A Rapid RP-HPLC Method development and Validation for the Analysis of Linagliptinin Bulk and Pharmaceutical Dosage Form

K. Vijaya Sri*, M. Anusha, S. Ravinder Reddy

Department of Pharmaceutical Analysis, Malla Reddy College of Pharmacy, Maisammaguda,

Secunderabad-500 014, Telangana, India.

*Corresponding Author E-mail: vijayasree_2002@yahoo.co.in

ABSTRACT:

A simple, precise, accurate and robust RP-HPLC-UV method was developed and validated for the determination of Linagliptinin tablet dosage forms. Reverse-phase chromatography was performed on Agilent (100 mm × 2.5 mm, 3μm) column of C18 HPLC with EZ Chrome software with UV detector. Methanol: water containing 0.1% ortho phosphoric acid (70:30) (v/v) was used as mobile phase at a flowrate of 0.8 mL min^-1 with UV detection at 296 nm. Linearity was observed in the concentration range of 2.5– 15 μg mL^-1 with correlation co-efficient (R² = 0.999). The method was validated as per ICH guidelines. The RSD for intra-day (0.254) and inter-day (0.2449) precision were found to be less than 2 %. The percentage recovery was in good agreement with the labelled amount in the pharmaceutical formulations and the method is simple, precise, accurate and robust for the determination of Linagliptinin pharmaceutical dosage forms.

KEYWORDS: Linagliptin; RP-HPLC-UV, Method validation, Tablets

INTRODUCTION:

Linagliptinis a di-amino pyrimidine derivative^1,2. Chemically, it is 4-[[4-[[4-[(E)-2-cyanoethenyl]- 2,6-dimethylphenyl] amino]-2-pyrimidinyl] amino] benzonitrile monohydrochloride structure are shown in Figure 1.

Figure No. 1 Structureof Linagliptin

A thorough literature survey has revealed that UV spectroscopy³, HPLC^{4, 5}methods for Linagliptin with combination of other drugs, for its estimation in bulk, pharmaceutical dosage forms and biological samples. The aim of present research work for the development and validation of HPLC method, an attempt was made to develop a simple, accurate, precise and rapid method for the estimation of linagliptin in bulk and pharmaceutical dosage form.

EXPERIMENTAL:

Materials and reagents:

Linagliptin was gift sample from Dr Reddy Labs. Methanol HPLC Grade was purchased from Merck Chemical Company. HPLC Grade water was purchased from Avantor performance materials limited. The 0.45 μm pump Nylon filter was obtained from Advanced Micro Devices (Ambala Cantt, India) and Whatman no 5 filter paper was obtained from Modern Science lab, (Nashik, India).Glass wares used were Class A grade.

HPLC instrumentation and conditions:

Chromatographic separation was achieved by using maintained at 25 ºC. Isocratic elution was performed using methanol: water containing 0.1% ortho phosphoric acid (70:30) (v/v)at a flow rate 0.80 mL min^-1 with UV detection

at 296 nm. The overall run time was 10 min and 10 μL of sample was injected into the HPLC system.

Preparation of stock solution:

Linagliptinstock solution (1000 μg mL^-1) was prepared by accurately weighing 25 mg of linagliptinin a 25 mL amber volumetric flask and making up to volume with mobile phase. Working solutions for HPLC injections were prepared on a daily basis from the stock solution in a solvent mixture of methanol: water containing 0.1% ortho phosphoric acid (70:30)v/v. Solutions were filtered through a 0.45 μm membrane filter prior to injection.

Development of calibration curve:

Appropriate aliquots of standard linagliptin stock solution were taken in different volumetric flasks and the resultant solution was diluted up to the mark with the mobile phase to obtain a final concentration of 1,2.5, 5, 7.5, 10, 12.5 and 15μg mL^-1. Each of the solutions was injected using isocratic mode with UV detection at 296nm and flow rate of 0.8mLmin^-1. From the resultant chromatogram obtained, peak area was used to plot the calibration curve.

Method Validation:

The method was validated for the following parameters: linearity, limit of quantitation (LOQ), limit of detection(LOD), precision, accuracy, system suitability androbustness^6-8.

Linearity:

Linearity test solutions were prepared from a stock solution at different concentrations (i.e.1-15 μg mL^-1). 5 μL of each solution was injected in to the HPLC system and the peak area of the chromatogram obtained was noted. Correlation coefficient (r) of the line, constructed by plotting mean of peak areas against corresponding concentration, was found to be 0.9998.

Accuracy:

To ensure accuracy of the method, recovery studies were performed by standard addition method at 50%, 100% and 150% level to pre-analyzed samples and subsequent solutions were re-analyzed. At each level three determinations were performed.

Precision:

Precision of the method was determined in terms of repeatability and intra-day and inter-day precisions. Repeatability of the method was determined by analyzing six samples of same concentrations of drug. Chromatographs were recorded and area of each chromatograph was measured. Results of this determination are reported in table 4.The intra-day precision method was evaluated by carrying out 3 concentrations of linagliptin at three concentration levels (5, 10 and 15 μgmL^-1) (n=3) against a qualified reference standard. The % RSD of three obtained assay values at three different concentration levels was calculated. The inter-day precision study was performed at three different concentration levels (5, 10 and 15 μg mL^-1) and each value is the average of three determinations (n=3). The % RSD was calculated.

System Suitability parameters:

To ensure the validity of the analytical procedure, a system suitability test was established. System suitability parameters for the developed HPLC method were determined by injecting six replicates of the standard solution (5μg/mL). Parameters such as a number of theoretical plates (N), tailing factor and retention time were calculated.

Robustness:

The drug solution was subjected to small, deliberate changes like flow rate, wavelength and change in mobile phase ratio. The results obtained were not affected by varying the conditions and were in accordance with the results in original conditions.

Ruggedness:

To determine ruggedness, two different analysts performed assay on pharmaceutical dosage form in similar operational and environmental conditions using developed method.

Limit of quantification (LOQ) and limit of detection (LOD):

The limit of quantification (LOQ) and limit of detection (LOD) were based on the residual standard deviation of the response and the slope of the constructed calibration curve (n=3), as described in International Conference on Harmonization guidelines Q2 (R1).

Analysis of pharmaceutical formulations:

Twenty tablets were weighed accurately and finely powdered. A powder equivalent to 5mg of linagliptin was transferred carefully to 25mL volumetric flask and about 15mL diluent was added. The mixture was sonicated for 10 minutes. The volume was made up to 25mL with diluent, filtered through Whatman no. 5 filter paper. From the filtrate further dilutions were made to obtain 5μg/mL. The final solution was injected in HPLC, chromatogram was recorded and area was measured.

RESULTS AND DISCUSSION:

Several mobile phases were tried for the analysis. The mobile phase methanol: water containing 0.1% ortho phosphoric acid 70:30 (v/v) showed good separation and good peak symmetry. By the proposed method, the retention time of was found to be 1.28 minutes. The resulting chromatogram obtained is shown in Figure 2.

Figure 2 Chromatogram of Linagliptin

Table 1: Results of recovery studies

Spiked level (%)	Formulation Conc (µg/ml)	Pure Drug Conc (µg/ml)	Amount found	% Recovery	% Mean recovery ±SD	%RSD
50	5	2.5	7.11	100.4	100.1±0.305	0.3055
	5	2.5	7.08	99.8
	5	2.5	7.09	100.2
100	5	5	10.1	100.1	100.2±0.208	0.2081
	5	5	10.2	100.5
	5	5	10.18	100.2
150	5	7.5	12.14	99.5	100.3±0.472	0.4724
	5	7.5	12.14	100.2
	5	7.5	12.09	100.4

Table 2: Repeatability of the method

Concentration [µg/ml]	AUC	RT	AUC Mean ±SD	%RSD
5	11777732	1.28	1175213±699462	0.2296
5	11775736	1.20
5	11726739	1.28
5	11748740	1.27
5	11726734	1.28
5	11756734	1.26

Table 3: Results of intra-day and inter-day precision

Concentration (µg/ml)	Intra-day precision		Inter-day precision
Concentration (µg/ml)	Area mean±S.D (n=3)	%RSD	Area mean±S.D (n=3)	%RSD
1	26883±68394	0.2544	258503±6332.09	0.25
5	118952±95572	0.8034	1186171±106064	0.89
10	59712±21886	0.8427	264245±202784	0.76

Accuracy:

Accuracy is determined by performing recovery studies at 3 levels in which known amount of analyte shall be added and recovery shall be carried out in three replicates of each concentration level and the % recovery was calculated. The mean recovery was found between 99.7-100.7 % and %RSD between 0.5-0.9. The accuracy results are shown in Table 1.

Repeatability was determined by analyzing 5.0 μg/ml concentration of Linagliptin for six times and % RSD was found to be < 2 which shown in Table no 2.

Intermediate Precision (reproducibility)

The intraday and interday precision studies were carried out and the mean percent relative standard deviation (% RSD) was calculated and it was found to be 0.25 and 0.89respectively, which is within the acceptable criteria of not more than 2.0. The results are shown in Table 3.

System Suitability parameters

The System suitability parameters like number of theoretical plates (N) was found to be3598, tailing factor 0.6, which indicates efficient performance of column. The results are shown in Table 4.

Table 4: System suitability parameters

Parameter	Results
Retention time	1.283
Tailing factor	0.6
Number of Theoretical plates	3598
HETP	0.027

Table 5: Results of ruggedness studies

Parameters

Area

R_T

Mean± S.D

(n=3)

RSD

Analyst-1

11787732

1.283

118791±1292

0.108

Analyst-2

11970480

1.294

117071±1117

0.954

Table 6: Results of Robustness study

Parameter	RT	Area mean ± S.D (n=3)	% RSD
Flow rate 0.6ml/min	1.713	34633951± 12300	0.355
Flow rate 1ml/min	1.707	21744834±198510	0.912
Wave length 294nm	1.283	26518929±113528	0.428
Wave length 298nm	1.383	25293944±97575	0.385

Ruggedness

Using developed method two different analysts performed assay on marketed tablets of the drug in similar operational and environmental conditions. The results obtained were not affected and were in accordance with the results in original conditions. This shows the method was rugged and results are shown in Table no.5.

Robustness

The drug solution was subjected to small, deliberate changes like flow rate and wavelength. The results obtained were not affected by varying the conditions and were in accordance with the results in original conditions. This shows the method was robust and results are shown in Table no.6.

Application of the Proposed Method for Pharmaceutical Dosage Form

Estimation of Linagliptinin tablet dosage forms were carried out and got 5.002 as assay value. The result of assay obtained was found to be in good agreement with the labeled claim, indicating the absence of interference of the excipients and results are shown in Table no.7 and chromatogram was recorded and area was measured which was shown in figure 4.

CONCLUSION:

A simple, rapid, precise, accurate and robust RP-HPLC-PDA method was developed, validated and applied for the determination of linagliptinin pharmaceutical dosage forms. No interference from any components of pharmaceutical dosage form and the method has been successfully used to perform long-term and accelerate stability studies of linagliptin formulations.

ACKNOWLEDGEMENTS:

The authors are grateful to Chairman, Malla Reddy College of Pharmacy for providing necessary research facilities to carry out the research work and to Dr Reddy Labs, India for providing the gift sample of the drug.

REFERENCES:

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Received on 02.02.2015 Accepted on 03.03.2015

Asian J. Pharm. Ana. 5(1): Jan.- March 2015; Page 16-20

DOI: 10.5958/2231-5675.2015.00003.4

Formulation	Label claim	Amount found	% Purity	%RSD
Trajenta (5mg)	5mg	5.002	100.05%	0.771