Author(s): Chirag D. Patel1, 2, Swati Guttikar1 and Bhavesh H Patel2*
The present study describes a novel liquid chromatographic-tandem mass spectrometric (LCMS/MS) method for the simultaneous estimation of Irbesartan (IRB) and hydrochlorothiazide (HCTZ) in human plasma using liquid-liquid extraction technique. This method made use of electro spray ionization in negative mode for IRB and HCTZ using triple quadrupole mass spectrometry where Irbesartan D4 and Hydrochlorothiazide 13C 15N2 D2 were used as an internal standard (IS) for IRB and HCTZ respectively. Analytes were recovered by di ethyl ether: dichloromethane (70:30) subsequently separated on an Ace 5 C18 column (100 mm × 4.6 mm, 5 µm) using methanol:0.1% formic acid in water (70:30) as a mobile phase, at a flow rate of 1.0 mL/min. Quantification of IRB, HCTZ and Irbesartan D4 and Hydrochlorothiazide 13C 15N2 D2 was performed using multi-reaction monitoring mode (MRM) where transition of m/z 427.1 → 193.0 (IRB), 431.1 → 193.0 (Irbesartan D4), 295.8 → 269.0 for HCTZ and 300.9 → 271.1 for Hydrochlorothiazide 13C 15N2 D2 in negative mode. The calibration curve was linear (r 2 > 0.99) over the concentration range of 10-5000 ng/mL for IRB and 1-500 ng/mL for HCTZ. The intraday and inter-day precisions were less than 15% and the accuracy was all within ±15% (at LLOQ level ±20%). The LC-MS/MS method was fully validated for all the other parameters such as selectivity, matrix effect, recovery and stability as well. In conclusion, the findings of the present study revealed the selectivity and sensitivity of this method for the simultaneous estimation of IRB and HCTZ in human plasma.