A new stability indicating RP-HPLC method for the estimation of Flucytosine in presence of an internal standard

 

Paladugu Venkata Naveen*, Seru Ganapaty

Department of Pharmaceutical Analysis & Quality Assurance

GITAM Institute of Pharmacy, Gandhi Institute of Technology and Management (Deemed to be University),

Visakhapatnam, Andhra pradesh-530045, India

*Corresponding Author E-mail: paladuguvenkatanaveen@gmail.com

 

ABSTRACT:

Flucytosine is fungicidal acting against Candida species but not against other common fungi. Flucytosine is structurally related to the cytostatic fluorouracil and to floxuridine. Flucytosine is chemically 6-amino-5-fluoro-1H-pyrimidin-2-one with molecular formula C4H4FN3O and molecular weight 129.09 g/mol. A new RP-HPLC method has been proposed for the quantification of Flucytosine in presence of internal standard Linezolid in pharmaceutical formulations and the method was validated as per ICH guidelines. Mobile phase consisting of a mixture of Tetra butyl ammonium hydrogen sulphate: Methanol (50:50, v/v) with a flow rate 1 mL/min and UV detection at 254 nm was used for the assay of Flucytosine in presence of internal standard Linezolid. Flucytosine was exposed to different stress conditions such as acidic, alkaline, oxidation and thermal degradation. Linearity was observed over the concentration range 1.0100 μg/mL with linear regression equation y = 0.0238x - 0.0024 (R = 0.9998). The LOQ was found to be 0.8937 μg/mL and the LOD was found to be 0.2938 μg/mL. The present method can be successfully applied for the pharmaceutical formulations, kinetics study and bioanalytical studies. 

 

KEYWORDS: Flucytosine, Linezolid, RP-HPLC, Stability indicating, Validation, ICH guidelines.

 

 


INTRODUCTION:

Flucytosine (Figure 1A) is the only available nucleoside analog, acts as an antifungal by disrupting pyrimidine metabolism in the fungal cell nucleus. Flucytosine (FLU) is a small, very water-soluble molecule and therefore it is rapidly and nearly completely absorbed from the intestine after oral administration. Flucytosine can cause bone marrow suppression and GI toxicity. Flucytosine does not have any significant drug interactions. Flucytosine acts mainly against Candida and Cryptococcus and also against some of the species Cladosporium and Phialophora1-3. Various analytical techniques have been developed till now for the determination of Flucytosine which include fluorimetry4, microbiological assay5, UFLC6, process related impurities7 RP-HPLC8, liquid chromatography in biological fluids9-11 and in injectable formulations. In the present study the authors have proposed a new stability indicating RP-HPLC method for the determination of Flucytosine in presence of internal standard (IS) Linezolid and the optimized method was validated12. Linezolid (Figure 1B) belongs to oxazolidinone class and it is a synthetic antibiotic used for the treatment of multi-resistant bacterial infections.

 

 

Figure 1A: Structure of Flucytosine (FLU)

 

 

Figure 1B: Structure of Linezolid (LIN)

 

 

 

MATERIALS AND METHODS:

Chromatographic conditions:

Shimadzu Model HPLC system (Shimadzu Co., Kyoto, Japan) equipped with PDA detector and C8 Luna column (250 mm 4.60 mm i. d. 5m particle size) was used for the chromatographic study. The system was maintained at 23C. Mobile phase consisting of Tetra butyl ammonium hydrogen sulphate: Methanol (50:50, v/v) with a flow rate 1 mL/min (UV detection at 254 nm) was used for the determination of Flucytosine for the present study. The mobile phase was sonicated and filtered through 0.45m membrane filter prior to use.

 

Materials and reagents:

Flucytosine was obtained from Zydus Cadila Healthcare Ltd. as gift sample. Flucytosine is available as tablets and capsules. Flucytosine is available with brand names BD-Cytosin-500, FC fungTM 500 (Glenmark) and CytoFlu (Jolly Healthcare) as tablets and capsules (Labelled claim: 500 mg).

 

Preparation of Flucytosine and Linezolid drug solutions

25 mg of each Flucytosine and Linezolid were accurately weighed and transferred in to two different clean 25 mL volumetric flasks and made up to volume with HPLC grade methanol (MERCK) (1000 μg/mL). Dilutions of Flucytosine were made on daily basis with mobile phase consisting of Linezolid (10 μg/mL) sonicated and filtered through 0.45μm membrane filter prior to injection.

 

Method validation

Linearity

Dilute solutions (1.0-100 μg/mL) of Flucytosine containing 10 μg/mL Linezolid were prepared from their stock solutions with mobile phase and 20μL of each were injected in to the HPLC system. The mean peak area ratio of FLU to that of Linezolid was calculated from the chromatograms obtained and a calibration curve was drawn by taking the concentration of FLU drug solution on the x-axis and the corresponding mean peak area ratio values on the y-axis.

 

Precision, Accuracy and Robustness

Intraday and inter-day precision were studied, three different concentrations of Flucytosine were chosen to study the Intraday (same day) and Inter-day precision (three consecutive days) respectively and the % RSD was calculated. The accuracy study was evaluated in triplicate at three concentration levels (50, 100 and 150 %), and the percentage recoveries were calculated. Standard addition and recovery experiments were conducted to determine the accuracy of the method for the quantification of the drug product and the percentage recovery was calculated. The robustness of the method was assessed by exposing the drug solution to different analytical conditions purposely changing from the original optimized conditions. The effects so obtained were summarized to calculate the % RSD and has to be less than 2.0% specifying that the proposed method was robust.

 

Stress degradation studies13

Flucytosine was exposed to stress conditions such as acidic, basic, oxidation, thermal treatment. Acidic degradation was performed by treating the drug solution with 1mL of 0.1N HCl, heated at 75C for about one hour on a water bath. The stressed sample is then cooled neutralized with 1mL 0.1N sodium hydroxide solution and the solution was made up to volume to the required concentration with the mobile phase only after the addition of internal standard just before injection into the system. 20μl of the solution was injected in to the HPLC system.

 

Alkaline degradation, was performed by treating the drug solution with 1mL 0.1 N NaOH heated at 75C for about one hour on a water bath. The solution is then cooled and neutralized with 1mL 0.1N hydrochloric acid and diluted with the mobile phase only after the addition of internal standard just before injection into the system. 20 l of the solution was injected in to the HPLC system.

 

Oxidation degradation was performed by treating the drug solution with 1ml of 30% v/v H2O2 heated at 75 C for about one hour on a water bath. The solution is then cooled and diluted with mobile phase only after the addition of internal standard just before injection into the system. 20μl of the solution was injected in to the HPLC system.

 

Thermal degradation was performed by heating the drug solution at 75C for about one hour on a water bath. The solution is then cooled and diluted with mobile phase only after the addition of internal standard just before injection into the system. 20μl of the solution was injected in to the HPLC system.

 

Assay of Flucytosine tablets

Twenty tablets containing each 500 mg were purchased from the local pharmacy store and the contents were powdered and powder equivalent to 50 mg Flucytosine was extracted using methanol and then with the mobile phase in a 50 ml volumetric flask. The solution was sonicated for half an hour and filtered through 0.45 mm membrane filter and 20 L of this solution was injected in to the HPLC system only after the addition of internal standard. The peak areas observed by Flucytosine and Linezolid were noted at their retention time from the resultant chromatogram and the mean peak area ratio was calculated (n=3). The values were substituted in the linear regression equation obtained earlier and the amount of Flucytosine present in the tablets were calculated.

 

RESULTS AND DISCUSSION:

Flucytosine was estimated using a new stability indicating reverse phase liquid chromatographic method using an internal standard, Linezolid and the method was validated. During the optimization of Flucytosine, trials were made simultaneously for Linezolid also and then a simple method was established where both the drugs were eluted with resolution greater than 2.0. Mobile phase consisting of a mixture of Tetra butyl ammonium hydrogen sulphate: Methanol (50: 50) (pH 3.5) (50:50, v/v) with a flow rate 1 mL/min and UV detection at 254 nm was used for the assay of Flucytosine using C8 Luna column (250 mm 4.60 mm i.d. 5m particle size). A symmetrical and sharp peak for Flucytosine was observed at 2.857 0.02 min and that of the internal standard, Linezolid at 7.0 0.1 min within a run time of 10 min. The proposed method was compared with the previously reported liquid chromatographic methods (Table 1). The chromatograms obtained for the mobile phase, Flucytosine (API), Flucytosine (API) in presence of internal standard Linezolid and that of the tablet formulation in presence of IS were shown in Figure 2.

 

 


 

Table 1: Literature survey

Method/Mobile phase (v/v)

Detection wavelength (nm)

Comment

Reference

 

Tetra butyl ammonium hydrogen sulphate: Acetonitrile (50: 50)

215

RP-UFLC

Stability indicating

6

Water: Methanol (95:5)

260

HPLC

Process related impurities

7

(Sodium dihydrogen phosphate : 1-octane Sulfonic acid) buffer: Methanol: Acetonitrile (50:20:30)

264

RP-HPLC

 

8

Tetra butyl ammonium hydrogen sulphate: Methanol (50: 50) (pH 3.5)

254

RP-HPLC

Stability indicating

Internal standard (Linezolid)

Present method

 


Method validation

Linearity

Flucytosine obeys Beer-Lamberts law and follows linearity over the concentration range 1.0100 μg/mL (Table 3) (Figure-2) (% RSD 0.16-1.45) and the linear regression equation was found to be y = 0.0238x - 0.0024 (r2 = 0.9998) (Figure 2). The LOD was found to be 0.2523 μg/mL and the LOQ was found to be 0.8291 μg/mL.

Table 3: Linearity

Conc. (g/mL)

*Mean peak area

Peak area ratio (FLU/LIN)

% RSD

FLU

LIN

FLU

LIN

1

10

39376

1665812

0.0236

0.85

5

10

196881

1672351

0.1177

1.23

10

10

393763

1678276

0.2346

1.45

20

10

787526

1663421

0.4734

0.66

30

10

1181289

1678325

0.7038

0.51

40

10

1575052

1651248

0.9538

0.77

60

10

2362578

1679235

1.4069

0.16

80

10

3150104

1658130

1.8997

0.73

100

10

3937630

1663815

2.3666

0.94

*mean of three replicates

 

Precision, Accuracy and Robustness

Intraday and inter-day precision were studied at three different concentration levels of Flucytosine on the same day and on three consecutive days respectively and the % RSD was found to be 0.14-0.29 (Intraday) (Table 4) and 0.24-0.97 (Inter day) (Table 5) respectively (<2.0) demonstrating that the method is precise. The accuracy of the method was proved by the standard addition method and the percentage RSD was <2.0 (Table 6). The robustness of the assay method was established by introducing small changes in the chromatographic conditions which include detection wavelength (252 and 256 nm), percentage of organic phase i.e. methanol in the mobile phase (48 and 52%) and flow rate ( 0.1 ml/min). Robustness of the method was studied using 10 μg/mL of Flucytosine (Table 7) and the % RSD was found to be 0.11-1.85 (<2.0).

 

 

 

Figure 2: Calibration of Flucytosine in presence of Linezolid (IS)

 


 

Table 4: Intraday precision study of Flucytosine in presence of Linezolid (IS)

Conc.(g/mL)

*Mean peak area

Statistical Analysis*Mean SD (% RSD)

FLU

LIN

FLU/LIN

10

393763

1679786

0.2344

0.2336 0.000653 (0.27)

 

10

392817

1687312

0.2328

10

393819

1663589

0.2367

20

787526

1663421

0.4734

0.4717 0.001388 (0.29)

20

788975

1678315

0.4700

20

786215

1661321

0.4732

30

1181289

1678325

0.7038

0.7050 0.001021 (0.14)

30

1193215

1689312

0.7063

30

1178516

1678171

0.7022

*mean of three replicates

 

Table 5: Interday precision study of Flucytosine in presence of Linezolid (IS)

Conc.

(g/mL)

*Mean peak area

Statistical Analysis *Mean SD (% RSD)

FLU

LIN

FLU/LIN

10

393714

1697321

0.2319

0.2347 0.002286 (0.97)

10

401290

1689375

0.2375

10

389789

1678614

0.2322

20

789527

1687854

0.4677

0.4691 0.001143 (0.24)

20

788346

1675431

0.4705

20

787978

1698121

0.4640

30

1198975

1691732

0.7087

0.7062 0.002 (0.28)

30

1189354

1689781

0.7038

30

1178631

1673879

0.7041

*mean of three replicates

 

 

 

 

Table 6: Accuracy study of Flucytosine

Spiked

Conc.((g/mL)

Formulation

( g/mL)

Total Conc.

( g/mL)

*Conc. found (g/mL)

SD (%RSD)

% Recovery

 

5 (50%)

10

10

10

15

15

15

 

14.93 0.0367 (0.24)

99.32

99.88

98.80

 

10 (100%)

10

10

10

20

20

20

 

19.95 0.0489 (0.24)

99.51

100.1

98.87

 

15 (150%)

10

10

10

25

25

25

 

25.05 0.0622 (0.24)

100.5

99.49

100.8

*mean of three replicates

 

Table 7: Robustness study of Flucytosine in presence of Linezolid (IS)

Parameter

Condition

*Mean peak area

*Mean peak area ratio

(FLU / LIN)

*Mean peak area ratio

SD (% RSD)

FLU

LIN

Flow rate

( 0.1 ml/min)

0.9

412109

1685249

0.2445

0.2440 0.0045

(1.85)

1.0

413763

1679985

0.2463

1.1

401539

1664596

0.2412

Detection wavelength

( 2 nm)

252

404984

1675341

0.2417

0.2382 0.0022

(0.92)

254

393763

1675229

0.2351

256

398685

1675297

0.2379

Mobile phase composition

(Aq : Org) ( 2 % v/v)

52:48

394872

1675309

0.2357

0.2359 0.0002

(0.11)

50:50

393763

1671364

0.2356

48:52

395961

1675317

0.2364

*Mean of three replicates

 


Stress degradation studies

The Flucytosine API was eluted as a sharp peak at 2.857 0.2 min. During the acidic degradation the drug was eluted at 3.059 min and in alkaline degradation Flucytosine was eluted at 3.015 min. During the oxidation along with the drug peak (3.009 min) one more degradant was eluted at 4.196 min and in thermal degradation the drug was eluted at 2.991 min. Flucytosine was totally very much resistant towards all degradation conditions and the degradents were well separated. In all the degradation studies less than 3% degradation was observed and therefore it is confirmed that the method is selective and specific. The system suitability parameters has shown that the tailing factor was (1.5-2.0) and the theoretical plates were more than 2000. (Table 8). The individual chromatograms obtained during the forced degradation studies were shown in Figure 3.

 


 

 

Table 8: Stress degradation studies of Flucytosine in presence of Linezolid (IS)

Stress condition

Medium/ Temp. / Time

%

Recovery

%Drug

degradation

Theoretical plates

Tailing factor

FLU

LIN

FLU

LIN

Standard drug

100

------

4321

5064

1.197

1.114

Acidic degradation

0.1N HCl/ 75C/ 1 hour

81.71

18.29

3631

3434

1.504

1.541

Alkaline degradation

0.1N NaOH/ 75C/ 1 hour

94.24

5.76

3040

3869

1.426

1.382

Oxidation

30%H2O2/ 75C/ 1 hour

81.16

18.84

3431

4240

1.458

1.635

Thermal degradation

Water/ 75C/ 1 hour

85.32

14.68

3049

3506

1.444

1.386

 


Assay of Flucytosine capsules

Assay was performed by using two brands of Flucytosine capsules consisting of 500 mg API and then it was found that the amount of Flucytosine was found to be 99.592-99.786 (Table 9) and there is no interference of excipients.

 

Table 9: Assay of Flucytosine tablets

Brand

Label claim

(mg)

Observed amount

(mg)

% Recovery*

I

500

498.93

99.786

II

500

497.96

99.592

*Mean of three replicates

 

CONCLUSIONS:

The RP-HPLC techniques were validated as per ICH guidelines and found to be simple, economical, simple and robust for the quantification of Flucytosine tablets.

 

ACKNOWLEDGEMENT: 

The authors are grateful to Lupin Pharmaceuticals (India) for supplying gift samples of Flucytosine and Zydus Cadila Healthcare Ltd. and Mankind Pharmaceuticals (India) for supplying gift samples of Linezolid. The authors declare no conflict of interest.

 

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Received on 28.09.2020 Modified on 20.11.2020

Accepted on 06.12.2020 RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(1):289-294.

DOI: 10.5958/0974-360X.2021.00052.4