Bioanalytical Method for Quantification of Torsemide in Human Urine Using LC-MS/MS

S. Satheshkumar*¹, V. Muruganantham^2
1Ph.D. Scholar, Department of Pharmaceutics, College of Pharmacy, Vinayaka Mission’s College of Pharmacy, Salem- 636008, Tamilnadu, India
²Professor, Department of Pharmaceutics, College of Pharmacy, Vinayaka Mission’s College of Pharmacy, Salem- 636008, Tamilnadu, India.

A B S T R A C T
Anaccurate and simple method was developed using high performance liquid chromatography with electron spray ionization tandem mass spectrometry (HPLC-ESI-MS) to quantify the concentration of torsemide in human urine. Stable isotobically labelled compound torsemide D7 was used as an internal standard (ISTD). The chromatographic analysis was conducted on a Zorbax C18 XDB column (100x 4.6mm,i.d5µm) within 3min, using methanol with 5mM ammonium acetate (70:30%, v/v) was used as mobile phase at the flow rate of 0.700mL/min under an isocratic condition. The ionization was performed on electron spray ionization interference with positive mode by multiple reactions monitoring (MRM). The mass transitions were 349.100→264.100 m/z for torsemide and 356.200→264.200 m/z for ISTD. Method validated as per USFDA guidelines and calibration curve was found to be linear in the range of 10.443-5000.411ng/mL. The results were within the acceptance limits. The extraction efficiency was 91.46% at the three quality control levels. The lower limit of detection (LLOQ) was found to be 10.443ng/mL. Stability studies demonstrated that torsemide was stable in urine during bench-top (3hr25min at room temperature), auto-sampler (23hr 20 min at 4^oC), freeze-thaw (5cycles) and long termanalyte stability in urine (48days at -20^oC).
Keywords: Torsemide, Human Urine, Stability, Protein Precipitation, Validation.