Validated LC Method for the Estimation of Dorzolamide HCl ( Carbonic Anhydrase Inhibitor ) in Ophthalmic Solutions

A reverse phase HPLC method is described for the determination of Dorzolamide in eye drops. Chromatography was carried on an Inertsil ODS 3V column using Acetonitrile : (0.02M) 1,Octane Sulphonic acid buffer (pH 3.5) (36:64 v/v) on isocratic mode at a flow rate of 1 mL/min with UV detection at 254 nm. The detector response was linear in the concentration range 4-720 μg/mL. The limit of detection and limit of quantification are found to be 0.7041 and 2.3483 μg/mL respectively. The method was validated as per the ICH guidelines. The proposed method is rapid, accurate and precise and can be applied for the routine analysis of dorzolamide in ophthalimic solutions.


Introduction
Dorzolamide [1][2][3] (Figure 1) is a carbonic anhydrase (CA) inhibitor.Dorzolamide is a sulfonamide and a highly specific carbonic anhydrase II (CA-II) inhibitor, which is the main CA iso-enzyme involved in aqueous humor secretion.Inhibition of CA-II in the ciliary processes of the eye decreases aqueous humor secretion, presumably by slowing the formation of bicarbonate ions with subsequent reduction in sodium and fluid transport.Literature survey reveals that liquid chromatographic [7][8] methods, voltametric 9 , spectrophotometric 10 and simultaneous determination of DRZ along with timolol was reported by TLC-densitometry 11 , Ratio Derivative Spectrophotometery 12 and capillary electrophoresis 13 in ophthalmic preparations.The aim of the present work is to develop a LC method for the determination of Dorzolamide in ophthalmic solutions and to validate as per the ICH guidelines.

Instrument
Quantitative HPLC was performed on a binary gradient HPLC with Waters Alliance (Waters Corporation, MA, USA) equipped with a Waters 2695 separations module and a Waters 2695 PDA detector.Data acquisition was performed by the Empower pro-software operated on a Pentium® IV microprocessor.An Inertsil ODS 3V column (250 x 4.0 mm, 5 µmwas used.

Chromatographic conditions
The mobile phase composed of Acetonitrile : (0.02M) 1-Octane Sulphonic acid buffer (pH adjusted to 3.5 with o-phosphoric acid) was used in the ratio of 36:64 v/v with flow rate of 1.0 ml/min.The mobile phase was degassed and filtered through 0.2 µm membrane filter before pumping into HPLC system .Analysis was carried out at 254 nm.

Preparation of standard stock and sample solutions
Accurately weighed 100 mg of Dorzolamide pure drug was taken and dissolved in acetonitrile in a 100 mL volumetric flask and sonicated.The stock solution was further diluted with the mobile phase to get a series of solutions containing 4.0-720.0μg/mL of DRZ.

Validation
Linearity 10 μl of each of the above prepared solutions (4.0-720.0μg/mL) were injected in to the HPLC system and the peak area (Table 1) was recorded from the respective chromatograms.A representative chromatogram is shown in Figure 2. A calibration graph was drawn by plotting the concentration of Dorzolamide (μg/mL) on x-axis and the corresponding peak area on the y-axis (Figure 3).

Assay of commercial formulations
DORTAS (2%, 3 ml), DORZOX (2%, 5 ml) and OCUDOR eye drops (2%, 5 ml) brands were purchased from the local market and required sample solutions were made using the mobile phase and 10 μL of these formulation solutions were injected into the HPLC system and the peak areas were recorded.The results are given in Table 2.

Precision
The precision of an analytical procedure expresses the degree of scatter between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.The repeatability (intra-day precision) refers to the use of analytical procedure within a laboratory over a short period of time using the same operator with the same equipment.Intermediate precision (inter-day precision) involves estimation of variations in analysis when a method is used within a laboratory on different days, by different analysts.The intra-day repeatability was investigated using three separate sample solutions each at three different levels (200, 400 and 600 µg mL -1 ) prepared as reported above, from the freshly reconstructed formulations.Each solution was injected in triplicate and the peak areas obtained were used to calculate means and RSD% values (Table 3).
The inter-day reproducibility was checked on three different days, by preparing and analyzing in triplicate four separate sample solutions from the reconstructed formulations at the same concentration level of intra-day repeatability; the means and RSD% values were calculated from peak areas.

Accuracy
The accuracy of an analytical method is the closeness of the test results to the true value.To assess accuracy, freshly prepared placebo of the DRZ pharmaceutical formulations were spiked with various amounts of pure DRZ at 80, 100 and 120%.Each solution was injected in triplicate and the peak areas were used to calculate means and RSD% values (Table 4) and compared with those obtained with standard DRZ solutions.

Results and Discussion
The analysis of the chromatogram of Dorzolamide revealed the following efficiencies of the column: for Dorzolamide N =8676 (where N represents theoretical plate number) and asymmetry 1.05.The limit of detection (LOD) and limit of quantification (LOQ) for Dorzolamide were found to be 0.7041 and 2.348 μg/mL respectively.The retention time of Dorzolamide was found to be 6.027 min.The linearity was followed in the concentration range of of 4.0-720.0μg/mL with regression equation y = 21855x + 27183 and correlation coefficient 0.999.The method is precise [0.822-1.021](Inter-day) and 0.144-0.673(Intraday)] and accurate (0.1792-0.3459) as the RSD values were less than 2 %.The percentage of purity of Dorzolamide in commercial formulations is found to be 99.33-99.58.

Conclusion
The proposed liquid chromatographic method is simple, rapid, reproducible and economical for the analysis of Dorzolamide in pharmaceutical formulations and can be used successfully applied for the quantitative analysis of Dorzolamide in pharmaceutical formulations.

Table 1 .
Calibration data of Dorzolamide.

Table 2 .
Analysis of commercial formulations (Eye Drops).