MATERIALS AND METHODS. Chemicals and Reagents. Glycopyrrolate reference standard The HPLC grade solvents used were of E-merck India. All other chemicals and reagents were of analytical reagent grade. HPLC grade h2o was prepared creating use of Millipore purification system.
Maxalt tablets containing 10 mg of Glycopyrrolate was used for analysis purpose. An Agilent liquid chromatography instrument equipped with a push in isocratic mode. Analytical column used was Bondapak C18 3. 9x300mm, 10m the system was connected together with the help of chemistration software in a computer system for data collection and processing Chromatographic conditions Chromatographic separation was carried out at space heat with Bondapak C18 3. 9x300mm, 10m column with 3.
Mobile phase containing of 1230 volumes of Phosphate buffer, seven volumes of 1N Sulphuric acid, 470 volumes of Acetonitrile and 300 volumes of Methanol. The ratio pH was located to be 6. Then finally filtered creating use of 0. 45 nylon membrane filter and degassed in sonicator for six minutes. The injection volume for standard and sample were 50 L and eluted at a flow rate of 2.
3 mL or min, the eluents were monitored at 222 nm Standard preparation. Accurately weigh and transfer about 50 mg of Glycopyrrolate into a 50 mL volumetric flask. Sum about 30 mL of diluent sonicate for 10 periods and dilute to volume with diluent and mix. 0 mL of above solution into 50 mL volumetric flask and dilute to volume with diluent and combine 40 mcg or mL of Glycopyrrolate. Then filter the solution through the 0.
This solution was injected into the column and the peak region and retention time was record as shown fig -1 Sample preparation:. Accurately weigh and transfer a quantity of powdered tablets equivalent to 10 mg of Glycopyrrolate about 1000 mg powder into a 250 mL volumetric flask, sum about 150 mL of diluent, and sonicate for 20 periods and cold to space heat then filter the solution through a 0. Duplicate preparation. Analysis of formulation. Twenty tablets each containing 10mg of Glycopyrrolate were weighed and average mass was calculated.
A quantity of fine powder equivalent to 10mg of Glycopyrrolate was weighed accurately, and transferred into 10ml volumetric flask, and created up to volume with mobile phase. Distant dilution of this sample stock solution within the linearity section were created creating use of mobile phase and filtered through 0. Then 50 L solutions were injected into column and chromatogram was recorded. All the determinations were conducted in triplicate. The described method was validated for the assay of Glycopyrrolate creating use of following parameters.
To determine the robustness regarding the developed method, experimental conditions were purposely altered and the retention was examined. In all the deliberate varied chromatographic conditions a flow rate 0. 05, mobile phase composition at different ratios 1230:6:470:300 v or v or v or v showed that the retention time of peak remains unaffected but for mini changes Table-1a, 1b, 1c. Accurately weighed and transferred about 50 mg of Glycopyrrolate working standard in to 50 mL volumetric flask. Sum bout 30 ml of diluent, sonicated for 10 periods and diluted to volume with diluent.
Transfer 4 ml regarding the above solution into a 50ml volumetric flask and diluted to volume with diluent. Then transferred the 5ml regarding the above solution into a volumetric flask and diluted to volume with diluent. Inject 100 L of standard preparation for six times into the chromatograph and recorded the system suitability parameters as per procedure the conclusions are provided table-2. The linearity of detector response is established by plotting a graph of concentration versus region of Glycopyrrolate standard and determined the correlation coefficient. A series of solutions of Glycopyrrolate standard solutions within the concentration section from about LOQ position 50% to about 150% regarding the target concentration were prepared and injected into the HPLC system.
The detector response was located to be linear from LOQ position 50% to150% of target concentration for Glycopyrrolate standard with a correlation coefficient values greater than 0. 999698 Table-3 Precision. The precision regarding the analytical method was studied by analysis of multiple six sampling of homogeneous sample. The precision expressed as % RSD. The %RSD was located to be 0.
50% within the conclusions of precision and the assay section regarding the six sample preparation is 101. Method was checked for the accuracy regarding the analytes covering the section of most Assay and Content Uniformity, % recovery of most analytes was tabulated in Table-5. RESULTS AND DISCUSSION The Glycopyrrolate peak within the sample was identified by comparing together with the Glycopyrrolate standard and the retention time was located to be 4. The estimation of Glycopyrrolate tablets was carried out by RP-HPLC creating use of mobile phase possessing a composition of 1230 volumes of phosphate buffer, seven volumes of 1N sulpuricacid 470 volumes of Acetonitrile, 300 volumes methanol. The pH was located to be 6.
then finally filtered creating use of 0. 45 membrane filter and degassed in sonicator for 10 minutes. The column used was Bondapak C18 3. 9x300mm, 10m column with 3. Flow rate of mobile phase was 2.
3 mL or minute, system suitability parameters for example RSD for six replicate injections were located to be fewer than 2%, theoretical plates and tailing factor 1. The quantitative estimation was carried out on tablete by receiving the similar to concentration as for standard solution and assay conclusions located to be the acceptance criteria of system suitability was RSD should not be higher than 2% and the method display system suitability 0. 70% which shows that method was repeatable. The acceptance criteria of method repeatability was RSD should not be higher than 2. 0% and the method display method repeatability 0.
50% which shows that the method was precise. The validation of developed method shows that the drug stability is well within the limits. The linearity regarding the detector response was located to be linear from 50 to 150 g or ml of target concentration for Glycopyrrolate standard with a correlation coefficient price is greater than 0. The correlation coefficient of r2 = 1, which shows that the method was capable of producing good response in UV-dictator. The accuracy limits is the % recovery should be within the section of 99.
The validation of developed method shows that the accuracy is well within the limit, which shows that the method is capable of showing good accuracy. Influence on Flow Variation Table-1a. Influence on variation of mobile phase Composition. Organic phase Acetonitrile Variation Table-1b. Low organic phase -5%.
High organic phase +5%. Organic phase Methanol Variation Table-1c. Low organic phase -5%. High organic phase +5%. System suitability Table-2.
System suitability parameters. Concentration mcg or mL. Correlation coefficient r2. 999698 Precision Table -4. Day two or Analyst two or Inst.
Day 3 or Analyst 3 or. 6 Accuracy Table -5 Sample No. Based on the above validation data, it is evident that the HPLC method documented within the protocol for the determination of % assay for Glycopyrrolate in Glycopyrrolate Tablets USP two mg is treated like a validated method. Hence it is recommended to be use for the determination of % assay of Glycopyrrolate in Glycopyrrolate tablets USP two mg.
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