INTRODUCTION: [8,2,3]

Chemically linagliptin is known as 8-[(3R)-3-aminopiperidin-1-yl]-7-(but-2-yn-1-yl)-3-methyl-1-[(4-methylquinazolin-2-yl)methyl]-2,3,6,7-tetrahydro-1H-purine-2,6-dione.Several method are reported for the Individual estimation of linagliptin and other methods are reported like High performance Liquid Chromatography with U.V detector (HPLC-UV) individually and in combination with other drugs. But U.V spectrophotometry method using Area under Curve (AUC) not reported.

Linagliptin is used with proper diet and exercise program and possibly control high blood sugar level. It is used by people with type 2 diabetes. It means type 2 diabetes its long term metabolic disorder that is characterized by high blood sugar level insulin resistance and relative lack of insulin.

Linagliptin is used with a proper diet and exercise program and possibly with other medication to control high blood sugar. It is used by people with type 2 diabetes. Controlling high blood sugar helps prevents kidney damage, blindness, nerve problems, loss of limbs, and sexual function problems. Proper control of diabetes may also lessen your risk of heart attack or stroke. Linagliptin is dipeptidyl peptidase-4 (DPP-4) inhibitor which is used in combination with diet and exercise in the therapy of type 2 diabetes, either alone alone or in combination with other oral hypoglycemic agents. Linagliptin has been linked to rare instances of clinically apparent liver injury.

The developed methods were validated for accuracy, precision, ruggedness and sensitivity. Accordingly, the objective of this study was to develop and validate the simple spectrophotometry method using (AUC) technique for Quantitative estimation of linagliptin bulk material and tablets as per ICH guidelines Q2 (R1).

Figure 1: Chemical structure of Linagliptin

8-[(3R)-3-aminopiperidin-1-yl]-7-(but-2-yn-1-yl)-3-methyl-1-[(4-methylquinazolin-2-yl) methyl]-2,3,6,7-tetrahydro-1H-purine-2,6-dione.

EXPERIMENTAL WORK:

Materials:

Linagliptin supplied as a gift sample by Macleods Pharmaceuticals Ltd., (Vapi, Gujarat). Tablet formulation (Tradjenta) was purchased from an Indian market, Containing Linagliptin 5 mg. All chemical and reagents used were of analytical grade.

Instrument [1,4]

A double beam UV-VIS spectrophotometer (UV-1700, Shimadzu, Japan) connected to computer loaded with spectra manager software UV Probe 2.21 with 10mm quartz cells was used. The spectra were obtained with the instrumental parameters as follows: Wavelength range: 200–400nm; scan speed: Medium; sampling interval: 1.0nm. All weights were taken on an electronic balance (Model Shimadzu AUX 120).

Preparation of standard stocksolution and selection of wavelengths [2]

A stock standard solution was prepared by dissolving 10 mg of linagliptin in a 100mL of 10% v/v methanol to obtain a concentration of 100μg/mL. From it, an appropriate concentration of 10μg/mL was prepared and scanned in the UV-visible range 400–200nm; linagliptin showed a maximum absorbance at 295nm. In Method A, Area Under Curve (Auc) of the zero-order spectrum was recorded between the 280 and 307nm. While, in Method B, C, zero-order spectra were derivatized into first and second-order AUC was recorded between 302 and 317 nm; 290 and 303nm.

Method A [2]

The Zero-order, absorption spectra linagliptin recorded using UV probe 2.21 with delta lambda 8 and scaling factor 16. In method A area under curve (AUC) of the zero-order spectrum was recorded between the 280nm 307nm. The calibration curve were constructed by plotting concentration 04-24μg/mL versus/AUC between selected wavelengths for method A respectively.

Method B [5]

The Zero-order, absorption spectra linagliptin were derivatized in first-order using UV probe 2.21 with delta lambda 4 and scaling factor 10. In method B area under curve (Auc) of the First-order spectrum was recorded between the 302 and 317nm. The calibration curve were constructed by plotting concentration 04-24 μg/mL versus/AUC between selected wavelengths for method B respectively.

Method C [5]

The Zero-order, absorption spectra linagliptin were derivatized in second-order using UV probe 2.21 with delta lambda 16 and scaling factor 250. In method C area under curve (AUC) of the Second-order spectrum was recorded between the 290nm and 303nm. The calibration curve were constructed by plotting concentration 04-24μg/mL versus/AUC between selected wavelengths for method C respectively.

Preparation of sample solution [7]

Five Linagliptin tablets (label claim 5mg) were weighed, transferred to a clean dry mortar, and grounded into fine powder using a pestle. Tablet powder equivalent to 10 mg of linagliptin was transferred to a 100ml volumetric flask and 50ml methanol was added. After ultrasonic vibration for 10 min, volume was made up to be marked with methanol and filtered through whatman filter paper. From the filtrate, an appropriate volume was taken and diluted with methanol to get the final concentration of 10μg/mL for methods A, B and C. the responses measured and concentration in the sample were determined from respective linearity equation.

Validation of the method [9]

The proposed method was validated as per ICH-Guidelines Q2 (R1).

Study of linearity curves:

From the stock standard solution, an appropriate amount of aliquots portion in the range of 0.4–2.4mL were transferred into a series of 10mL volumetric flasks and diluted up to mark using the same solvent to obtain a concentration in the range of4-24μg/mL. The solutions were scanned on a spectrophotometer in the range of 400–200nm. The calibration curves were plotted concentrations versus AUC. The linearity of the “methods A, B and C” was evaluated by analysis of six standard solution of linagliptin of concentration 04, 08, 12, 16, 20, 24μg/mL.

Accuracy [8]

The accuracy of all methods was evaluated by measurements of recovery. To the reanalyzed sample solution (10μg/mL methods A, B and C). Known amounts of stock standard solution were added at different level that is 80%, 100% and 120%. The solution were reanalyzed by the proposed methods. The experiments were repeated for three times at each level method.

Precision [7]

Precision of the methods was studied as intra-day and inter-day variations. In Method A, precision was determined by analyzing the 8, 12, and 16μg/mL of linagliptin solutions as intra-day and inter-day variations. In Method B, and C precision was determined by analyzing the 8, 12, and 16μg/mL of linagliptin solutions as intra-day and inter-day variations.

Sensitivity [6]

The sensitivity of measurements of linagliptin by the use of the proposed methods was estimated in terms of the limit of quantification (LOQ) and the limit of detection (LOD). The LOQ and LOD were calculated using equation LOD = 3.3 × N/B and LOQ=10 × N/B, where ‘N’ is the standard deviation of the AUC of the drugs (n=3), taken as a measure of noise, and ‘B’ is the slope of the corresponding calibration curve.

Repeatability [6]

Repeatability was determined by analyzing 6 and 16 μg/mL concentration of linagliptin solution for six times for Methods A, B and C respectively.

Ruggedness [6]

Ruggedness of the proposed methods was determined for 12μg/mL concentrations of linagliptin by analysis of aliquots from a homogenous slot by two analysts using the same operational and environmental conditions for Methods A, B and C respectively.

Analysis of the tablet formulation [8]

Five tablets were weighed accurately and powdered. Powder equivalent to 10mg of linagliptin was weighed and transferred to 100mL volumetric flask, then dissolved in 50mL of methanol by shaking the flask for 15 min with the help of sonicator, and volume was made up to mark with methanol. The solution was filtered through whatman filter paper no. 41. An appropriate volume 1.0mL was transferred into a 10mL volumetric flask and the volume was adjusted to the mark to obtain the desired concentration of 10μg/mL. The AUC was recorded at selected wavelengths for Method A. While in Method B, C, AUC of the first and second-order derivative spectrum was recorded in between selected wavelength ranges. The concentration of the drug was determined from the respective linear regression equations.

Figure 2: Zero-order derivative spectrum of linagliptin in 10% v/v methanol.

Selected U.V range in 280 nm to 307 nm. (Method A)

Figure 3: First-order derivative spectrum of linagliptin in 10% v/v methanol.

Selected U.V. range 302 nm to 317 nm. (Method B)

Figure 4: Second-order derivative spectrum of linagliptin in 10% v/v methanol

Selected U.V range 290 nm to 303 nm. (Method C)

RESULTS AND DISCUSSION:

The molecular structure of the Linagliptin is presented in Fig.1. Methanol was selected as the solvent for Linagliptin because provides good solubility and other characteristics for AUC measurements. The absorption spectrum of Linagliptin in methanol for the method is indicated in Fig. 2, 3 and 4.

Method Validation [7,8,10]

The proposed method was validated as per ICH guidelines Q2 (R1).

Selection of wavelengths:

Figures 2, 3 and 4 show the selection of wavelengths in Methods A, B and C respectively. The selection of wavelengths in both the methods is based on the reproducibility of the results.

Linearity studies:

The linear regression data for the calibration curves showed a good linear relationship over the concentration range 4–24μg/mL for Method A, B and C. The results are expressed in Table 1.

Accuracy:

The pre-analyzed sample used in Methods A, B and C was 16μg/mL, respectively. In Method A, B and C the mean % recovery was found to be in the range of 90.8 – 98.09%, 79.62 – 88.08%, 63.78 – 80.60% [Table 2]

Precision:

The precision of the developed method was expressed in terms of % relative standard deviation (% RSD). These results show reproducibility of the assay. The % RSD values found to be less than 2 indicate that the methods were precise for the determination of drugs in formulation [Table 3].

Sensitivity:

The LOD and LOQ for linagliptin were found to be 1.05 and 3.18μg, respectively, for Method A. For Method B and C they were found to be 0.0820 and 0.2486μg, and 0.1372 and 0.4157μg respectively.

Table 1: Optical characteristics and linearity data of linagliptin

Parameters	Method A	Method B	Method C
Linearity range (μg/mL)	4-24	4-24	4-24
Selected range (nm) for AUC	280.00-307.00	302.00-317.00	290.00-303.00
Slope	0.207	0.0684	0.864
Intercept	0.039	0.0223	0.0285
Correlation coefficient	0.9997	0.9991	0.999
Limit of detection (μg)	1.05	0.0820	0.1372
Limit of quantitation (μg)	3.18	0.2486	0.4157

Table 2: Accuracy

% Value	Method A		Method B		Method C
	% Recovery	% RSD	% Recovery	% RSD	% Recovery	% RSDs
80	90.08	1.1319	79.62	1.722	63.78	0.216
100	91.84	1.9401	87.56	0.150	78.27	0.480
120	98.09	1.4757	88.08	1.228	80.60	0.448

Table 3: Precision

	Conc. (μg/m)			Intra-day			Inter-day
Method A	Method B	Method C	% RSD Method A	Method B	Method C	% RSD Method A	Method B	Method C
8	8	8	0.94	1.26	0.472	1.31	0.92	1.300
12	12	12	1.36	0.84	1.107	1.04	0.72	0.817
16	16	16	0.79	0.59	0.454	1.27	0.56	0.945

Table 4: Ruggedness

Methods	Analysts	Amount found [%]	% RSD
Method A	1	96.99	1.047
Method A	2	95.39	1.897
Method B	1	88.75	1.432
Method B	2	88.73	1.347
Method C	1	68.93	1.681
Method C	2	67.68	1.567

Repeatability:

Repeatability was determined by analyzing 16 μg/mL (Method A, B and C) concentrations of Linagliptin solution for six times and the % amount determined with % RSD<2 for both the methods.

Ruggedness:

The peak area was measured for the sameconcentration solutions, six times for both methods. The results were in the acceptable range for both the drugs. The results showed that the % RSD was less than 2% [Table 4].

CONCLUSION:

The UV spectrophotometric method developed for determination of linagliptinbulk and tablet was based on different analytical techniques, AUC method for Zero order, first order and second order derivative. The method was validated and found to be simple, sensitive, accurate, and precise in compliance to the limits stated in the ICH guidelines Q2 (R1). Hence, we conclude that the method can be used successfully for routine analysis of linagliptin in bulk and in tablet. The proposed spectrophotometric (AUC) method is not reported in literature. Hence, the present methodcould be available for the analysis of Linagliptin. However, it can be served as an alternative where advancedare not available. This single and simultaneous method could beexplored for routine analysis.

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Received on 30.03.2020 Modified on 14.04.2020

Asian J. Research Chem. 2020; 13(3):228-232.

DOI: 10.5958/0974-4150.2020.00044.9