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Research Article

METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION of

VILDAGLIPTIN AND METFORMIN IN TABLET DOSAGE FORM BY RP-HPLC

K.PUSHPA LATHA AND D.RAMACHANDRAN*

Acharya Nagarjuna University, Nagarjuna nagar-522510, Guntur dist, Andhrapradesh, India. Email: [email protected]

Received: 04 Dec 2012, Revised and Accepted: 21 Dec 2012

ABSTRACT

A simple reverse phase liquid chromatographic method has been developed and subsequently validated for simultaneous determination of

vildagliptin and metformin in combination. The separation was carried out using a mobile phase consisting of 2mM phosphate buffer and

Acetonitrile with pH 3.5 adjusted with ortho phosphoric acid in the ratio of 70: 30%v/v. The column used was Phenomenex C18, (250 mm x 4.6 mm

i.d, 5µm) with flow rate of 1 ml / min using PDA detection at 293 nm. The described method was linear over a concentration range of 5-50 µg/ml

and 12.5-125 µg/ml for the assay of Vildagliptin and Metformin respectively. Gatifloxacin (50 µg/ml) was used as internal standard. The retention

times of Vildagliptin, Metformin and Gatifloxacin were found to be 2.1, 2.5 and 5.5min respectively. Results of analysis were validated statistically

and by recovery studies. The limit of detection (LOD) and the limit of quantification (LOQ) for Vildagliptin and Metformin were found to be 5 and10

μg/ml 10 and 25 μg/ml respectively. The results of the study showed that the proposed RP-HPLC method is simple, rapid, precise and accurate,

which is useful for the routine determination of Vildagliptin and Metformin bulk drug and in its pharmaceutical dosage form.

Keywords: Vildagliptin, Metformin, RP-HPLC, Pharmaceutical dosage form.

INTRODUCTION

Vildagliptin (S)-1-[N-(3-hydroxy-1-adamantyl) glycyl] pyrrolidine-2- carbonitrile Figure 1 is an oral anti-hyperglycemic agent (anti-

diabetic drug) of the new dipeptidyl peptidase-4 (DPP-4) inhibitor class of drugs. Vildagliptin inhibits the inactivation of GLP-

1[1,2] and GIP by DPP-4, allowing GLP-1 and GIP to potentiate the secretion of insulin in the beta cells and suppress glucagon release

by the alpha cells of the islets of Langerhans in the pancreas. Vildagliptin has been shown to reduce hyperglycemia in type 2

diabetes mellitus. Novartis has since withdrawn its intent to submit vildagliptin to the FDA, as of July 2008 [3]. The Food and Drug

Administration had demanded additional clinical data before it could approve vildagliptin including extra evidence that skin lesions

and kidney impairment seen during an early study on animals have not occurred in human trials. While the drug is still not approved for

use in the US, it was approved in Feb 2008 by European Medicines

Agency for use within the EU [4] and is listed on the Australian PBS with certain restrictions [5]. The EMEA has also approved a new oral

treatment released by Novartis, called Eucreas, a combination of vildagliptin and metformin [6].

The DPP-4 enzyme is known to be involved in the suppression of

certain malignancies, particularly in limiting the tissue invasion of

these tumours. Inhibiting the DPP-4 enzymes may allow some cancers

to progress [7, 8]. A study of DPP-4 inhibition in human non-small cell

lung cancer (NSCLC) concluded that "DPPIV functions as a tumor

suppressor, and its down regulation may contribute to the loss of

growth control in NSCLC cells [9]. The risk of cancer suppression with

DPP-4 down-regulation applies to all the DPP-4 inhibitors on the

market in addition to vildagliptin (sitagliptin and saxagliptin)

Metformin (N, N-dimethylimidodicarbonimidic diamide) is an

oral antidiabetic drug in the biguanide class. It is the first-line drug

of choice for the treatment of type 2 diabetes, in particular,

in overweight and obese people and those with normal kidney

function [10-13]. Its use in gestational diabetes has been limited by

safety concerns. It is also used in the treatment of polycystic ovary

syndrome, and has been investigated for other diseases

where insulin resistance may be an important factor. Metformin

works by suppressing glucose production by the liver.

Metformin is the only antidiabetic drug that has been conclusively

shown to prevent the cardiovascular complications of diabetes. It

helps reduce LDL cholesterol and triglyceride levels, and is not

associated with weight gain. As of 2010, metformin is one of only

two oral antidiabetics in the World Health Organization Model List

of Essential Medicines (the other being glibenclamide) [4]. When

prescribed appropriately, metformin causes few adverse effects (the

most common is gastrointestinal upset) and is associated with a low

risk of hypoglycemia. Lactic acidosis (a buildup of lactate in the

blood) can be a serious concern in overdose and when it is

prescribed to people with contraindications, but otherwise, there is

no significant risk.

First synthesized and found to reduce blood sugar in the 1920s,

metformin was forgotten for the next two decades as research

shifted to insulin and other antidiabetic drugs. Interest in

metformin was rekindled in the late 1940s after several reports

that it could reduce blood sugar levels in people, and in 1957,

French physician Jean Sterne published the first clinical trial of

metformin as a treatment for diabetes. It was introduced to the

United Kingdom in 1958, Canada in 1972, and the United States in

1995. Metformin is now believed to be the most widely prescribed

antidiabetic drug in the world; in the United States alone, more

than 48 million prescriptions were filled in 2010 for its generic

formulations [5, 6].

HO NH

N

O

C

H

N

N NH

NH2

NH NH

Fig. 1: Chemical structures of vildagliptin and metformin

International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 5, Issue 1, 2013

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Ramachandran et al.

Int J Pharm Pharm Sci, Vol 5, Issue 1, 459-463

460

MATERIALS AND METHODS

Chemicals and Reagents

Samples of Vildagliptin, Metformin and Gatifloxacin confirmed by

spectral characterization and SOR (specific optical rotation) were

obtained from Process Research Department of Emmanthi

Laboratories Ltd, Hyderabad, India. HPLC-grade Acetonitrile was

procured from Sigma Aldrich, India.

Instrumentation

Chromatographic system consisted of a Waters Model Alliance 2695

separation module equipped with auto sampler Photodiode array

ultraviolet (UV) detector. The data recorded using empower

software. The separation was carried out using a mobile phase

consisting of 2mM phosphate buffer and Acetonitrile with pH 3.5

adjusted with ortho phosphoric acid in the ratio of 70: 30%v/v. The

column used was Phenomenex C18, (250 mm x 4.6 mm i.d, 5µm)

with flow rate of 1 ml / min using PDA detection at 293 nm.

Preparation of stock, working standard solutions, and sample

solution

A stock solution of vildagliptin and metformin (100 μg/mL) was

prepared, by taking 10 mg of each drug, accurately weighed, in

separate 100-mL volumetric flasks. They were dissolved in 25 mL of

mobile phase and then the volume was made up to the mark to get

100 μg/mL. The internal standard solution was prepared by taking

10 mg of gatifloxacin in a 100 ml standard flask. It is dissolved by

adding 25 ml of mobile phase, shaken for few minutes to get a clear

solution and the final volume was made up to 100 ml. For each drug,

appropriate aliquots were pipetted out from the standard stock

solution into a series of 10 mL volumetric flasks to get a

concentration of 5, 10,20,30,40 and 50 µg/ml of vildagliptin, 12.5,

25, 50, 75,100 and 125 µg/ml of metformin and 50 µg/ml of

gatifloxacin (Internal Standard).

Development and validation of HPLC Method

Present study was conducted to obtain a new, affordable, cost-

effective and convenient method for HPLC determination of vildagliptin and metformin in tablet dosage form. The experiment

was carried out according to the official specifications of USP–30, ICH- 1996, and Global Quality Guidelines-2002. The method was

validated for the parameters like system suitability, selectivity, linearity, accuracy, precision, and robustness.

System suitability

The system suitability was assessed by six replicate analysis of

vildagliptin and metformin at a 100% level to verify the resolution and reproducibility of the chromatographic system adequate for the

analysis to be done. This method was evaluated by analyzing the repeatability of retention time, peak area for both vildagliptin and

metformin tailing factor, theoretical plates (Tangent) of the column and resolution between the peaks of vildagliptin and metformin.

Selectivity

Selectivity was determined in the presence of common excipients

used in the tablet formulation. Sample containing 100% vildagliptin

and metformin was injected first. Then the samples mixed with

three different placebo formulations were injected to find out the

selectivity of the method

Linearity

Linearity of the method was determined by constructing calibration

curves. Standard solutions of vildagliptin and metformin at different

concentrations level (80%, 90%, 100%, 110%, and 120%) were

used for this purpose. Before injection of the solutions, the column

was equilibrated for at least 30 min with the mobile phase. Each

measurement was carried out in six replicates to verify the

reproducibility of the detector response at each concentration level.

The peak areas of the chromatograms were plotted against the

concentrations of vildagliptin and metformin to obtain the

calibration curves. The five concentrations of the standard were

subjected to regression analysis to calculate calibration equation

and correlation coefficients.

Accuracy

The accuracy is the closeness of agreement between the true value

and test result. Accuracy was determined by means of recovery

experiments, by addition of active drug to placebo formulations. The

accuracy was calculated from the test results as the percentage of

the analyte recovered by the assay.

Precision

The precision of the method was determined by repeatability (intra-

day) and intermediate precision (inter-day) study. Repeatability was

determined by performing four repeated analysis of the three

standard solutions (90%, 100% and 110% of target concentration)

of vildagliptin and metformin on the same day, under the same

experimental conditions. The intermediate precision of the method

was assessed by carrying out the analysis of previous standard

solutions on three different days (inter-day) in the same laboratory.

The relative standard deviation (% RSD) was determined in order to

assess the precision of the method.

Robustness

The robustness of the method was assessed by altering the some

experimental conditions such as, by changing the flow rate from 0.9

to1.1 ml/min, amount of acetonitrile (10% to 15%,) the temperature

of the column (28 °C to 32 °C) and PH of the mobile phase.

RESULTS AND DISCUSSION

The developed method has been validated as per ICH guidelines10.

Every 20 μL of the working standard solution of vildagliptin in the

mass concentration range of 5 to 50 μg/mL, and that for metformin

in the mass concentration range of 12.5 to 125 μg/mL were injected

into the chromatographic system. The chromatograms were

developed and the peak area was determined for each concentration

of the drug solution. Calibration curves of vildagliptin and

metformin were obtained by plotting the peak area ratio versus the

applied concentrations of vildagliptin and metformin. The linear

regression coefficients were found to be 0.9998 and 0.9991for

vildagliptin and metformin respectively.

Repeatability of the method was checked by injecting replicate

injections of the solution 10 μg/mL and 25 μg/mL of vildagliptin and

metformin respectively and the RSD was found to be 0.326% and

0.435%. Variability of the method was studied by analyzing the

solution on the same day (intra-day precision) and on three different

days (inter- day precision). The results obtained for intra-day

precision (RSDs) were 0.879 %& 0.945 % respectively, at n = 6, for

both vildagliptin and metformin. The inter-day precisions (RSDs)

were 0.254 % and 0.364 %, respectively, at n = 6, for both

vildagliptin and metformin.

Accuracy of the method was tested by carrying out recovery studies at

different spiked levels. The estimation was carried out as described

earlier. At each level, three determinations were performed and

results obtained. The amounts recovered and the values of percent

recovery were calculated, results are shown in Table 1.

The specificity of the method was checked for the interference of

impurities in the analysis of a blank solution (without any sample)

and then a drug solution of 20 μg/mL was injected into the column,

under optimized chromatographic conditions, to demonstrate the

separation of both vildagliptin and metformin from any of the

impurities, if present. As there was no interference of impurities and

also no change in the retention time, the method was found to be

specific and also confirmed with the results of analysis of

formulation.

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

calculated as 3.3 ∂/S and 10 ∂/S, respectively as per ICH guidelines,

where ∂ is the standard deviation of the response (y-intercept) and S

is the slope of the calibration plot. The results of validation parameters

and System suitability parameters were shown in Table 2.

Ramachandran et al.


461

Table 1: Recovery studies of vildagliptin and metformin.

Drug Conc. Of Std. (µg/mL) Conc. Of solution (µg/mL) Amount found (µg/mL) % Recovery %RSD

Vildagliptin 10 10 19.90 99.50 0.324

10 20 30.14 100.46 0.687

10 30 40.87 102.17 0.652

Metformin 25 25 49.69 100.53 0.517

25 50 74.82 99.38 0.674

25 75 99.83 99.83 0.285

Table 2: System suitability of parameters

Validation parameters Vildagliptin Metformin

Linearity range (µg/mL) 5-50 12.5-125

LOD (ng/mL) 5 10

LOQ (ng/mL) 10 25

Intraday (%RSD) 0.879 0.945

Inter day (%RSD) 0.254 0.364

Repeatability (%RSD) 0.3251 0.4250

Accuracy 99.50-102.17 99.38-100.53

Peak purity index 1.0000 1.0000

Resolution factor (Rs) - 19.618

r 0.9998 0.9991

Asymmetry factor (As) 0.95

No. of theoretical plates (N) 3059 12500

Capacity factor (K) - 0.330

High equivalent to theoretical 49.029 11.999

Plates (HETP)

Tailing factor 1.149 1.140

Separation factor - 7.902

Analysis of Formulation

Twenty tablets of vildagliptin and metformin in combination were

weighed, their average weight was determined, and finally they

were crushed to a fine powder. The tablet powder equivalent to 20

mg of vildagliptin and 50 mg of metformin was weighed and

transferred to a 100 mL volumetric flask, first dissolved in 50 mL of

mobile phase, and then the volume was made up to the mark with

the mobile phase. The content was ultrasonicated for 30 min for

complete dissolution. The solution was then what Mann’s filter

paper No-41. The selection of the mixed sample solution for analysis

was carried out by the optimization of various dilutions of the tablet

dosage form, considering the label claim. The mixed sample solution

of 10 μg/mL of vildagliptin and 25 μg/mL of metformin, which was

falling in the Beer’s-Lamberts range with 50 μg/mL internal

standards, showed good results and was selected for the entire

analysis. The results of tablet analysis (n = 6) were found to be 99.81

and 99.44 for vildagliptin and metformin respectively.

From the typical chromatogram of vildagliptin, metformin and

Gatifloxacin (Internal standard) (Fig. 2), it was found that the

retention time of vildagliptin was 2.1 min, Gatifloxacin was 2.5 min

and metformin was 5.5 min, which were well-resolved peaks with a

resolution factor of 3.3 for Gatifloxacin and 19.618 for metformin. As

there was no interference of impurities and also no change in the

retention time, the method was found to be specific and the

respective peak purity curve and overlay UV Spectrum were shown

in Fig 3, 4, & 5. The results analysis was shown in Table – 3.

Fig. 2: Typical chromatogram of vildagliptin, Gatifloxacin and metformin.

Ramachandran et al.


462

Fig. 3: Peak purity curve of vildagliptin

Fig. 4: Peak purity curve of metformin

Fig. 5: Overlaid UV spectrum of Vildagliptin, Gatifloxacin and metformin

Ramachandran et al.


463

Table 3: Analysis of formulation

Drugs Labeled amount % lable claim Amount taken(mg) Amount found for assay (µg/mL) (mg)

Vildagliptin 20 10 9.981±0.153 99.81

Metformin 50 25 24.86±1.025 99.44

CONCLUSION

The developed method was validated in terms of accuracy,

repeatability, and precision. A good linear relationship was observed

for vildagliptin and metformin in the concentration ranges of 5 –50

μg/mL and 12.5– 125 μg/mL respectively. The correlation

coefficient for vildagliptin was found to be 0.9998 and that for

metformin was 0.9991. The inter-day and intra-day precision results

were good enough to indicate that the proposed method was precise

and reproducible. The assay experiment showed that the contents of

vildagliptin and metformin estimated in the tablet dosage form were

free from the interference of excipients. This demonstrated that the

developed HPLC method was simple, linear, precise, and accurate,

and could be conveniently adopted for the routine quality control

analysis of vildagliptin and metformin simultaneously, from its

pharmaceutical formulations and bulk drug.

ACNOWLEDGEMENT

Thanks to Emmanthi Laboratories Ltd, Hyderabad, India for

providing lab facilities

REFERENCES

1. Ahren, B; Landin-Olsson, M; Jansson, PA; Svensson, M; Holmes,

D; Schweizer, A (2004). "Inhibition of dipeptidyl peptidase - 4 reduces glycemia, sustains insulin levels, and reduces glucagon

levels in type 2 diabetes”. The Journal of Clinical Endocrinology and Metabolism 89 (5): 2078–84.

2. Mentlein, R; Gallwitz, B; Schmidt, WE (1993). "Dipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide,

glucagon-like peptide-1(7-36) amide, peptide histidine

methionine and is responsible for their degradation in human serum". European journal of biochemistry / FEBS 214 (3):

829–35.

3. Focus on Health: Drug makers say FDA slows U.S. pipeline

Emphasis on safety, side effects grows; swing of pendulum? By

Avery Johnson and Ron Winslow 2180 words 2 July 2008 The

Wall Street Journal Europe

4. EU approves Novartis diabetes drug Galvus. Reuters, February

01, 2008

5. NPS - better medicines, better health (August 2010). "Vildagliptin

for type 2 diabetes". Retrieved 27 August 2010.

6. EU approves Novartis's Eucreas diabetes drug. Reuters,

February 25, 2008.

7. Pro B, Dang NH (October 2004). "CD26/dipeptidyl peptidase IV

and its role in cancer". Histol. Histopathol. 19 (4): 1345–51.

8. Wesley UV, McGroarty M, Homoyouni A (February 2005).

"Dipeptidyl peptidase inhibits malignant phenotype of prostate

cancer cells by blocking basic fibroblast growth factor signaling

pathway". Cancer Res. 65 (4): 1325–34.

9. Wesley, U; et al (2004). "Role for dipeptidyl peptidase IV in

tumor suppression of human non small cell lung carcinoma

cells." Int J Cancer 109 (6): 855–866.

10. Clinical Guidelines Task Force, International Diabetes

Federation (2005). "Glucose control: oral therapy". In: Global

Guideline for Type 2 Diabetes. Brussels: International Diabetes

Federation, 35–8. Retrieved on November 6, 2007.

11. National Collaborating Centre for Chronic Conditions. Type 2

diabetes: national clinical guideline for management in primary

and secondary care (update) London: Royal College of

Physicians; 2008. p. 86.

12. American Diabetes Association. Standards of medical care in

diabetes—2009. Diabetes Care. 2009; 32 Suppl 1:S13–61.

13. (March 2010) WHO Model List of Essential Medicines, 16th

edition, World Health Organization, p. 24. Retrieved on 22

December 2010.

14. Bailey CJ, Day C. Metformin: its botanical background. Practical

Diabetes International. 2004; 21(3):115–7.

15. "The Use of Medicines in the United States: Review of 2010”.

IMS Institute for Healthcare Informatics (April 2011).

Retrieved on April 28, 2011.

International Journal of ChemTech ResearchCODEN( USA): IJCRGG ISSN : 0974-4290Vol.5, No.5, pp 2429-2435, July-Sept 2013

Validation Of HPLC Method For Determination Of AtorvastatinIn Tablets And Identify Diketone Impurity By LC-Mass

K.Pushpa Latha* and D.Ramachandran

Acharya Nagarjuna University, Nagarjuna nagar-522510, Guntur dist,Andhrapradesh, India.

*Corres.author: [email protected]

Abstract: A rapid high performance liquid chromatographic method was developed and validated fordetermination of atorvastatin in pharmaceutical dosage forms and for evaluation of its stability in thesolid phase. The identify the diketone impurity by the LC-MS. Separation of atorvastatin wassuccessfully achieved on a C-18 column utilizing the mobile phase A contains a mixture of buffer,acetonitrile and tetrahydrofuran in the ratio of 70:25:5 v/v/v and mobile phase B contains a mixture ofbuffer, acetonitrile and tetrahydrofuran in the ratio of 25:70:5 v/v/v, 1.54 gm of ammonium acetate in1000 mL of water and adjust pH to 4.0±0.05 with glacial acetic acid, this solution used as a buffer. Thedetection wavelength was 248 nm. The method was validated and the response was found to be linearin the drug concentration range of 0.04 mg/mL 0.4 mg/mL. The mean values ± RSD of the slope andthe correlation coefficient were 8.192 ± 0.260 and 0.999, respectively. The RSD values for intra- andinterday precision were < 1.00% and 0.90%, respectively.

Introduction

Atorvastatin (ATO) is chemically (βR, δR)-2-(4- fluorophenyl)-β, δ-dihydroxy-5-(1-methylethyl)-3- phenyl-4-[(phenylamino)carbonyl]-lH-pyrrole-1-heptanoic acid, calcium salt (2:1) trihydrate and diketo impurity (Figure1). Atorvastatin is an inhibitor of 3-hydroxy-3-methylglutaryl- coenzyme A (HMG-CoA) reductase. Thisenzyme catalyzes the conversion of HMG-CoA to mevalonate, an early and rate-limiting step in cholesterolbiosynthesis. Atorvastatin is administered as the calcium salt of the active hydroxyl acid and is used between 10and 80 mg per day to reduce the raised lipid levels in patients with primary hyperlipidemia (familial and nonfamilial) or combined hyperlipidemia [1-4]. HPLC has been the analytical method of choice for the kineticalstudy of ATO. Several procedures of chromatographic techniques such as LC/MS/MS, microbore LC/ESI-MS/MS, HPLC with electrospray tandem mass spectrometry and LC methods with UV detector have beentested for the determination of ATO in biological fluids [5-10] and pharmaceutical dosage forms [11, 12].However, any generally recommended or rapid analytical method for the determination of ATO andsimultaneously, for evaluation of its stability in solid state has not yet been described in any pharmacopoeia andliterature.

In the present study, a new rapid, validated for determination of atorvastatin in pharmaceutical dosage forms andfor evaluation of its stability in the solid phase. The identify the diketone impurity by the LC-MS.

K.Pushpa Latha et al /Int.J.ChemTech Res.2013,5(5) 2430

N

OH

OOC

HO

HN

O

F

2

Ca++

-

Atorvastatin

CONH

OO

F

Diketo impurity

Fig. 1: Chemical structures of a) Atorvastatin b) Diketo impurity

Experimental

Chemicals and reagents

Atorvastatin (amorphous form) was obtained from Zydus Cadila, tablets of atorvastatin (10 mg of atorvastatinper tablet) were obtained from Parke- Davis, oxazepam and HPLC grade methanol, acetonitrile were purchasedfrom Aldrich.

Instrumentation

The chromatographic system consisted of a pump, Hypersil BDS, C18, 250X4.6mm, 5µm column, UV at 248nm detectors and analysis was performed on a chromatographic system waters, alliance 2695 HPLC systemhaving 2487 UV detector with empower chromatography software.

Analytical Procedure

Stock and standard calibration solutions Stock solution of ATO (1.2 mg/mL) was prepared in methanol. Thissolution could be stored at 268 K for over 1 month with no evidence of decomposition. Standards solutions ofATO were prepared with methanol in the range of 0.04 mg/mL 0.4 mg/mL, maintaining the concentration of ISat a constant level of 0.01 mg/mL. Hundred microliters of each solution was injected into the column andchromatograms were recorded. The calibration curve for the HPLC analysis was constructed by plotting theratio of peak normalization of ATO to IS against concentration. Internal standard (IS; oxazepam) solution 10.0mg of oxazepam in methanol diluted to 100.0 mL with the same solvent.

Analysis of tablets

Ten tablets were weighed to get the average weight and then powdered. The fine powder, equivalent to 10 mg ofATO, was weighed and transferred into a 25 mL calibrated flask and dissolved using methanol. This mixturewas sonicated (15 min) and then filtered through a 0.45 mm membrane filter. After filtration, the appropriatevolume (1.0 mL) was taken into a 10 mL flask added to 1.0 mL of IS (oxazepam). All determinations wereconducted in triplicate. The amount of ATO was calculated from the related linear regression equations.Conditions of kinetic studies Kinetic studies of conditions were in compliance with recommendations of theInternational Commission of Harmonisation [13, 14]. Samples of ATO (10.00 mg) were accurately weighed into5 mL vials. The vials with ATO were placed in desiccators containing aqueous saturated solutions of sodiumchloride (relative humidity RH = 76.4%) and inserted in heat chamber set at 363 K. After definite time intervals,determined by rate of degradation, the respective vials were taken out of the chamber, cooled to roomtemperature, and the contents dissolved in methanol. The so obtained solution was quantitatively transferred into


a measuring flask and made up to total volume of 25.0 mL with methanol. To 1.0 mL of the solution, 1.0 mL ofsolution of IS was added. The chromatograms were interpreted using the following dependence: PATO/PIS = f(t); where PATO is the area of ATO signal and PIS represents the values of IS (oxazepam).

Results And Discussion

Validation of the method

Validation of HPLC method was in compliance with recommendations of the International Commission ofHarmonisation [15-17].

Selectivity

This method was selective for the ATO, as well as for the internal standard (Oxazepam), in the presence ofdegradation product. The selectivity of HPLC method is illustrated in Figure 2a. The typical excipients includedin the drug formulation do not interfere with selectivity of the method (Figure 2b). The analysis of thechromatogram of ATO, its degradation product and IS, revealed the following efficiencies of the column: forATO N = 2600, degradation product N = 4986, and IS N = 1494 (where N represents theoretical plate number).The separation factors between ATO and oxazepam (IS) = 3.15; ATO and product of degradation = 16.9.

Figure 2a. HPLC chromatogram for the analysis of degradation solution of ATO (363 K, RH 76,4%). Peak 1:ATO; peak 2: internal standard (oxazepam); peak 3: product of degradation. Chromatographic conditions aredescribed in the text.

Figure 2b. HPLC chromatogram of an extract of tablets. Peak 1: ATO; peak 2: internal standard (oxazepam).Chromatographic conditions are described in the text.


Precision and accuracy

The repeatability of the method was examined by injecting the solution consisting of ATO (0.1mg/mL, 0.2mg/mL and 0.4 mg/mL) and IS (0.1 mg/mL) into the HPLC system for three consecutive days. The results aregiven in Table 1. The repeatability of the HPLC method was good and precise, and the RSD values wereobtained between 0.70% and 1.00%.

Table 1. Accuracy and precision for ATO assay.

Parameter Intraday Inter-day

Day 1 (n = 10) Day 2 (n = 10) Day 3 (n = 10) (n = 30)Added concentration0.1000 mg/mLMeasured concentration(mg/mL)

0.1009 ± 0.0007 0.1007 ± 0.0006 0.1001 ± 0.0006 0.1006 ± 0.0006

Recovery (%) 100.91 ± 0.73 100.72 ± 0.59 100.13 ± 0.58 100.59 ± 0.63SD 0.0010 0.0008 0.0009 0.0009RSD (%) 0.983 0.776 0.885 0.881Added concentration0.2000 mg/mL

0.1999 ± 0.001 0.2001 ± 0.001 0.1998 ± 0.001 0.1999 ± 0.001

Recovery (%) 99.95 ± 0.37 100.05 ± 0.54 99.91 ± 0.58 99.91 ± 0.58SD 0.0013 0.0015 0.0016 0.0015RSD (%) 0.668 0.731 0.812 0.737

Linearity

The linearity of the method was determined in terms of the correlation coefficient between its ATO and the ratioof peak normalization of ATO to that of IS. The internal standard was added to the solutions under investigationat a constant concentration of 0.1 mg/mL. The calibration range was between 0.04 mg/mL 0.4 mg/mL presentedwith the equation of

y = ac + b = (8.192 ± 0.260) × c + (0.00232 ± 0.0010).

The intercept b is very small, statistically non-significant and the correlation coefficient closed to r = 0.999. Thevalues obtained showed good linearity. The intraday and inter-day accuracy of method was also examined. Theevaluated data are given in Table 2.

Sensitivity

To calculate the limit of quantitation (LOQ) and limit of detection (LOD), signal to noise ratio of 10 and 3,respectively, were used. The results are shown in Table 2.

Table 2. The intraday and inter-day accuracy and linearity of proposed method.

IntradayParameter

Day 1 (n = 10) Day 2 (n = 10) Day 3 (n = 10)

Inter-day (n = 10)

Slope (a ± a) 8.132 ± 0.392 8.222 ± 0.172 8.223 ± 0.217 8.192 ± 0.260intercept (b ± b) 0.00302± 0.0006 0.00319±0.0019 (-0.75 ± 0.6)10-3 0.00232 ± 0.0010Correlationcoefficient (r)

0.998 0.999 0.999 0.999

RSD of slope a 0.170 0.074 0.094 0.113RSD of intercept b 0.0422 0.0185 0.0233 0.084N 10 10 10 10LOQ (mg/mL) 0.015 0.015 0.015 0.015LOD (mg/mL) 0.035 0.035 0.035 0.035


Solution stability

The stability of standard and sample preparations was determined over 48 h. Standard and sample preparationswere stored at ambient temperature under laboratory light conditions. Solutions were analyzed at 0, 24, and 48h. The results were evaluated for the percent difference from time zero. Less than 1.0% difference wasobserved, which demonstrates that the standard and sample preparations were stable for up to 48 h, when storedat ambient temperature under laboratory light conditions.

Application to tablets

The present method was applied to the analysis of ATO in three batches of tablets. The results presented inTable 3 are in good agreement with the labeled content. All data represent the average of ten determinations.

Table 3. Results obtained in determination of ATO in tablets.

LC-Mass conditions

System suitability

Accurately weigh and transfer about 100 mg of diketone standard into 100 mL volumetric flask dissolve anddilute to volume with diluents. Further dilute 5 mL to 50 mL with diluent

Sample preparation

Accurately weigh and transfer about 100 mg of sample into 100 mL volumetric flask dissolve and dilute tovolume with diluent. Further dilute 5 mL to 50 mL with diluent.

Chromatographic conditions

Column : Hypersil BDS, C18, 250X4.6mm, 5 µm

Detector : UV at 248nm

The diketone impurity on shows the HPLC chromatogram in Figure 3a and 3b .

Fig 3a. HPLC chromatogram of Diketone impurity

Batch Mean (mg) ± SD1 10.44 ± 0.0812 10.21 ± 0.0453 10.32 ± 0.026


LC-parameters

Buffer : 0.02M ammonium acetate pH 4.0 with acetic acidColumn : Hypersil BDS, C18, 250X4.6mm, 5 µmWavelength : UV at 248nmInjection volume : 20 µlMS/MS parametersSystem : Applied biosystemProbe : ESIPolarity : positiveScan range : 50-1000 m/zD.P : 56 voltsF.P : 260 voltsEntrance potential : 10 volts

Fig 3b. DAD Spectral data

Conclusion

In summary, a new rapid, validated for determination of atorvastatin in pharmaceutical dosage forms and forevaluation of its stability in the solid phase and identify the diketone impurity by the LC-MS.

Acknowledgments

The authors express their thanks to colleagues in the Nagarjuna University, for providing analytical and spectraldata.


References

1. Sparks D.L., Sabbagh M.N., Connor D.J., Lopez J., Launer L.J., Petanceska S., Browne P.: Curr.Alzheimer Res. 2, 343 (2005).

2. Gee M., Hasson N.K., Hahn T., Ryono R.: J. Manag. Care Pharm. 8, 453 (2002).3. Castano G., Mas R., Fernandez L., Illnait J., Mesa M., Alvarez E., Lezcay M.: Drugs Aging 20, 153

(2003).4. Zajac M., Pawelczyk E.: In-hibitory hydroxyl methyl glutaryl co enzyme A (I-HMGCoA). In Chemia

lekuw, pp. 401-403, A.M. Poznao, 2000.5. Hermann M., Cristensen H., Rebsaet J.L.: Anal. Bioanal. Chem. 382, 1242 (2005).6. Van Pelt C.K., Corso T.N., Schultz G.A., Lowes S., Henion J.: Anal. Chem. 73, 582 (2001).7. Miao X.S., Metcalfe C.D.: J. Mass Spectrom. 38, 27 (2003).8. Jemal M., Ouyang Z., Chen B.C., Teitz D.: Rapid Commun. Mass Spectrom. 13, 1003 (1999).9. Altuntas G.T., Erk N.: J. Liq. Chromatogr. Relat. Technol. 27, 83 (2004).10. Bullen W.W., Miller R.A., Hayes, R.N.: J. Am. Soc. Mass Spectrom. 10, 55 (1999).11. Koytchev R., Ozalp Y., Erenmemisoglu A., van der Meer M.J., Alpan R.S.: Arzneimittel- Forschung

54, 573 (2004).12. Erturk S., Sevinc-Aktas E., Ersoy L., Ficicioglu S.: J. Pharm. Biomed. Anal. 33, 1017 (2003).13. ICH Steering Committee, in: European Agency for the Evaluation of Medicinal Products, International

Commission on Harmonisation, London (CPMP/ICH/381/95), 1994.14. ICH Steering Committee, ICH Q2B Validation of Analytical Procedures: methodology, European

Agency for the Evaluation of Medicinal Products, International Commission on Harmonisation, London(CPMP/ICH/281/ 95), 1996.

15. Papadoyannis N.I., Samanidou F.V.: J. Liq. Chromatogr. Relat. Technol. 27, 753 (2004).16. Validation of Analytical Methods: Methodology ICH Topic Q2B, Pharm Europa., 1996, 8, 108.17. Huber, L. Good Laboratory Practice and Current Good Manufacturing Practice, p. 152, Hewlett-

Packard, Germany 1994.

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Development and validation of RP-HPLC method for simultaneous estimation of metformin HCl and sitagliptin in bulk and its pharmaceutical formulations

K. Pushpa Latha* and D. Ramachandran

Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar, Andhra Pradesh, India

_____________________________________________________________________________________________ ABSTRACT A new, simple, rapid, selective, precise and accurate isocratic reverse phase high performance liquid Chromatography assay method has been developed for simultaneous estimation of Metformin HCl and Sitagliptin in tablet formulations. The separation was achieved by using ACE column C18 150 x 4.6 mm, 5.0 µm particle size, in mobile phase Orthophosphoric acid buffer and Methanol in the ratio of 600:400v/v. The flow rate was 1.0 mL.min-1 and the separated drugs were detected using UV detector at the wavelength of 228 nm. The retention time of Metformin HCl, and Sitagliptin, was noted to be 3.00 and 4.50, respectively, indicative of rather shorter analysis time. The method was validated as per ICH guidelines. The proposed method was found to be accurate, reproducible, and consistent. Keywords: Liquid Chromatography; Metformin HCl, Sitagliptin, Combined dosage forms; Simultaneous estimation, Validation _____________________________________________________________________________________________

INTRODUCTION Metformin [1] belongs to the bi-guanide class of anti-diabetic drug which is extensively used in the treatment of type II diabetes mellitus [Non-insulin-dependent diabetes mellitus (NIDDM)].The antihyperglycemic effects of Metformin are not only the inhibition of intestinal glucose absorption and the improvement of peripheral and hepatic insulin sensitivity but also the reduction of hepatic glucose production and the enhancement of peripheral glucose utilization. Molecular formula C4H11N5.HCL, molecular weight 165.62g/mol, white to off-white crystalline powder and hygroscopic. Freely soluble in water and slightly soluble in ethanol (95%). Metformin declines hyperglycemia primarily by suppressing glucose production by the liver (hepatic gluconeogenesis) [2]. Metformin activates AMP-activated protein kinase (AMPK), an enzyme that plays an important role in insulin signaling, whole body energy balance, and the metabolism of glucose and fats [2]. Activation of AMPK was required for Metformin's inhibitory effect on the production of glucose by liver cells [3]. Which in turn inhibits the expression of the hepatic gluconeogenic genes PEPCK and Glc-6-Pase [4]. The mechanism by which biguanide increase the activity of AMPK remains uncertain. Peak plasma concentrations (Cmax) are reached within one to three hours of taking immediate-release Metformin and four to eight hours with extended release formulations[5-6]. The plasma protein binding of Metformin is negligible, as reflected by its very high apparent volume of distribution (300–1000 L after a single dose). Steady state was usually reached in one or two days [7-8]. Sitagliptin [9] is an oral dipeptidyl peptidase-4 (DPP-4) inhibitor, which improves glycaemic control by inhibiting DPP-4 inactivation of the incretin hormones glucagon-like peptide-1 and glucose dependent insulinotropic polypeptide. This increases active incretin and insulin levels and decreases glucagon levels and post-glucose-load glucose excursion. (R)-4-oxo-4-[3-(trifluoromethyl)-5,dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl) butan-2-amine. Molecular formula C16H15F6N5O, molecular weight 407.314 g/mol. White to off-white powder. Soluble in water and in N, N-dimethyl formamide. Sitagliptin improves the ability of alpha cells to suppress glucagon secretion (by enhancing active incretin levels), which results in reduced glycogen breakdown and

K.Pushpa Latha and D. Ramachandran Der Pharmacia Lettre, 2014, 6 (4):515-521 ______________________________________________________________________________


glucose synthesis. Sitagliptin increases insulin synthesis and release from pancreatic beta cells, which helps to reduce hepatic glucose over production [10-12]. Sitagliptin does not undergo extensive metabolism. In vitro studies indicate that the primary enzyme responsible for the limited metabolism of Sitagliptin was Cytochrome P3A4 (oxidation), with contribution from Cytochrome P2C8 [13-14]. Somatropin antagonize the hypoglycaemic effect of Sitagliptin [15-17]. Monitor for changes in fasting and postprandial blood sugars.

NCH3

NH

NH

NH2

CH3

NH

N

N

O

N

N

F

F

F

N H2

F

FF

A. Metformin HCl B. Sitagliptin

Fig.1.The structures of Metformin HCl and Sitagliptin

Literature survey revealed a few HPLC methods for the estimation of Metformin HCl and Sitagliptin. In the present study, an attempt has been made to develop a method for the simultaneous estimation of two drugs- Metformin HCl and Sitagliptin. It can also be applied for routine analysis of either one or of any combinations of in these drugs dosage forms.

MATERIALS AND METHODS 2.1. Chemicals and Reagents Milli-Q Water, Methanol (HPLC Grade), Orthophosphoric acid (AR Grade), was obtained from Qualigens Ltd., Mumbai. All other chemical of analytical grade were procured from local sources unless specified. All dilutions were performed in standard class-A, volumetric glassware. 2.2. Instrumentation and Chromatographic Conditions Instrumentation The analysis of the drug was carried out on a waters LC system equipped with 2695pump and 2996 photodiode array detector was used and a Reverse phase HPLC column ACE column C18 150 mmx4.6 mm I.D; particle size 5 µm)) was used. The output of signal was monitored and integrated using waters Empower 2 software. Buffer preparation 1.0 ml of orthophosphoric acid was mixed with 500 ml of Milli-Q water, and shaken for 15 min and degassed. Filter the solution through 0.45µm membrane filter. Mobile phase preparation Prepare a filtered and degassed mixture of Buffer and Methanol in the ratio of 600:400 v/v. Filter the solution through 0.45µm membrane filter. Diluent preparation Mobile Phase is used as diluent. Standard preparation: 500 mg of Metformin HCl and 50 mg of Sitagliptin was accurately weighed and transferred into a 100 ml volumetric flask; 70 ml of diluent was added and sonicated. Volume was made with diluent and mixed well. 5 ml of the above stock solution was diluted to 25 ml with diluent to produce drug in the concentration of 1000 µg/ml of Metformin HCl and 100 µg/ml of Sitagliptin. Sample preparation: 10 tablets were weighed accurately and finely powdered. Tablet powder equivalent to 500 mg of Metformin HCl and 50 mg of Sitagliptin was weighed and transferred into a 100 ml volumetric flask and 70 ml of diluent was added. Sonicated for 30 minutes with intermittent shaking and cooled to room temperature. Volume was made with diluent and



mixed well.5 ml of the above sonicated solution was transferred into 25 ml volumetric flask. Volume was made with diluent and mixed well. Chromatographic conditions An ACE column C18 (150 mmx4.6 mm I.D; particle size 5µm) Column was used for analysis at 30°C column temperature. The mobile phase was pumped through the column at a flow rate of 1.0 mL/min. The sample injection volume was 10 µL. The photodiode array detector was set to a wavelength of 228nm for the detection and Chromatographic runtime was 10 minutes.

RESULTS AND DISCUSSION Method development To develop a suitable and robust LC method for the determination of Metformin HCl and Sitagliptin, different mobile phases were employed to achieve the best separation and resolution. The method development was started with Agilent Zorbax C-18, (150 × 3.4 mm, 5 µ) with the following mobile phase. Accurately transfer about Orthophosphoric acid buffer. Filter the solution through 0.45µm membrane filter. Prepare a filtered and degassed mixture of Buffer and Methanol in the ratio of 600:400 v/v respectively. It was observed that Tailing was not satisfactory for both Metformin hydrochloride and Sitagliptin and the retention time of Metformin hydrochloride and Sitagliptin were found to be 3.0 and 4.5 minutes respectively. For next trial the Column was changed. An ACE column C18 (150 mmx4.6 mm I.D; particle size 5µm) Column was used. The mobile phase composition was Buffer and Methanol in the ratio of 600:400 v/v. above trail the Tailing was satisfactory. The retention time of Metformin hydrochloride is 3.00 minutes and Sitagliptin is about 4.50 (Fig.2) and the peak shape for these two was good. The chromatogram of Metformin hydrochloride and Sitagliptin standard using the proposed method is shown in (Fig.2) System suitability results of the method are presented in Table-1. A typical chromatogram for simultaneous estimation of Metformin hydrochloride and Sitagliptin obtained by using the aforementioned mobile phase from 10 µL of the assay preparation is illustrated in Fig.2.

Fig.2: A typical HPLC Chromatogram showing the peak of Metformin hydrochloride and Sitagliptin Method validation The developed RP-HPLC method extensively validated for assay of Metformin hydrochloride and Sitagliptin using the following Parameters. Specificity Blank and Placebo interference A study to establish the interference of blank and placebo were conducted. Diluent and placebo was injected into the chromatograph in the defined above chromatographic conditions and the blank and placebo chromatograms were recorded. Chromatogram of Blank solution (Fig.3) showed no peaks at the retention time of Metformin hydrochloride and Sitagliptin peak. This indicates that the diluent solution used in sample preparation do not interfere in estimation of Metformin hydrochloride and Sitagliptin in tablets. Similarly Chromatogram of Placebo solution (Fig.4) showed no peaks at the retention time of Metformin hydrochloride and Sitagliptin peak. This



indicates that the Placebo used in sample preparation do not interfere in estimation of Metformin hydrochloride and Sitagliptin in Metformin hydrochloride and Sitagliptin tablets. The chromatogram of Metformin hydrochloride and Sitagliptin Blank using the proposed method is shown in Fig.3.

Fig. 3: A typical HPLC Chromatogram showing the no interference of diluent for Metformin hydrochloride and Sitagliptin The chromatogram of Metformin hydrochloride and Sitagliptin Placebo using the proposed method is shown in Fig.4.

Fig.4: A typical HPLC Chromatogram showing the no interference of placebo for Metformin hydrochloride and Sitagliptin

Table 1: System suitability parameters for Metformin hydrochloride and Sitagliptin by proposed method

Name of the Compound Retention Time Theoretical plate Tailing factor USP Resolution Metformin hydrochloride 3.096 11677 1.193 - Sitagliptin 4.558 8485 1.296 9.156

Precision In the study of the instrumental system precision where, a RSD of 0.05% was obtained for the standard area of Metformin hydrochloride and 0.06 for Sitagliptin obtained corresponding to the first day, Similarly being 0.06% for Metformin hydrochloride and 0.07% for Sitagliptin for the second day, respectively. The method precision study for six sample preparations in marketed samples showed a RSD of 0.5% and the 95% confidence interval of 0.5 with the assay range of 97-103 for Metformin hydrochloride. Similarly the method precision study for six sample preparations in marketed samples showed a RSD of 0.7% and the 95% confidence interval of 0.7 with the assay range of 97-103 for Sitagliptin.



For the intermediate precision, a study carried out by the same analyst working on different day. The results calculated as inter-day RSD corresponded to 0.1 % (For Standard of Metformin hydrochloride) and 0.12% (For Standard of Sitagliptin). Both results together with the individual results are showing that the proposed analytical technique has a good intermediate precision.

Table 2: Method Precision studies for Metformin hydrochloride and Sitagliptin by proposed method

S.No Sample Weight Sample Area-1 Sample Area-2 % Assay % Assay 1 709.00 7598155 6639604 100 100 2 709.00 7592155 6635941 100 100 3 709.00 7597921 6636540 100 100 4 709.00 7597248 6631039 100 100 5 709.00 7599568 6638302 100 100 6 709.00 7591258 6630047 100 100

Avarage Assay: 100 100 STD 0.05 0.06

%RSD 0.05 0.06

The accuracy of the method was determined on three concentration levels by recovery experiments. The recovery studies were carried out in triplicate preparations on composite blend collected from 20 tablets of Metformin hydrochloride and Sitagliptin, analyzed as per the proposed method. The percentage recoveries with found in the range of 99.7 to 103 with an overall %RSD of 0.11 for Metformin hydrochloride and The percentage recoveries with found in the range of 101.0 with an overall %RSD of 0.05 for Sitagliptin. From the data obtained which given in Table-3 and Table-4 the method was found to be accurate.

Table 3: Recovery studies for Metformin hydrochloride by proposed method

% Level Recovery Range % RSD at each level Over all %RSD

50 101 0.06 0.11 100 101 0.02

150 101 0.25

Table 4: Recovery studies for Sitagliptin by proposed method

% Level Recovery Range % RSD at each level Over all %RSD 50 101 0.08

0.05 100 101 0.06 150 101 0.02

Linearity of detector response The standard curve was obtained in the concentration range of 500-1500µg/ml for Metformin hydrochloride and 50-150µg/mL for Sitagliptin. The linearity of this method was evaluated by linear regression analysis. Slope, intercept and correlation coefficient [r2] of standard curve were calculated and given in Fig.5 for Metformin hydrochloride and Fig.6 For Sitagliptin to demonstrate the linearity of the proposed method. From the data obtained which given in Table-5 For Metformin hydrochloride and Table-6 For Sitagliptin the method was found to be linear within the proposed range.

Table 5: Linearity studies for Metformin hydrochloride by proposed method

METFORMIN HCL CONC% Area ug/ml

50 3799300 500 75 5697363 750.00 100 7590726 1000.00 125 9492497 1250 150 11377545 1500



Fig.5: Calibration curve for Metformin hydrochloride

Table 6: Linearity studies for Sitagliptin by proposed method

SITAGLIPTIN % level Area Concentration (ug/ml)

50 3312342 50 75 4973473 75 100 6636842 100 125 8296822 125 150 9953590 150.00

Fig. 6: Calibration curve for Sitagliptin

CONCLUSION An RP-HPLC method for simultaneous estimation of Metformin hydrochloride and Sitagliptin was developed and validated as per ICH guidelines. The results obtained indicate that the proposed method is rapid, accurate, selective, and reproducible. Linearity was observed over a concentration range of 500-1500µg/ml for Metformin hydrochloride and 50-150µg/mL for Sitagliptin. The method has been successfully applied for the analysis of marketed tablets. It can be used for the routine analysis of formulations containing any one of the above drugs or their combinations without any alteration in the assay. The main advantage of the method is the common chromatographic conditions adopted for all formulations. Therefore, the proposed method reduces the time required for switch over of chromatographic conditions, equilibration of column and post column flushing that are typically associated when different formulations and their individual drug substances are analyzed. We have developed a fast,



simple and reliable analytical method for determination of Sitagliptin in pharmaceutical preparation using RP-HPLC. As there is no interference of blank and placebo at the retention time of Sitagliptin. Validation of this method was accomplished, getting results meeting all requirements. This method is simple, very fast, reproducible, with a good accuracy and precision. It allows reliably the analysis of Metformin hydrochloride and Sitagliptin in bulk, its different pharmaceutical dosage forms. Acknowledgment Authors thank full to Sri Siddhardha Pharmacy College, Nuzvid, for providing Instruments and analytical support.

REFERENCES [1] The Merck index, N.J., USA: Merck & Co Inc, 2006, P.773. [2]Bolen S, Feldman L, Vassy J, Ann Intern Med. 2007, Vol 147(6), P. 386-99. PMID 17638715. [3] Kirpichnikov D, McFarlane SI, Sowers JR, Ann Intern Med, 2002, Vol 137(1), P. 25–33. [4]Somogyi A, Stockley C, Keal J, Rolan P, Bochner F, Br J Clin Pharmacol, 1987, Vol 23(5): P.545–51. [5] Bristol-Myers Squibb, U Label Information, 2008. [6] Kim YD, Park KG, Lee YS, 2008; Vol 57(2), P.306–14. [7] Wesley UV, McGroarty M, Homoyouni A, Cancer. Res, 2005, Vol 65 (4): P.1325-1334. [8] Sonia K, Prasad babu K, , IJPSR. 2013; Vol 4(4), P.1469-1474. [9] Kurra Neelesh, Int J Pharm Ind Res, 2013, Vol 3(2), P.114–120. [10]Sachin L Patil, Jayant R Bhinge, Chetan M Bhalgat, Int J Pharm Biomed Sci, Vol 2(4), P.244-248. [11] Sadaf momin, Vishakha kulkarni, Kishor butte, Sudha rathod, Int J Adv Pharm Res, 2013, Vol 4(5), P. 1796 – 1802. [12]Shailaja B, Jadhav, Swati K, Kupkar et al, Int J Pharm Ed Res.2013, Vol 47(1), P.13-16. [13]Sujani PV, Phanindra SS, Anasuya P, Padamanabha Reddy Y, Devanna N, J Pharm Sci. 2013, Vol 2(2), P.143-148. [14]Anil dubala, Rizwanbasha khatwal, Jayasankar kosaraju, Venkat meda, Malay K. Samantal, J Pharm Sci. 2012; Vol 4(2): P. 691-694. [15] Lakshmana Rao A, Raja T, Int J Pharm Chem, 2012, Vol 2(4), P.696-702.

[16]Loni AB, 3rd

International Conference and Exhibition on Analytical & Bioanalytical Techniques, 2012, Vol 3(7), P.77-82. [17] Malleswararao CS, MV Suryanarayana, Sci pharm, 2012, Vol 80 (1), P.139-52.

Research Article

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR THE ESTIMATION OF PANTOPRAZOLE

K.PUSHPA LATHA* AND D.RAMACHANDRAN

Acharya Nagarjuna University, Nagarjuna Nagar 522510, Guntur dist, Andhrapradesh, India. Email: [email protected]

Received: 09 March 2013, Revised and Accepted: 25 March 2013

ABSTRACT

A simple, selective, linear, precise and accurate RP-HPLC method was developed and validated for rapid assay of Pantaprazole in pharmaceutical dosage form. Isocratic elution at a flow rate of 1.0 mL min -1 was employed on a Symmetry C18 column at ambient temperature. The mobile phase consisted of acetonitrile: phosphate buffer 60:40 (v/v) and the detection wavelength were at 234 nm. Linearity was observed in concentration range of 50-175 μg/mL. The retention time for Pantaprazole was 2.28 min. The method was validated as per the ICH guidelines. The proposed method can be successfully applied for the estimation of Pantaprazole in pharmaceutical dosage forms.

Keywords: Estimation, Method development, Pantaprazole, RP-HPLC, Validation.

INTRODUCTION

The proton-pump inhibitor pantoprazole inhibit gastric acid by blocking the H+/K+-adenosine triphosphatase enzyme system (the proton pump) of the gastric parietal cell [1]. It is used for short-term treatment of erosion and ulceration of the esophagus[2]. The pantoprazole oral dosage forms are supplied in enteric-coated tablets.Different analytical methods are reported in the literature for the assay of pantoprazole in dosage forms and in biological fluids including spectrophotometry[3-8], TLC9, HPTLC[10-12]. Pantoprazole sodium is chemically Sodium 5-(difluoromethoxy)-2-[[(3,4-dimethoxy-2- pyridinyl) methyl] sulfinyl]-1H-benzimidazole sesquihydrate. It has an empirical formula of C16H15F2N3O4S and molecular weight of 383.37. The aim of this work was to develop new and validated, simple and reproducible RP-HPLC method allowing the estimation of in dosage forms and human plasma samples.

Instrumental and analytical conditions

The HPLC analysis was carried out on Waters HPLC system (2695 module) equipped with 2487 dual lambda detector with auto Sampler and running on Waters Empower software. The column used is Symmetry C18 (150 × 4.6 mm, packed with 5 μm) and detection was performed at 234 nm. The injection volume of sample was 20 μL and the run time was 6 minutes. An isocratic mobile phase containing acetonitrile and 0.02 M phosphate buffer at 60: 40 (v/v) at the pH 3.5 was carried with the flow rate at 1.0mL min-1. The mobile phase was filtered through 0.45μm membrane filter and degassed before use.

Reagents and chemicals

Pantaprazole working standard was obtained from Merck (India). Tablets were purchased from local pharmacy manufactured by Novartis. Ultra pure water was obtained from a millipore system. HPLC grade acetonitrile was obtained from Merck (India) limited. All other chemicals used were AR grade.

Preparation of mobile phase

Dissolved 2.72 g of Potassium Di hydrogen orthophosphate in 1000 mL of water and mixed, pH adjusted to 3.5 using ortho phosphoric acid, sonicated to degas the buffer. Transferred 600 volumes of acetonitrile and 400 volumes of buffer into a 1000 volumes mobile phase bottle and mixed. Then sonicated up to 15 minutes for degas the mobile phase and filtered through 0.45 μm filter under vacuum. The same mobile phase was used as diluent.

Preparation of Standard Solution

Accurately weighed about 10 mg of Pantaprazole and transferred into a 10mL volumetric flask and 7 mL of diluent was

added and sonicate to dissolve it completely and the volume was adjusted with the mobile phase to get stock solution of 1000 μg/mL. Then 1 mL of stock solution is transferred into 10 ml volumetric flask and make up to volume with mobile phase and filter through 0.45 μm filters, which gives a solution of strength 100 μg/mL.

Preparation of sample solution

Weigh 20 Pantaprazole tablets and calculate the average weight. Accurately weigh and transfer the sample equivalent to 50 mg of Pantaprazole into a 50 ml volumetric flask. Add about 25ml of diluent, sonicate to dissolve it completely and make volume up to the mark with diluent. Mix well and filter through 0.45 μm filter. Further pipette 1 ml of the above stock solution into a 10 ml volumetric flask and dilute up to the mark with diluent. Mix well and filter through 0.45 μm filter.

Method Validation

The objective of the method validation is to demonstrate that the method is suitable for its intended purpose as it is stated in ICH guidelines. The method was validated for linearity, precision, accuracy, specificity, limit of detection, limit of quantification, robustness and system suitability.

Linearity

From the standard stock solution, the various dilutions of Pantaprazole in the concentration of 50, 75, 100, 125, 150 and 175μg/mL were prepared. The solutions were injected using 20 μL injection volumes in to the chromatographic system at the flow rate of 1.0 mLmin-1 and the effluents were monitored at 234 nm, chromatograms were recorded. Calibration curve of Pantaprazole was obtained by plotting the peak area ratio versus the applied concentrations of Pantaprazole, given in table 1. The linear correlation coefficient was found to be 1, shown in figure2.

Precision

Repeatability of the method was checked by injecting replicate injections of 100 μg/mL of the solution for six times on the same day as intraday precision study of Pantaprazole and the % RSD was found to be 0.15, given in table 2.

Accuracy

Pantaprazole reference standards were accurately weighed and added to a mixture of the tablets excipients, at three different concentration levels (50%, 100% and 150%). At each level, samples were prepared in triplicate and the recovery percentage was determined and presented in table 3.

International Journal of Current Pharmaceutical Research

ISSN- 0975-7066 Vol 5, Issue 2, 2013

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Latha et al. Int J Curr Pharm Res, Vol 5, Issue 2, 119-121

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Specificity

Spectral purities of Pantaprazole chromatographic peaks were evaluated for the interference of the tablet excipients as per the methodology. In the work, a solution containing a mixture of the tablet excipients was prepared using the sample preparation procedure to evaluate possible interfering peaks and no interference peaks were observed.

Robustness

To determine the robustness of the method, two parameters (flow rate, composition of mobile phase) from the optimized chromatographic conditions were varied. Statistical analysis showed no significant difference between results obtained employing the analytical conditions established for the method and those obtained in the experiments in which variations of parameters were introduced. Thus the method showed to be robust which is shown in table 4.

Ruggedness

Inter day variations were performed by using six replicate injections of standard and sample solutions of concentrations which were prepared and analyzed by different analyst on three different days over a period of one week. Ruggedness also expressed in terms of percentage relative standard deviation and statistical analysis showed no significant difference between results obtained employing different analyst.

Detection and quantitation limits

According to the determined signal-to-noise ratio, Pantaprazole presented limits of detection of 0.06μg/mL and limits of quantitation of 0.8μg/mL, where the compounds proportion found in the sample solutions injected on to the chromatograph. However, the objective of the method is the quantitation of Pantaprazole so that the values obtained should be considered as the limit of method sensitivity.

System Suitability

System suitability tests were carried out on freshly prepared standard stock solutions of Pantaprazole and it was calculated by determining the standard deviation by injecting standards in six replicates at 6 minutes interval and the values were recorded and the system suitability parameters are shown in table 5.

RESULTS AND DISCUSSION

The nature of the sample, its molecular weight and solubility decides the proper selection of the stationary phase. The drug Pantaprazole was preferably analyzed by reverse phase chromatography and accordingly C18 column was selected. The elution of the compound from the column was influenced by polar mobile phase. The ratio of the acetonitrile to phosphate buffer was optimized to give symmetric peak with short run time. Different mobile phases were tried but satisfactory separation, well resolved and good symmetrical peaks were obtained with the mobile phase of acetonitrile: phosphate buffer at the ratio of 60:40 (v/v). The

retention time of Pantaprazole was found to be 2.28 min, which indicates a good base line. The RSD values for accuracy and precision studies obtained were less than 2% which revealed that developed method was accurate and precise. The system suitability parameters are given in Table 5. A typical chromatogram showing the separation of Pantaprazole is shown in figure 3.

Table 1: Linearity of Pantaprazole

Concentration (μg/mL) Average area 50 641426 75 969169 100 1286672 125 1607741 150 1932957 175 2257921

Fig. 1: Structure of Pantaprazole

Fig. 2: Linearity curve of Pantaprazole

Table 2: Precision of Pantaprazole

Injections Area 1 1278827 2 1280763 3 1283837 4 1280931 5 1283027 6 1279532 Mean 1281153 SD 1947.051 %RSD 0.1517

Table 3: Accuracy of Pantaprazole

% Conc Amount added (mg) Amount found (mg) % Recovery Mean Recovery 50% 5.0 4.96 99.2% 99.17% 100% 10.0 9.96 99.6% 150% 15.5 14.8 98.7%

Table 4: Robustness of Pantaprazole

Parameters Adjusted to Average Area Rt SD % RSD Flow rate as per method 1.0mL/min 0.8 mL/min 1295159 2.291 5918.5 0.45

1.0mL/min 1289713 2.289 4993.7 0.39 1.2ml/min 1297130 2.284 3887.0 0.30

Mobile phase composition Acetonitrile: Buffer (60:40) Acetonitrile: Buffer (55:45) 1297717 2.279 3475.6 0.28 Acetonitrile: Buffer (60:40) 1294644 2.282 4987.4 0.38 Acetonitrile: Buffer (65:35) 1303208 2.284 5215.3 0.40

Latha et al. Int J Curr Pharm Res, Vol 5, Issue 2, 119-121

121

Table 5: System Suitability for Pantaprazole

Concentration Injection Area Rt 100 μg/mL Injection-1 1287115 2.285

Injection-2 1283496 2.287 Injection-3 1290171 2.288 Injection-4 1289663 2.286 Injection-5 1289921 2.289 Injection-6 1290228 2.288

Statistical Analysis

Mean 1288432 2.287167 SD 2686.291 0.001472 %RSD 0.21 0.06 Tailing Factor 1.6 Plate Count 2496.6

Fig. 3: Standard Chromatogram of Pantaprazole

CONCLUSION

A validated RP-HPLC method has been developed for the determination of Pantaprazole in tablet dosage form. The proposed method is simple, rapid, accurate, precise and specific. Therefore, it is suitable for the routine analysis of Pantaprazole in pharmaceutical dosage form.

ACKNOWLEDGEMENTS

The authors are thankful to emanthi laboratories limited, Hyderabad for providing gift sample of Pantaprazole and for providing necessary facilities to carry out the research work.

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