ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF HYDROCORTISONE AND MICONAZOLE SIMULTANIOUS IN TOPICAL DOSAGE FORM BY RP HPLC

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ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF

HYDROCORTISONE AND MICONAZOLE SIMULTANIOUS IN

TOPICAL DOSAGE FORM BY RP-HPLC

A. Yadagiri Naga Manikanta*

[1]

Department of Pharmaceutical Sciences, Acharya Nagarjuna University, Nagarjuna Nagar,

Guntur, Andhra Pradesh- 522 510.

[2]

M.A.M. College Of Pharmacy, Kesanupalli, Narsaraopet, Guntur, A.P.

ABSTRACT

A simple reverse phase high perfomance liquid chromatographic

method has been developed and subsequently validated for

simultaneous determination of Hydrocortisone and miconazole in

combined dosage form. The separation was carried out using a mobile

phase consisting of methanol and mono basic potassium phosphate

buffer in the ratio of 80:20 v/v. The column used was Inertsil –ODS

C18 (250 × 4.6 mm, 5μ) with flow rate of1.0ml/min using PDA detection at 239nm. The described method was linear over a

concentration range of 20ppm to 80ppm for the assay of

Hydrocortisone and miconazole respectively. The retention times of

Hydrocortisone and miconazole were found to be 3.942min and

2.869min respectively. Results of analysis were validated statistically

and by recovery studies. The limit of quantification (LOQ) for

Hydrocortisone and miconazole were found to be 1.69mg/ml and

1.74mg/ml respectively. Then the limit of detection (LOD) for Hydrocortisone and

miconazole were found to be 0.56 mg/ml and 0.57 mg/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 Hydrocortisone and miconazole bulk drug

and in its pharmaceutical dosage form.

KEYWORDS: Hydrocortisone, miconazole, methanol.

Volume 4, Issue 8, 2063-2080. Research Article ISSN 2277– 7105

Article Received on 05 June 2015,

Revised on 29 June 2015, Accepted on 22 July 2015

*Correspondence for Author

A . Yadagiri Naga Manikanta

Department of

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INTRODUCTION

Hydrocortisone/miconazole isa combination drug, often consisting of

1% hydrocortisone (a classI topical steroid) with 2% miconazole (a broad

spectrum antifungal). This combination drug is sold as Daktacort .[1] Hydrocortisone binds to the cytosolic glucocorticoid receptor. After binding the receptor the newly formed

receptor-ligand complex translocates itself into the cell nucleus, where it binds to many glucocorticoid

response elements (GRE) in the promoter region of the target genes. The DNA bound

receptor then interacts with basic transcription factors, causing the increase in expression of

specific target genes. The anti-inflammatory actions of corticosteroids are thought to involve

lipocortins, phospholipase A2 inhibitory proteins which, through inhibition arachidonic acid,

control the biosynthesis of prostaglandins and leukotrienes.[2] Miconazole is an anti-fungal

medication related to fluconazole (Diflucan), ketoconazole (Nizoral), itraconazole

(Sporanox), and clotrimazole (Lotrimin, Mycelex). It is used either on the skin or in the

vagina for fungal infections. Miconazole was approved by the FDA in 1974. Miconazole

prevents fungal organisms from producing vital substances required for growth and function.

This medication is effective only for infections caused by fungal organisms. It will not work

for bacterial or viral infections.[3]

Miconazole Hydrocortisone

MATERIALS AND METHOD Chemicals and solvents

Hydrocortisone, miconazole as gift samples from Mylan Laboratories Limited, Hyderabad,

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hydrocortisone, miconazole-2%W/W (manufactured by Johnson & johnson) were procured

from local pharmacy. Potassium dihydrogen phosphate – AR grade (SD.Fine chem. Ltd,

mumdai), Acetonitrile-HPLC grade (Merck India), Methanol – HPLC grade (Merck India).

INSTRUMENTATION

The chromatographic separations were performed using HPLC-Waters alliance

(Model-2690/5) consisting of an in-built auto sampler, a column oven and Waters 996 PDA detector.

The data was acquired through Empower-2-software. The column used was Inertsil ODS

(250×4.6 mm, 5μ). Meltronics sonicator was used for enhancing dissolution of the

compounds. Elico pH meter was used for adjusting the pH of buffer solution. All weighing

was done on Sartorious balance (model AE-160).

Chromatographic conditions

The mobile phase consists ofMethanol : phosphate buffer in the ratio of 80:20 v/v. The

mobile phase was pumped from solvent reservoir in the ratio of 80:20 v/v to the column in

the flow rate of 1.0 ml/min whereas run time set was 10 min. The separation was performed

on Inertsil ODS-3V 250mm x 4.6mm, 5μm column and the column was maintained the

temperature ambient and the volume of each injection was 20µl. Prior to injection, the

column was equilibrated for at least 30 min with mobile phase flowing through the system.

The eluents were monitored at 239 nm.

OPTIMISED METHOD Mobile Phase

Degassed Methanol and Buffer in the ratio of 80:20 V/V.

Preparation of (KH2PO4 0.1M) buffer

Weight 3.8954g of di-sodium hydrogen phosphate and 3.4023 of potassium dihydrogen

phosphate in to a beaker containing 1000ml of distilled water and dissolve completely. Then

ph is adjusted with orthophosphoric acid and then filtered through 0.45µm membrane filter.

Preparation of stock solution

Reference solution: The solution was prepared by dissolving 20.0 mg of accurately weighed

Miconazole Nitrate and 25.0 mg Hydrocortisone in Mobile phase, in two 100.0 mL

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from each solution into a 50.0 mL volumetric flask and then makeup with mobile phase and

sonicate for 10min.

Preparation of working standard solution

The stock solutions equivalent to 20ppm to 80ppm with respect to both drugs were prepared

in combination of Miconazole Nitrate and Hydrocortisone above, sonicated and filtered

through 0.45µ membrane

Optimized chromatographic conditions

M

ic

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a

z

o

le

N

it

ra

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2

.8

6

9

H

y

d

ro

c

o

rt

is

o

n

e

3

.9

4

2

AU

0.00 0.02 0.04 0.06 0.08

Minutes

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Inference

Got chromatogram at RT’s of 2.869min toMiconazole Nitrate and 3.942min to

Hydrocortisone.

Parameters Method

Stationary phase (column) Inertsil -ODS C18(250 x 4.6 mm, 5 µ)

Mobile Phase Methanol and Buffer in the ratio of _{80:20 V/V.}

Flow rate (ml/min) 1.0 ml/min

Run time (minutes) 10 min

Column temperature (°C) Ambient

Volume of injection loop (l) 20

Detection wavelength (nm) 239nm

Drug RT (min) 2.869min for Miconazole Nitrateand

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METHOD VALIDATION

The developed method was validated as per the ICH (International Conference on

Harmonization) guidelines with respect to System suitability, Specificity,

Linearity,presission, Accuracy, Limit of detection and Limit of quantification.

1.SYSTEM SUITABILITY

A Standard solution was prepared by using Miconazole Nitrate and Hydrocortisone working

standards as per test method and was injected Five times into the HPLC system.

The system suitability parameters were evaluated from standard chromatograms by

calculating the % RSD from five replicate injections for Miconazole Nitrate and

Hydrocortisone, retention times and peak areas.

ACCEPTANCE CRITERIA

1. The % RSD for the retention times of principal peak from 5 replicate injections of each

Standard solution should be not more than 2.0 %

2. The % RSD for the peak area responses of principal peak from 5 replicate injections of

each standard Solution should be not more than 2.0%.

3. The number of theoretical plates (N) for the Miconazole Nitrate and Hydrocortisone peaks

is NLT 3000.

4. The Tailing factor (T) for the Miconazole Nitrate and Hydrocortisone

peaks is NMT 2.0

TABLE- 1: Data of System Suitability for Miconazole Nitrate and Hydrocortisone

Miconazole Hydrocortisone

Injection RT Peak area

USP plate count

USP

tailling RT

Peak area

USP platecount

USP trailling

1 2.869 674753 10953.609752 1.153539 3.942 1218805 9478.317159 0.899633

2 2.867 674261 10951.014286 1.155271 3.942 1214014 9452.196217 0.893423

3 2.871 675298 10003.278630 1.157740 3.944 1215474 9569.928335 0.894443

4 2.868 679221 10986.906427 1.159499 3.940 1227655 9619.633847 0.882222

5 2.872 688636 10846.878423 1.152820 3.943 1267019 9749.907462 0.892316

MEAN 2.870

241

678433

.8 10768.34 1.155774

3.943

212 1228593 9573.997 0.892407

SD 0.001

817

6031.1 35

0.007 07

122124. 007

%RSD 0.052

21

0.8889 79

0.025 353

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M ic o n a z o le N it ra te 2 .8 6 9 H y d ro c o rt is o n e 3 .9 4 2 AU 0.00 0.02 0.04 0.06 0.08 Minutes

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Fig: 1-5 Chromatograms of system suitability (standards 1-5)

Inference: System suitability Chromatogram for standard – 1

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

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1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Inference: System suitability Chromatogram for standard - 4 M ic o n a z o le N it ra te 2 .8 7 2 H y d ro c o rt is o n e 3 .9 4 3 AU 0.00 0.02 0.04 0.06 0.08 Minutes

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

OBSERVATION

The %RSD for retention times and peak areas were found to be within the limit. Refer table:

8 As shown in fig 1-5.

2.SPECIFICITY

Miconazole Nitrate and Hydrocortisone

Solutions of standard and sample were prepared as per the test method are injected into

chromatographic system.

ACCEPTENCE CRITERIA

Chromatograms of standard and sample should be identical with near Retention time.

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1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

OBSERVATION

The chromatograms of Standard and Sample were same identical with same retention time.

As shown in fig: Got a peak for standard at an Rt of 2.872min for Miconazole Nitrate and

3.943min for Hydrocortisone.

3.LINEARITY OF TEST METHOD

A Series of solutions are prepared using Miconazole Nitrate and Hydrocortisone working

standards at concentration levels from 20ppm to 80 ppm of target concentration .Measure the

peak area response of solution at Level 1 and Level 6 six times and Level 2 to Level 5 two

times.

Correlation Coefficient should be not less than 0.9990.

% of y- Intercept should be ±2.0.

[image:8.595.80.540.72.238.2]

% of RSD for level 1 and Level 6 should be not more than 2.0%.

TABLE 2: Data of Linearity (Miconazole Nitrate) and (Hydrocortisone) Concentration

(ppm)

Average Area

Statistical Analysis of miconazole

Average Area

Statistical analysis of hydrocortisone

0 0 Slope 18600 0 slope 5140

20 632546 y-Intercept 276.2 1202965 y-intersept 114.7

30 658296 Correlation

Coefficient 1 1254371

Correlation

Coefficient 1

40 694400 1295856

50 730308 1297167

60 916282 1308577

70 9402046 1359903

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Fig: 7(a) Linearity Plot (Concentration Vs Response) of Miconazole Nitrat.

Fig: 7(b) Linearity Plot (Concentration Vs Response) of Hydrocortisone

OBSERVATION

The linear fit of the system was illustrated graphically. The results are presented in table2.

4. PRECISION 4.1Repeatability

a. System precision: Standard solution prepared as per test method and injected five times.

b. Method precision: Prepared six sample preparations individually using single as per test method and injected each solution.

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The individual assays of Miconazole Nitrate and Hydrocortisone should be not less than 98%

and not more than 102.0%.

OBSERVATION

Test results are showing that the test method is precise. Refer tables 3and 4 for system

precision and for method precision.

Table 3: Data of Repeatability (System precision) for Miconazole Nitrate and hydrocortisone

Concentration 40ppm

injection Peak area of

miconazole. %Assay

Peak area of

hydrocortisone %Assay

1 674753 98.66 1218805 99.95

3 675298 101.53 1215474 100.06

4 679221 100.53 1227655 99.30

5 688636 99.98 1267019 100.00

Mean 678433.8 100.00 1228593 99.91

Statistical Analysis

SD 6031.135 1.107678 22124.07 0.35819

%RSD 0.888979 1.10 1.800764 0.35

b)Method precision

Injection

Peaks areas of Miconazole

%Assay Peaks areas of

hydrocortisone %Assay

1 633495 98.55 1202110 98.6

2 635992 98.88 1203700 99.02

3 639828 99.40 1201851 98.12

4 639098 99.30 1202255 98.31

5 648289 100.53 1203283 98.81

6 631322 98.28 1202349 98.36

Statistical Analysis

Mean 637312 99.278 1202687.6 98.48

SD 5988.879 0.827236 771.5483 0.352647

%RSD 0.0891 0.83 0.1358 0.35

4.2Intermediate precision (analyst to analyst variability)

A study was conducted by two analysts as per test method.

ACCEPTENCE CRITERIA

The individual assays of Miconazole Nitrate and Hydrocortisone should be not less than 98%

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Table 5: Data of Intermediate precision (Analyst 2) for Miconazole Nitrate and hydrocortisone

Injection Peak Areas of

miconazole %Assay

Peak areas of

hydrocortisone %Assay

1 636792 99.99 1205267 99.78

2 634360 99.66 1205625 99.95

3 655696 101.53 1205840 100

4 644147 99.98 1202735 98.55

5 644127 99.97 1208991 101.50

6 652525 101.10 1208543 101.37

Statistical Analysis

Mean 644607.8 100.37 1206333.5 100.19

SD 6392.59 0.753536 12572.599 1.100898

% RSD 1.183 0.75 1.24 1.09

OBSERVATION

Individual %assays and %RSD of Assay are within limit and passes the intermediate

precision, Refer table: 5

5. ACCURACY (RECOVERY)

A study of Accuracy was conducted. Drug Assay was performed in triplicate as per test

method with equivalent amount of Miconazole Nitrate and Hydrocortisone into each

volumetric flask for each spike level to get the concentration of Miconazole Nitrate and

Hydrocortisone equivalent to 50%, 100%, and 150% of the labeled amount as per the test

method. The average % recovery of Miconazole Nitrate and Hydrocortisone were calculated.

The mean % recovery of the Miconazole Nitrate and Hydrocortisone at each spike level

should be not less than 98.0% and not more than 102.0% for both the drugs separately.

OBSERVATION

Amount found

%Recovery = --- × 100

Amount added

The recovery results indicating that the test method has an acceptable level of accuracy.

[image:11.595.43.556.102.285.2]

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TABLE-6: (i)Data of Accuracy for Miconazole Nitrate Concentration

% of spiked level

Amount added (ppm)

Amount found (ppm)

% Recovery

Statistical Analysis of % Recovery

50%

Injection 1 20 20.04 100.22 MEAN 100.06

50%

Injection 2 20 19.97 99.85

50%

Injection 3 20 20.02 100.11 %RSD 0.18

100 %

Injection 1 40 40.01 100.02 MEAN 100.04

100 %

Injection 2 40 40.05 100.14

100%

Injection 3 40 39.98 99.96 %RSD 0.091

150%

Injection 1 60 60.08 100.14 MEAN 100.02

150%

Injection 2 60 59.97 99.96

150%

Injection 3 60 59.98 99.98 %RSD 0.09

(ii)Data of Accuracy for Hydrocortisone Concentration

% of spiked level

Amount added (ppm)

Amount found (ppm)

% Recovery

Statistical Analysis of % Recovery

50%

Injection 1 20 20.15 100.75 MEAN 99.69333

50%

Injection 2 20 19.86 99.31

50%

Injection 3 20 19.80 99.02 %RSD 0.92

100 %

Injection 1 40 39.88 99.70 MEAN 99.83333

100 %

Injection 2 40 40.12 100.30

100%

Injection 3 40 39.80 99.50 %RSD 0.41

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Injection 1

150%

Injection 2 60 59.76 99.61

150%

Injection 3 60 60.06 100.10 %RSD 0.31

6. RUGGEDNESS OF TEST METHOD a) System to system variability

System to system variability study was conducted on different HPLC systems, under similar

conditions at different times. Six samples were prepared and each was analyzed as per test

method.

Comparison of both the results obtained on two different HPLC systems, shows that the assay

test method are rugged for System to system variability.

The % relative standard deviation of Miconazole Nitrate and Hydrocortisone from the six

sample preparations should be not more than 2.0%.

The % assay of Miconazole Nitrate and Hydrocortisone should be between 98.0%-102.0%.

OBSERVATION

The % RSD was found within the limit. Ref tables: 14 .

b) column to column variability

Column to column variability study was conducted by using different columns. Six samples

were prepared and each was analysed as per test method

The %RSD of Miconazole Nitrate and Hydrocortisone tablets should be NMT2.0%. The

%assay of Miconazole Nitrate and Hydrocortisone should be between 98.0% and 102.0% for

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[image:14.595.103.493.115.273.2]

Table 7:Data of system to system variability (Miconazole Nitrate And Hydrocortisone) System-2

OBSERVATION

The results obtained by comparing with both two types were within limit.

7. ROBUSTNESS

a) Effect of variation of flow rate

A study was conducted to determine the effect of variation in flow rate. Standard solution

prepared as per the test method was injected into the HPLC system using flow rates,

1.0ml/min and1.2ml/min. The system suitability parameters were evaluated and found to be

within the limits for 1.0ml/min and 1.2ml/min flow.

Miconazole Nitrate and Hydrocortisone and was resolved from all other peaks and the

retention times were comparable with those obtained for mobile phase having flow rates

1.0ml/min.

The Tailing Factor of Miconazole Nitrate and Hydrocortisone standards should be NMT 2.0

for Variation in Flow.

TABLE: 8(i) Data for Effect of variation in flow rate (Miconazole Nitrate):

Flow 0.8 ml

Std Area

Tailing factor

Flow 1.0 ml

Std Area

Flow 1.2 ml

Std Area

620286 1.322089 634322 1.604878 602077 1.285372

619282 1.331920 635792 1.584354 601854 1.319385

621337 1.296438 634360 1.543805 602403 1.292055

620456 1.315454 635696 1.568590 603421 1.304561

620765 1.326551 633147 1.559986 602465 1.294621

S.NO: Peak area

Assay % of Miconazole

Nitrate Peak area

Assay % of hydrocortisone

1 634360 98.65 1203625 99.98

2 634098 98.63 1202225 99.30

3 635696 98.86 1202840 98.60

4 633289 98.52 1204283 99.30

5 634147 98.63 1202735 98.55

6 633495 98.55 1203110 98.73

Mean 634180.8 98.64 1203136.3 99.07667

[image:14.595.68.534.654.760.2]

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Avg 620425 1.31849 Avg 634663.4 1.572323 Avg 602444 1.299199

SD 754.0018 0.013728 SD 1100.917 0.023367 SD 599.8833 0.013223

%RSD 0.086 1.04 %RSD 0.184 1.48 %RSD 0.09 1.01

TABLE: 8(ii) Data for Effect of variation in flow rate (Hydrocortisone)

Flow 0.8 ml

Std Area Tailing factor

Flow 1.0 ml

Flow 1.2 ml

1273707 1.362089 1206349 1.280574 1266195 1.285372

1273211 1.352617 1205267 1.279932 1265885 1.299385

1273948 1.376926 1205625 1.261721 1266303 1.308063

1273465 1.345752 1205840 1.276089 1267243 1.274662

1273862 1.374925 1205735 1.250640 1265762 1.267630

Avg 1273638.6 1.362462 Avg 1205763.2 1.269791 Avg 166277.6 1.287022

SD 3301.369 0.013609 SD 392.1635 0.01314 SD 582.9758 0.016786

%RSD 1.041 0.99 %RSD 0.19 1.03 %RSD 0.35 1.3

OBSERVATION

The tailing factor for Miconazole Nitrate and Hydrocortisone was found to be within the

limits. As shown in table 8.

8. LIMIT OF DETECTION AND QUANTITATION (LOD and LOQ) Miconazole Nitrate

From the linearity plot the LOD and LOQ are calculated:

LOD = 3.3 σ

S

3.3×3244.904

= --- = 0.57

18600

LOQ = 10 σ

S

10×3244.904

= --- = 1.74

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Hydrocortisone

From the linearity plot the LOD and LOQ are calculated:

LOD = 3.3 σ

S

3.3×867.0705

= --- = 0.56

5140

LOQ = 10 σ

S

10×867.0705

= --- = 1.69

5140

SOMMARY AND CONCLUSSION

The analytical method was developed by studying different parameters. First of all, maximum

absorbance was found to be at 272nm Miconazole Nitratefor and 248nm for Hydrocortisone.

Common wavelength will be 239nm and the peaks purity was excellent. Injection volume

was selected to be 20µl which gave a good peak area. The column used for study was Inertsil

C18, ODS chosen good peak shape. Ambient temperature was found to be suitable for the

nature of drug solution. The flow rate was fixed at 1.0ml/min because of good peak area,

satisfactory retention time and good resolution. Different ratios of mobile phase were studied,

mobile phase with ratio of Methanol and Buffer in the ratio of 80:20 V/V was fixed due to

good symmetrical peaks and for good resolution. So this mobile phase was used for the

proposed study.

The present recovery was found to be 98.0-101.50 was linear and precise over the same

range. Both system and method precision was found to be accurate and well within range.

Detection limit was found to be 0.57 Miconazole Nitrateand 0.56 for Hydrocortisone.

Linearity study was, correlation coefficient and curve fitting was found to be. The analytical

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the drugs. The analytical passed both robustness and ruggedness tests. On both cases, relative

standard deviation was well satisfactory.

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