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1339

Padmalatha et al. World Journal of Pharmacy and Pharmaceutical Sciences

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF

RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATON OF

METFORMIN AND SITAGLIPTIN

H.Padmalatha* and M.Harish,

Department of Pharmaceutical Analysis, Gyana Jyothi College of Pharmacy, Gyana Jyothi

Nagar, Uppal Bus Depot, Hyderabad, India.

ABSTRACT

A New, precise, rapid, accurate RP-HPLC method was developed for

the Simultaneous Estimation of Metformin and Sitagliptin in tablet

dosage form. After optimization the good chromatographic separation

was achieved by Isocratic mode with a mixture of Phosphate

Buffer(PH 3.3) and Methanol in the ratio of 80:20 v/v as the mobile

phase with Alltima C18 (150 x 4.6 mm, 5m) column as stationary

phase at flow rate of 1.0 mL/min and detection wavelength of 210 nm.

The retention times for Metformin and Sitgliptin found to be 2.35 min

and 3.04 min respectively. The linearity of this method was found in

the concentration range of 50 μg/mL to 300 μg/mL for Metformin and

5 μg/mL to 30 μg/mL Sitgliptin. The correlation coefficient R2 value

is found to be 0.9998 for Metformin and 0.9993 for Sitgliptin. The

LOD and LOQ for Metformin were found to be 0.38 μg/mL and 1.14

μg/mL repectively and 0.19μg/mL and 0.58μg/mL for Sitagliptin. The Proposed method was

found to be Linear, precise and accurate for the quantitative estimation Metformin and

Sitagliptin in Bulk and Tablet dosage form and can be used for commercial purposes.

KEYWORDS: Metformin, Sitagliptin, RP-HPLC ethod,Simultaneous estimations.

MATERIALS AND METHODS

Instruments used: HPLC. WATERS 2695 separation module With PDA WATERS 2998

detector. Software: EMPOWER Reagents: All reagents used were of analytical grade and

were obtained from s.d. fine chemicals, Mumbai.(India).

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Article Received on

07 Jan 2015,

Revised on 01 Feb 2015,

Accepted on 25 Feb 2015

*Correspondence for

Author

H.Padmalatha

Department of

Pharmaceutical Analysis,

Gyana Jyothi College of

Pharmacy, Gyana Jyothi

Nagar, Uppal Bus Depot,

Hyderabad, India.


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Experimental Work

Mobile Phase Optimization

Buffer

Accurately weighed and transferred 1.36gr of Potassium di-hydrogen phosphate in 1000ml of

Volumetric flask add about 900ml of milli-Q water added and degas to sonicate and finally

make up the volume with water and PH adjusted to 3.3 with dil. Orthophosphoric acid

solution.

Mobile phase

Buffer and Methanol taken in the ratio 80:20.

Optimized Chromatographic conditions:

Instrument Waters HPLC 2695 with PDA

detector & EMPOWER software

Temperature 30°C

Column Alltima 150 x 4.6 mm, 5.

Buffer 2 mL of ortho phosphoric acid in 1000

mL of water; pH was adjusted to 2.4

Mobile phase 80% Buffer, 20% Methanol

Flow rate 1 ml per min

Wavelength 210 nm

Injection volume 10 l

Run time 6 min

Diluent Water

3.3.3: Preparation of buffer and mobile phase

Preparation of ortho Phosphoric acid buffer

Accurately weighed 2 mL of ortho phosphoric acid was taken in a 1000 mL volumetric flask

& add 500 mL of HPLC water & sonicate for 10 min.Filter through 0.45 µ filter under

vaccum filtration unit & make upto 1000 mL with HPLC water and then the pH was adjusted

to 2.4.

Preparation of mobile phase

Accurately measured 800mL (40%) of above buffer and 200 mL (60%) of methanol HPLC

grade were mixed and degassed in an ultrasonic water bath for 10 minutes and then filtered

through 0.45 µ filter under vacuum filtration

Diluent Preparation

Used the water as Diluent


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3.3.4Preparation of the Metformin and Sitagliptin Standard & Sample Solution:

Standard Solution Preparation

Accurately Weighed and transferred 20mg of Metformin and 5mg of Sitagliptine working

Standards into a 10 ml&25ml clean dry volumetric flask, add 7ml& 20ml of diluent,

sonicated for 5 minutes and make up to the final volume with diluents.

Sample Solution Preparation

5 tablets were weighed and calculate the average weight of each tablet then the weight

equivalent to 5 tablets was transferred into a 100mL volumetric flask, 70mL of diluent added

and sonicated for 25 min, further the volume made up with diluent and filtered. From the

filtered solution 1ml was pipeted out into a 10 ml volumetric flask and made upto 10ml with

diluent. 0.8ml from the secondary stock solution was pipette out and made up to 10ml with

same diluents.

Procedure

Inject 20 L of the standard, sample into the chromatographic system and measure the areas

for the Metformin and Sitagliptin peaks

System Suitability

Tailing factor for the peaks due to Metformin and Sitagliptin in Standard solution Should not

be more than2.

Theoretical plates for the Metformin and Sitagliptin peaks in Standard solution should not be

less than 2500

RESULTS AND DISCUSSION

The developed method was validated based on ICH guidelines to detect and quantitate both

drugs in dosage form with use of HPLC system equipped with PDA detector.

Method development and optimization of chromatographic parameters

Trial 1

Mobile phase : Buffer (pH-3.3) and methanol in ratio 40:60 (v/v)

Column : Altima C18 column

Flow rate : 1.0 mL/min

Detector wavelength : 200-400 nm

Column temperature : 300C


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Injection volume : 10 L

Run time : 20 min

Fig. No. 4.1 Chromatogram for Trial 1

Trial 2

Mobile phase : Buffer (pH-3.3) and Methanol in ratio 85:15 (v/v)

Column : Altima C18





Run time : 20 min

Name Retention Time Area %Area USP Plate count USP Tailing

1 Metformin 2.912 3744723 85.56 8110 1.12

2 Sitagliptin 3.851 631802 14.44 5861 1.34


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Trial 3

Mobile phase : Buffer (pH-2.4) and solvent mixture the ratio 80:20 (v/v)

Column : Altima C18 (4.6 x 250mm, 5m)





Run time : 6 min



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Trial 4

Mobile phase : Buffer (pH-3.3) and solvent mixture the ratio 40:60 (v/v)

Column : Hiber C18 (4.6 x 250mm, 5m, Make: Agilent)




Injection volume : 10L

Run time : 20min


Trial 5

Mobile phase : Buffer (pH-3.3) and methanol in the ratio 60:40 (v/v)






Run time : 20 min


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Trial-6







Run time : 20 min


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Trial-7


Column : ODS C18 (4.6 x 250mm, 5m)





Run time : 20 min

4.2 SYSTEM SUITABILITY

Results of system suitability parameters for Metformin and Sitagliptin

Theoretical plates must be not less than 2500

Tailing factor must be not less than 0.9 and not more than 2.


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Theoritical plate was obtained for the first standard Injection for Metformin is 5118and

Sitagliptin is 3429.

It was found from above data that all the system suitability parameters for developed method

were within the limit

VALIDATION PARAMETERS

The developed method was validated according to the ICH guidelines as follows:

SPECIFICITY

The system suitability for specificity was carried out to determine whether there is any

interferences of any impurities in retention time of analytical peak. The study was performed

by injecting blank.

Blank solution

Fig No. 4.1Chromatogram Showing Blank Preparation

Placebo Solution

Fig No. 4.2 Chromatogram Showing Placebo Preparation


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LINEARITY&RANGE

The linearity study was performed for the concentration of 25% to 150% level. Each level

was injected into chromatographic system .The area of each level was used for calculation of

correlation coefficient. The range was calculated from the calibration graphs constructed by

concentration versus peak areas.

Linearity level-1 (25%)

Fig.No4.3 Chromatogram Showing Linearity Level-1 (25%)

Fig .No. 4.4 Chromatogram Showing Linearity Level-2 (50%)


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Fig .No. 4.5 Chromatogram Showing Linearity Level-3 (75%)

Fig .No. 4.6 Chromatogram Showing Linearity Level-4(100%)

Fig .No. 4.7 Chromatogram Showing Linearity Level-5(150%)


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Table No. 4.4 Showing Results from Linearity Study

S.No Pipetted from

stock (mL)

Volume of

flask (mL)

Concentration

in ppm(Met)

Concentration

in ppm(Sita)

%Linearity

Level

1 0.25 10 50 5 25

2 0.5 10 100 10 50

3 0.75 10 150 15 75

4 1 10 200 20 100

5 1.25 10 250 25 125

6 1.5 10 300 30 150

Metformin and Sitagliptin

CONC% Area PPM CONC% Area PPM

25 851677 50 25 2303841 5

50 1703667 100 50 3458006 10

75 2536530 150 75 4605307 15

100 3445469 200 100 5763958 20

125 4212414 250 125 6912558 25

150 5110999 300 150 879262 30

Plotting of Calibration Curves Metformin and Sitagliptin

Fig. No.4.8 Calibration Curve of Metformin

Fig. No.4.9 Calibration Curve of Sitagliptin


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The linearity study was performed the correlation coefficient of Metformin and Sitagliptin

were found to be 0.9998 and 0.9993 respectively (NLT 0.999).

The % RSD peak areas of 6 replicate injections for lower and higher levels a NMT 2%

respectively.

The calibration curves were linear in the range 50- 300 µg/ml for Metformin, 5-30 µg/ml for

Sitagliptin

ACCURACY

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

150% level was injected in triplicate into chromatographic system. The area of each level

was used for calculation of %recovery. Chromatograms are shown in Fig.No.7.18-31 and

results are tabulated in Table. No.7.4.

Fig .No. 4.10 Chromatogram Showing Accuracy-50% Injection-1



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Fig .No. 4.15Chromatogram Showing Accuracy-100% Injection-3

Fig .No.4.16 Chromatogram Showing Accuracy-150% Injection-1



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Fig .No.4.18 Chromatogram Showing Accuracy-150% Injection-3

4.5

METFORMIN

% Level Sample wt Sample area % recovery Mean

50 2000 1773138 100.98

100.40 50 2000 1767958 100.68

50 2000 1747958 99.54

100 4000 3534152 100.63

100.05 100 4000 3505007 99.80

100 4000 3502288 99.72

150 6000 5278027 100.19

99.79 150 6000 5266040 99.96

150 6000 5223785 99.16

SITAGLIPTIN

% Level Sample wt Sample area % recovery Mean

50 2000 363467 99.84

99.90 50 2000 364639 100.16

50 2000 363018 99.71

100 4000 723620 99.38

99.73 100 4000 720825 99.00

100 4000 733925 99.47

150 6000 1091062 100.45

100.00 150 6000 1088000 99.62

150 6000 1091505 99.44

The accuracy study was performed for % recovery. The % recovery of Metformin was found

to be 100% and the % recovery of Sitagliptin was found to be 100%. (NLT 97% and NMT

103%).


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PRECISION

Repeatability

The sample solution was injected for six times and measured the area for all six injections in

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

limits. The precision study was performed for six injections of Metformin and Sitagliptin

samples. Each sample injection was injected into chromatographic system. The area of each

sample injection was used for calculation of % RSD.

Fig .No.4.19 Chromatogram Showing Precision Injection-1



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Fig .No.4. 22Chromatogram Showing Precision Injection-4

Fig .No.4.23Chromatogram Showing Precision Injection-5


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Fig .No. 4.24Chromatogram Showing Precision Injection-6


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Table Showing Results from Precision Study

S.No Sample Wt Area-1 Area-2 % Assay % Assay

1 800 644703 3491590 99.45 99.42

2 800 654248 3517193 100.92 100.15

3 800 649003 3541636 100.11 100.84

4 800 642210 3507289 99.06 99.87

5 800 650134 3492480 100.28 99.44

6 800 648668 3482248 100.7 99.15

Avg 99.98 99.81

Std 0.65 0.62

%Rsd 0.70 0.60

The % RSD values were within 2 and the method was found to be precise.

LIMIT OF DETECTION AND QUANTIFICATION

Detection Limit of Metformin:

The Detection Limit of an individual analytical procedure is the lowest amount of analyte in

a sample which can be detected but not necessarily quantitated as an exact value.

S/N Ratio of standard preparation of Metformin was found to be 320.6.

Quantitation Limit of Metformin

The Quantitation limit of an analytical procedure is the lowest amount of analyte in a sample

which can be quantitatively determined with suitable precision and accuracy.

S/N Ratio of standard preparation of Metformin was found to be 335.2.

Detection Limit of Sitagliptin

The Detection Limit of an individual analytical procedure is the lowest amount of analyte in

a sample which can be detected but not necessarily qualtitated as an exact value.

S/N Ratio of standard preparation of Sitagliptin was found to be 22.9.

Quantitation Limit of Sitagliptin

The Quantitation limit of an analytical procedure is the lowest amount of analyte in a sample

which can be quantitatively determined with suitable precision and accuracy.

S/N Ratio of standard preparation of Sitagliptin was found to be 31.2.


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Fig .No.4.25 Chromatogram Showing Limit of Detection of Metformin and Sitagliptin

Fig .No.4.26Chromatogram Showing Limit of Quantification of Metformin and

Sitagliptin

4.7: LOD values of Metformin and Sitagliptin


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4.8: LOQ Values of Metformin and sitagliptin

ROBUSTNESS

Influence on Flow Variation

Demonstrated robustness of assay method by changing the flow rate for 0.8 mL/min and 1.2

mL/min instead of specified flow rate (1.0 mL/min). By injecting the replicate injections of

samples in 0.8 mL/min and 1.2 mL/min flow rate and found that system suitability

parameters were passed. The % RSD of peak area, tailing factor and theoretical plates of

Metformin and Sitagliptin samples was found within the limits.

Fig .No.4.27 Chromatogram Showing Variation of Flow Rate 0.8 mL/min


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Fig .No.4.28 Chromatogram Showing Variation of Flow Rate 1.2 mL/min

Influence on variation of Column Temperature

Robustness of assay method by changing the column temperature for 25°C and 35°C instead

of specified column temperature (30°C) was demonstrated. By injecting the replicate

injections of standard in 25°C column temperature and 35°C column temperature and found

that system suitability parameters were passed. The % RSD of peak area, tailing factor and

theoretical plates of Metformin and Sitagliptin was found within the limits.


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Fig .No.4.29 Chromatograms Showing Variation of Temperature 250c

Fig .No.4.30 Chromatogram Showing Variation of Temperature 350c


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Table No. 10. Showing Results from Robustness Study of Metformin and Sitagliptin

Showing Results from Robustness Study

PEAK NAME:METFORIN

S.No Sample

Name Peak Name Rt Area

Usp

Tailing

Usp Plate

Count

1 Temp1 Metformin 2.282 3481825 1.18 5908

2 Temp2 Metformin 2.285 3470483 1.15 5527

3 Temp3 Metformin 2.281 3422130 1.19 5502

4 Temp4 Metformin 2.285 3436022 1.15 5471

5 Flow1 Metformin 2.515 3882427 1.20 6019

6 Flow2 Metformin 2.516 3903616 1.19 6125

7 Flow3 Metformin 2.290 3510630 1.16 5831

8 Flow4 Metformin 2.290 3488709 1.18 5776

Sitagliptin

S.No Sample

name

Peak

name RT Area

USP

Tailing

USP plate

count

1 Temp1 Sitagliptin 2.864 495394 1.37 3859




5 Flow1 Sitagliptin 3.169 686971 1.39 3825




The analytical method was found to be robust with respect to change in flow rate and

Temperature

SYSTEM SUITABILITY

The system suitability studies were done with the 20 mg of Metformin and 5 mg of

Sitagliptin standard drugs. The % RSD values are below 2%, theoretical plate count is above

2500 and tailing factor is less than 2, indicating that the method is suitable. The

chromatograms were recorded for all injections are shown below


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Chromatogram Showing System Suitability: Injection-1




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Fig .No.4.35 Chromatogram Showing System Suitability: Injection-5

Fig.No.4.36 Chromatogram showing System Suitability : Injection -6


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Table No. 7.9. Showing Results from System Suitability Study Sitagliptin

CONCLUSION

In this present study, an attempt was made to develop an analytical method for the

simultaneous estimation of Metformin and Sitagliptin in tablet dosage form which is

accurate, simple and fast.

The RP-HPLC method was developed by using a mobile phase composition of Ortho

phosphoric acid buffer and Methanol in ratio of 80:20.


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The analysis is resolved by using a Alltima C18 (4.6 x 250mm, 5m) or equivalent, in

isocratic mode, with mobile phase containing Buffer and Methanol with ratio of 80:20 was

used. The flow rate was 1.0ml/min and the analyte was monitored at 210 nm. The retention

time for Metformin was 2.35min and Sitagliptin it is 3.04 mins respectively.

The proposed method was subjected to validation parameters as per ICH guidelines and

found within the limit.It can be employed as stability-indicating one. The results are found to

be complying with the acceptance criteria for each of the parameter.

The method was validated for precision, accuracy, linearity, specificity, robustness,

ruggedness.

The system suitability parameters were within limit, hence it was concluded that the method

was suitable to perform the assay.

Linearity was obtained in the concentration range of 50-300µg/ml for Metformin and 5-30

µg/ml Sitagliptin with correlation coefficient of 0.9998 for Metformin and 0.9993 for

Sitagliptin drug. The percentage recovery of both drugs was found to be in the range of 98%-

102%.The method was rugged as observed from the analysis being performed by different

analysts, on different systems.

Assay was performed using the validated method and the formulation taken for analysis was

found to contain more than 99% of the each drug as mentioned in label claim.

Therefore it was concluded that the proposed method can be used for routine analysis of

Metformin and Sitagliptin in its tablet dosage form.

SUMMARY AND CONCLUSION

In this present study, an attempt was made to develop an analytical method for the

simultaneous estimation of Metformin and Sitagliptin in tablet dosage form which is

accurate, simple and fast.

The RP-HPLC method was developed by using a mobile phase composition of Ortho

phosphoric acid buffer and Methanol in ratio of 80:20.

The analysis is resolved by using a Alltima C18 (4.6 x 250mm, 5m) or equivalent, in

isocratic mode, with mobile phase containing Buffer and Methanol with ratio of 80:20 was


1368


used. The flow rate was 1.0ml/min and the analyte was monitored at 210 nm. The retention

time for Metformin was 2.35min and Sitagliptin it is 3.04 mins respectively.

The proposed method was subjected to validation parameters as per ICH guidelines and

found within the limit.It can be employed as stability-indicating one. The results are found to

be complying with the acceptance criteria for each of the parameter.

The method was validated for precision, accuracy, linearity, specificity, robustness,

ruggedness.

The system suitability parameters were within limit, hence it was concluded that the method

was suitable to perform the assay.

Linearity was obtained in the concentration range of 50-300µg/ml for Metformin and 5-30

µg/ml Sitagliptin with correlation coefficient of 0.9998 for Metformin and 0.9993 for

Sitagliptin drug. The percentage recovery of both drugs was found to be in the range of 98%-

102%.The method was rugged as observed from the analysis being performed by different

analysts, on different systems.

Assay was performed using the validated method and the formulation taken for analysis was

found to contain more than 99% of the each drug as mentioned in label claim.

Therefore it was concluded that the proposed method can be used for routine analysis of

Metformin and Sitagliptin in its tablet dosage form.

ACKNOWLEDGEMENTS

Authors are thankful to VASUDHA PHARMA drugs Pvt. Ltd. (India). for providing the gift

samples of drug and also thankful to Chairman, Department of pharmaceutical Analysis,

GNANA JYOTHI COLLEGE OF PHARMACY, Hyderabad for providing laboratory

facilities to carry out this research work.

REFERENCES

1. T. Raja et al, validated rp-hplc method for simultaneous estimation of metformin and

sitagliptin in bulk drug and pharmaceutical formulation ijpcbs, 2012; 2(4): 696-702,

Lakshmana Rao et al. ISSN, 2249-9504.


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2. Ramanjaneyulu Juvvigunta et al,l Method Development and Validation for Simultaneous

Estim ation of Sitagliptin and Metformin Hydrochloride in Tablet Dosage form by RP-

HPLC International Journal of Pharma Sciences, 2013; 3(5): 360-364, 2320-6810.

3. Loni et al. method development and validation for simultaneous determination of

sitagliptin phosphate and metformin hydrochloride by rp-hplc in bulk and tablet dosage

form AJPSR, Aug.2012; 2(8): 2249-4898.

4. Nancy Veronica B et al; Development and validation of a new simple RP-HPLC method

for estimation of Metformin HCl and Sitagliptin phosphate simultaneously in bulk and

dosage forms Int J Adv Pharm Gen Res, 2014; 2(1): 1-14, 2347-5102.

5. Shyamala M. et al., Validated RP-HPLC For Simultaneous Estimation of Sitagliptin

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PharmTech Research, 2011; 19(2): 2249-3387.

6. K.L. Goa and E.M. Sorkin. Metformin. Drugs, 1993; 46: 409–427

7. Sachin L Patil1, Jayant R Bhinge and Chetan M Bhalgat Development and Validation of

a Stability Indicating RP-HPLC Method for Simultaneous determination of Sitagliptin

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Pharmaceutical and Biomedical Sciences ISSN, 2229-3701

8. Dubala, Anil; Khatwal, Rizwanbasha; Kosaraju, Jayasankar; Meda, Venkat; Samanta,

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rp-hplc and its application to pharmacokinetic study International Journal of Pharmacy &

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9. R. Lavanya, Md. Yunoos Development and Validation of RP-HPLC method for the

estimation of Sitagliptin Phosphate in Bulk Journal of Advanced Pharmacy Education &

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