Simultaneous spectrophotometric determination of compounds: Application to an anti-diabetic formulation of Teneligliptin and Metformin
Mukthinuthalapati Mathrusri Annapurna*, Raghu Raj Naik, Sistla Mounica Pratyusha
Department of Pharmaceutical Analysis, GITAM Institute of Pharmacy,
GITAM (Deemed to be) University, Visakhapatnam-530045, India
*Corresponding Author E-mail: mannapurna.mukthinuthalapati@gitam.edu
ABSTRACT:
Teneligliptin and Metformin are antidiabetic drugs. Teneligliptin is a novel oral DPP-4 inhibitor classified as peptidomimetic and approved in Japan in September 2012 for the management of Type 2 diabetes mellitus. Type 2 diabetes mellitus is a chronic progressive disease and involves multiple systems. Metformin is biguanide derivative which decreases hepatic glucose production as well as intestinal absorption of glucose and improves insulin sensitivity by increasing peripheral glucose uptake and utilization. Teneligliptin has been systematically evaluated in as monotherapy with diet and exercise and in combination with metformin, glimepiride, pioglitazone, and insulin in short-term (12 weeks) and long-term (52 weeks) studies. In the present study three new spectrophotometric methods such as Dual wavelength method (Method A), Graphical absorbance ratio method (Method B) and First derivative (Method C) method were proposed for the simultaneous determination of Teneligliptin and Metformin tablets using hydrochloric acid. Linearity was observed 1-30 µg/ml and 1-80 µg/ml in both Method A and Method B and 1-30 µg/ml and 1-100 µg/ml in Method C for Metformin and Teneligliptin respectively. The methods were validated as per ICH guidelines and applied for the simultaneous determination of Teneligliptin and Metformin tablets.
KEYWORDS: Teneligliptin, Metformin, Dual wavelength method (Simultaneous equation method), Graphical absorbance ratio method (Q-Analysis), First derivative method, Validation.
INTRODUCTION:
MATERIALS AND METHODS:
UV-1800 double beam UV-VIS spectrophotometer (Shimadzu) with quartz cells was used for the study and all the sample solutions were scanned with medium scanning speed (200-400 nm). Stock solutions of both Teneligliptin and Metformin were prepared separately in methanol and a series of dilutions were made using 0.1N hydrochloric acid solution for the present study.
Method validation
A series of Metformin and Teneligliptin solutions were prepared from their stock solutions and scanned against their reagent blank i.e. 0.1N hydrochloric acid. The absorbance at the selected wavelengths was taken and their absorptivity values were calculated for Method A and Method B and by substituting these absorptivity values in the equations, the individual concentrations of both drugs were determined. In Method C the concentration of each drug was calculated at the zero crossing point (ZCP) of the other drug (minima observed) from the first derivative spectra and vice versa. A calibration curve was drawn by taking the concentration of the drug solution on the x- axis and the corresponding absorbance values were taken on the y-axis for Method A and Method B and for Method C the derivative absorbance was taken.
The intra-day and inter-day precision studies were performed on the same day and on three different days at three different concentration levels (5, 10 and 20 µg/mL) and finally the % RSD was calculated. Accuracy studies were carried out by spiking the formulation solution with the pure drug (50%, 100%, and 150%) and the % recovery was calculated for all the three methods A, B and C.
Assay of formulations (Tablets)
Twenty tablets of two brands containing both Teneligliptin and Metformin available were procured from the local pharmacy store (20 mg Teneligliptin and 500 mg Metformin) and extracted with methanol. The filtered stock solutions of the two brands were diluted with 0.1N hydrochloric acid solution as per the requirement for Method A, B and C.
RESULTS AND DISCUSSION:
Three new spectrophotometric methods-Dual wavelength method (Simultaneous equation method (Method A), Graphical absorbance ratio method (Q– Analysis) (Method B) and First derivative (Method C) spectrophotometric methods were proposed for the estimation of Teneligliptin and Metformin HCl combined dosage forms (Tablets) in 0.1N hydrochloric acid solution.
Method validation
Dual wavelength method (Method A)
In Method A (Dual wavelength method) the absorption maxima of both Metformin and Teneligliptin HCl were selected. Metformin HCl has shown absorption maxima (λmax) at 215.80 nm and that of Teneligliptin at 244.60 nm (Figure 1). The absorptivity values were calculated from their absorbance recorded at the selected wave lengths of their individual spectra (Table 1) and substituted in the simultaneous equations for both the drugs.
A1 and A2 represent the absorbance of the formulation solution at 215.80 nm and 244.60 nm respectively; CMET and CTENE are the concentrations of Metformin HCl and Teneligliptin (g/100 ml)
At 215.80 nm, A1 = 638.4 CMET + 254 CTENE
At 244.60 nm, A2 = 122.7 CMET + 318.7 CTENE
Table 1: Linearity of Metformin and Teneligliptin (Method A & Method B)
|
Metformin |
||||||
|
Conc. (µg/ml) |
Absorbance (215.80 nm) |
Ɛ 215.80 nm |
Absorbance (244.60 nm) |
Ɛ 244.60 nm |
Absorbance 228.02 nm |
Ɛ 228.02 nm |
|
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
1 |
0.069 |
690 |
0.012 |
120 |
0.052 |
520 |
|
2 |
0.138 |
690 |
0.025 |
125 |
0.105 |
525 |
|
4 |
0.26 |
650 |
0.049 |
122 |
0.211 |
527.5 |
|
8 |
0.5 |
625 |
0.097 |
121 |
0.420 |
525 |
|
10 |
0.614 |
614 |
0.12 |
120 |
0.520 |
520 |
|
20 |
1.201 |
600.5 |
0.251 |
125 |
1.050 |
525 |
|
30 |
1.8 |
600 |
0.38 |
126 |
1.580 |
526.6 |
|
Teneligliptin |
||||||
|
Conc. (µg/ml) |
Absorbance (215.80 nm) |
Ɛ 215.80 nm |
Absorbance (244.60 nm) |
Ɛ 244.60 nm |
Absorbance 228.02 nm |
Ɛ 228.02 nm |
|
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
10 |
0.273 |
273 |
0.332 |
332 |
0.263 |
263 |
|
20 |
0.497 |
248 |
0.656 |
328 |
0.520 |
260 |
|
30 |
0.776 |
258 |
0.968 |
322 |
0.780 |
260 |
|
40 |
1.002 |
250 |
1.261 |
315 |
1.070 |
267.5 |
|
50 |
1.241 |
248 |
1.555 |
311 |
1.310 |
262 |
|
60 |
1.51 |
251 |
1.882 |
313 |
1.590 |
265 |
|
80 |
2.00 |
250 |
2.486 |
310 |
2.110 |
263.7 |
Graphical absorbance ratio method (Method B)
In Method B the isosbestic point i.e. 228.02 nm and the absorption maxima of one of the drugs (i.e. λmax Metformin HCl = 215.80 nm) were chosen. The overlay absorption spectra of Teneligliptin (Y) and Metformin HCl (X) were shown in Figure 1. The absorptivity (Ɛ) values obtained at the selected wavelengths (Table 1) were substituted in the equation given below.
Cx 
Cy 
Cx = Concentration of Metformin HCl
Cy = Concentration of Teneligliptin
A1= Absorbance of the sample at iso-absorptive wavelength 228.02 nm.
A2 = Absorbance of the sample at wavelength 215.80 nm.
ax1 = Mean absorptivity of Metformin HCl at 228.02 nm. = 524.1
ay1 = Mean absorptivity of Teneligliptin at 228.02 nm. = 263.0
Qm = Absorbance of formulation solution at 228.02 nm / Absorbance of formulation solution at 215.80 nm
Qx = Absorptivity of Metformin HCl at 215.80 nm / Absorptivity of Metformin HCl
at 228.02 nm = 1.21
Qy = Absorptivity of Teneligliptin at 215.80 nm / Absorptivity of Teneligliptin
at 228.02 nm = 0.965
Beer-Lambert’s law was obeyed over the concentration range 1-30 µg/ml and 1-80 µg/ml in both Method A and Method B for Metformin and Teneligliptin respectively. Calibration curves were drawn by taking the concentration on the x-axis and the corresponding absorbance values on the y axis for Method A and Method B (Figure 2). The % RSD in precision and accuracy studies for the simultaneous determination of Metformin and Teneligliptin in Methods A and B was found to be less than 2.0 indicating that methods are precise and accurate.
First derivative method (Method C)
In first derivative method (Method C) the individual zero order absorption spectra obtained for Metformin and Teneligliptin were transformed in to their first order derivative spectra (Figure 2) with the help of inbuilt software (Figure 3). Teneligliptin has shown zero crossing point at 270.67 nm and Metformin has shown zero crossing points at 211.61, 219.60, 233.53 and 267.99 nm respectively. Teneligliptin was estimated at 219.60 nm (selected zero crossing point of Metformin) and Metformin was calculated at 223.33 nm (selected zero crossing point of Teneligliptin).
Beer-Lambert’s law was obeyed over the concentration range 1-30 µg/ml and 1-100 µg/ml in Method C for Metformin and Teneligliptin respectively. Calibration curves were drawn by taking the concentration on the x-axis and the corresponding derivative absorbance values on the y axis (Figure 4). The % RSD in precision and accuracy studies of the simultaneous determination of Teneligliptin and Metformin in three methods A, B and C was found to be less than 2.0 indicating that the three methods are precise and accurate.
Table 2: Linearity of Teneligliptin and Metformin (Method C)
|
Teneligliptin (ZCP of Metformin 219.60 nm) |
Metformin (ZCP of Teneligliptin 223.33 nm) |
||
|
Conc. (µg/ml) |
Absorbance |
Conc. (µg/ml) |
Absorbance |
|
0 |
0 |
0 |
0 |
|
10 |
0.234 |
1 |
0.073 |
|
20 |
0.461 |
2 |
0.127 |
|
30 |
0.670 |
4 |
0.216 |
|
40 |
0.884 |
8 |
0.407 |
|
50 |
1.110 |
10 |
0.484 |
|
60 |
1.310 |
20 |
0.972 |
|
80 |
1.710 |
30 |
1.44 |
|
100 |
2.130 |
- |
- |
Assay of Teneligliptin and Metformin tablets
The percentage purity was found to be 99.50-99.85 and 99.71-99.97 for Teneligliptin and Metformin respectively (Table 3) in Method A, Method B and Method C. The three methods can be successfully applied for the simultaneous determination of Teneligliptin and Metformin.
Table 3: Assay of Metformin and Teneligliptin tablets
|
Brand |
Drug |
Label claim (mg) |
*Amount found (mg) |
*% Recovery |
||||
|
Method A |
Method B |
Method C |
Method A |
Method B |
Method C |
|||
|
I |
Teneligliptin Metformin |
20 500 |
19.95 498.61 |
19.97 499.13 |
19.93 499.29 |
99.75 99.72 |
99.85 99.83 |
99.65 99.86 |
|
II |
Teneligliptin Metformin |
20 500 |
19.94 498.56 |
19.90 499.83 |
19.96 499.67 |
99.70 99.71 |
99.50 99.97 |
99.80 99.93 |
CONCLUSION:
The three spectrophotometric methods are simple, economical, precise and accurate for the assay of Teneligliptin and Metformin tablets.
ACKNOWLEDGEMENT:
The authors are grateful to M/s GITAM (Deemed to be) University, Visakhapatnam for providing the research facilities. There is no conflict of interest.
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Received on 02.01.2020 Modified on 09.02.2020
Accepted on 01.03.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2020; 13(4): 1936-1940.
DOI: 10.5958/0974-360X.2020.00349.2