Instruments
HPLC system consisted of binary pump (Model Waters 515 HPLC pump), rheodyne loop injector and PDA detector (Waters 2998). Empower- version 2 software is used for data collection and analysis. Phenomenex C18 column (250 mm × 4.6 mm, 5 µm) is used for separation through isocratic elution using Acetonitrile: Methanol: Water in a ratio (27: 20: 53, v/v/v) pH 4 adjusted with 1% Ortho-phosphoric acid as mobile phase at flow rate 1 ml/min. The photo diode array (PDA) detector is used for detection monitoring at common wavelength 223 nm and the each injection volume was 20 µl. The calibrated Sartorius CP124S (Sartorius Corporation, United State) instrument is used for weighing of all the active pharmaceutical ingredients, excipients and chemicals which is having weighing sensitivity 0.01 mg.
Chemicals and reagents
Analytically pure active pharmaceutical ingredients EMPA (99.23%), LINA (98.92%), and MET (99.12%) was procured as a gratis samples from Torrent Pharmaceutical Pvt. Ltd., Ahmedabad, Gujarat, India, Zydus Cadila Healthcare Ltd., Ahmedabad, Gujarat, India and Sun Pharmaceutical Pvt. Ltd., Vadodara, Gujarat, India respectively. Methanol (HPLC grade) and acetonitrile (HPLC grade) were obtained from SRL Pvt. Ltd. Mumbai, India, HPLC grade water—Milli Q integral Water purification system, Merck KGaA, Darmstadt, Germany, and Ortho-phosphoric acid (AR grade) obtained from SRL Pvt. Ltd. Mumbai, India.
Chromatographic system
Preparation of mobile phase
By taking premix Acetonitrile: Methanol: Water (27:20:53, v/v/v) pH 4.0 adjusted with 0.6 ml of 1% O—phosphoric acid in 200 ml reservoir was used as a mobile phase. For degassing the mobile phase prior to use, the reservoir was sonicated for 20 min and mixtures of solvent were used as mobile phase.
Preparation of standard stock solution
Precisely weigh 100 mg of MET, 2.5 mg of LINA and 5 mg of EMPA and transfer into the three different 10 ml volumetric flask containing 2.0 ml of methanol and the volume was made upto the mark with methanol which gives the stock solution having concentrations 10000 µg/ml of MET, 250 µg/ml of LINA and 500 µg/ml of EMPA, respectively. Pipette out the 2.5 ml of MET, 0.5 ml of LINA and 0.5 ml of EMPA from the above stock solution, transfer into volumetric flask of 50 ml and make up the volume with methanol to acquire the concentration of working standard stock solution 500 µg/ml of MET, 2.5 µg/ml of LINA and 5 µg/ml of EMPA.
Calibration curve
From above working standard stock solution, pipette out appropriate volume of aliquots of MET (500 µg/ml), LINA (2.5 µg/ml) and EMPA (5 µg/ml) are transferred to different 10 ml volumetric flask and volume was adjusted up to the mark with the mobile phase to give a final concentration range of 50, 100, 200, 300, 400 and 500 µg/ml for MET, 0.25, 0.5, 1, 1.5, 2 and 2.5 µg/ml for LINA and 0.5, 1, 2, 3, 4 and 5 µg/ml for EMPA. By using the proposed chromatographic conditions, each solution was analyzed and the chromatogram was recorded. Calibration curves were constructed by plotting peak area v/s concentration and regression equations were computed.
Validation
According to the International Conference on Harmonization (ICH) guidelines Q2 (R1) guidelines [15], validation of the proposed developed RP–LC method was carried out.
Linearity
Linearity was studied by preparing standard solution of 7 different concentrations of 50, 100, 200, 300, 400 and 500 µg/ml for MET, 0.25, 0.5, 1, 1.5, 2 and 2.5 µg/ml for LINA and 0.5, 1, 2, 3, 4 and 5 µg/ml for EMPA. The chromatogram of each concentration was recorded 5 times from freshly prepared seven different concentrations for MET, LINA and EMPA. The terms of slope, intercept and correlation coefficient of MET, LINA and EMPA was used for the assessment of linearity. The calibration curves were developed by plotting concentration v/s peak area (n = 5).
Precision
Precision study was designed in expressions of intra-day and inter-day precisions. Intraday and Interday precision was carried out by analyzing sample solution at three levels covering low, medium and high concentration of the calibration curve three times on the same day for intraday precision study and over a period of three different days (n = 3) for interday precision study, 50, 200 and 500 µg/ml for MET, 0.25, 1 and 2.5 µg/ml for LINA and, 0.5, 2 and 5 µg/ml for EMPA. The chromatogram was recorded and peak areas obtained were used to calculate mean and % RSD values. The repeatability studies were carried out by estimating the response at 200 µg/ml for MET, 1.0 µg/ml for LINA and 2.0 µg/ml for EMPA 6 times and % RSD of area are reported.
Accuracy
The accuracy study was performed by calculating recovery of MET, LINA and EMPA with the help of standard addition method. Known amount of MET, LINA and EMPA at 80, 100, and 120% levels were spiked to pre quantified sample solution and the amount of MET, LINA and EMPA were estimated by putting the value of peak area to the regression equation of calibration curve.
Limit of detection and limit of quantification
The limit of detection (LOD) is defined as the lowest concentration of an analyte that can detect. The limit of quantification (LOQ) is the lowest amount of analyte that can be quantitatively determined with appropriate precision and accuracy. As per ICH guideline, LOD and LOQ were calculated using the following equation:
$${\text{LOD = 3}}{\text{.3 }} \times \sigma /{\text{S}}$$
$${\text{LOQ = 10 }} \times \sigma /{\text{S}}$$
where, σ is the standard deviation of y-intercepts of regression lines and S is the slope of the calibration curve.
Robustness
The robustness study has been carried out by a deliberate change in the proposed RP–LC chromatographic condition parameters like detection wavelength, flow-rate of mobile phase and the mobile phase solvent make on the results were examined for the concentration of 200 μg/ml for MET, 1 μg/ml for LINA and 2 μg/ml for EMPA. The average and %RSD of peak retention time were calculated.
Specificity
Specificity is the ability to assess unequivocally the analyte in the presence of components, which may be expected to be present. Typically these might be including impurities, degradants, preservatives, and excipients for checking the interference which are used in synthetic mixture. The developed method was found to be specific.
System suitability
The adequate performance of liquid chromatographic system can be assessed by System suitability parameters. A system suitability test requires ensuring that a given operating system may be normally applicable because of normal variation in equipment, supplies and techniques. System suitability also require for the verification of the resolution and reproducibility of the chromatographic system which are adequate for the analysis to be done.
Analysis of synthetic mixture [16]
The synthetic mixture of MET, LINA and EMPA was prepared in ratio of 500 mg: 2.5 mg: 5 mg respectively. Common excipients like Hydroxypropyl methylcellulose 0.34 g [HPMC], Polyethylene glycol 0.24 g [PEG], Magnesium stearate 0.15 g, Talc 0.18 g were weighed accurately and transfer into motor pestle along with 0.1 g of EMPA, 0.05 g of LINA, and 10 g of MET which is equivalent to 20 tablets. Synthetic mixture powder (0.554 g) was accurately weighed and transferred into 100 ml volumetric flask containing 20 ml of methanol. It was sonicated for 15 min and solution was filtered using Whatman filter paper No.42. Filtrate was collected in another 100 ml volumetric flask and the residue was washed with few amount of methanol, the filtrate and residue was combined and methanol was added upto the mark. From the above solution, 0.4 ml of aliquot was pipette out into the 10 ml volumetric flask and methanol was added up to the mark to obtain absolute concentration of 200 μg/ml for MET, 1.0 μg/ml for LINA and 2.0 μg/ml for EMPA respectively. The chromatogram was recorded at 223 nm and the quantification was carried out by keeping these values in the regression equation of calibration curve.