INTRODUCTION:

Linagliptin3, chemically, 8- [(3R)- 3 -aminopiperidin-1-yl ] -7- (but-2-yn-1-yl) -3-methyl-1-[(4-methylquinazolin-2-yl)methyl]-3,7-dihydro-1H-purine-2,6-dione is an DPP-4 inhibitor developed byBoehringer Ingelheim for treatment of type-II diabetes[Fig.-1A]. It is an inhibitor of DPP-4, an enzyme that degrades the incretion hormones glucagon –like Peptide (GLP-1) and glucose –dependent insulin tropic polypeptide (GLP-1)^[15-17].

Both GLP-1 and GLP-1 increase insulin biosynthesis and secretion from pancreatic beta cells in the presence of normal and elevated blood glucose levels. GLP-1 also reduces glucagon secretion from pancreatic alpha cells, resulting in a reduction in hepatic glucose output^[18].

FIGURE 1A: Linagliptin

Metformin HCl (MET) is 3-(diaminomethylidene)-1, 1-dimethylguanidine (fig. 1B). It is an oral anti-diabetic drug which is the first line drug of choice for the treatment of type 2 diabetes, particularly in overweight or obese people and those with normal kidney function. Metformin improves hyperglycaemia, primarily through its suppressive action on production of hepatic glucose (hepatic gluconeogenesis)^(9,14).

FIGURE 1B: Metformin hydrochloride

Several method were reported for the simultaneous estimation of Metformin HCl in combination and in alone with other drugs viz. UV- Spectrophotometry^{[1, 2, 3, 4, 9, 10, 13]}, estimation in plasma and urine using HPLC ^{[7, 14]}, assay using RP-HPLC ^[12]A comprehensive literature research reveals the lack of a spectrophotometric analytical method for simultaneous estimation of Linagliptin and Metformin HCl in pharmaceutical formulations. A successful attempt was made to develop, accurate, precise and simple method of analysis for estimation of both the drugs in combined dosage form.

EXPERIMENTAL:

INSTRUMENTATION:

A SHIMADZU double beam UV/Visible spectrophotometer (Model: 1800) and Borosil glass wares were used for the study. Calibrated electronic single pan balances SHIMADZU AY220, ultra-sonication were also used during the analysis.

CHEMICAL AND REAGENT:

Linagliptin and Metformin hydrochloride was generous gift samples from Mylan laboratories, Hyderabad, India were used in the study. The pharmaceutical dosage form used in this study was Ondero MET^TM(Boehringer lngelheim Pharmaceuticals, Inc.) labeled to contain 500mg of MET and 2.5mg of LIN. All other chemicals used were of analytical grade.

Preparation of stock solution and selection of wavelength for analysis:

Standard stock solutions of Linagliptin and Metformin HCl were prepared separately by adding 10 mg of drug to 5mlmethanol taken in 100 ml volumetric flasks and then sonicated for 10 minutes and the volume was made up to the mark with water. The resulting solutions contain 1mg/ml of the drug. The stock solutions of Linagliptin and Metformin HCl were further diluted with water to obtain the concentration of 30μg/ml. The resulting solutions were then scanned in UV spectrophotometer from 400 to 200nm ^(13,14). From the resulting spectra λmax for Linagliptin and Metformin HCl were calculated separately. The overlay spectra of Linagliptin and Metformin HCl were also recorded. From the overlay spectra the absorptive point of Linagliptin and Metformin HCl was calculated.

Method: Simultaneous equation method (Vierodt’s Method):

If a sample contains two drugs having absorbance at least at one wavelength, then it is possible to determine the drugs by simultaneous equation method (Vierodt’s Method). Two equations are constructed based on the fact that the absorbance at a particular λmax is sum of individual absorbance of two components. The scanning spectra of 30μg/ml solution of Linagliptin and Metformin HCl show clear peaks at 227nm and 232nmrespectively for Linagliptin and Metformin HCl .The λmax of each drug was selected for analysis .The stock solution of Linagliptin and Metformin HCl was then diluted in the range of 1-11µg/ml and 3-13 µg/ml and the absorbance of these solutions was measured at 232nm to verify the Beer’s law and the absorptivity values. Two simultaneous equations as given

A₁= ax₁C₁ + ay₁C₂ and A₂=ax₂C₁ + ay₂C₂

Where

A₁= (87.55) C₁+ (42.24) C₂ (I)

A₂= (72.82) C₁+ (48.76) C₂ (II)

C₁ and C₂ are the concentrations of Linagliptin and Metformin Hydrochloride in gm/100ml respectively in sample solution.A₁andA₂ are the absorbance of mixture at 227nm and 232nm respectively. Solving equation 1 and 2, C₁ and C₂ are calculated as absorbance’s of mixture at 227nm and 232nm.

C₁ = A₂ ay₁- A₁ ay₂ / ax₂ ay₁ – ax₁ ay₂

C₂= A₁ ax₂ – A₂ ax₁ / ax₂ay₁ –ax₁ay₂

C₁= A₂ (42.24) - A₁ (48.76)/- 1193.0212 (III)

C₂= A₁ (72.82) - A₂(87.55)/ -1193.0212 (IV)

Preparation and assay of tablet formulation:

Fixed dose combination of Linagliptin and Metformin Hydrochloride is approved for marketing as a (Ondero Met tablets) containing Linagliptin and Metformin Hydrochloride. 20 Ondero Met tablets were weighed and triturated in a mortar pestle and quantity of sample equivalent to 2.5 mg of Linagliptinand 500 mg of Metformin Hydrochloride was transferred into 100 ml volumetric flask containing 5ml of methanol and sonicated for 20 min Final volume was made up to the mark and filtered through Whatman filter paper (No. 41). The absorbance was taken at 227nm and 232 nm against blank .The concentrations of Linagliptin and Metformin Hydrochloride was calculated by equation III, IV. The results are reported in the Table 1.

METHOD VALIDATION:

The UV Spectrophotometric method was validated as per ICH guidelines for method validation. The performance parameters like linearity, precision and accuracy were evaluated.

Linearity:

Linearity was studied by diluting standard stock solution of Linagliptin 1 -11μg/ml and Metformin Hydrochloride 3-13 μg/ml concentrations. Calibration curves with concentration verses absorbance were plotted at their respective Wavelengths and the obtained data was subjected to regression analysis using the least square method. The standard curves for Linagliptin and Metformin Hydrochloride are shown in (Fig. 3, 4) respectively and data is presented in Table 2.

Accuracy:

To check the accuracy of the developed methods and to study interference of formulation additives, analytical recovery experiments were carried out by using standard addition method. Tablet samples solution of each drug was added to Reference standard at three different concentrations level (80, 100 and 120%). At each level, samples were prepared in triplicate and the mean percentage recoveries and % RSD value were calculated. Table .6 shows the result for accuracy of the method.

Precision:

Repeatability: A mixture containing 10μg/ml each of Linagliptin and Metformin Hydrochloride was prepared and analyzed both by method A and B. The data is represented in Table 3. Intermediate precision: intermediate precision is studied in terms of intraday and inter-day precision. Three concentrations of Linagliptin and Metformin Hydrochloride was selected in a mixture and analyzed by method A and B. For intraday, the analysis was carried out at different intervals on the same day and for inter day, the analysis was carried on different days. Table 4 and 5 give the results for intraday and inter-day studies respectively.

Ruggedness:

Ruggedness of the proposed method is determined by analysis of aliquots from homogenous slots by different analysts using similar operational and environmental conditions.

CONCENTRATION

Fig.3.Standard curve of Linagliptin at 227nm

CONCENTRATION

Fig.4.Standard curve of Metformin Hydrochloride at 232nm

Table 1: Optical characteristics and linearity data

Parameter	Linagliptin	Metformin HCl
Absorption maximum (nm)	227	232
Beer’s law limit (µg/ml)	1-11	3-13
Correlation coefficient	0.9881	0.9944
Regression equation Y=mX+C	0.1036x+0.1158	0.0742x + 0.0127
Intercept	0.1181	0.0755
Slope (m)	0.1036	0.0742

Table 2: *E (1%, 1 cm) for Linagliptin and Metformin Hydrochloride

Drug	*E(1%,1cm) at 227nm±SD	*E(1%,1cm) at 232nm±SD
Linagliptin	Ax1 =87.55±0.43	Ax2=72.82±0.31
Metformin Hydrochloride	Ay1=42.24±0.26	Ay2=48.76±0.29

Table 3: Repeatability study data for Linagliptin and Metformin Hydrochloride (n=6)

Drug	Concentration taken (µg/ml)	% Found	% RSD
Linagliptin	07	104.55±0.0075	0.85
Metformin Hydrochloride	07	99.61±0.013	0.25

Table 4: Intraday Precision data for Linagliptin and Metformin Hydrochloride (n=3)

Drug	Concentration (µg/ml)	% Found	% RSD
Linagliptin	3	116.6±0.0017	0.35
Linagliptin	7	103.9±0.0026	0.29
Linagliptin	11	98.56±0.0017	0.13
Metformin Hydrochloride	5	120.58±0.0015	0.30
Metformin Hydrochloride	9	115.21±0.0014	0.17
Metformin Hydrochloride	13	109.38±0.001	0.093

Table 5: Interday Precision data for Linagliptin and Metformin Hydrochloride (n=3)

Drug	Concentration (µg/ml)	% Found	% RSD
Linagliptin	3	110.25±0.002	0.54
Linagliptin	7	103.01±0.0036	0.47
Linagliptin	11	98.12±0.0017	0.15
Metformin Hydrochloride	5	103.59±0.001	0.25
Metformin Hydrochloride	9	97.49±0.0017	0.25
Metformin Hydrochloride	13	100.03±0.0015	0.15

RESULTS AND DISCUSSION:

The methods discussed in the present work provide a convenient, precise and accurate way for simultaneous analysis of Linagliptin and Metformin Hydrochloride in its bulk and pharmaceutical dosage form. Absorbance maxima of Linagliptin at 227nm and Metformin Hydrochloride at 232nm were selected for the analysis. Regression analysis shows linearity over the concentration range of Linagliptin at 1-11 µg/ml and Metformin Hydrochloride at 3-13 µg/ml with respective correlation coefficients of 0.9881 and 0.9944 respectively. The % RSD for repeatability (n=3), intraday and interday (n=3) precision was found to be less than 2% indicating the precision of method. The amount of drugs estimated by the proposed methods was in good agreement with the label claim. The proposed methods were validated. The Accuracy of the proposed methods was ascertained by recovery studies and the results are expressed as % recovery. Recovery experiments indicated the absence of interference from commonly encountered pharmaceutical additives. % Recovery for A Linagliptin and Metformin Hydrochloride was found within the range of 99.61% and 100.80%. Values of standard deviation and coefficient of variation were satisfactorily low indicating the accuracy of both the methods. The % RSD value for both Linagliptin and Metformin Hydrochloride was found to be less than 2%. The results did not show any statistical difference between operators suggesting that methods developed were rugged. All statistical data proves validity of the methods and can be used for routine analysis of pharmaceutical formulations containing both these drugs.

CONCLUSION:

Based on the results obtained, it is found that the developed UV -Spectrophotometric technique is quite simple, accurate, precise, reproducible, sensitive and economical. They can become effective analytical tools for routine quality control of Linagliptin and Metformin Hydrochloride bulk drug combinations and their combined pharmaceutical dosage form without any prior separation of components.

REFERENCES:

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Received on 01.02.2017 Accepted on 10.05.2017

Asian J. Pharm. Ana. 2017; 7(2): 79-83.

DOI: 10.5958/2231-5675.2017.00013.8

Sample	Concentration (%)	Amount Recovered (µg/ml)	Recovery (%)	%RSD
Linagliptin	80	4	99.04±0.002	0.46
Linagliptin	100	5	99.33±0.002	0.36
Linagliptin	120	6	99.21±0.001	0.22
Metformin Hydrochloride	80	4	98.61±0.001	0.39
Metformin Hydrochloride	100	4.9	100.73±0.001	0.33
Metformin Hydrochloride	120	6	99.29±0.002	0.48