Instrumentation and chromatograph
The HPLC chromatograph used was Agilent Infinity 1260 series (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with 1260 binary pump VL (400 bar), 1260 manual injector (600 bar), Rheodyne 7725i seven-port sample injection valve with 20-μL fixed loop, ZORBAX Eclipse Plus C18 (250 mm × 4.6 mm, 5 μm), 1260 DAD VL, 20-Hz detector, standard flow cell (10 mm, 13 μL, 120 bar), OpenLab CDS EZChrom Ed. Workstation and syringe (50.0 μL, FN, LC tip). All weighing for analysis was performed on Shimadzu electronic analytical balance AX-200 (Kyoto, Japan). Water used for analysis was prepared by triple distillation assembly. All dilutions, mobile phase and other solutions that were used for the analysis were filtered through a 0.2-μm nylon filter (Ultipor®N66 Nylon 6,6 membrane, Pall Sciences, Pall India Pvt. Ltd., Mumbai, India).
Chemicals and reagent
The working standard used was OLO which was supplied by Ranbaxy Laboratories Limited (Gurgaon, Harayana, India) as a gift sample. Formic acid and methanol were procured from Merck Specialties Private Limited (Mumbai, India). The mobile phase was prepared from the combination of formic acid (0.1%) and methanol.
Accurately weighed 50 mg of OLO was dissolved in 50% aqueous methanol to prepare stock I (1,000 μg/mL) in a 50-mL volumetric flask. Stock II (100 μg/mL) was prepared from stock I, which was used to prepare further dilutions containing 1, 5, 10, 15 and 20 μg/mL. All dilutions were filtered through the 0.2-μm nylon filter (Ultipor®N66 Nylon 6,6 membrane, Pall Sciences) and chromatographed by a set of conditions on Agilent Infinity 1260 series. The mixture of 0.1% formic acid and methanol (35:65) was used as the mobile phase for the elution of the drug on Zorbax Eclipse Plus C18 column (250 mm × 4.6 mm, 5 μm) at 1.0 mL/min flow rate. OLO was successfully eluted at 3.77 min with a run time of 7 min, and detection was performed using a photodiode array detector (PDA) at 300 nm.
According to the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceutical for Human Use (ICH) guidelines (ICH 2000a, b), the developed method was validated to assure the reliability of the results of analysis for the different parameters, viz. linearity, range, accuracy, precision, robustness, limit of quantization (LOQ), limit of detection (LOD) and specificity. Linearity was determined by serial dilutions (1 to 20 μg/mL) of the OLO in 50% aqueous methanol in triplicate. The range of OLO was validated between 5 and 15 μg/mL with triplicates of dilutions. The recovery method was used to determine the accuracy of the developed method by spiking the standard solution to the pre-analysed samples (5, 10 and 15 μg/mL), which was repeated six times. Repeatability and intermediate precision were studied to assure the precision of the method. Six replicates of the standard dilution (5 μg/mL) were chromatographed subsequently to assure repeatability, which were further chromatographed after 5 h to assure the stability of the drug in the solvent system. The standard dilutions were analysed by different analysts in subsequent days to study intermediate precision in the linearity range. A change in temperature (20°C, 25°C and 30°C) and acidic content of the aqueous part of the mobile phase (5% change in 0.1% aqueous formic acid) was observed. Different serial dilutions of OLO (0.1 to 1,000 ng/mL) were chromatographed to calculate the signal-to-noise ratio to determine LOD and LOQ. Alkaline-degraded OLO samples were chromatographed to ascertain the specificity of the developed method for OLO.
Analysis of pharmaceutical products
As per the ‘Chromatography’ subsection, the samples of raw material have been prepared and analysed in the set of condition of analysis, which was repeated in six batches. The response to chromatographic analysis was used to calculate the concentration with the help of a regression equation.
Powdered tablets were weighed equivalently to 50 mg of OLO and sonicated to dissolve the drug content in methanol to extract the complete drug content from the tablet powder. The sonicated solution was filtered through Whatman filter no. 41 to remove the un-dissolved excipients of the tablet dosage form, and different serial dilutions were prepared by subsequent dilution with 50% aqueous methanol. All dilutions were filtered through the 0.2 μm nylon filter and chromatographed. The drug content was determined from the regression equation.
An accurately measured volume of ophthalmic solution (Olodin, FDC Limited, Aurangabad, India), equivalent to about 10 mg of OLO, was transferred to a 100 mL 163 volumetric flask and diluted with diluent to the volume. The solution was mixed and sonicated for 10 min. An appropriate dilution was prepared from the stock solution and filtered (stock P, 100 μg/mL). Aliquots of stock P were diluted to get sample concentrations (5, 10 and 15 μg/mL) in the range of linearity for the developed method and filtered through the 0.2 μm nylon filter. All these dilutions were chromatographed, and the area under the curve (AUC) of the peak of OLO was placed in the regression equation to get the concentration of the samples.