ABOUT AUTHORS:
Sahoo.U1*, Sethy.S.P1, Biswal.S1, Patro.S.K1, Banerjee.M1, Sundeep Kumar.H.K.S1, Patel.D2
*1Department of Pharmaceutical Chemistry
Institute of Pharmacy and Technology, Salipur, Cuttack, Odisha-754202
2ZYG Pharma, Pvt, Ltd, Pithampur, Madhya Pradesh
sarada9439504350@gmail.com
ABSTRACT-
A mobile phase consisting of Acetonitrile and water in the ratio of 60:40 gave a well resolved and sharp peak for Zinc-Pyrithione with a retention time of 11.61 mins. The flow rate was 1 ml/min and effluent was monitored at 322 nm. Zobrax C18 column, run time of 30 min and ambient temperatures were found to be optimum for the analysis. System suitability was performed by injecting five replicate injections of working standard solution. The System suitability results obey all the parameters and found within the acceptable range. The precision study was found to be less than 1 (% RSD). So, it indicates the method is precise. The recovery study was found to be 98 to 102 % and % RSD was found to be less than % 1. So, the method is more accurate, precise, and sensitive.
REFERENCE ID: PHARMATUTOR-ART-1935
INTRODUCTION- Ketoconazole
Chemically ketoconazole is 1-[4-(4-{[(2R, 4S)-2-(2, 4-Dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1, 3-dioxolan-4-yl] methoxy} phenyl) piperazin-1-yl] ethan-1-one
Ketoconazole is a synthetic antifungal drug used to prevent and treat fungal skin infections, especially in immune compromised patients such as those with AIDS or those on chemotherapy. Ketoconazole is sold commercially as an anti-dandruff shampoo, topical cream, and oraltablet. Ketoconazole is very lipophilic, which leads to accumulation in fatty tissues. The less toxic and more effective triazole compounds fluconazole and itraconazole are sometimes preferred for internal use. Ketoconazole is best absorbed at highly acidic levels, so antacids or other causes of decreased stomach acid levels will lower the drug's absorption when taken orally. Absorption can be increased by taking it with an acidic beverage, such as cola.[1] ketoconazole is structurally similar to imidazole, and interferes with the fungal synthesis of ergo sterol, a constituent of fungal cell membranes, as well as certain enzymes. As with all azoles antifungal agents, ketoconazole works principally by inhibiting the enzyme cytochrome P450 14-alpha-demethylase (P45014DM).[2-6]
Chemically ZincPyrithione isbis (2-pyridylthio) zinc 1, 1'-dioxide. Zinc pyrithione is a coordination complex of zinc. This colorless solid is used as an antifungal and antibacterial agent. The pyrithione ligands, which are formally monoamines, are cheated to Zn2+ via oxygen and sulfur centers. In the crystalline state, Zinc pyrithione exists as a centrosymmetric dimmer (see figure), where each zinc is bonded to two sulfur and three oxygen centers.[7]In solution, however, the dimmers dissociate via scission of one Zn-O bond.
Zinc pyrithione is best known for its use in treating dandruff and seborrhea dermatitis [8].It also has antibacterial properties and is effective against many pathogens from the streptococcusand staphylococcusclass. Its other medical applications include treatments of psoriasis, eczema, ringworm, fungus, athletes foot, dry skin, atopic dermatitis, tinea, and vitiligo.
Due to its low solubility in water(8 ppm at neutral pH), zinc pyrithione is suitable for use in outdoor paints and other products that provide protection against mildew and algae. It is an effective algaecide. It is chemically incompatible with paints relying on metal carboxyl ate curing agents. When used in latex paints with water containing high amount of iron, a sequestering agent that will preferentially bind the iron ions is needed. Its decomposition by ultraviolet light is slow, providing years of protection even against direct sunlight.
Zinc pyrithioneproduces its antifungal activity by deriving from of its ability to disrupt membrane transport by blocking the proton pump that energizes the transport mechanism.[9]
From the literature review we identified that, there around 7 to 8 works, related to our paper were done. But our work was little difference from them. First we prepared the shampoo formulation according to the marketing formulation. Then changing some parameters like (mobile phase, column, Temp.) to performed the assay which provided good results in comparison to others. So we choose this method to determined Zinc Pyrithione in Ketoconazole Shampoo Preparation by using Reverse Phase High Performance Liquid Chromatography. [10-18]
METHODOLOGY-
PREPARATION OF KETOCONAZOLE SHAMPOO FORMULATION
Procedure
- Add 10 gm of Benzyl alcohol, 25 gm of Polysorbate-80, 20 gm of Propylene glycol,10 gm of Ketoconazole,5 gm of Glycol distearate and 75 gm of SLES in to a oily phase kettle and was heated in a (50*c) for 15 minutes.
- In an aqueous phase container, 1 gm of HPMC was mixed with purified water. Then it was dispersed with (25 gm of SLS+2.5 gm of Poly quaterium+1 gm of EDTA, which were prepared by previously in another container). The pH was adjusted with sodium hydroxide (5.5-6) and was heated in a (50*c) for 20 minutes.
- In another container add 30 gm of CAPB, 15 gm of dimethicone and 10 gm of Zinc pyrithione. Then it was homonized in a homonizer.Then citric acid was added to adjust the pH (5.5-6).
- The oil phase content was added with aq.phase content in a compounding kettle, fitted with homonizer & homonized. The content (CAPB, ZnPTO, dimethicone mixture, which were previously prepared) were added to compounding kettle. Then it was mixed and homonized properly and adjusted to the pH 5.5-6.
Determination of λmax of the Zinc Pyrithione by UV spectrophotometer.
• Preparation of standard stock solution: Weigh accurately 100mg of Zinc Pyrithione and transferred into a 100 mL volumetric flask containing 30 mL of methanol and then sonicated for 3 minutes and then the volume was made up to the mark with methanol to produce 1mg/mL.
• Preparation of working standard stock solution: 10 mL of standard stock solution + 20 ml cupric sulphate solution were transferred into another 100 ml volumetric flask and left for 1 hour for complex formation then diluted up to the mark with chloroform in order to produce 100 µg/mL.
• 1 ml working standard stock solution was transferred into a 10ml volumetric flask and then the volume was made up to the mark with the methanol to produce 10 µg/mL. It was run in the spectrophotometer to determine the λmax of the Zinc Pyrithione.
Drug-ZPTO.
Solvent- Methanol, Chloroform, & Cupric sulphate solution.
Absorbance Range -0.00A to 1.00A.
Wave length Range- 200nm to 400nm.
Mode- Spectrum. Mode.
• Result: UV spectrum of an analyte comprising two nos λmax (one for lower absorbance & other for higher absorbance)
Table No: 1
|
Types |
λmax |
Absorbance |
|
Higher |
322nm |
0.076 |
|
Lower |
247nm |
0.166 |
From the above spectrum we concluded that, two spectrums were determined which have two λmax value. One is 322nm (higher wave length) having absorbance 0.070 & other is 247nm (lower wavelength) having absorbance 0.166.
So we choose the λmax 322nm (higher wave length) and setting Absorbance Zero (0), to determined the calibration curve of ZPTO, also setting the instrument in Absorbance mode instead of Spectrum mode. By using serial dilution method, to determined the calibration curve of ZPTO.
From the calibration curve, we determined the concentration of ZPTO in sample.
Fig No 3: UV Spectrum of Zinc Pyrithione
OPTIMIZED CHROMATOGRAPHIC CONDITION:-
Instrument Name: Agilent Series 1200
Instrument ID: 108
Column Name: C-18 (250 ×4.6 mm) & 5µ Particle size (Zorbax)
Set Wave length : 322 nm
Flow rate: 1 ml/min
Injection Volume: 10µl
Temperature: 20ºC
Run Time: 30 minutes.
Standard Preparation : Weigh accurately 20.8 mg of Zinc Pyrithione and transferred into a 100 mL volumetric flask containing 30 mL of methanol and then sonicated for 3 minutes and then the volume was made up to the mark with methanol.
• 10 mL of above solution + 20 ml cupric sulphate solution were transferred into another 100 ml volumetric flask and left for 1 hour for complex formation then diluted up to the mark with chloroform for extraction and shake for 10 minutes.
• Pipette out 5 ml of lower layer and volume make up to 100 ml with methanol.
Sample Preparation : Weigh accurately 1.9502gm of sample (Shampoo) and transferred into a 100 mL volumetric flask containing 30 mL of methanol and then sonicated for 3 minutes and then the volume was made up to the mark with methanol.
10 mL of above solution + 20 ml cupric sulphate solution were transferred into another 100 ml volumetric flask and left for 1 hour for complex formation then diluted up to the mark with chloroform for extraction and shake for 10 minutes Pipette out 5ml of lower layer and volume make up to 100 ml with methanol
After preparation of standard & sample solution & also mobile phase selection, then choose the method.
In this step, setting the all parameters like Temperature, Pressure, Purging valve, column, Injection volume, Run Time etc. Then save the Method.
Validation According to USP Guidelines[19], [20]
Method validation
After method development, validation of the current method was performed in accordance with USP requirements for assay determination (Category-I: Analytical methods for quantization of active ingredients in finished pharmaceutical products) which include accuracy, precision, selectivity, linearity and range
Linearity and range:
The drug concentrations were prepared as per given in the Table. The calibration curve was constructed by taking concentration on X-axis and Area on Y- axis. The linearity data is shown in the Table No: 2
|
Sl. No. |
Concentration (mcg/mL) |
Area |
|
1 |
0 |
0 |
|
2 |
1.04 |
9729720.4 |
|
3 |
2.08 |
19419931 |
|
4 |
3.12 |
28189301 |
|
5 |
4.16 |
38918782 |
|
6 |
5.2 |
47535692 |
|
7 |
6.24 |
57368320 |
Table No: Linearity data of Zinc-pyrithione
Fig No: 4 Calibration curve of Zinc-Pyrithione
Precision: Inter-day precision wasdetermined by injecting the standard solution (1.04 mcg/ml) of the analyte for six times. The % RSD of peak areas of Zinc pyrithione for the six replicates was found to be less than 1. The result of precision is shown in the Table.
Table No: 3 Inter-day-precision
|
Inter-day Precision |
% result ± % RSD |
|
1 |
98.12 0.62 |
|
2 |
98.24 ± 0.71 |
|
3 |
98.36 ±0.87 |
|
4 |
98.41 ± 0.92 |
|
5 |
98.26 ±0.76 |
|
6 |
99.03 ± 0.26 |
Selectivity: Selectivity of the current method was demonstrated by good separation of zinc pyrithione. Furthermore, zinc pyrithione is good separated from the excipients of the shampoo preparation as seen in Figure.
Fig No: 5 Good Separation of Zinc Pyrithione from excipients (Standard)
Fig No: 6 Good Separation of Zinc Pyrithione from excipients (Sample)
System suitability:The System suitability test was performed by five replicate injections of 1.04 µg/mL respectively and the results are shown in Table.
TableNo: 4 Results of System Suitability parameter
|
Parameters |
Results |
|
Retention time ( Rt) |
11.614 |
|
Theoretical Plate |
3060.8 |
|
Asymmetric factor |
0.97 |
|
Area (% RSD) |
0.84939 |
Specificity: The peak purity of Zinc pyrithione was assessed by comparing the retention time (Rt) of standard Zinc Pyrithione. Good correlation was found between the retention time of standard and sample of Zinc Pyrithione.
Accuracy: It was found by recovery study using standard addition method. Known amounts of standard Zinc Pyrithione was added to pre-analyzed samples at a level from 50%, 100% and 150% and then subjected to the proposed RP-HPLC method. Results of recovery studies are shown in Table No: 5
Table No: 5- Accuracy data of Zinc Pyrithione
|
Recovery data |
Statistical Analysis |
||||||
|
% Level of |
Formulation |
Amount of pure (µg/ml) |
Amount of (µg/ml) |
% Result of |
Mean |
SD |
% RSD |
|
50 |
2.08 |
1.04 |
1.06 |
101.9231 |
101.3462
|
1.458
|
1.438
|
|
2.08 |
1.04 |
1.07 |
102.8846 |
||||
|
2.08 |
1.04 |
1.05 |
100.9615 |
||||
|
2.08 |
1.04 |
1.03 |
99.03846 |
||||
|
2.08 |
1.04 |
1.06 |
101.9231 |
||||
|
100 |
2.08 |
2.08 |
2.06 |
99.03846 |
100.1923
|
1.686
|
1.682
|
|
2.08 |
2.08 |
2.09 |
100.4808 |
||||
|
2.08 |
2.08 |
2.1 |
100.9615 |
||||
|
2.08 |
2.08 |
2.13 |
102.4038 |
||||
|
2.08 |
2.08 |
2.04 |
98.07692 |
||||
|
150 |
2.08 |
3.12 |
3.1 |
99.35897 |
101.0897
|
1.146
|
1.134
|
|
2.08 |
3.12 |
3.14 |
100.641 |
||||
|
2.08 |
3.12 |
3.18 |
101.9231 |
||||
|
2.08 |
3.12 |
3.16 |
101.2821 |
||||
|
2.08 |
3.12 |
3.19 |
102.2436 |
||||
Result & Discussion:
A mobile phase consisting of Acetonitrile and water in the ratio of 60:40 gave a well resolved and sharp peak for Zinc-Pyrithione with a retention time of 11.61 mins. The flow rate was 1 ml/min and effluent was monitored at 322 nm. Zobrax C18 column, run time of 30 min and ambient temperatures were found to be optimum for the analysis. System suitability was performed by injecting five replicate injections of working standard solution. The System suitability results obey all the parameters and found within the acceptable range. The precision study was found to be less than 1 (% RSD). So, it indicates the method is precise. The recovery study was found to be 98 to 102 % and % RSD was found to be less than % 1. So, the method is more accurate. There is no additional peaks in the chromatogram, so it indicates that the there is no interference from the excipients present in the shampoo formulation. So, the proposed method can be regularly used for the quality control test for the determination of zinc-Pyrithione in Ketoconazole shampoo formulation. So, the proposed method is simple, accurate, selective and precise. The method was validated as per USP category 1.
Acknowledgments
We would like to thanks ZYG Pharma Company and Institute of Pharmacy & Technology, Salipur, Cuttack, Odisha for their encouragement, cooperation, help and providing all facilities
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