Development and validation of a new analytical RP-HPLC method for the determination of flavoxate hydrochloride in bulk form and pharmaceutical dosage form

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Introduction

Flavoxate is a muscarinic antagonist and spasmolytic used for the symptomatic relief of conditions associated with lack of muscle control in the bladder, such as dysuria, urgency, and nocturia. A drug that has been used in various urinary syndromes and as an antispasmodic. Its therapeutic usefulness and its mechanism of action are not clear. It may have local anesthetic activity and direct relaxing effects on smooth muscle as well as some activity as a muscarinic antagonist. Flavoxate Hydrochloride [1] is the hydrochloride salt form of Flavoxate, a parasympatholytic agent with antispasmodic activity. Flavoxate hydrochloride competitively binds to muscarinic receptors, thereby preventing the actions of acetylcholine. This relaxes vascular smooth muscle, mainly of the urinary tract, and prevents smooth muscle contractions. Flavoxate is a spasmolytic flavone derivative that acts by relaxing the smooth muscle in the urinary tract. Flavoxate [2] is a competitive muscarinic receptor antagonist indicated for the treatment of overactive bladder with symptoms of urge urinary incontinence, urgency, and urinary frequency.Flavoxate [3] (flay vox' ate) is a synthetic quaternary ammonium anticholinergic which inhibits the muscarinic actions of acetylcholine on autonomic nerve endings, decreasing the smooth muscle tone of bladder and gastrointestinal tract. Flavoxate hydrochloride [4] is contraindicated in patients who have any of the following obstructive conditions: pyloric or duodenal obstruction, obstructive intestinal lesions or ileus, achalasia, gastrointestinal hemorrhage and obstructive uropathies of the lower urinary tract. The IUPAC Name of Flavoxate HCL [5] is 2-piperidin-1-ylethyl 3-methyl-4-oxo-2-phenylchromene-8-carboxylate; hydrochloride. The Chemical Structure of Flavoxate HCL is as follows Fig 1: Chemical Structure of Flavoxate HCL Several methods [27-30] have been reported for the determination of Flavoxate HCL by UV-visible Spectrophotometry and by HPLC, UPLC in bulk and also in human serum. In this context, a simple, sensitive and economic reversed phase HPLC method has been developed and validated for the quantification of Flavoxate HCL in bulk and pharmaceutical dosage forms.

Materials and Methods

Instruments Used Table 1: Instruments used S. No. Instruments and Glass wares Model

1 HPLC

WATERS Alliance 2695 separation module, Software:

Empower 2, PDA 996 Detector.

7 Digital ultra sonicator Labman

Chemicals Used Table 2: Chemicals Used S.No. Chemical Brand names

3 Acetonitrile for HPLC Merck

HPLC Method Development Preparation of Standard Solution add about 7ml of Methanol and sonicate to dissolve and removal of air completely and make volume up to the mark with the same Methanol. solutions6 into a 10ml volumetric flask and dilute up to the mark with Methanol.

Procedure Inject the samples by changing the chromatographic conditions and record the chromatograms, note the conditions of proper peak elution for performing validation parameters as per ICH guidelines [7-10].

Mobile Phase Optimization Initially the mobile phase tried was methanol: Water and Acetonitrile: Water with varying proportions. Finally, the mobile phase [11] was optimized to Methanol and Water in proportion 45:55 v/v respectively.

Optimization of Column The method was performed with various C18columns like ODS column, Xterra, and X Bridge C18 column. Symmetry C18 (4.6 x 150mm, 5m) was found to be ideal as it gave good peak shape and resolution [12] at 1ml/min flow.

Preparation of Mobile Phase Accurately measured 450 ml (45%) of HPLC Methanol and 550 ml of HPLC Water (55%) were mixed and degassed in a digital ultrasonicator for 10 minutes and then filtered through 0.45 µ filter under vacuum filtration [13].

Diluent Preparation The Mobile phase was used as the diluent.

Method Validation Parameters System Suitability mark with diluents [14].

Procedure The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits [15].

Specificity Preparation of Standard Solution solvent. (Stock solution). solutions into a 10ml volumetric flask and dilute up to the Preparation of Sample Solution Take average weight of the Tablet and crush in a mortar by using pestle and weight 10 mg equivalent weight of Flavoxate HCL sample into a 10mL clean dry volumetric flask and add about 7mL of Diluent [16] and sonicate to dissolve it solvent.

Further pipette 0.72ml of Flavoxate HCL above stock mark with diluent.

Procedure Inject the three replicate injections of standard and sample solutions and calculate the assay [17] by using formula:

Linearity solvent18. (Stock solution)

Preparation of Level – I (24ppm of Flavoxate HCL) Preparation of Level – II (48ppm of Flavoxate HCL): Preparation of Level – III (72ppm of Flavoxate HCL): Preparation of Level – IV (96ppm of Flavoxate HCL): Preparation of Level – V (120ppm of Flavoxate HCL):

Procedure Inject each level into the chromatographic system19 and measure the peak area.

Plot a graph of peak area versus concentration (on X-axis concentration and on Y-axis Peak area) and calculate the correlation coefficient [20].

Precision Repeatability Preparation of Flavoxate HCL Product Solution for Precision Further pipette 0.72 ml of the above Flavoxate HCL stock solutions into a 10ml volumetric flask and dilute up to the The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within Intermediate Precision To evaluate the intermediate precision (also known as Ruggedness) of the method, Precision was performed on different days by maintaining same conditions.

Procedure Day 1 The standard solution was injected for six times and measured the area for all six injections in HPLC. The %RSD for the area of six replicate injections was found to be within Day 2 The standard solution was injected for six times and measured the area for all six injections in HPLC. The %RSD for the area of six replicate injections was found to be within Accuracy Procedure Inject the Three replicate injections of individual concentrations (50%, 100%, 150%) were made under the optimized conditions. Recorded the chromatograms and measured the peak responses. Calculate the Amount found and Amount added for Flavoxate HCL and calculate the individual recovery and mean recovery values.

Robustness The analysis was performed in different conditions to find the variability of test results. The following conditions are checked for variation of results.

For Preparation of Standard Solution Effect of Variation of Flow Conditions The sample was analyzed at 0.7 ml/min and 0.9 ml/min instead of 0.8ml/min, remaining conditions are same. 10µl of the above sample was injected and chromatograms were recorded Effect of Variation of Mobile Phase Organic Composition The sample was analyzed by variation of mobile phase i.e. Methanol: Water was taken in the ratio and 40:60, 50:50 instead of 45:55, remaining conditions are same.10µl of the above sample was injected and chromatograms were recorded.

Results and Discussion

Development of a Method Different chromatographic conditions were tried for better separation and resolution. Symmetry C18 (4.6×150mm) 5µmcolumn was found satisfactory. Peak purity of Flavoxate HCL was checked using UV detector and 260 nm was considered satisfactory for detecting the drug with adequate sensitivity. A number of solvents in the different ratio over a wide range of pH were tried, but either peak shape was broad or resolution was not good. Repeated trials to obtain good, sharp peak with an efficient resolution between two peak of Flavoxate HCL done on a C18 column in isocratic HPLC gave satisfactory results. The run time was good in isocratic trial with mobile phase consisting of Methanol and Water (45:55% v/v) and Symmetry C18 (4.6×150mm) 5µm Column, flow rate 0.8 ml/min and detection wavelength 260 nm gave the satisfactory results in terms of retention time, resolution, symmetry and sensitivity. A typical RP-HPLC chromatogram for determination of Symmetry C18 (4.6×150mm) 5µmfrom bulk and standard preparation and from pharmaceutical formulation was shown in (fig-2).

Optimized Chromatographic Conditions:

5µm Column temperature : 40ºC Wavelength : 260nm Flow rate : 0.8ml/min Injection volume : 10µl Run time : 6minutes Fig 2: Optimized Chromatographic Condition Validation of an Analytical Method The developed RP-HPLC method was validated for parameters like system suitability, linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ) and robustness according to ICH guidelines [21].

System Suitability Standard solutions were prepared as per the test method and injected into the chromatographic system. The system suitability parameters like theoretical plates, resolution and asymmetric factor were evaluated. The system suitability parameters [22] were tabulated in table 3. All the parameters were found to be within the limits.

Table 3: Results of system suitability for Flavoxate HCL S. No. Peak Name RT Area (µV*sec) Height (µV) USP Plate Count USP Tailing Std. Dev. 31827.08 Specificity The ICH documents define specificity as the ability to assess unequivocally the analyte in the presence of components that may be expected to be present, such as impurities, degradation products, and matrix components. Analytical method was tested for specificity [22] to measure accurately quantitates Flavoxate HCL in drug product.

The % purity of Flavoxate HCL in pharmaceutical dosage form was found to be 99.7%.

Linearity The linearity of the test solutions for the assay method was prepared from Flavoxate HCL standard stock solution at five concentration levels from 24µg/ml to 120µg/ml of assay concentration. The peak area versus concentration data was treated by least-squares linear regression analysis (fig-3). The results have shown an excellent correlation between peak areas and concentration within the concentration range of 24– 120 μg/ml for Flavoxate HCL (table-4). The correlation coefficient was found to be 0.9989 for the drug, which meet the method validation acceptance criteria and hence the method was said to be linear for both the drugs.

Chromatographic Data for Linearity Study Table 4: Linearity Data of Flavoxate HCL Concentration Level (%)

Concentration g/ml Average Peak Area Fig 3: Calibration Curve of Flavoxate HCL Linearity Plot The plot of Concentration (x) versus the Average Peak Area (y) data of Flavoxate HCL is a straight line. Y = mx + c Slope (m) = 31709 Intercept (c) = 34216 Correlation Coefficient (r) = 0.998 Validation Criteria: The response linearity is verified if the Correlation Coefficient is 0.99 or greater. Conclusion: Correlation Coefficient (r) is 0.99, and the intercept is 34216. These values meet the validation criteria.

Precision The precision [23] of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.

Repeatability Obtained Five (5) replicates of 100% accuracy solution as per experimental conditions. Recorded the peak areas and calculated % RSD.

Table 5: Results of repeatability for Flavoxate HCL: S. No. Peak Name Retention time Area(µV*sec) Height (µV) USP Plate Count USP Tailing Std.Dev 9476.485 Intermediate Precision Analyst 1 Table 6: Results of Intermediate Precision for Flavoxate HCL S.No. PeakName RT Area (µV*sec) Height (µV) USPP late count USPT ailing Std.Dev. 4333.851 Analyst 2 Table 7: Results of Intermediate Precision Analyst 2 for Flavoxate HCL S.No. PeakName RT Area (µV*sec) Height (µV) USPPlate Count USPTailing Std.Dev. 9988.458 Accuracy Accuracy at different concentrations (50%, 100%, and 150%) was prepared and the % recovery24 was calculated.

Table 8: The Accuracy Results for Flavoxate HCL %Concentration (at specification Level)

Amount Found (ppm) % Recovery Mean Recovery 50% 1172485 36 35.8 99.4 99.5% 100% 2314753 72 71.6 99.4 150% 3480210 108 107.9 99.9 Limit of Detection for Flavoxate HCL The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.

LOD= 3.3 × σ / s Where σ = Standard deviation of the response S = Slope of the calibration curve Result =5.5µg/ml Quantitation Limit The quantitation limit25 of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined.

LOQ=10×σ/S Where, σ = Standard deviation of the response S = Slope of the calibration curve Result =16.7µg/ml Robustness The robustness was performed for the flow rate variations from 0.7 ml/min to 0.9ml/min and mobile phase ratio variation from more organic phase to less organic phase ratio for Flavoxate HCL. The method is robust26 only in less flow condition and the method is robust even by change in the Mobile phase ±5%. The standard and samples of Flavoxate HCL were injected by changing the conditions of chromatography. There was no significant change in the parameters like resolution, tailing factor, asymmetric factor, and plate count.

Summary and Conclusion

The analytical method was developed by studying different parameters. First of all, maximum absorbance was found to be at 260nm and the peak purity was excellent. Injection volume was selected to be 10µl which gave a good peak area. The column used for study was Symmetry C18 because it was giving good peak.40 º C temperatures was found to be suitable for the nature of drug solution. The flow rate was fixed at 0.8ml/min because of good peak area and satisfactory retention time. Mobile phase is Methanol: water was fixed due to good symmetrical peak. So this mobile phase was used for the proposed study. Methanol: water was selected because of maximum extraction sonication time was fixed to be 10min at which all the drug particles were completely soluble and showed good recovery. Run time was selected to be 6min because analyze gave peak around 2.3 and also to reduce the total run time. The percent recovery was found to be 98.0- 102 was linear and precise over the same range. Both system and method precision was found to be accurate and well within range. The analytical method was found linearity over the range of 24-120ppm of the Flavoxate HCL target concentration. The analytical passed both robustness and ruggedness tests. On both cases, relative standard deviation was well satisfactory. The %RSD values were within 2 and the method was found to be precise. The results expressed in Tables for RP-HPLC method was promising. The RP-HPLC method is more sensitive, accurate and precise compared to the Spectrophotometric methods. This method can be used for the routine determination of Flavoxate HCL in bulk drug and in Pharmaceutical dosage forms.