New RP-HPLC Method of Miglitol in Tablet Dosage form Including Forced Degradation Studies and Estimation in Spiked Rabbit Plasma

A simple, accurate, precise, economic, and speciÞ c stability indicating the RP-HPLC method has been developed for the estimation of Miglitol in tablet dosage form and spiked rabbit plasma. Chromatographic separation was achieved using Lachrom HPLC with Lichrospher, ODS, (250х 4.6) mm, ф 5 column at ambient temperature with 0.05 M ammonium acetate as a mobile phase at a ß ow rate of 0.5 ml/min and 216 nm was selected as a wave length for detection of a method. The retention time was 6.45 min. The standard curve was found to be linear (r2= 0.996) in the concentration range 800-1200 μg/ml using 1000 μg/ml as the test concentration. Apparent recovery was 98.338-101.704 % with RSD 0.942-0.964 for two brands. The method was repeatable with RSD 0.736. The intraday and interday precisions were RSD 0.957 and 1.019, respectively. LOD and LOQ of the method were 20 and 70 μg/ml, respectively. The presented study separates degradents and peaks appeared after spiking drug with plasma with a resolution of more than 1.5.

[6][7] The literature survey reveals that there are methods for the estimation of Miglitol in bulk and dosage forms including capillary electrophoresis, [8] tendom mass liquid chromatography, [9,10] and complexometric method, [11] but no RP-HPLC method was found with UV detection.The aim of this study is to develop a precise, accurate, economic, and simplest stability indicating the RP-HPLC method for the estimation of Miglitol (MGL) in tablet dosage forms and spiked plasma.

Apparatus and chemicals
The chromatographic system consists of an L-7110

INTRODUCTION
Miglitol, (2R, 3R, 4R, 5S)-1-(2-hydroxyethyl)-2-(hydroxymethyl) piperidine-3,4,5-triol [Figure 1], an oral anti-diabetic drug acts by inhibiting the ability of the patient to breakdown complex carbohydrates into glucose and slows the digestion of sugars so our body has time to store extra sugar. [1]e development of analytical method and validation followed by stability testing forms an important part of the process of drug product development. [2,3]The assay of drug product in stability test sample needs to be determined using a stability-indicating method, as recommended by the International Conference on Harmonization (ICH) solvent delivery system (Merk Hitachi), L-7400 doublebeam UV detector (Merk Hitachi), L-7500 integrator, and a rheodyne injector valve bracket Þ tted with a 20 µl sample loop.Chromatographic separation was performed on a stainless steel Lichrocart ODS Column, (250×4.6 mm) packed with 5 µ particle diameter, Lichrocart HPLC guard cartridge system, and a winchrom software.All of the chemicals and reagents were of analytical grade.Solvents of HPLC grade purchased from Merck Ltd, Mumbai.Reference standard Miglitol was kindly gifted by Glenmark Pharmaceuticals, Mumbai.Misobit and Mignar tablet of Lupin Pharma and Glenmark Pharmaceuticals, respectively, were purchased from the local market.

Chromatographic conditions
The mobile phase consisting of a 0.05 M ammonium acetate solution prepared by 3.85 g ammonium acetate was accurately weighed and dissolved in 1000 ml water.The mobile phase was degassed and Þ ltered by passing through a 0.45 µ millipore Þ lter prior to use.The elution was carried out under isocratic condition at a ß ow rate of 0.5 ml/min., with UV detection at 216 nm at ambient temperature.The chromatogram of Þ nally optimized chromatographic condition has been shown in Figure 2.
Preparation of standard solution and calibration curve 500 mg pure Miglitol was weighed and dissolved in water in 50 ml volumetric ß ask and diluted up to the mark with water to get a 10 mg/ml solution.Suitable aliquots of standard stock solution of Miglitol were taken in the concentration range and diluted with the mobile phase to obtain working standard solutions of suitable concentrations.Triplicate 20 µl injections were made for each concentration and are chromatographed under the above-mentioned conditions.The average peak area of the three replicates was plotted against the corresponding concentration to obtain the calibration curve [Figure 3], regression equation was found to be (y =21185x +3E+06) with a 0.9996 correlation coefÞ cient.

Validation of the proposed method
The developed method was validated for specificity, linearity, precision, accuracy, LOD, LOQ, and system suitability testing.

SpeciÞ city
Various generally used excipients such as lactose, talc, starch, and magnesium stearate were mixed in proportion approximately 90 mg, 28 mg, 30 mg, and 2 mg, respectively.They were mixed in 10 ml water in a volumetric ß ask, diluted up to the mark and Þ ltered. 2 ml of this Þ ltrate was diluted to 10 ml with mobile phase. 2 ml of the Þ ltrate was mixed with a 2 ml standard stock solution in 10 ml volumetric ß ask and diluted up to the mark to produce 1000 µg/ml.One dilution of a 1000 µg/ml standard solution was also prepared.All the solutions were injected in the order of excipients mixture, 1000 µg/ml standard solution, and then excipients-drug mixture.
Overlay of excipients, pure drug, and excipients [Figure 4] with drug shows no peak of the excipients at the RT of the drug.Excipient peak shows a resolution of 2.5 with the drug peak, hence the method is speciÞ c.

Linearity
Linearity is accessed by visualizing the of the calibration curve.The points in the calibration curve distributed equally above and below the trend line show linearity.

Range
Linearity range: It is the interval in which the response is directly proportional to the concentration between the upper and lower levels (which is generally ± 5% of the intercept having slope equal to zero) and was 800-1200 µg/ml, respectively.
Working range: It begins from limit of quantiÞ cation to the maximum concentration used for the development of the analytical method and in this case it is 70-1200.

Precision
Repeatability: Repeatability was accessed by six replicate injections of 1000 µg/ml solution of drug.% RSD found was found to be 0.736 showing that the method is repeatable.
Intraday and intraday precision: 0.8 ml, 1 ml, and 1.2 ml was taken out from the standard stock solution and diluted to 10 ml to make 800 µg/ml, 1000 µg/ml, and 1200 µg/ml, respectively.Three replicates of each dilution were injected three times a day for intraday and three replicates for 3 days for interday.% RSD found to be in this case were 0.957 and 1.019, respectively.

Accuracy
Recovery studies were performed with two brands Misobit (Lupin Ltd) and Mignar (Glenmark pharmaceuticals Ltd).
Powdered Misobit tablets (Lupin Ltd.) equivalent to 100 mg MGL was transferred to a 10 ml volumetric ß ask and ultrasonication was done for 20 min with approximately 5 ml water.Solution was then diluted up to the mark with water and Þ ltered through 0.45 µ Þ lter.0.8 ml of this solution was spiked in three different 10 ml volumetric ß asks with 0.1, 0.2, and 0.3 ml of previously analyzed standard stock solution.Finally, volume was made up to the mark with mobile phase and estimation of drug content was done by proposed method.
Same procedure was followed for Mignar tablets except ultrasonication time was kept for 15 min.The results of recovery studies are shown in Tables 1 and 2

System suitability testing
For system suitability testing six replicates of 1000 µg/ml were injected and result obtained given in Table 3.

Optimization of extraction time
Four dilutions of 1000 µg/ml of the formulation were prepared in the mobile phase and sonicated for 10, 15, 20, 25, and 30 min and 20 µl was injected immediately.

Procedure for estimation of MGL in tablet dosage form
Powdered Misobit tablet equivalent to 50 mg of MGL was taken in 50 ml volumetric ß ask and ultrasonication was done using approximately 25 ml water and diluted up to the mark with mobile phase.The content of drug in tablet was found by using equation.The same procedure was followed for Mignar tablets except ultrasonication time used is 20 min.The results of estimation of purity of Misobit and Mignar are given Table 4.

Forced degradation studies
Acidic degradation 5 ml of standard stock solution was transferred in 50 ml volumetric ß ask and 1 ml concentrated HCl was added to it and kept for 2 h at ambient temperature then diluted to 25 ml with water.The pH of the solution was adjusted to 6.5 with 1 M NaOH.The solution was Þ nally diluted to 50 ml with mobile phase, and 20 µl volume was injected.The same procedure was repeated at 50 and 100 ºC, but no were degradents observed.
Alkali degradation 5 ml of the standard stock solution was transferred in a 50 ml volumetric ß ask and 1 ml 10 M NaOH was added to it and kept for 2 h then diluted to 25 ml with water.The pH of the solution was adjusted to 6.5 with 1 N HCl.The solution was Þ nally diluted to 50 ml with mobile phase and 20 µl volume was injected.The same procedure was repeated at 50 and 100 o C, but no degradents were observed.
Oxidative degradation 5 ml of the standard stock solution was transferred in a 50 ml volumetric ß ask.1 ml 30% H 2 O 2 was added to it and kept for 2 h.It was diluted till 25 ml with water.The solution was Þ nally diluted to 50 ml with mobile phase and 20 µl volume was injected.The same procedure was repeated at 50 o C and 100 o C, but no degradents were observed in the chromatogram.
Degradation by UV light 5 ml of the standard stock solution was transferred in a 50 ml volumetric ß ask in UV chamber for 24 h then diluted up to the mark with mobile phase to produce 1000 µg/ml and 20 µl was injected but again no degradents were observed.

Thermal degradation
Wet heat: 5 ml of the standard stock solution was transferred to two different 50 ml volumetric ß asks and kept on water bath for 3 and 6 h, respectively, at 80 o C. It was diluted up to the mark with mobile phase to produce 1000 µg/ml and 20 µl was injected.Figure 6 represents the chromatogram obtained due to wet heat.
Dry heat: 50 mg accurately weighed drug was taken in china dish and kept it in oven for 48 h at 100 o C. The drug was transferred to a 50 ml volumetric ß ask and diluted approximately 25 ml with water then volume was made up to the mark with mobile phase to produce1000 µg/ ml and 20 µl volume was injected.Figure 7      The overall summary of degradation studies is presented in Table 5.

Estimation of miglitol in spiked plasma
Preparation of sample Approximately 1.5 ml blood was taken by cutting the marginal ear vein of rabbit and collected in eppendrof tubes with previously added EDTA.This was stored at −20 o C and centrifuged for 2 min at 8000 rpm for 10 min to separate plasma and again stored at 20 o C. 0.2 ml plasma was spiked with 0.1 ml of the standard stock solution and 0.6 ml mobile phase was added.Finally, the components were mixed thoroughly using cyclo mixture and then centrifuged for 5 min at 5000 rpm.The chromatogram obtained after injecting prepared sample is shown in Figure 8.
Recovery study of miglitol in spiked plasma 0.1 ml of 8000 µg/ml drug solution prepared by a standard stock solution and spiked with 0.2 ml plasma and volume was made 1 ml with mobile phase and analyzed.0.1 ml of this solution was spiked with 0.1, 0.2, and 0.3 ml of 1000 µg/ml previously analyzed drug solution in three separate eppendrof tubes, and volume was made up to 1 ml with mobile phase.The results are shown in Table 6.

CONCLUSION
In the present work, a HPLC method for the estimation of Miglitol in tablet dosage form has been developed.The proposed method is simple, precise, and accurate and do not suffer from any interferences due to common excipients.The degradation studies and results obtained after spiking drug in rabbit plasma showed separation of drug peak and other peaks with a resolution of more than 1.5.The newly developed method can be used in pharmaceutical industry for routine analysis of Miglitol in tablet dosage form.The summary of validation parameters is given in Table 7.The presented study can be further proceeded for the development of the bioanalytical method for the estimation of Miglitol in biological ß uids.

Figure 2 :Figure 4 :
Figure 2: Chromatogram of optimized condition of the proposed method

Figure 5 :
Figure 5: RSD V/S concentration (µg/ml) graph for the determination of LOQ and LOD of the proposed method

Figure 7 :
Figure 7: Chromatogram of dry heat degradation

Figure 6 :
Figure 6: Chromatogram of wet heat degradation

Figure 8 :
Figure 8: Chromatogram of MGL with spiked plasma

Table 7 : Summary of validation parameters Parameters Results SpeciÞ
city Resolution of degradants and excipients with drug peak > 1.5, hence method is speciÞ c.