Estimation of Nateglinide by using MBTH as a Chromogenic Reagent
N. Raghavendra Babu*1, Y. Padmavathi1, R. Swethan Babu2, Amreen Unnisa1, M. Bhavana1,
P. Ravi Kumar1
1Department of Pharmaceutical Analysis, G. Pulla Reddy College of Pharmacy, Mehdipatnam, Hyderabad, Telangana
2Department of Pharmaceutical Chemistry, SRM University, Chennai, Tamil nadu.
*Corresponding Author E-mail: nayakaraghavendrababu@gmail.com
ABSTRACT:
Nateglinide is used as an oral anti-diabetic drug. The aim of the present study is to develop spectrophotometric method for the estimation of nateglinide by using MBTH reagent. Visible spectrophotometric method has been developed for quantification of nateglinide. The absorbance of green colored chromogen was measured at 634nm against appropriate reagent blank and the graph of absorbance against concentration was plotted. Beer’s law was obeyed in the concentration range of 50-300µg/mL. The LOD and LOQ were found to be 11.78 μg/mL and 35.7 μg/mL respectively with coefficient of correlation 0.999. The proposed method has been validated for linearity, accuracy and precision as per ICH guidelines and applied for quantitative analysis of nateglinide using chromogenic reagent.
KEYWORDS: Nateglinide, MBTH reagent, Visible spectrophotometry, Beer’s law, ICH guidelines.
INTRODUCTION:
Nateglinide is used alone or in combination with other medications to treat type 2 diabetes (condition in which the body does not use insulin normally and therefore cannot control the amount of sugar in the blood) in people whose diabetes cannot be controlled by diet and exercise alone. Nateglinide belongs to a class of drugs called meglitinides. Nateglinide helps your body regulate the amount of glucose (sugar) in your blood. It decreases the amount of glucose by stimulating the pancreas to release insulin. Nateglinide may be used in combination with metformin as an adjunct to diet and exercise for the management of type 2 diabetes mellitus in patients who do not achieve adequate glycemic control with metformin monotherapy.
Invitro pharmacological studies suggest that nateglinide stimulates insulin secretion by blocking ATP sensitive potassium channel in pancreatic beta-cells, causing cell membrane depolarization which results in calcium influx and insulin secretion1-3.
MBTH Reagent:
Besthron has synthesized 3-methyl-2-benzothiazolinone hydrazone (MBTH). Although MBTH was first prepared in 1910 but its analytical abilities were evidenced only in 1957. Then it was introduced as a reagent for colorimetic determination in 1961 since, it has become a versatile analytical reagent for determination of amines and phenolic types of drugs. MBTH was originally introduced as a reagent for aldehydes, later its use got extended to a variety of organic compounds like phenols, aryl amines and different N and S heterocyclic compounds. The reagent is used in the form of an aqueous solution and the reaction products can be extracted into chloroform.
Literature survey4-20 revealed that there are only very few UV spectrometric and RP-HPLC methods were reported for the determination of nateglinide in tablet and bulk dosage form. There were no visible spectrophotometric method has been developed, which was simple and economical when compared to other UV methods. Thus, the present study includes development of fast, accurate and sensitive visible spectrophotometric method for the estimation of nateglinide as an active pharmaceutical ingredient as well as in pharmaceutical formulation by using MBTH as a chromogenic reagent. Now a days, no one are using simple conventional reagent such as 3-methyl-2-benzothiazolinone hydrazone (MBTH) due to lack of sensitivity by chemometric methods.
In the present study, an attempt has been made for estimation of Nateglinide, MBTH looses two electrons and one proton to form an electrophlic intermediate which is highly coupling species, then reacts (or) couples with nateglinide to form a coloured chromogen (apple green colour) which is measured at 634 nm.
Chemicals and Reagents:
MBTH (3-methyl-2-benzothiazoline hydrazone), Sodium hydroxide, Ferric chloride anhydrous, Potassium chloride, Methanol.
Instruments:
UV- Visible spectrophotometer (Shimadzu UV-1800), pH meter (ELICO, LI 127), Shimadzu BL Digital Balance, Sonica ultrasonicator, Refrigerated Centrifuge, Hot Air Oven.
Standards and Sample:
Nateglinide (BIOCON), Glinate-120mg (Glenmark pharmaceuticals).
Method development:
Preparation of MBTH (0.5%w/v):
Dissolve 50mg of MBTH is dissolved in 10mL water.
Preparation of 1% Ferric chloride:
Fresh aliquots of nateglinide ranging from 0.5-3mL from stock solution (1000µg/mL) were transferred into a series of 10mL volumetric flasks to provide final concentration range of 50-300µg/mL. To each flask 1mL of MBTH (0.5%w/v solution) was added followed by 1mL of Ferric chloride solution (1%). The solution in each tube was made up to the mark with distilled water. The absorbance of green colored chromogen was measured at 634nm against appropriate reagent blank and the graph of absorbance against concentration was plotted as shown in the (Fig.6). The regression equation and coefficient correlation was given in the table.
A Visible spectrophotometric method has been developed for quantitative estimation of nateglinide using MBTH reagent. The method developed was validated according to the ICH Guidelines Q2 (R1): Validation of Analytical Procedures: Text and Methodology. Validation of method was performed to ensure that the performance characteristic of the method meets the requirements for the intended analytical applications.
Method validation:
Linearity and Range:
The linearity of analytical method is ability to elicit test results that are directly proportional to the concentration of analyte in the sample within the range. The range of analytical method is the interval between upper and lower levels of analyte that have been demonstrated within a suitable level of precision, linearity and accuracy. Under the optimum conditions, the calibration curve for the determination of nateglinide by its reaction with MBTH was constructed by plotting the absorbance as a function of corresponding concentrations which are shown in (Table 1).
Limit of Detection and Limit of Quantification:
The sensitivity of proposed method for measurement of nateglinide was estimated in terms of LOD & LOQ. The limit of detection (LOD) and the limit of quantification (LOQ) were determined according to the International Conference Of Harmonization (ICH) guidelines for the validation of analytical procedure. The following formulae were used:
LOD=𝟑.𝟑𝛔/𝐒
LOQ=𝟏𝟎𝛔/𝐒
Where,
σ = standard deviation of the response (intercept) S = slope of the calibration curve
The mean of the slope and standard deviation of response were obtained after plotting three calibration curves.
Optical conditions and Statistical Data of Regression equation:
The optimum conditions such as Beer’s Law limits, Molar absorptivity, Sandell’s sensitivity and other regression characteristics like slope (m), intercept (c), correlation coefficient were calculated and presented in (Table 2).
Precision:
The precision of an analytical method is the degree of agreement among individual test results, when the method is applied repeatedly to multiple samplings of homogeneous sample. It provides an indication of random errors results and expressed as Relative Standard Deviation (% RSD).
Repeatability:
Repeatability assessment of an analytical method is performed by analyzing six replicates of single concentration that is 150μg/mL. Absorbances of samples were recorded at 634nm. The % relative standard deviation (RSD) was calculated. The results obtained were shown in (Table 3).
Intraday and Inter day precision:
Variations of results within the same day (intraday) and variation of results between days (inter day) were analyzed.
The intra-assay precision of the proposed method was determined on samples of drug solutions at varying concentration levels (120µg/mL, 150µg/mL, 180µg/mL) by analyzing three replicates of each sample as a batch in a single assay run at 634nm. The %RSD was calculated and results were in (Table 4).
The Inter-assay precision was determined by analyzing the same samples (120µg/mL, 150µg/mL, 180µg/mL) in three consecutive days at 634nm. The %RSD was calculated and results were in (Table 5).
Accuracy:
Accuracy is the closeness of test results obtained by the method to the true value.
Accuracy for drug substance was determined on samples of drug solutions at varying concentration levels in the range of 80%-120% (120μg/mL, 150μg/mL, 180μg/mL) by analyzing three replicates of each sample as a batch in a single assay. The %RSD was calculated and reported in (Table 6).
To study the accuracy of drug product, 10 tablets were weighed, powdered and estimation was carried out. Recovery studies were carried out by adding known amount of standard drug (120, 150, 180µg/mL) to the sample solution (150µg/mL). The %recovery was calculated and reported in the (Table 7).
Estimation of nateglinide in dosage forms using MBTH Reagent:
Twenty tablets of nateglinide (Glinate manufactured by Glenmark Pharmaceuticals Ltd., India, contains 120mg of nateglinide) were weighed and finely powdered. The powder equivalent to 100mg was weighed and transferred to 100mL volumetric flask containing 25mL methanol and sonicated for 30mins. The flask was shaken and volume was made up to mark with methanol to obtain a solution of 1000µg/mL. The solution was centrifuged at 2500rpm for 15 mins.
From the above solution (1000µg/ml) 1.5mL of sample was pipette into 10mL graduated tube followed by addition of 1mL of MBTH reagent (0.5%w/v) and 1mL of Ferric chloride solution (1%) and solution was made to 10mL using distilled water. The absorbance of resulting green colored solution was measured at 634nm against appropriate reagent blank. The obtained results are given in (Table 8).
RESULTS AND DISSCUSSION:
Determination of Absorption maximum (λ max):
The absorption spectra was recorded in wavelength region of 400-800nm (visible region).
|
S. No |
Concentration (μg/ml) |
Absorbance* |
|
1 |
50 |
0.062 |
|
2 |
100 |
0.148 |
|
3 |
150 |
0.246 |
|
4 |
200 |
0.354 |
|
5 |
250 |
0.468 |
|
6 |
300 |
0.564 |
*Average of three determinations
Fig.6: Calibration curve for nateglinide at 634nm with MBTH
Limit of Detection and Limit of Quantitation:
The sensitivity of proposed method for measurement of nateglinide was estimated in terms of LOD & LOQ was found to be 11.78 μg/mL and 35.7 μg/mL.The calculate values of LOD and LOQ were found to be and respectively. This indicates sensitivity of the method.
Optical conditions and Statistical Data of Regression equation:
The optimum conditions such as Beer’s Law limits, Molar absorptivity, Sandell’s sensitivity and other regression characteristics like slope (m), intercept (c), Correlation coefficient were calculated and presented in (Table 2).
Table 2: Optimum conditions and Spectral data
|
λ max |
634nm |
|
Colour |
Apple Green |
|
Beer’s law range |
50-300µg/mL |
|
Sandell’s sensitivity (µg/cm2/0.001 absorbance units) |
3.98 |
|
Molar absorptivity (litre/moles/cm) |
1.75x10-3 |
|
Limit of detection (µg/mL) |
11.78 |
|
Limit of quantification (µg/mL) |
35.7 |
|
Regression equation (y) |
y=0.002x-0.046 |
|
Slope (a) |
0.002 |
|
Intercept (c) |
-0.046 |
|
Correlation coefficient (r2) |
0.998 |
y= mx+c
where c is the concentration of drug in µg/ml and y is the absorbance measured at λ max. The results obtained with the proposed method confirm that this method is linear and sensitive.
Precision:
The precision of the developed analytical method was assessed by checking repeatability, intra-day precision and inter-day precision.
Repeatability:
Repeatability results obtained for six replicates of standard solutions of nateglinide were shown in (Table 3).
|
S. No |
Concentration (µg/ml) |
Absorbance |
Mean* ±Standard deviation |
% RSD |
|
1 |
150 |
0.242 |
0.24±0.003 |
1.24 |
|
2 |
150 |
0.239 |
||
|
3 |
150 |
0.243 |
||
|
4 |
150 |
0.247 |
|
|
|
5 |
150 |
0.240 |
||
|
6 |
150 |
0.245 |
*Average of six determinations
The results reveal that %RSD values were within the limits hence the method is repeatable.
Intermediate Precision Intra-day precision:
The intraday precision of the proposed method was determined by analyzing the corresponding concentration (80%, 120%, 150%) 2 times on the same day and the results were reported in terms of relative standard deviation. The results were shown in (Table 4).
Inter-day precision:
The inter-day precision of the proposed method was determined by analyzing the corresponding concentrations (80%, 120%, 150%) on 3 different days and the results were reported in terms of relative standard deviation. The results were shown in (Table 5).
|
S. No |
Concentration (µg/ml)) |
Mean Absorbance* |
Mean ±Standard deviation |
% RSD |
|
|
1 |
120 |
Day-1 |
0.213 |
0.217 + 0.004 |
2.0 |
|
Day-2 |
0.218 |
||||
|
Day-3 |
0.222 |
||||
|
2 |
150 |
Day-1 |
0.247 |
0.256 + 0.005 |
1.99 |
|
Day-2 |
0.250 |
||||
|
Day-3 |
0.257 |
||||
|
3 |
180 |
Day-1 |
0.305 |
0.311 + 0.006+ |
1.93 |
|
Day-2 |
0.323 |
||||
|
Day-3 |
0.351 |
||||
*Average of 3 determinations
|
S. No |
Concentration (µg/ml)) |
Mean Absorbance* |
Mean ±Standard deviation |
% RSD |
|
|
1 |
120 |
Morning |
0.210 |
0.212± 0.002 |
1.18 |
|
Afternoon |
0.212 |
||||
|
Evening |
0.215 |
||||
|
2 |
150 |
Morning |
0.238 |
0.241± 0.004 |
1.66 |
|
Afternoon |
0.243 |
||||
|
Evening |
0.246 |
||||
|
3 |
180 |
Morning |
0.295 |
0.304± 0.005 |
1.70 |
|
Afternoon |
0.302 |
||||
|
Evening |
0.305 |
||||
*Average of 3 determinations
The results shows that all the calculated % RSD values for both interday and intraday precision are below 2, therefore the method is precise.
Accuracy:
The analytical accuracy is the nearness of the results obtained against the real values at each level of nateglinide concentration. The results of obtained for accuracy studies for the drug substance and drug product were reported in terms of % RSD and % recovery respectively.
For drug substance:
From the below data it was found that the %RSD (acceptance criteria <2%) values are within the acceptance limits. The results were shown in (Table 6).
|
S. No |
Concentration (µg/ml) |
Mean ±Standard deviation* |
%RSD |
|
1 |
120 |
0.207 ± 0.004 |
1.51 |
|
2 |
150 |
0.241 ± 0.004 |
1.85 |
|
3 |
180 |
0.304 ± 0.005 |
1.94 |
*Average of six replicates
For drug product (Recovery study):
The recovery was assessed by determining the agreement between the measured standard concentration and added known concentration to the sample. The test was done by spiking the pre-analysed tablet powder with pure nateglinide at three different levels (80%, 100% and 120%). The results were shown in (Table 7).
|
Tablet used |
Amount of sample added (µg/mL) |
Amount of standard added (µg/mL) |
Amount recovered (µg/mL) |
%Recovery ± Standard Deviation* |
|
Glinate |
150 |
120 |
273 |
101.1 ± 1.064 |
|
150 |
150 |
297.33 |
99.1 ± 1.2 |
|
|
150 |
180 |
325.6 |
98.6 ± 1.28 |
* Average of three replicates
From the above data it was found that recovery values are within the acceptance limits.
The proposed method was then applied for the determination of nateglinide in marketed formulations (tablets) Glinate manufactured by Glenmark Pharmaceutical Ltd., India, contains 120mg of nateglinide. The % purity of the drug was presented in (Table 8).
|
Tablet used |
Label Claim (mg) |
Amount found (mg)** |
% Purity |
|
Glinate |
120 |
113.2 |
94.7 |
*Average of three Replicates
Assay limits: 89.8 to 111.3
From the above data it was found that recovery values are within the acceptance limits.
Color stability:
The stability of the color of the drug substance as well as the drug product was checked and reported in (Table 9). The color for drug product and standard were found to be stable for 24 hrs.
Table 9: Color stability data for nateglinide at 634nm using MBTH method
|
Concentration (µg/ml) |
Time (hrs) |
Absorbance |
|
150 |
4 |
0.247 |
|
8 |
0.247 |
|
|
12 |
0.247 |
|
|
16 |
0.247 |
|
|
20 |
0.247 |
|
|
24 |
0.247 |
|
|
28 |
0.210 |
|
|
32 |
0.109 |
CONCLUSION:
A new simple spectrophotometric method using chromogenic reagent was developed and validated for estimation of nateglinide in dosage forms. This method was developed by using MBTH as chromogenic reagent with ferric chloride as oxidising agent. The developed method was validated as per ICH guidelines and the validation parameters were found to be within the acceptance limits. The proposed method was found to be linear, accurate and reproducible therefore used for routine quality control of nateglinide dosage forms. This method can be validated in plasma for bioanalytical applications.
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Received on 26.07.2019 Accepted on 21.08.2019
© Asian Pharma Press All Right Reserved
Asian J. Pharm. Ana. 2019; 9(4):199-204.
DOI: 10.5958/2231-5675.2019.00033.4