world journal of pharmaceutical research jif impact factor

www.wjpr.net Vol 6, Issue 17, 2017.

365

Sudharani et al. World Journal of Pharmaceutical Research

A VALIDATED RP_HPLC METHOD FOR THE SIMULTANEOUS

ESTIMATION OF METFORMIN HYDROCHLORIDE & LINGLIPTIN

IN ITS BULK AND PHARMACEUTICAL DOSAGE FORM

N. Sudharani*1, Manasa Bollepally

2 and Raveendra Babu Konduri

3

1Asst. Professor, Dept of Pharmaceutical Analysis, Anurag Pharmacy College, Kodad.

2,3Asst. Prof. Anurag Pharmacy College Kodad.

ABSTRACT

A new method was established for simultaneous estimations of

Metformin and Linagliptin by RP-HPLC method. The

chromatographic conditions were successfully developed for the

separation of Metformin and Linagliptin by using Agilent C18 column

(4.6 x 150 mm) 5μ, flow rate was 1ml/min, mobile phase ration was

(60:40 v/v) Methanol : Ammonium acetate buffer (KH2PO4 and

K2HPO4) pH is 3 (pH was adjusted with orthophosphoricacid),

detection wavelength was 254nm. The instrument used was WATER

HPLC auto sampler, separation module 2695, photo diodearrey

detector 996, Empower-Software version-2. The retention time was

found to be 2.344 min and 3.284 mins, the % purity of Metformin and Linagliptin was found

to be 101.27% and 99.97% respectively. The system suitability parameters for Metformin and

Linagliptin such as theoretical plates and tailing factor were found to be 4668, 1.3 and 6089

and 1.2, the resolution was found to be 6.0. The method was precise and rousted.

KEYWORDS: Metformin, Linagliptin, HPLC, Precision, Methanol.

1. INTRODUCTION

Metformin, chemically N, N-Dimethylimidodicarbonimidic diamide is an oral antidiabetic

drug in the biguanide class [Fig.-1A]. It is the first-line drug of choice for the treatment of

type-II diabetes. Metformin suppresses glucose production by the liver. It helps in reducing

LDL cholesterol and triglyceride levels. Linagliptin, 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 by Boehringer Ingelheim for treatment of

World Journal of Pharmaceutical Research JIF Impact Factor 7.523

Volume 6, Issue 17, 365-380. Review Article ISSN 2277–7105

*Corresponding Author

N. Sudharani

Asst. Professor, Dept of

Pharmaceutical Analysis,

Anurag Pharmacy College,

Kodad.

Article Received on

30 Oct. 2017,

Revised on 20 Nov. 2017,

Accepted on 10 Dec. 2017,

DOI: 10.20959/wjpr201717-10383


366


type-II diabetes [Fig.-1]. Linagliptin is an inhibitor of DPP-4. It stimulates the release of

insulin in a glucose- dependent manner and decreases the levels of glucagon in the

circulation. Fig.-1A: Metformin [Fig.-2:] Linagliptin The detailed survey of literature

revealed that several Spectrophotometric methods5-9, HPLC methods10-12, Stability

indicating methods13,14 and Plasma extraction methods15,16 were reported for the

determination these drugs individually or in combination with other drugs in pharmaceutical

dosage forms. A few HPLC methods are available with the combination of above-cited

drugs17-21, with lower linearity range and or having longer retention times. The author made

an attempt to develop and validate a cost-effective RP-HPLC assay method for estimation of

Metformin and Linagliptin from formulated dosage form. The developed method is validated

as per ICH and all relevant guidelines 22-27 for broad linearity range than other available

methods and with better retention times.

1. Metformin. 2. Linagliptin.

2. PLAN OF WORK

To develop a new analytical method for the simultaneous estimation of Metformin HCL and

Linagliptin by RP-HPLC.


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3.1 AIM AND OBJECTIVE

The analytical method for the simultaneous estimation of Metformin HCL and Linagliptin

will be developed by RP-HPLC method by optimizing the chromatographic conditions.

The developed method is validated according to ICH guidelines for various parameters

specified in ICH guidelines, Q2 (R1).

Instrumentation, chemicals & Reagents

2996 series of Waters Photodiode array detector attached to 2995 series of Waters HPLC,

which is having Hamilton syringe and autosampler opted for chromatography. A degasser to

remove the dissolved air and column oven to maintain the desired temperature is also

available in the system. Mobile phase with a composition of Acetonitrile: 0.02 M phosphate

buffer (pH5.0): 35:65 v/v with 1.0 mL flow rate and Waters Xbridge C18, 4.6*150mm, 5μ as

a stationary phase with an injection volume of 10 μL were selected as chromatographic

conditions. Detector wavelength was fixed at 225 nm. Reagents and Chemicals Working

standards of Metformin and Linagliptin obtained from Spectrum Pharma Research Solutions,

Hyderabad as gift samples. HPLC grade, Water and Acetonitrile and ortho phosporic acid

and methanol procured from Merck Chemical Division, Mumbai. JENTADUETO tablets

containing 500mg of Metformin and 2.5 mg of Linagliptin were purchased from the

pharmacy.

Working Standard Stock Solution Preparation: 10 mg of Metformin and 1 mg of

Linagliptin working standards were accurately weighed, and these samples were transferred

to 10mL and 100mL volumetric flasks respectively, containing diluent. The mixture was

sonicated for 5 minutes to aid dissolution and finally made up to the volume with the same

diluent. The below table indicates the dilutions and the concentration of the stock solution.

phosphate buffer was prepared by 2.95 grams of Ammonium acetate dissolved and diluted to

1000ml with HPLC water and pH was adjusted to 4.5 with orthophosphoric acid.

Preparation of mobile phase: Mix a mixture of above buffer 40 ml (40%) and 60 ml of

Acetonitrile (HPLC grade-60%) and degassed in ultrasonic water bath for 5 minutes. Filter

through 0.22 µ filter under vacuum filtration.

Procedure: 10L of the blank, standard and sample were injected into the chromatographic

system and areas for the Metformin and Linagliptin the peaks were used for calculating the %

assay by using the formulae.


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System suitability

Tailing factor for the peaks due to Metformin and Linagliptin in standard solution should

not be more than 1.5.

Theoretical plates for the Metformin and Linagliptin peaks in standard solution should not

be less than 2000.

Assay calculation

Where: Avg.wt = average weight of tablets P= Percentage purity of working standard

LC= Label Claim of metformin mg/ml.

3.2 ANALYTICAL METHOD VALIDATION

1. Specificity: The system suitability for specificity was carried out to determine whether

there is any interference of any impurities in retention time of analytical peak. The study was

performed by injecting blank.

Figure 1: Chromatogram showing standard injection.

The specificity test was performed it was found that there was no interference of impurities in

retention time of analytical peak.

2. Linearity Preparation of stock solution: 10 mg of metformin and 1 mg of Linagliptin

working standard were accurately weighed and were transferred into a 10ml clean dry

volumetric flask, add about 2ml of diluent and sonicate to dissolve it completely and make

volume up to the mark with the same solvent.


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* Preparation of Level – I (50ppm of metformin and 5 ppm of Linagliptin)

* Preparation of Level – II (100ppm of metformin and 10ppm of Linagliptin)

* Preparation of Level – III (150ppm of metformin and 15ppm of Linagliptin)

* Preparation of Level – IV (200 ppm of metformin and 20ppm of Linagliptin)

* Preparation of Level – V (250 ppm of metformin and 25ppm of Linagliptin)

Procedure

Each level was injected into the chromatographic system and peak area was measured. Plot a

graph of peak area versus concentration (on X-axis concentration and on Y-axis Peak area)

and the correlation coefficient was calculated.

Acceptance criteria

Correlation coefficient should be not less than 0.999.

The linearity study was performed for the concentration of 50 ppm to 250 ppm and 5ppm

to 25 ppm level. Each level was injected into chromatographic system. The area of each level

was used for calculation of correlation coefficient. The chromatograms are shown in Figure

and results are tabulated in Table. No.1 & 2 Calibration graph for MET and LINA are shown

in Figure.

Table. 1: Linearity Results for Metformin.

Metformin r2

= 0.999.

S.No Linearity Level Concentration Area

1 I 50 ppm 471543

2 II 100 ppm 656277

3 III 150 ppm 794999

4 IV 200 ppm 946124

5 V 250 ppm 1002139

Correlation Coefficient 0.999


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Table. 2: Linearity Results for Linagliptin.

S. No Linearity Level Concentration Area

1 I 5 ppm 56472

2 II 10 pmm 73841

3 III 15 ppm 92655

4 IV 20 ppm 111541

5 V 25 ppm 130567

Correlation Coefficient 0.999

Fig. 2: Showing calibration graph for Linagliptin.

Name: Linagliptin: Processing Method: Linagliptin Curve: R^2:0.999747

Linagliptin r2

= 0.999

3. Range

Based on precision, linearity and accuracy data it can be concluded that the assay method is

precise, linear and accurate in the range of 50µg/ml-250µg/ml and 5µg/ml-25µg/ml of

Metformin and Linagliptin respectively.

4. Accuracy

Preparation of standard stock solution: 10mg of metformin and 1mg of Linagliptin

working standard were accurately weighed and transferred into a 10ml clean dry volumetric

flask add about 2ml of diluent and sonicate to dissolve it completely and make volume up to

the mark with the same solvent (Stock solution).Further pipette out 1 ml of the above stock

solution into a 10 ml volumetric flask and was diluted up to the mark with diluents.

Preparation of sample solutions

For preparation of 50% solution(5mg of metformin and 0.5 mg of Linagliptin working

standard were accurately and dissolved in stock solution Further pipette out 10 ml of the


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above stock solution into a 100ml volumetric flask and was diluted up to the mark with

diluents).

for preparation of 100% solution (10 mg of metformin and 1 mg of Linagliptin working



diluents).

for preparation of 150% solution (15mg of metformin and 2 mg of Linagliptin working



diluents).

Procedure: The standard solutions of accuracy 50%, 100% and 150%were injected into

chromatographic system. Calculate the amount found and amount added for Metformin and

Linagliptin and calculate the individual % recovery and mean % recovery values.

Acceptance criteria

The % recovery for each level should be between 98.0 to 102.0%.

The accuracy study was performed for 50%, 100% and 150% for metformin

and Linagliptin. Each level was injected in triplicate into chromatographic system. The area o

f each level was used for calculation of % recovery. Chromatograms are shown in Fig.No.3

and results are tabulated in Table.

Fig. 3. Chromatograms showing accuracy-50% injection-1, 2, 3.


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Peak Name: Metformin

Peak Name RT Area (μV*Sec) Height (μV)

1 Metformin 2.343 659856 125163

2 Metformin 2.343 650507 124410

3 Metformin 2.344 659615 125429

Mean

656659.5

Std.Dev.

5329.9

%RSD

0.8

Peak Name: Linagliptin

Peak Name RT Area (μV*Sec) Height (μV)

1 Linagliptin 3.282 65167 10054

2 Linagliptin 3.284 65177 10007

3 Linagliptin 3.284 65593 10017

mean

65312.5

Std.Dev.

243.3

%RSD

0.4

Accuracy -100%.

Fig.. Chromatogram showing accuracy -100% injection-1, 2, 3.


S. No Peak Name RT Area (μV*Sec) Height (μV)

1 Metformin 2.343 1300848 246191

2 Metformin 2.343 1303077 246044

3 Metformin 2.344 1308849 247851

mean

1304258

Std.Dev.

4129.2

%RSD

0.3


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Peak Name: linagliptin


1 Linagliptin 3.282 124926 19256

2 Linagliptin 3.283 124041 19253

3 Linagliptin 3.286 124560 19420

mean

124509

Std.Dev.

444.6

%RSD

0.4

Accuracy 150%.

Fig. Chromatogram showing accuracy -150 % injection-1, 2, 3.


S. NO Peak Name RT Area (μV*Sec) Height (μV)

1 Metformin 2.343 1852144 348534

2 Metformin 2.344 1856224 348167

3 Metformin 2.345 1855458 348256

mean

1854608.3

Std.Dev.

2168.5

%RSD

0.1


374




1 Linagliptin 3.282 178268 27699

2 Linagliptin 3.282 178708 27684

3 Linagliptin 3.282 178576 27779

Mean

178517.2

Std.Dev.

225.3

%RSD

0.1

Table. Showing accuracy results for metformin

%Concentration

(at specification level)

Average

area

Amount

added(mg)

Amount

found(mg) Recovery

Mean

recovery

50% 656659 5 4.96 99.91%

99.56% 100% 1304258 10 9.98 99.18%

150% 1854608 15 15.02 99.60%

Table. Showing accuracy results for Linagliptin

%Concentration

(at specification level)

Average

area

Amount

added (mg)

Amount

found (mg)

%

Recovery

Mean

recovery

50% 65312 0.5 0.99 99.53%

99.47% 100% 124509 1.0 1.05 99.38%

150% 178517 1.5 1.495 99.52%

The accuracy study was performed for % recovery of Metformin and Linagliptin. The %

recovery was found to be 99.56% and 99.47% respectively (NLT 98% and NMT 102%).

Repeatability

Intermediate Precision

5. Repeatability: The standard solution was injected for five times and measured the area for all

five injections in HPLC. The %RSD for the area of five replicate injections was found to be

within the specified limits.

Intermediate precision/Ruggedness The standard solution was injected for five times and

measured the area for all five injections in HPLC. The %RSD for the area of five replicate

injections was found to be within the specified limits.

Repeatability

The precision study was performed for five injections of metformin and Linagliptin. each

standard injection was injected into cheomatographic system. the area of each standard

injection was used for calculation of %RSD. The chromatograms are shown in Fig. No. and

result are tabulated in table.


375


Fig. Chromatograms showing precision.

Table. Showing % RSD results for Metformin



1 Metformin 2.343 1302729 248455

2 Metformin 2.344 1309759 248699

3 Metformin 2.344 1302947 249526

4 Metformin 2.345 1303977 246695

5 Metformin 2.345 1303236 250012

mean

1304529.8

Std.Dev.

2961.1

%RSD

0.2

Table. showing % RSD results for Linagliptin



1 Linagliptin 3.285 124263 19458

2 Linagliptin 3.287 124487 19634

3 Linagliptin 3.287 124175 19600

4 Linagliptin 3.288 124894 19327

5 Linagliptin 3.288 124495 19540

mean

124462.7

Std.Dev.

278.6

%RSD

0.2

6. Intermediate precision/Ruggedness

The intermediate precision study was performed for five injections of metformin and

Linagliptin. Each standard injection was injected into chromatographic system. The area of

each standard injection was used for calculation of % RSD. The chromatograms are shown in

Fig. And results are tabulated in Table.


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Table showing results for intermediate precision of Metformin



1 Metfomin 2.342 1305937 247870

2 Metfomin 2.343 1306476 246764

3 Metfomin 2.344 1304520 245696

4 Metfomin 2.344 1300148 247140

5 Metfomin 2.345 1308271 247280

mean

1305070.2

Std.Dev.

3061.8

%RSD

0.02

Table Showing results for intermediate precision of Linagliptin



1 Linagliptin 3.278 122962 19165

2 Linagliptin 3.281 122487 19115

3 Linagliptin 3.281 122632 19073

4 Linagliptin 3.281 122626 19003

5 Linagliptin 3.283 122702 19123

mean

122681.8

Std.Dev.

174.8

%RSD

0.1

7. Precision

Repeatability

Preparation of stock solution

10 mg of metformin and 1 mg of Linagliptin working standard were accurately weighed and

transferred into a 10ml clean dry volumetric flask add about 2ml of diluent and sonicate to

dissolve it completely and make volume up to the mark with the same solvent. Further pipette

out 1ml of the above stock solution into a 10ml volumetric flask and was diluted up to the

mark with diluent.

Procedure The standard solution was injected for five times and measured the area for all five

injections in HPLC. The %RSD for the area of five replicate injections was found to be within the

specified limits.

Acceptance criteria

The % RSD for the area of five standard injections results should not be more than 2.


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Intermediate Precision/Ruggedness

To evaluate the intermediate precision (also known as ruggedness) of the method, precision

was performed on different days by using different make column of same dimensions.

Acceptance criteria

The % RSD for the area of five sample injections results should not be more than 2%.

8. Limit of detection (LOD)

LOD’s can be calculated based on the standard deviation of the response (SD) and the slope

of the calibration curve (S) at levels approximating the LOD according to the formula. The

standard deviation of the response can be determined based on the standard deviation of y-

intercepts of regression lines.

Formula:

Where - Standard deviation (SD) S - Slope

9. Limit of quantification

LOQ’s can be calculated based on the standard deviation of the response (SD) and the slope

of the calibration curve (S) according to the formula. Again, the standard deviation of the

response can be determined based on the standard deviation of y-intercepts of regression

lines.

Formula

Where - Standard deviation S - Slope

10. Robustness

As part of the robustness, deliberate change in the flow rate, mobile phase composition was

made to evaluate the impact on the method.

a) The flow rate was varied at 0.4ml/min to 0.6 ml/min. Standard solution 150 ppm of

metformin and 15 ppm of Linagliptinwas prepared and analysed using the varied flow rates

along with method flow rate.

b) The organic composition in themobile phase was varied from 65% to 75% standard

solution 150 µg/ml of metformin and15 µg/ml of Linagliptin were prepared and analysed


378


using the varied mobile phase composition along with the actual mobile phase composition in

the method.

System suitability

10 mg of metformin and 1 mg of Linagliptin working standard was accurately weighed and

transferred into a 10ml clean dry volumetric flask and add about 2ml of diluent and sonicate

to dissolve it completely and make volume up to the mark with the same solvent (Stock

solution).Further pipette out 1ml of Metformin and Linagliptin from the above stock solution

into a 10ml volumetric flask and was diluted up to the mark with diluent.

Assay calculation for Metformin and Linagliptin

The assay study was performed for the metformin and Linagliptin. Each three injections of

sample and standard were injected into chromatographic system. The chromatograms are

shown in Fig. No.5.5 & 5.6 and results are tabulated in Table.No.5.

Table. 5: Showing assay results.

S. No Name of compound Label claim Amount taken %purity

1 Metformin 500 754.7 99.24

2 Linagliptin 2.5 735.6 101.04

4. CONCLUSION

A new method was established for simultaneous estimation of Metformin and Linagliptin by

RP-HPLC method. The chromatographic conditions were successfully developed for the

separation of Metformin and Linagliptin by using Agilent C18 column (4.6 X 150 mm) 5μ,

flow rate was 1 ml / min, mobile phase ratio was (60:40 v/v) methanol: Ammonium acetate

buffer (KH2PO4 and K2HPO4) pH=3 (pH was adjusted with ortho phosphoric acid), detected

wave length was 254 mm. the instrument used was Waters HPLC Auto sampler module

2695, photo diode array detector 996, Empower –Software version -2. The retention times

were found to be 2.344 mins and 3.284 mins. The percentage purity of Metformin and

Linagliptin Was found to be 101.27% and 99.97% respectively. The system suitability

parameter for Metformin and Linagliptin such as theoretical plates and tailing factor were

found to be 4668, 1.3 and 6089 and 1.2, the resolution was found to be 6.0. The analytical

method was validated according to ICH guidelines (ICH, Q2 (R1)). The linearity study of

Metformin and Linagliptin was found in concentration range of 50μg-250μg and 5μg -50μg

and correlation coefficient (r2) was found to be 0.999 and 0.999% recovery was found to be

99.56% and 99.48%, % RSD for repeatability was 0.2 and 0.2, %RSD for intermediate


379


precision was 0.2 and 0.1 respectively. The precision study was precise, robust, and

repetable.LOD value was 3.17 and 5.68, and LOQ value was 0.0172 and 0.2125 respectively.

Hence the suggested RP-HPLC method can be used for routine analysis of Metformin and

Linagliptin in API and Pharmaceutical dosage form.

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