Simultaneous Quantification of Mesalamine and Its Metabolite N-Acetyl Mesalamine in Human Plasma by LC-MS/MS and Its Application to a Bioequivalence Study
Kanchanamala Kanala *
Research and Development, Jawaharlal Nehru Technological University Anantapur Andhrapradesh, India and Department of Pharmaceutics, Ratnam Institute of Pharmacy, Pidatapolur, Muthukur, Nellore, Andhrapradesh, India.
Nagiat T. Hwisa
Faculty of Pharmacy, University of Zawia, Al-Zawia, Libya.
Babu Rao Chandu
Faculty of Pharmacy, University of Zawia, Al-Zawia, Libya.
Fathi H. Assaleh
Faculty of Pharmacy, University of Zawia, Al-Zawia, Libya.
K. Mukkanti
Research and Development, Jawaharlal Nehru Technological University Hyderabad, Andhrapradesh, India.
Prakash Katakam
Faculty of Pharmacy, University of Zawia, Al-Zawia, Libya.
Bala Sekhara Reddy Challa
Department of Pharmaceutical Analysis, Vagdevi College of Pharmacy, Gurazala, Guntur, Andhrapradesh, India.
*Author to whom correspondence should be addressed.
Abstract
Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was used for simultaneous quantification of mesalamine and its metabolite N-acetyl mesalamine in human plasma with N-acetyl mesalamine D3 as an internal standard (IS). Chromatographic separation was performed on a Thermo, HyPURITY C18 (150 x 4.6 mm, 5 ïm) column with an isocratic mobile phase composed of 10 mM ammonium acetate and methanol in the ratio of 85:15 (%v/v), at the flowrate of 0.6 mL/min. The drug, metabolite and internal standard were extracted by liquid-liquid extraction. The method was validated over a linear concentration range of 2-1500 ng/mL for mesalamine and 10-2000 ng/ml for N-acetyl mesalamine, which demonstrated intra and inter-day precision ranging from 1.60 to 8.63% and 2.14 to 8.67% for mesalamine and 0.99 to 5.67% and 1.72 to 4.89% for N-acetyl mesalamine respectively. Similarly, the intra- and inter-day accuracy varied from 102.70 to 105.48% and 100.64 to 103.87% for mesalamine, 99.64 to 106.22% and 100.71 to 104.27% for N-acetyl mesalamine respectively. Both analytes were found to be stable throughout freeze–thawing cycles, bench top and postoperative stability studies. The method was successfully applied to support a bioequivalance study of healthy subjects.
Keywords: Mesalamine, N-Acetyl mesalamine, LC/MS/MS, bioequivalence, pharmacokinetics