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Stability indicating RP-HPLC Method for Simultaneous Determination of L- Methyl folate and Escitalopram in Bulk and Pharmaceutical Formulation

Bhairam Bhoomaiah*1, Anireddy Jayasree2  and Danavena Rambabu3

1Centre for Chemical Sciences and Technology, Institute of Science and Technology ,Jawaharlal Nehru Technological University Hyderabad, India.

2Centre for Chemical Sciences and Technology, Institute of Science and Technology ,Jawaharlal Nehru Technological University Hyderabad, India.

3Hetero drugs Limited, Hyderabad, Telangana., India.

Corresponding Author E-mail: bhoomesh.analyst@gmail.com

DOI : http://dx.doi.org/10.13005/ojc/320643

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ABSTRACT:

Stabiliy indicating Reverse phase -HPLC Methodbeen described for the simultaneous estimation of L-Methyl folate and Escitalopram in combined tablet formulation form. Chromatographic separation was carried out by using reversed phase HPLC and the method was achieved on a ODS column with UV detection. The mobile phase was optimized with Acetonitrile : 0.01% H3PO4in water 35:65 (%V/V) Flow rate of 1.0ml/min and The wavelength was selected at 212nm. The drug was stressed by acidic, alkaline, oxidative, thermal and photolytic conditions and the degradation samples were analyzed by the proposed method. Degradation studies showed that all the two drugs were degraded under oxidative, acidic, alkaline and thermal conditions, Minor degradation observed under photolytic and hydrolysis condition. Analytical Method validation parameters such as specificity, linearity, accuracy, precision, Ruggedness and Robustness were determined and System suitability of all the parameters was passed. Hence this method was stability indicating method, It can be used for the routine and Stability analysis of  L-Methyl folate and Escitalopram in pharmaceutical dosage forms.

KEYWORDS:

RP-HPLC; L-Methyl folate; Escitalopram

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Bhoomaiah B, Jayasree A, Rambabu D. Stability indicating RP-HPLC Method for Simultaneous Determination of L- Methyl folate and Escitalopram in Bulk and Pharmaceutical Formulation. Orient J Chem 2016;32(6).


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Bhoomaiah B, Jayasree A, Rambabu D. Stability indicating RP-HPLC Method for Simultaneous Determination of L- Methyl folate and Escitalopram in Bulk and Pharmaceutical Formulation. Orient J Chem 2016;32(6). Available from: http://www.orientjchem.org/?p=25839


Introduction

Levomefolicacid(L-Methyl folate)

Levomefolic acid (fig- 1) was primary biologically active form of folic acid used at the cellular level for DNA reproduction. A-vitamin (B9) essential to human health and function. One of its most notable functions is its role in creating key neurotransmitters or brain chemicals that regulate human mood, cognitive ability and arousal. The three primary brain chemicals are dopamine, norepinephrine and serotonin. Chemically, these brain chemicals are referred to as monoamines. Abnormal amounts (either too much or too little) of these chemicals can cause various forms of mental illness and disease including depression, schizophrenia and attention deficit disorder.

Our body does not create its own supply of folic acid (also known as folate), it must be acquired from the diet by eating foods or taking supplements containing this b-vitamin. Your body then takes the dietary folic acid and uses the enzyme MTHFR (methylenetetrahydrofolatereductase) to transform it into levomefolic acid [1].  Your body can then use it to carry out a specific range of reactions and function. Levomefolic acid is used to produce the neurotransmitter nor epinephrineLevomefolic acid (and folic acid in turn) has been proposed for treatment of cardiovascular disease[2][3] and advanced cancers such as breast and colorectal cancers[citation needed] A lack (deficiency) of folate in the human body can be caused by certain diseases, by taking certain medications, or by not getting enough folate in your diet. Folate deficiency can lead to decreased red blood cells, or anemia. Folate deficiency can also cause high levels of a certain amino acid in the blood, a condition called hyperhomocysteinemia (HYE-per-HOE-moe-sis-tin-EE-mee-a).L-methylfolate is not an antidepressant or anti-psychotic medication. However, l-methylfolate may enhance the effects of antidepressant medications.  Literature survey reveals that there were less number of analytical methods for  L-methylfolate or in combination with other drugs including spectroscopic and chromatographic methods are reported.

Figure 1: Structure of Levomefolic acid

Figure 1: Structure of Levomefolic acid


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Figure 2: Structure of Escitalopram

Figure 2: Structure of Escitalopram



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Escitalopram

Escitalopram (trade names Lexapro, Cipralex) (Fig 2) is an antidepressant of the selective serotonin reuptake inhibitor (SSRI) class. It is approved by the U.S. Food and Drug Administration (FDA) for the treatment of major depressive disorder and generalized anxiety disorder in adults; other indications include social anxiety disorder, panic disorder and obsessive-compulsive disorder. Escitalopram is the S-stereoisomer (enantiomer) of the earlier Lundbeck drug citalopram (Celexa), hence the name escitalopram. Escitalopram is noted for its high selectivity of serotonin reuptake inhibition and, as a result has fewer side effects not related to its serotonergic activity. According to a meta-analysis of 12 new-generation antidepressants, escitalopram and sertraline (Zoloft) are the best in terms of efficacy and acceptability in the acute-phase treatment of adults with major depression; however, sertraline may be a better choice because of the lower cost.escitalopram exhibits superior therapeutic properties to citalopram or merely represents an example of “ever greening” is controversial

The utility of antidepressant drugs in the treatment of mild-to-moderate depression is itself controversial. In those with very severe depression there is a large benefit[4]. The most recent Reviews concluded (with caveats in some cases) that escitalopram is modestly superior to citalopram in efficacy and/or tolerability[5]. Literature survey reveals that there were many analytical methods for escitalopram or in combination with other drugs including spectroscopic and chromatographic methods are reported Simultaneous  estimation [6]  of  escitalopram  and  single  estimation[7]  of  escitalopram has  been  done  previously  in  UV  and  also  in  Colourometry[8]

Simultaneous  determination  of  escitalopram oxalate and clonazepam in combined tablets by HPTLC[9] and RP‐HPL has also been performed.but there is no method established  for the stability indicating RP-HPLC under stress for this combination.The present work describes the development of stability indicating RP-HPLC method, which can quantify these components simultaneously from a combined dosage formThe present RP-HPLC method was validated [10][11] and applied under stressed conditions according to International conference on harmonization (ICH) guidelineThere stability indicating assay methods helps in establishing the inherent stability of the drug which provides assurance on detection changes in identity, purity and potency of the product on exposure to various conditions.In the present study, Levomefolic acid, Escitalopram is exposed to a variety of stress like acidic, basic, thermal, photolytic and oxidative stress conditions[12]. According to ICH guidelines the stress testing of the drug substances helps in identifying the likely degradation products.The aim of the present work was to develop stability indicating method for determination of all the two drugs in presence of its degradation products.

Materials and methods

Chemicals and reagents

L-Methyl folate andEscitalopram standard was obtained from reputed companies,. HPLC grade Methanol, Water, Ortho phosphoric acid and Acetonitrile were purchased from Merck Pvt limited, Mumbai.  0.45μm nylon membrane filter papers were purchased from Pall Life Sciences, Mumbai. A combined dosage tablet lefodap plus was purchased from Med plus Pharmacy.

Instrumentation

In the present study Performed with Waters 2998 PDA detector and Waters Empower2 software was used Shimadzu double beam UV-Visible spectrophotometer was used for spectral analysis and the data was generated and recorded by UV probe software. Sonicator (1.5L) Ultrasonicator was used to sonicating the mobile phase and samples. Standard and sample drugs were and weighed by using Denver electronic analytical balance andsartorious analytical balanace (SI-234).

Determination of maximum absorbance

L-Methyl folate and Escitalopram standard solution was scanned in the range of 200-400 nm against mobile phase as blank. L-Methyl folate and Escitalopram shows maximum absorbance at 212nm.The wave length selected for the determination of L-Methyl folat and Escitalopram is 212nm.

Diluent

Water: Methanol (50:50)

Preparation of standard stock solution

6mg of L- Methyl folate and 8mg Escitalopramweighed in to 10ml volumetric flasks separately. Add 3/4ml of diluents, sonicated to up to dissolve the  drugs and make up to the mark with diluents, finally 600μg/ml of L- Methyl folate and 800μg/ml of Escitalopram Standard stock solutions of concentration were prepared using diluent. Fromtake 1ml of  standard stock solution, in to a 10ml Volumetric flask and and added dilent mixed well dilueted to get 60μg/ml of L- Methyl folate and 80μg/ml of Escitalopram with diluent.

Preparation of Tablet formulation

Taken weight of 20 tablets of lefodapplus and calculate the average weight then crush the tablets and  weighted the equivalent to 1 tablet taken into a 25ml volumetric flask, added the 10ml of diluents and sonicated for 30 min, further diluetedup volume  with diluent and filtered through 0.45µ filter .Then taken 2ml of above filter solution into a 10 ml volumetric flask and made upto volume with diluent. The finally sample solution concentrations of 60μg /ml of L-Methyl folate and 80µg/ml of Escitalopram was obtained.

Chromatographic conditions

The present study explains the development and validation for the estimation of L-Methyl folate and Escitalopramin Bulk and tablet forms using the most commonly employed ODS 250mm x 4.6 mm, 5m.column with UV detection. The mobile phase was optimized with Acetonitrile : 0.01% H3PO4in water 35:65 (%V/V). The wavelength was detected and selected at 212nm, Iso-absorptive point for both the drugs. Good resolution was carried out at 212nm and both drugs showed good absorbance at this wavelength with minimum interference of the other drug. Analysis was carried out at  column temperature 30°C temperature. Compounds were separated with a mobile phase consisting of Acetonitrile : 0.01% H3PO4in water 35:65 (%V/V). Flow rate of 1.0 ml/min and detection of wavelength at 212nm. The mobile phase was filtered by using 0.45µm membrane filter paper and solicited and Degassed in Ultrasonicator for 10min.Optimized chromatographic conditions were shown in table-1 Standard and blank and formulation chromatograms were shown in figure no.3, 4 and 5. All parameters of method was validated as per the USFD and ICH guidelines.

Table 1: Optimized chromatographic conditions for L-Methyl folate and Escitalopram

S.NO

Parameter

Results

1

Mobile Phase

Acetonitrile : 0.01% H3PO4in water 35:65 (%V/V

2

Wavelength

212nm

3

column

ODS 250mm x 4.6 mm, 5m.

4

Injection volume

10mL

5

Flow Rate

1.0ml/min

6

Pump Mode

Isocratic

7

Run time

10 min

Figure 3: Blank chromatogram of L-Methyl folate and Escitalopram

Figure 3: Blank chromatogram of L-Methyl folate and Escitalopram



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Figure 4: Standard chromatogram of L-Methyl folate and Escitalopram Figure 4: Standard chromatogram of L-Methyl folate and Escitalopram 


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Figure 5: Formulation chromatogram of L-Methyl folate and Escitalopram

Figure 5: Formulation chromatogram of L-Methyl folate and Escitalopram



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Analytical Validation of the method

Assay Analytical method Validation of L-Methyl folate and Escitalopram by HPLC was carried out with respect to the following parameters.

System suitability

System suitability test was performed before each validation parameter.system suitability result shown in table-2. In the system suitability test L-Methyl folate and Escitalopram peaks were separated with good resolution, such as tailing factors, theoretical plates, and repeatability against the specifications set for the method. The tailing factor was within ≤ 2, theoretical plate number of ≥ 2000, which satisfied the acceptance criteria.

Table 2: System suitability Results  forL-Methylfolate and Escitalopram

S.NO

Parameter

Results

1

Api Concentration

L- Methyl folate– 60 µg/ml

Escitalopram – 80 µg/ml

2

RT

L- Methyl folate – 2.280min

Escitalopram – 5.697 min

3

Resolution

L- Methyl folate – ……..

Escitalopram -10.81

4

Area

L- Methyl folate – 517514

Escitalopram – 730351

5

Theoretical Plates

L- Methyl folate – 1161

Escitalopram – 4374

6

Tailing Factor

L- Methyl folate – 1.32

Escitalopram -1.41

 

Linearity and range

Linearity of L-Methyl folate and Escitalopram detector response of assay method was found by injecting seven standard solutions with concentration ranging from 15µg/ml to 90µg/ml for L-Methyl folate and 20µg/ml to 120µg/ml for Escitalopram of the test concentration and a graph was plotted for concentration versus peak area. Good linear relation was observed within the concentrations and the study. Regression equation was found to be y= 8862.x + 204.1 (r2=1.000) for L-Methyl folate and y= 9273.x + 1391 (r2=0.999) for Escitalopram

Y=slope, m=intercept, c=correlation coefficient. The results were shown in Table-3,Table-4, fig-6 and fig -7

Table 3: Results for linearity of L-Methyl folate

No

Concentration(µg/ml)

Peak area

1

0

0

2

15

132701

3

30

264945

4

45

400021

5

60

534130

6

75

665262

7

90

795920

Table 4: Results for linearity of Escitalopram

S.No

Concentration

(µg/ml)

Peak area

1

0

0

2

20

183208

3

40

372861

4

60

562544

5

80

745938

6

100

933617

7

120

1106405

 

Figure 6: Linearity of L-Methyl folate

Figure 6: Linearity of L-Methyl folate



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Figure 7: Linearity of Escitalopram

Figure 7: Linearity of Escitalopram



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Precision

Repeatability of results called as Precision. In this parameter injecting 6 eplicate injections of the solution 60μg/ml L-Methyl folate and 80μg/ml of Escitalopramrespectively.The%RSD was below 2.0% there found to be 0.87% and 0.79. Variability of the method was Checkedby analyzing the solution on the same day (intra-day precision) and on other different day (inter- day precision). The results were obtained for intra-day precision  %RSDs were below 2.0%  they were  0.87 % and 0.79 % respectively. The inter-day precisions %RSDs were 0.66% and 0.36 %, respectively it is also below 2.0%. The results were shown in table-5.

Table 5: Precision Results for of L-Methyl folate and Escitalopram

S.No

L-Methyl folate

Escitalopram

L-Methyl folate

Escitalopram

 

Intraday precision

Interday precision

1

519055

730022

510325

726226

2

513781

724488

509391

722452

3

513863

725274

506107

722991

4

516973

720769

502404

719327

5

506829

734154

511588

725318

6

518129

735207

509145

721058

RSD

0.87

0.79

0.66

0.36

 

Accuracy

Accuracy of the method was Studiedby internal standard addition method. A known concentration amount of standard drug was added to the fixed amount of pre-analyzed tablet solution. Percent of recovery was calculated by comparing the area before and after the addition of the standard drug. As per ICH Guidelines Accuracy parameter performed at least 3 levels. The Recovery of  method was performed at 50%, 100%and 150% levels of standard concentration for both L-Methyl folate and Escitalopram drugs. We were prepared above given 3 levels solutions in triplicate and injected as per the proposed method. % recovery was calculated  for each levela found to be within the acceptance criteria of 98-102% for both drugs as per guidelines. This Resultsshowed that the recoveries of L-Methyl folate and Escitalopram by the proposed methods are satisfactory. %RSD accepted within the limit of 2.0%. The results are shown in table-6 and table-7

Table 6: Results for Accuracy ofL-Methyl folate

S.NO

%Recovery

Concentration in µg/ml

Amount Found

% recovery

%RSD

Target

Spiked

Total

1

50%

60

30

90

30.08

100.27

0.38

2

60

30

90

29.90

99.68

3

60

30

90

30.12

100.39

4

100%

60

60

120

59.71

99.52

0.94

5

60

60

120

60.17

100.29

6

60

60

120

59.06

98.43

7

150%

60

90

150

89.88

99.87

1.02

8

60

90

150

90.93

101.03

9

60

90

150

89.09

98.99

 

Table 7: Results for Accuracy of Escitalopram 

S.NO

%Recovery

Concentration in µg/ml

Amount Found

% recovery

%RSD

Target

Spiked

Total

1

50%

80

40

120

39.40

98.50

1.49

2

80

40

120

40.55

101.38

3

80

40

120

39.71

99.28

4

100%

80

80

160

79.70

99.63

1.14

5

80

80

160

79.16

98.96

6

80

80

160

80.94

101.18

7

150%

80

120

200

119.22

99.35

0.90

8

80

120

200

120.17

100.14

9

80

120

200

118.03

98.36

 

Ruggedness

Ruggedness was performed by the analyst to analyst variability, % RSD) was calculated. The %RSD was below 2.0%, Cumulative %RSD both analysts also found below 2.0%. The results are shown in table-5and table-8.

Table 8: Result for Ruggedness of L-Methyl folate and Escitalopram

S.NO

L-Methyl folate

Escitalopram

1

510325

726226

2

509391

722452

3

506107

722991

4

502404

719327

5

511588

725318

6

509145

721058

RSD

0.66

0.36

 

Robustness

To perform the robustness of the method, Experimental Chromatographic conditions such as the  Flow rate ,temperature  and composition of the mobile phase.The small changes weretested The results are obtained small variations which do not  effected on the results It indicates that the Analytical method was  robust. The results were shown on the Table-9

Table 9: Results for robustness of the L-Methyl folate and Escitalopram

S.NO

Condition

Change

L-Methyl folate

Escitalopram

Mean Area

%RSD

Mean Area

%RSD

1

Standard

……..

514471

0.9

730082

0.8

2

MP 1

40:60(%V/V)

513547

1.1

734666

1.1

3

MP 2

30:70(%V/V)

502632

0.9

725802

1.4

4

Flow-1

0.9 mL/min

589325

1.1

834351

1.4

5

Flow-2

1.1 mL/min

471802

0.9

675146

1.5

6

Oven Temp-1

25°C

496975

1.2

709669

1.2

7

Oven Temp-2

35°C

499556

0.7

713144

1.2

 

Limit of Detection and Quantification Limit

Determination of the Limit of Detection and Limit of Quantification was performed by standard deviation method.Standard with low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected. Limit of Detection is generally considered acceptable 3.3*σ/slop for estimating the detection limit. The quantification limit 10.0*σ/slop is generally determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be quantified with acceptable accuracy and precision. The results are shown on the table-10

Table 10: results for LOD and LOQ

Parameter

L-Methyl folate (µg/ml)

Escitalopram (µg/ml)

LOD

0.06

0.26

LOQ

0.18

0.79

 

Formulation

We were prepared assay sample solution injected into the HPLC. The assay results were calculated against standard peak areas.the calculated % assay was found to be 99.41% for L-Methyl folate and 99.91 % for Escitalopram. Hence the method can use for the simultaneous estimation of L-Methyl folate and Escitalopram in pharmaceutical formulation and stability sample analysis. Results of the formulation analysis were shown in table -11.

Table 11: result for formulation of L-Methyl folate and Escitalopram

S.NO

Drug

Brand

Dosage

Amount Prepared

Amount Found

%Assay

1

L-Methyl folate

Lefodep Plus

7.5mg

60

59.65

99.41

2

Escitalopram

10.0mg

80

79.93

99.91

 

Solution stability

The standard and samplessolutions  ofL-Methyl folate and Escitalopram does not change their concentration up to 24hrs. So standard and samples solution ofL-Methyl folate and Escitalopram Stable up to  24 hours. The results were shown in table-12.

Table 12: Results for stability

Time in hrs

PeakArea

L-Methyl folate

Escitalopram

0

502700

717606

24

508145

722895

 

Force Degradation Studies

Forced degradation study undertaken to demonstrate the specificity of method, when developing the stability- indicating methods, particularly when little information is available about potential degradationof  products. These studies also provide information about the degradation pathways and degradation products that could form during storage period in different conditions . Forced degradation studies may help facilitate pharmaceutical development as well in areas such as formulation development manufacturing and packaging in which knowledge of chemical behavior can be used improve a drug product

Oxidative Stress

To 1 ml of sample stock solution of L-Methyl folate and Escitalopram and 1 ml of 20% hydrogen peroxide (H2O2) was added separately. The solutions were kept for 2hours at 60ºc. For HPLC Analysis , TheResultant solution was diluted to Required concentration  60µg/ml of L-Methyl folate& 80µg/ml of Escitalopram solution and  injected into the HPLC system  Recorded the chromatograms to assess the stability of sample

Acid Degradation Studies

To 1 ml of stock s solution L-Methyl folate and Escitalopram, 1ml of 2N Hydrochloric acid was added and refluxed for 30min at 60ºc. TheResultant solution was diluted to Required concentration 60µg/ml of L-Methyl folate& 80µg/ml of Escitalopram solution and injected into the HPLC system.Recorded the chromatograms to assess the stability of sample

Base Degradation Studies

To 1 ml of stock solution L-Methyl folate and Escitalopram, 1ml of 2N sodium hydroxide was added and refluxed for 30mins at  60ºc. TheResultant solution was diluted to Required concentration 60µg/ml of L-Methyl folate& 80µg/ml of Escitalopramsolution andinjected into the HPLC system.Recorded the chromatograms to assess the stability of sample

Thermal Degradation Studies

The thermal degradation carried out by sample solution was kept in oven at 105ºcfor 6 hours. For HPLC analysis, TheResultant solution was diluted to Required concentration  60µg/ml of L-Methyl folate& 80µg/ml of Escitalopram solution and  injected into the HPLC system Recorded the chromatograms to assess the stability of sample.

Photolytic degradation studies

The photochemical degradation was also studied by exposing the tablet powder to UV Light by kept the beaker cointaining tablet powder in UV Chamber for 7days or 200 Watt hours/min in photo stability chamber. For HPLC Analysis, TheResultant solution was diluted to Required concentration  60µg/ml of L-Methyl folate& 80µg/ml of Escitalopram solution and  injected into the HPLC system.Recorded the chromatograms to assess the stability of sample.

Hydrolysis Degradation Studies

Hydrolysis Degradation Study carried out by refluxing the drug in water for 6hrs at a temperature of 60ºC for HPLC analysis, TheResultant solution was diluted to Required concentration  60µg/ml of L-Methyl folate& 80µg/ml of Escitalopram solution and  injected into the HPLC system   recorded the chromatograms to assess the stability of sample. The Degradation results shown in table-13

Table 13: Result for Degrdation of L-Methyl folate and Escitalopram

Degradation

Assay of

L-Methyl folate

Assay of Escitalopram

water

98.89

99.00

UV

98.66

97.43

Thermal

96.09

96.08

Acid

94.08

94.09

Base

93.14

93.35

Peroxide

91.97

91.97

 

Results and Discussion

The present experiment was aimed to develop and validated stability indicating RP- HPLC method forsimultaneous  estimation ofL-Methyl folate and Escitalopram.for selection of mobile phase ,After several Experiment trails with different mobile phase compositions, a Acetonitrile : 0.01% H3PO4in water 35:65 (%V/V) was found to be desired composition. This ratio was optimized for the simultaneous estimation of all the two drugs.

The analytical data obtained from the linearity parameter ,linear regression analysis showed a linear relationship in peak areas and concentration in the range of 15µg/ml to 90µg/ml for L-Methyl folate and 20µg/ml to 120µg/ml for Escitalopram of the test concentration. The method was  precise, repeatable results were obtained.The %RSD values for intra-day and inter-day precision less than 2.0%. The developed method was accurate recovery obtained at each levelas per guide lines

The limit of detection ofL-Methyl folate and Escitalopram was found to be 0.06μg/mL and 0.26μg/mL ,limit of quantification was  0.18μg/mLand0.79μg/mL .While studying  the robustness, the system suitability results of all the two drugs were within acceptance criteria,

The established method was robust  system suitability parameter passed  affected after varying the parameters like flow rate and Temparature. The assay of commercial tablets was established with present chromatographic condition developed and it was found to be more accurate and reliable. The percentage label claim present in tablet formulationwas found to be 99.41, 99.91 for L-Methyl folate and Escitalopramrespectively.

The drugs were exposed to acidic, base, oxidative, thermal and photolytic conditions and the stressed samples were analyzed by the proposed method. Degradation studies showed that all the two drugs were degraded under, thermal, Acid, Base and peroxide conditions. The drug peak areas decreased sufficiently with drastic change in the Rt values.

Thus the developed RP-HPLC Stability inicating method was found to be simple, rapid, sensitive, accurate, precise and specific for the simultaneous estimation of two drugs in bulk and pharmaceutical dosage forms and for routine analysis in stability studies of these drugs.

Conclusion

The Stability indicating RP-HPLC method was developed and validated  simultaneousestmation and quantitative determination of L-Methyl folate and Escitalopramfrom in  its formulation. All the validation parameters were found to be within the limits according to the ICH guidelines. The proposed method was found to be specific for the drugs of interest irrespective of the excipients present and the method was found to be suitable for the routine and stability analysis of the marketed formulation

Acknowledgements

The author, B. Bhoomaiah, expresses his sincere thanks toProf. A. Jayasree, Director of CCST, JNTU Hyderabad, for her excellence guidance and encouragement throughout the project. The author also expresses his deep sense of gratitude to AurobindoPharma Pvt Ltd, Bachually Hyderabad for giving an opportunity to work in that organization and providing facilities in the laboratory.

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