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• Losartan Cmax(ng/mL) AUC0−48(h. ng/mL) AUC0−inf(h. ng/mL) Tmax(h) t1/2(h) Kel (1/h) EXP-3174 Cmax(ng/mL) AUC0−48(h. ng/mL) AUC0−inf(h. ng/mL) Tmax(h) t1/2(h) Kel (1/h) Hydrochlorothiazide Cmax(ng/mL) AUC0−48(h. ng/mL) AUC0−inf(h. ng/mL) Tmax(h) t1/2(h)
• Kel (1/h) 243± 53 517± 194 535± 209 1.46± 0.29 2.12± 1.05 0.322± 0.007 0.301± 0.006 – – – – 455± 152 2147± 784 2235± 739 3.28± 0.33 4.05± 0.84 0.176± 0.021 0.140± 0.015 – – – – 65.32± 23.4 417± 134 455± 139 2.40± 0.18 9.12± 1.20 0.0760± 0.003 0.0763± 0.004 – – – –
• Cmax: Maximum plasma concentration; AUC0−t: Area under the plasma concentration-time curve from zero hour to 48 h;
• AUC0−inf: Area under the plasma concentration-time curve from zero hour to infinity; Tmax: Time point of maximum plasma concentration;
• t1/2: Half life of drug elimination during the terminal phase; Kel: Elimination rate constant; SD: Standard deviation; CI: confidence interval;
• CV: coefficient of variation 3. Experimental
• Chemicals and materials
• Figure 6. Mean plasma concentration-time profiles of losartan, EXP-3174, and hydrochlorothiazide after oral adminis- tration of 50 mg of losartan potassium and 12.5 mg of hydrochlorothiazide hydrochloride fixed dose tablet formulation to 65 healthy Indian male subjects under fasting.
• Liquid chromatography and mass spectrometric conditions
• The chromatographic analysis of LOS, EXP-3174, and HCTZ was carried out on a Waters Acquity UPLC system (Milford, MA, USA) employing a BEH C18 (50× 2.1 mm, 1.7 µm) column, maintained at 35 ◦
• C. The mobile phase was prepared in premixed solvents consisting of 1.0 % (v/v) formic acid in water and acetonitrile (15:85, v/v). The mobile phase was pumped at a flow rate of 0.350 mL/min. The injection volume was set at 10 µ L. The sample manager temperature was maintained at 5 ◦
• C with an alarm band of±3 ◦
• C and the average pressure of the system was 6000 psi. Detection and quantitation of analytes and ISs was carried out using multiple reaction monitoring (MRM) for deprotonated precursor
• → product ion transitions on a Quattro Premier XE mass spectrometer from Waters – Micro Mass Technologies (Milford, MA, USA), in the negative ionization mode. Source dependent and compound dependent mass parameters optimized and MRM transitions for analytes and ISs are summarized in Table 1. MassLynx software version 4.1 was used to control all parameters of UPLC and MS.
• Calibrators and quality control samples
• C, while CSs and QC samples in plasma were kept at –70 ◦ C until use.
• Sample extraction protocol
• Pa positive pressure of nitrogen at 2.4 L/min flow rate.
• Elution of analytes and ISs from the cartridges was carried out with 500 µ L of acetonitrile:water (90:10, v/v) into prelabeled tubes. The eluate was evaporated to dryness in a thermostatically controlled water bath maintained at 40 ◦
• C under a gentle stream of nitrogen for 5 min. After drying, the residue was reconstituted in 100 µ L of reconstitution solution [10 mM ammonium formate: acetonitrile (20:80, v/v)] and 10 µ L was used for injection in the chromatographic system.
• Validation procedures
• The standard stock solutions of analytes and ISs were evaluated for short-term and long-term stability ◦ at 25 C and 5 ◦
• C, respectively. The analyte stability in spiked plasma samples was evaluated by measuring the area ratio response (analyte/IS) of stability samples against freshly prepared standards having identical concentration. The % change was determined using the expression % Change =
• Mean stability samples - Mean comparison samples
• Mean comparison samples × 100
• Bench top (at room temperature), wet extract at 5 ◦ C, dry extract at 5 ◦
• C, processed sample stability at room temperature, and freeze-thaw (–20 ◦ C and –70 ◦
• C) and long-term (–20 ◦ C and –70 ◦
• C) stability of analytes in plasma were studied at three QC levels using six replicates. The stability samples were quantified against freshly prepared quality control samples. Stability data were acceptable if the % CV of the replicate determinations did not exceed 15.0% and the mean accuracy value was within±15.0% of the nominal value.
• Bioequivalence study and incurred sample reanalysis
• 10 min and plasma was separated and stored at –70
• The method reproducibility was verified by reanalysis of 213 incurred samples and the results were compared with original study samples. The acceptance criterion was based on two-thirds of the original results and repeat results should be within 20% of each other.48 3.7. Conclusion