concise review of bioanalytical methods of small molecule immuno‐oncology drugs in cancer therapy

“…Chromatographic separation was achieved using a Synergi™ Hydro-RP C 18 column and an isocratic mobile phase was consisting of methanol and 10 mM ammonium formate at pH 4.0 (90:10, v/v) delivered at a flow rate of 0.9 ml/min (Bhadoriya, Dasandi, Parmar, Shah, & Shrivastav, 2018). Kertys, Urbanova, and Mokry (2018) water, which was delivered at a flow rate 0.5 ml/min (Kertys, Urbanova, & Mokry, 2018). Er et al 2019 The matrix effect was ≤10%, which demonstrates a very low effect of matrix and that it did not have any adverse impact on the analysis.…”