Development of UV- Spectrophotometric Method for the Simultaneous Estimation of Nateglinide and Piperine in Combined Tablet dosage form
Sama Venkatesh, Sirimalla Jyothi, Rajeswari Chiluka, Yenumula Padmavathi
Department of Pharmacognosy, G. Pulla Reddy College of Pharmacy, Mehdipatnam, Hyderabad, Telangana.
*Corresponding Author E-mail: rajeswari930@gmail.com
ABSTRACT:
KEYWORDS: Piperine, UV-Spectrophotometry, Nateglinide, Estimation, ICH guidelines.
1. INTRODUCTION:
Nateglinide is a phenylalanine and cyclohexane derivative that acts as a hypoglycemic agent by stimulating the release of insulin from the pancreas. It is a shortacting, insulinotropic anti-diabetic agent used for the treatment of type II diabetes mellitus. Nateglinide stimulates insulin secretion by blocking ATP sensitive potassium channels in pancreatic beta-cells, causing cell membrane depolarization which results in calcium influx and insulin secretion[1,2,3].
Piperine is the pungent alkaloid present in Piper nigrum has been shown to possess diverse biochemical and pharmacological activities and is known to modify the biotransformation of drugs. Piperine acts by suppressing P-glycoprotein and CYP450 enzymes, which counter act the metabolism of nateglinide via these proteins, thus enhancing the oral bioavailability of nateglinide.[4,5]
Literature survey may not reveal any RP-HPLC method or UV- spectrophotmetry method for the simultaneous estimation of nateglinide and piperine in combined tablet dosage form. Simultaneous estimation of nateglinide and piperine combined pharmaceutical dosage form is not official in any pharmacopoeia, hence no official method is available for their estimation. Hence an attempt has been made to develop a simple, accurate, sensitive, rapid and economic method for the simultaneous estimation of nateglinide and piperine in combined tablet dosage form using UV- Spectrophotmetry. For the purpose of simultaneous estimation the tablets of nateglinide along with piperine will be prepared and analysed.[6,7,8,9]
Fig.1: Nateglinide([N(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine])
Fig.2 Piperine (1-[(2E, 4E)-5-(1,3-benzodioxol-5-yl)-1-oxo-2, 4-pentadienyl] piperidine)
2. EXPERIMENTAL:
2.1. Chemicals and reagents:
A gift sample of nateglinide was obtained from Dr. Reddy’s laboratory, Hyderabad, gift sample of piperine was obtained from Aldrich Sigma, Hyderabad, HPLC grade methanol and water were purchased from S.D Fine Chemicals, Mumbai, India. All other chemicals/reagents were of research grade and used without further purification.
2.2. Instruments:
Instruments used were Shimadzu UV-1800 UV-Visible Spectrophotometer, Remik Tablet Punching Machine, Shimadzu BL-220H Digital Balance, PCI Analytics 6.5 li200H Ultra Sonic Bath Sonicator
2.3. Standard solutions:
Standard piperine of 1000μg/mL solution was prepared by dissolving 100mg of piperine working standard in 100mL of methanol and mixed well. From this 100 μg/ml solution was prepared by taking 10mL of piperine standard solution in 100mL methanol. Standard nateglinide of 1000μg/mL solution was prepared by dissolving 100mg of nateglinide working standard in 100mL methanol.
2.4 Selection of analytical wavelength:
10µg/mL solutions of piperine and nateglinide were prepared from the stock standard solutions of seperately using methanol as solvent and the solutions were scanned agianst blank (methanol) in the entire UV range to determine the λmax values. Clear peaks were observed at 258nm and 244nm for nateglinide and piperine respectively. Hence these wavelengths were choosen as the λmax values for nateglinide and piperine respectively. By the overlay graph it was evident that both the drugs absorb at λmax of each other drug. So those particular wavelengths were choosen as λmax.
2.5. Calibration curve for piperine (5-25µg/ml):
From the working standard stock solution of piperine (100µg/ml), appropriate aliquots like 0.5, 1.0, 1.5, 2.0, 2.5ml solutions were transfered in 10ml graduated tubes. The solution in each tube was made up with methanol to obtain working standard concentration ranging from 5-25µg/ml. The absorbance of these solutions was measured agianst blank (methanol) at 244 nm.[10,11]
2.6. Calibration curve for nateglinide (50-250 µg/ml):
From the working standard stock solution of nateglinide (1000µg/ml) appropriate aliquots like 0.5, 1.0, 1.5, 2.0, 2.5ml solutions were transfered in 10 ml graduated tubes. The solution in each tube was made up with methanol to obtain working standard concentration ranging from 50-250µg/ml. The absorbance of these solutions was measured agianst blank (methanol) at 258 nm.
2.7. Validation of simultaneous estimation method for the estimation of piperine and nateglinide:
The method developed was validated according to the ICH Guidelines Q2 (R1): Validation of Analytical Procedures: Text and Methodology. Method was validated for various parameters such as Linearity and Range, LOD (Limit of Detection), LOQ (Limit of Quantification), Precision to ensure that the performance characteristic of the method meets the requirements for the intended analytical applications.[12,13]
2.8 Formulation of Combined Dosage Form Tablets:
Table 1.
Ingredients for the formulation of combined dosage form tablets
|
S. No |
Drugs and Excipients |
Manufacturers |
Quantity (mg) |
|
1 |
Nateglinide (Drug) |
Dr. Reddy’s laboratory, Hyd |
100 |
|
2 |
Piperine (Drug) |
Aldrich Sigma |
10 |
|
3 |
Sodium starch glycollate |
S d fine chem limited, Mumbai |
5 |
|
4 |
Talcum powder |
S d fine chem limited, Mumbai |
3 |
|
5 |
Magnesium powder |
S d fine chem limited, Mumbai |
2 |
|
6 |
Dibasic calcium phosphate |
NR Chem, Mumbai |
20 |
|
7 |
Starch |
S d fine chem limited, Mumbai |
10% |
|
8 |
Lactose |
S d fine chem limited, Mumbai |
40 |
2.9. ANALYSIS OF COMBINED DOSAGE FORM TABLETS [14,15,16,17]:
Sample preparation:
10 tablets were weighed; average weight was calculated and crushed into fine powder in a cleaned and dried mortar with a pestle. Tablet powder equivalent to 100 mg of piperine and nateglinide was weighed and transferred into a 100ml volumetric flask, to this 10mL of methanol was added and sonicated for 15 minutes with intermediate shaking by obtaining a clear solution with maximum sediments settled at the bottom of the tube. Then it is diluted and made up with methanol after being achieved to room temperature and mixed well and sonicated further for 2 minutes. Then the graduated tube with the test solution is kept aside for 20 minutes by closing it with a glass stopper or lid. This solution was filtered through Whatmann filter paper (no.41) and collected in another test tube.
150μg/ml solution of nateglinide and 15μg/ml solution of piperine was prepared by transfering 1.5ml from the prepared tablet stock solution to 10ml volumetric flask and the volume was made upto mark with methanol, the absorbance is checked at 244nm and 258nm.
% assay of piperine and nateglinide tablets was calculated using the formula.
% assay X 100
3. RESULTS AND DISCUSSION:
New UV spectrophotometric method was developed and optimised by Shimadzu UV-1800 instrument using simultaneous equation method.
3.1. DEVELOPMENT AND OPTIMISATION:
3.1.1. Absorption maximum of Piperine:
The absorption maximum of 244nm was taken from absorption spectrum and selected as wavelengeth for spectrophtometric determinations. The difference absorption spectrum was shown in Fig. 3
Fig. 3: Absorption spectrum of piperine
3.1.2. Absorption maximum of nateglinide:
The absorption maximum of 258nm was taken from absorption spectrum and selected as wavelengeth for spectrophtometric determinations. The difference absorption spectrum was shown in Fig.4
Fig. 4: Absorption spectrum of nateglinide
3.1.3. Overlay Spectrum of nateglinide and piperine:
Absorption maximum for nateglinide and piperine was found to be 258nm and 244nm respectively from the overlay spectrum as shown in Fig. 5
Fig. 5: Overlay Spectrum of nateglinide and piperine
3.1.4. Selection of analytical concentration range and verification of Beers law:
In the concentration range, 5-25μg/ml and 50-250μg/ml there was a linear relationship between concentration and absorbance values for piperine and nateglinide respectively. This range was selected for construction of calibration curve. The optimised instrumental conditions are shown in table. 2
Table 2. Optimized UV conditions
|
Drugs |
Piperine |
Nateglinide |
|
λmax |
244 nm |
258 nm |
|
Beer’s law range |
5-25 μg/ml |
50-250 μg/ml |
|
Regression equation(y) |
y=0.070x+0.012 |
y =0.007x+0.009 |
|
Slope |
0.070 |
0.007 |
|
Intercept |
0.012 |
0.009 |
|
Coefficient of determination (r2) |
0.999 |
0.999 |
3.1.5. Simultaneous equation method:
Standard solutions of nateglinide and piperine in combination were prepared in methanol and the absorbance of these solutions was measured at 258nm and 244nm. Calibration curves were plotted to verify the beer’s law and absorptivity values calculated at the respective wavelengths for both the drugs. Two simultaneous equations as below were formed using these absorptivity values, A (1%,1cm)
Cx = A2ay1 – A1ay2 / ax2ay1 – ax1ay2 Cy
= A1ax2 – A2ax1 / ax2ay1 – ax1ay2
Where, Cx and Cy are the concentrations of nateglinide (NTG) and piperine (PIPE) measured using sample solutions.
A1 and A2 are absorbance of the diluted sample at λ1 and λ2 respectively.
ax1, ax2 are the absorptivity values of X at λ1 and λ2
ay1, ay2 are the absorptivity values of Y at λ1 and λ2
The obtained values: A1= 0.543; A2= 0.538; ax1= 0.933; ax2 = 0.6; ay1= 62.8; ay2 = 64.8
By substituting the values in the above equations Cx and Cy were determined.
3.2. Method validation:
The developed method was validated for the following parameters
3.2.1. Linearity:
Linearity was obtained in the 5-25μg/ml and 50-250μg/ml concentration range for piperine and nateglinide respectively and the standard calibration curve data is shown in table.3 for piperine and nateglinide and calibration curve in fig.6 for piperine and in fig.7 for nateglinide.
Table 3: Standard calibration curve data for piperine and nateglinide
|
S. No |
Volume of stock solution transferred (ml) |
Final volume (mL) |
Conc μg/ml (PIPE) |
Conc μg/ml (NTG) |
Abs* (244 nm) |
Abs* (258 nm) |
|
1 |
0.5 |
10 |
5 |
50 |
0.367 |
0.371 |
|
2 |
1.0 |
10 |
10 |
100 |
0.72 |
0.731 |
|
3 |
1.5 |
10 |
15 |
150 |
1.08 |
1.096 |
|
4 |
2.0 |
10 |
20 |
200 |
1.422 |
1.444 |
|
5 |
2.5 |
10 |
25 |
250 |
1.788 |
1.818 |
*Average of three determinations
Fig. 6: Standard calibration curve for piperine
Fig. 7: Standard calibration curve for nateglinide
The response of the drug was found to be linear in the investigation concentration range and the linear regression equation was y= 0.070x + 0.012, y= 0.007x + 0.009 for piperine and nateglinide respectively and correlation coefficient was 0.999 for piperine and nateglinide respectively.
3.2.2: Limit of Detection (LOD) and Limit of quantitation (LOQ):
The LOQ and LOD reported as a means to analyse the sensitivity. LOD (nateglinide) calculated by using standard deviation method was found to be 4.2μg/mL. LOQ calculated by using standard deviation method was found to be 12.5μg/mL. LOD (piperine) calculated by using standard deviation method was found to be 1.5 μg/mL. LOQ calculated by using standard deviation method was found to be 3.8μg/mL
LOQ = 10σ/S LOD= 3.3σ/S
3.2.3. Precision:
The precision of the developed analytical method was assessed by checking repeatability, intra-day precision and inter-day precision.
3.2.3.1 Repeatability:
The precision of the instrument was checked by repeatedly checking (n=6) solution of piperine (15 μg/ml) and nateglinide (150 μg/ml) and measuring respective absorbance. The results were reported in terms of relative standard deviation and were found to be not more than 2 %. The results were shown in table 4.
Table 4: Repeatability data of piperine and nateglinide
|
S. No |
Conc NTG (µg/mL) |
Conc PIPE (µg/mL) |
Absorbance (258 nm) |
Absorbance (244 nm) |
%RSD (258 nm) |
%RSD (244 nm) |
|
1 |
150 |
15 |
1.168 |
1.164 |
0.15 |
0.18 |
|
2 |
150 |
15 |
1.168 |
1.152 |
||
|
3 |
150 |
15 |
1.169 |
1.169 |
||
|
4 |
150 |
15 |
1.167 |
1.166 |
||
|
5 |
150 |
15 |
1.166 |
1.168 |
||
|
6 |
150 |
15 |
1.164 |
1.169 |
*Average of six determinations
The developed method was precise as the absorbance values of six replicates are close. The calculated %RSD values are within the limits.
3.2.3.2. Intra-day precision:
The intraday precision of the proposed method was determined by analyzing the corresponding concentration (80%,120%.150%) 3 times on the same day and the results were reported in terms of relative standard deviation. The results were shown in table 5.
3.2.3.3 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.
Table 5: Inter-day and intra-day precision data for piperine and nateglinide
|
S. No |
Concentration (NTG) (μg/mL) |
Concentration (PIPE) (μg/mL) |
%RSD (258 nm) |
%RSD (244 nm) |
|
1 |
120 |
12 |
1.5 |
1.71 |
|
2 |
150 |
15 |
0.47 |
0.74 |
|
3 |
180 |
18 |
0.43 |
1.35 |
*Average of 3 determinations
The results shows that all the calculated % RSD values are below 2, therefore the method may be precise.
3.2.4. Accuracy:
The analytical accuracy is the nearness of the results obtained against the real values. The results of obtained for accuracy studies for the drug substance and drug product were reported in terms of % RSD and % recovery respectively.
3.2.4.1.
For drug substance:
Accuracy data for piperine and nateglinide drug substance was shown in table 6 and 7
Table 6: Accuracy data for drug substance at 258 nm
|
S. No |
Accuracy level |
Concentration (NTG+PIPE) (μg/ml) |
Absorbance (258 nm) |
Mean ±SD |
%RSD
|
|
1 |
80% |
120+12 |
0.861 |
0.8564±0.0064
|
0.74
|
|
0.849 |
|||||
|
0.859 |
|||||
|
2 |
100% |
150+15 |
0.949 |
0.9436±0.0050 |
0.53 |
|
0.939 |
|||||
|
0.943 |
|||||
|
3 |
120% |
180+18 |
1.266 |
1.255±0.00953
|
0.72 |
|
1.249 |
|||||
|
1.25 |
Table 7: Accuracy data for drug substance at 244nm
|
S. No |
Accuracy level |
Concentration (NTG+PIPE) (μg/ml) |
Absorbance (244 nm) |
Mean ±SD |
%RSD
|
|
1 |
80% |
120+12 |
0.766 |
0.7666±0.0020
|
0.26 |
|
0.769 |
|||||
|
0.765 |
|||||
|
2 |
100% |
150+15 |
0.953 |
0.9563±0.0030
|
0.3 |
|
0.959 |
|||||
|
0.957 |
|||||
|
3 |
120% |
180+18 |
1.246 |
1.234±0.0102
|
0.81 |
|
1.227 |
|||||
|
1.23 |
From the above data it was found that the %RSD (acceptance criteria <2%) values are within the acceptance limits.
The analytical accuracy is the nearness of the results obtained against the real values. At each level of drug product concentration. The results of obtained for accuracy studies for the drug product were reported in terms of % RSD and % recovery respectively.
3.2.4.2.
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 piperine and nateglinide at three different levels (80%, 100% and 120%). The results were shown in table 8.
From the data of table-8, it was found that the %recovery values are within the limits.
From the data of table-8, it was found that the %RSD (acceptance criteria <2%) values are within the acceptance limits.
3.2.5 Assay of piperine and nateglinide tablets:
The calculated percent assay of piperine and nateglinide combined dosage form tablets were found to be 98.6% and 99.3% for piperine and nateglinide respectively and the values were shown in table 9.
Table 8: Accuracy data for piperine and nateglinide drug substance
|
Tablet used |
Amt of sample added (NTG+PIPE) (µg/mL) |
Amt of standard added (NTG+PIPE) (µg/mL) |
Total amt added (µg/mL) |
Amt recovered (µg/mL) |
%Recovery ±Standard deviation (NTG) |
%Recovery ±Standard deviation (PIPE) |
|
180 mg (NTG and PIPE) |
150+15 |
120+12 |
270+27 |
268.65+26.79 |
98.81 ± 0.82 |
99.23±0.94 |
|
150+15 |
150+15 |
300+30 |
301.46+29.67 |
100.49± 1.25 |
98.9± 0.86 |
|
|
150+15 |
180+18 |
330+33 |
327.33+32.68 |
99.22± 0.83 |
99.09± 0.91 |
*Average of 3 determinations
Table 9: Assay values of piperine and nateglinide combined dosage form tablets
|
Tablet used |
Label claim (mg) |
Amount found (µg/mL) |
% Assay |
|||
|
Piperine |
Nateglinide |
Piperine |
Nateglinide |
Piperine |
Nateglinide |
|
|
Piperine and nateglinide combined tablet dosage form |
10 |
100 |
14.8 |
149 |
98.6 |
99.3 |
4. CONCLUSION:
A new, simple and reproducible simultaneous equation method (Vierdott’s method) was developed and validated for the quantitative estimation of nateglinide and piperine in combined tablet dosage form using Schimadzu UV1800 instrument with UV Probe 4.0 software. The analysis of samples was carried out by using quartz cuvettes as sample cells. The method was developed and optimised with instrumental conditions, absorbance mode as measurement mode. Wavelengths 258nm and 244nm were selected as absorption maximum for nateglinide and piperine respectively.
Simultaneous equation method was developed by considering the absorbance measurements of nateglinide and piperine in methanol. The analytical methods were validated as per ICH Q2 (R1) guidelines with respect to various parameters linearity, accuracy, precision, LOQ and LOD. Simultaneous equation method was linear over a concentration range of 50-250μg/ml and 5-25 μg/ml, for nateglinide and piperine respectively. Limit of detection was found to be 4.2μg/mL and limit of quantification was found to be 12.5μg/mL for nateglinide and limit of detection was found to be 1.5 μg/mL and limit of quantification was found to be 3.8 μg/mL for piperine. Relative standard deviation for intra-day precision and inter-day precision were within the acceptable limits. The mean recovery of the combined tablet dosage form of nateglinide and piperine with the developed method was found to be 99.5% and 99.07% respectively.
From the results, it can be concluded that the developed method was effective for quantitative determination of nateglinide and piperine in bulk and combined tablet dosage form without any interference of other constitute in the formulation. Tablets were prepared and analyzed by the proposed method and assay of the drug was calculated. The developed method could be readily used for the quantitative estimation of nateglinide and piperine in their combined dosage form.
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Received on 31.03.2020 Modified on 21.04.2020
Accepted on 18.05.2020 ©Asian Pharma Press All Right Reserved
Asian J. Pharm. Ana. 2020; 10(3):134-140.
DOI: 10.5958/2231-5675.2020.00024.1