A Novel Stability Indicating Analytical Development and Validation of an RP-HPLC Assay Method for the Quantification of Voglibose in Bulk and its Formulation

Md. Nazmul Sardar¹, Ekhlass Uddin², Md. Shahin Reza³, Shajahan Talukdar Sunny⁴,

Md. Faysal Khan Shuvo⁴, Md. Mamun Hossain⁵, Md. Faruk Hossen², Md. Ali Asraf²,

Md. Kudrat-E-Zahan⁶*

¹Department of Pharmacy, University of Rajshahi, Bangladesh.

²Department of Chemistry, University of Rajshahi, Bangladesh.

³Department of Chemistry, Tennessee State University, Nashville, United States.

⁴Department of Pharmacy, Southeast University, Bangladesh.

⁵Department of Pharmacy, Rajshahi University, Rajshahi-6205, Bangladesh.

⁶Professor, Department of Chemistry, Rajshahi University, Bangladesh.

*Corresponding Author E-mail: kudrat.chem@ru.ac.bd, nazmulsardar91@gmail.com, ekhlassuddin@gmail.com

ABSTRACT:

The investigation developed a new stability-indicating Reverse phase high-pressure liquid chromatographic (RP-HPLC) assay method to quantify Voglibose in bulk and pharmaceutical dosage form. The technique used isocratic elution on an Amino (NH₂) column (250x4.6mm, 5µ particle size) with a mobile phase of Phosphate Buffer (pH 6.7) and Acetonitrile (27:73 v/v). The separation was monitored using UV detection at 210 nm. The method was validated, meeting ICH guidelines. The retention time of Voglibose was 6.38 minutes, and the linearity was between 24-56 µg/ml. The limit of detection and quantitation were 0.70 and 2.11 µg/ml, respectively. Analytical performance parameters were determined according to ICH guidelines. The method was simple, precise, accurate, robust, and rapid, making it suitable for quantifying Voglibose in bulk and tablet formulation.

GRAPHICAL ABSTRACT:

KEYWORDS: Reverse Phase HPLC, Voglibose, Method development and validation, ICH Guidelines.

INTRODUCTION

Under the trade name BASEN, Voglibose was initially introduced in Japan in 1994 and was developed in 1981 to improve postprandial hyperglycemia in individuals with diabetes mellitus.^1,2 In patients with diabetes mellitus, voglibose (Figure 1), a strong α-glucosidase inhibitor with the chemical formula C₁₀H₂₁NO₇ and a molecular weight of 267.27 g/mol, is used to reduce postprandial blood glucose levels.^3,4 Voglibose's chemical name is [5-(1, 3-dihydroxy propane 2-yl-amino)-1-(hydroxymethyl)cyclohexane-1, 2, 3, 4-tetrol], which is comparable to the IUPAC designation for carbohydrates, which is (1S,2S,3R,4S,5S).cyclohexane-1,2,3,4-tetraol-5-(1,3-dihydroxypropan-2-ylamino)-1-(hydroxymethyl). The substance has the appearance of an off-white to white crystalline powder that dissolves easily in water. Normally, complex carbohydrates are broken down into monosaccharides, or simple sugars, that the intestines can absorb.⁵

Therefore, complex carbs have less of an effect on blood sugar when alpha-glucosidase inhibitors are taken.^6-8 Acarbose, miglitol, and voglibose are the three glucosidase inhibitors that are most frequently employed.⁹ The most efficient and safest of them all is Voglibose. No LC method that detects refractive index has been reported for the analysis of voglibose and that may be used to analyze the substance in tablets that are sold commercially. Several analytical techniques, such as spectrophotometric methods and HPLC, have been reported for the determination of Voglibose.¹⁰ The most popular method for determining the amount of Voglibose in human blood cells, plasma, saliva, and cerebrospinal fluid as well as for researching drug metabolites in urine is high-performance liquid chromatography (HPLC).^11-13 Voglibose assays using the previously recommended HPLC methods are quite expensive and need advanced equipment prior to ICH guidelines.^14-17

The present study aims to develop a simple, accurate, and precise RP-HPLC method for the estimation of Voglibose in bulk and pharmaceutical dosage forms. In this research, a new RP-HPLC method was developed for the estimation of Voglibose in pharmaceutical formulations, which showed high reproducibility, sensitivity, and economics according to ICH guidelines.

Figure 1: The chemical structure of Voglibose.

MATERIALS AND METHODS:

Instrument and chromatographic conditions:

The liquid chromatographic system employed to develop this technique was Shimadzu HPLC 2050 consisting PDA detector and autosampler. Chromatographic separation was achieved on Amino (NH₂) column (250x4.6mm, 5µ particle size) with mobile Phosphate Buffer (pH 6.7) and Acetonitrile (27:73 v/v) at 50°C column temperature. The flow rate was 1.5 ml/min and the detector wavelength was kept at 210 nm for monitoring the elution. The injection volume was 100 µL and total run time was 10 min.

Reagents and solutions:

Pure sample, working standard, and Placebo of Voglibose were kindly supplied by Radiant Pharmaceuticals, Dhaka, Bangladesh. Acetonitrile and purified water used were of HPLC grade. Reagent grade reagents were all other reagents employed in this investigation.

Mobile phase preparation:

Buffer: 0.60g monobasic potassium phosphate and 0.35g dibasic sodium phosphate were dissolved into 1000 ml purified water and adjusted pH to 6.7 with 1 N sodium hydroxide solution. The mobile phase was a mixture of Phosphate buffer (pH 6.7) and Acetonitrile (27:73% v/v). The solution was filtered through a 0.45 mm membrane filter under vacuum filtration.

Preparation of standard solution:

20mg of Voglibose working standard was weighed and transferred to a 50mL volumetric flask with 30mL diluent (mobile phase). The solution was kept for sonication for 10 min and cool the solution to room temperature. The final volume of the stock solution was made up to the mark with the diluent. Furthermore, 5ml of this solution was transferred to a 50ml volumetric solution and volume up to mark with diluent. The final concentration is represented as 40 µg/ml. The solution was passed through a Nylon Disc filter having 0.45 µm or finer porosity.

Preparation of sample solution (Assay):

Twenty tablets, each containing 0.2 mg of Voglibose were weighed, the average weight was calculated, and a quantity equivalent to 4mg of Voglibose was weighed accurately and transferred to a 100ml volumetric flask, added 2-3 ml water to disintegrate the whole tablet, then added 60ml diluent (mobile phase). The solution was kept for sonication for 10 min and cooled the solution to room temperature. The final volume of the stock solution was made up to the mark with the diluent and the sample was centrifuged at 4000 rpm for 10 minutes. The solution was passed through a Nylon Disc filter having 0.45 µm or finer porosity.

Method development:

A variety of mobile phases were investigated in the development of a stability-indicating RP-HPLC method for the analysis of Voglibose in tablet dosage form. The selectivity and sensitivity of the assay were used to determine whether the mobile phase was appropriate.

Method validation:

Once the HPLC method development was over, the method was validated in terms of parameters like System suitability, Specificity, precision, accuracy, linearity and range, LOD, LOQ, ruggedness, robustness, stability, etc. Values for the percentage relative standard deviation were computed for every parameter. The suggested HPLC method was validated by ICH regulations.

System suitability:

System suitability parameters were studied to verify the system performance. Five replicate samples containing Voglibose [40 µg/ml] were analyzed using the developed method. When the applicability of the system was evaluated, variables such as retention duration, percent relative standard deviation [%RSD] of peak area, theoretical plate count, and tailing factor were taken into account. The developed method has produced a theoretical plate above 2000 for Voglibose with a tailing factor of less than 2. Similarly, the percent relative standard deviation [%RSD] of peak area and retention time was less than 2, which ensures the suitability of the developed method. Results are shown in Table 1.

Table 1: System suitability conditions of the developed method.

Test Parameters	Observation	Specification	Result (Pass/Fail)
%RSD of the area of 5 replicate injection of Standard Solution	0.48	≤ 2.00	Pass
Theoretical Plate Count	3126	NLT 2000	Pass
Tailing Factor	1.17	NMT 2.0	Pass

Method precision:

Six independent sample preparations of a single formulation lot were used to assess precision. The sample preparation instructions were followed to prepare the sample solution. Percentage relative standard deviation (%RSD) was found to be less than 2% for the method, which proves that the method is precise. Results are shown in Table 2.

Intermediate precision:

Another analyst carried the Intermediate method precision on another day using another column and instruments. The standard and six sample solutions were prepared by following in the standard and sample preparation. The result of the % RSD of six samples was found to be less than 2.0%. Results are shown in Table 2.

Table 2: Method precision and intermediate precision for Assay (%) determination.

Precision of Voglibose (% of Assay)
Sample ID	Method Precision (%)	Intermediate Precision (%)
Sample-1	100.25	100.41
Sample-2	99.23	100.31
Sample-3	100.63	99.80
Sample-4	99.49	99.38
Sample-5	100.64	100.94
Sample-6	100.21	99..36
STDEV	0.59	0.63
%RSD	0.59	0.63
Average of 12 samples: 100.05
STDEV of 12 sample: 0.60
% RSD of 12 samples: 0.60

Accuracy:

The accuracy of the method was determined by interpolation of replicate (n = 3) peak areas of three accuracy standards (20, 40, and 60 µg/ml). The percentage of recovery was computed for every instance. The mean % recoveries obtained were 99.56, 99.55, and 99.84 %, for 50, 100 and 150 % concentration levels respectively. The range of 99.8–101.20 percent for the mean recoveries indicates that there is no interference from excipients. The outcomes appear in Table 3.

Linearity:

The calibration curve constructed for Voglibose was linear over the concentration range of 24-56 µg/ml. Peak areas of Voglibose were plotted versus its concentration and linear regression analysis was performed on the resultant curve. The correlation coefficients of R² = 1.000, prove linear regression analysis. Typically, the regression equation for the calibration curve was found to be y = 3744.808x – 306.395. The linearity curve is shown in Figure 2 and the results of Linearity are shown in Table 4.

Figure 2: Linearity Curve (Concentration verses Area: R² = 1.000).

Specificity:

The condition of the HPLC method like the percentage of organic solvent in the mobile phase, pH of buffer flow rate, etc. was changed. Despite the aforementioned modifications, shift retention times and slight alterations in peak forms were observed, but no new peaks were discovered. Retention time and Placebo interference are given in Table 5. Peaks of diluent, placebo, standard and sample solution are shown in separate Figures from 3-6.

Table 4: Linearity Result of Voglibose.

Concentration (µg/ml)	Area
4	15034
8	29173
16	59638
24	90138
32	118673
40	149361
48	180371
56	208977

Table 5: Specificity result of Voglibose.

Sample ID	Observation
Sample ID	Retention Time	Remarks
Diluent	No interfere	No diluent Peak
Placebo	No interfere	No Placebo Interference
Standard Solution	6.38	Passed
Sample Solution	6.38	Passed

Sensitivity:

The sensitivity of the proposed method was estimated in terms of LOD (Limit of detection) and LOQ (limit of quantification) by using standard deviation and slope of the calibration curve.

The LOD and LOQ were determined by using flowing equation:

LOD: 3.3 x σ/S

LOQ: 10 x σ/S

σ = Average standard deviation

S = Slope of the curve

LOD: 0.70 (µg/ml)

LOQ: 2.11 (µg/ml)

Solution stability:

This study indicates that the standard and samples are stable at 15°C temperature when standard and samples vial are stored at sample tray in HPLC and there were no significant changes in assay value and the results were found within ±2% deviation. The results of Stability studies are shown in Table 6.

Table 6: Solution stability for standard and sample solution of Voglibose.

Time Point (hour)	Standard Solution (Peak Area)	% Deviation	Sample Solution (Peak Area)	% Deviation
0	148568	Not applicable	156895	Not applicable
4^th hour	147361	0.81	158931	-1.30
8^th hour	150132	-1.05	154938	1.25
12^th hour	145995	1.73	157398	-0.32
16^th hour	149058	-0.33	158907	-1.28
20^th hour	149938	-0.92	155909	0.63
24^th hour	148039	0.36	156391	0.32

Robustness: Small but intentional adjustments to the technique parameters had no negative impact on the method's performance, indicating that the approach was found to be robust. As expected, the retention time of analytes decreased with increasing mobile phase flow rate and vice versa. A slight decrease in retention time of the analytes was observed with increasing column oven temperature. While standard and samples were often evaluated simultaneously and at the same flow rate and temperature for routine quality control analysis, flow rate and temperature had no negative effects on the procedure. The results of robustness were shown in Table 7.

Application of Validated Method for Assay of Voglibose in Pharmaceutical Dosage Form:

The developed method was successfully implemented in the assay of Voglibose in Pharmaceutical dosage form in which the assay result was found to be 100.07%. According to ICH Q2R1 criteria, the technique validation results were good. The peak areas were found to be linear over the concentration range of 24-56 µg/ml with a correlation coefficient of 1.000. Method specificity can be proved using the “Peak purity” parameter in the ‘Lab Solution’ software of HPLC. The unaffected assay of the drug in the blend confirms the non-interference by any excipients. Difference between method precision and Intermediate precision was less than 2%. Percent recovery in accuracy study was within the limit of 98 to 102%.

Table 7: Solution stability for standard and sample solution of Voglibose.

Robustness Parameter	Variation	Retention time	USP Plate Count	USP Tailing
Flow rate	0.8 ml/min	5.06	3196	1.15
	1.0 ml/min	6.38	3126	1.17
	1.2 ml/min	7.81	3153	1.18
Column Temperature	48 °C	5.97	3098	1.19
	50 °C	6.38	3126	1.17
	52 °C	6.89	3260	1.15
Mobile Phase composition	Buffer: ACN (22:78)	4.91	2975	1.12
	Buffer: ACN (27: 73)	6.38	3126	1.17
	Buffer: ACN (32:68)	8.23	3187	1.20

CONCLUSION:

An isocratic liquid chromatographic method has been described and validated for qualitative and quantitative determination of Voglibose in bulk and its formulation in this investigation. The method showed acceptable assay precision (<2% RSD) and the Mean %Recovery (100.02 %) obtained at 60-140% of the target analytical concentration was within limit. In addition to its high sensitivity and robustness, the intended RP-HPLC method proved reliable for stability indicating analysis of Voglibose. So this proposed method can be used for routine quality control analysis of dosage forms and stability samples in pharmaceutical science.

CONFLICT OF INTEREST:

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGMENT:

The authors are thankful to Radiant Pharmaceuticals Ltd. Bangladesh for providing chemicals and instrumentation facilities.

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Received on 28.09.2024 Revised on 16.10.2024

Accepted on 14.11.2024 Published on 25.11.2024

Available online from December 27, 2024

Asian J. Research Chem. 2024; 17(6):344-350.

DOI: 10.52711/0974-4150.2024.00058

Accuracy Level	Sample ID	% of Recovery	Average (%)	SD	% RSD	Average (%)	% RSD
60 %	Sample 1	99.18	100.10	0.90	0.89	100.02	0.70
	Sample 2	100.97
	Sample 3	100.14
100%	Sample 1	100.31	100.08	0.22	0.22
	Sample 2	99.88
	Sample 3	100.05
140%	Sample 1	99.15	99.89	0.70	0.70
	Sample 2	100.53
	Sample 3	99.99