Development and validation of bioanalytical method for simultaneous estimation of ramipril and hydrochlorothiazide in human plasma using liquid chromatography-tandem mass spectrometry

• We developed a rapid and highly sensitive LC-MS/MS method for simultaneous estimation of ramipril and hydrochlorothiazide in human plasma.
• The method is accurate, precise and meets validation requirements by guideline.
• Low detection limits of 2 ng/mL for ramipril and 8 ng/mL for hydrochlorothiazide.
• Method utilizes highly selective liquid–liquid extraction technique with a sample volume of 200 μL.

The present study describes a novel liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the simultaneous estimation of ramipril (RAM) and hydrochlorothiazide (HCTZ) in human plasma using liquid–liquid extraction technique. This method made use of electrospray ionization in positive mode for RAM and in negative mode for HCTZ using triple quadrupole mass spectrometry where carbamazepine was used as an internal standard (IS). Analytes were recovered by methyl tertiary butyl ether:dichloromethane (85:15) subsequently separated on an Enable C18 G column (150 mm × 4.6 mm, 5 μm) using methanol:0.1% formic acid in water (85:15) as a mobile phase, at a flow rate of 0.5 mL/min. Quantification of RAM, HCTZ and IS was performed using multi-reaction monitoring mode (MRM) where transition of m/z 417.2 → 234.1 (RAM) and 237.0 → 194.0 (IS) in positive mode and 296.1 → 205.0 for HCTZ in negative mode. The calibration curve was linear (r2 > 0.99) over the concentration range of 2–170 ng/mL for RAM and 8–680 ng/mL for HCTZ. The intra-day and inter-day precisions were <15% and the accuracy was all within ±15% (at LLOQ level ±20%). Additionally, 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 RAM and HCTZ in human plasma.