Analytical Method Validation Protocol for Nystatin

INTRODUCTION

An analytical procedure is developed to test a defined characteristic of the drug substance or drug product against established acceptance criteria for that characteristic. In the development of a new analytical procedure, the choice of analytical instrumentation & methodology should be selected based on the intended purpose & scope of the analytical method. Parameters that may be evaluated during method development are specificity, linearity, limits of detection (LOD) and limits of quantitation (LOQ), range, accuracy, and precision.

OBJECTIVE

The documented evidence of whether the analytical method is suitable for its intended purpose to establish the assay of Nystatin.

SCOPE

The scope of this protocol is to evaluate the acceptability of the analytical method used for the determination of Nystatin content by HPLC.

RESPONSIBILITY

1. It is the responsibility of the validation officer to validate the assay method for Nystatin by HPLC.
2. It is the responsibility of the QC Executive to follow the method for routine analysis.
3. The Manager - Q.C, should ensure that methods of analysis are available and that the equipment to be used for analysis is properly working and calibrated.
4. The Q.A. Manager should ensure that the method validated for the finished product should meet the acceptance criteria and maintain records for further activity.

METHODOLOGY

Equipment
HPLC	:		A liquid chromatograph capable of gradient program is equipped with a variable wavelength UV detector & controlled by valid software.
Column	:		Cosmosil, 250 mm × 4.6 mm, 5 µm. C18
Analytical Balance	:		An analytical balance is sufficiently sensitive to detect the changes of 0.1mg.
Chemicals
Methanol		:			Merck (HPLC Grade)
Dimethylformamide		:			Merck
Glassware
Volumetric Flasks		:				Calibrated Volumetric Flasks
Pipettes		:				Calibrated Pipettes
Beakers		:				Calibrated Beakers
Other Glassware		:				Borosil or Equivalent
Documents
Standard Operating Procedure				:			For HPLC & Analytical Balance.
Calibration Documents				:
Guidelines				:			ICH guidance Q2(R1) Validation of Analytical Procedures: Text and Methodology for developing & validating analytical methods.

DATA ELEMENTS REQUIRED FOR ASSAY METHOD VALIDATION

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SELECTION OF ANALYTICAL PERFORMANCE PARAMETER:

ANALYTICAL METHOD FOR NYSTATIN:

Chromatographic Conditions:
Column: 250 mm × 4.6 mm, 5 µm. C18
Flow Rate: 1.0 mL/minute
Column Temperature: 30°C
Detection Wavelength: 305 nm
Injection Volume: 10 µL
Mobile Phase: Water : Methanol : Dimethylformamide ; ( 15 : 70 : 15, v/v )

Standard Solution:

Weigh accurately 20 mg of Nystatin working standard and transfer into 50 mL volumetric flask add 30mL mobile phase, sonicate for 5 minutes, make up the volume with mobile phase & mix. Filter through 0.45µ filter.

Sample Preparation:

Weigh accurately about 20 mg of sample and transferred in to 50 mL volumetric flask, add 30 mL mobile phase & sonicate for 5 minutes, dilute with mobile phase to volume & mix. Filter through 0.45µ filter.

Calculation:
Calculate the amount of Nystatin:

Where,

Ru : Area of Sample

Rs : Average Area of Standard
Ws: Weight of Nystatin working standard
Wu : Weight of Sample
Nystatin units/mg of standard 

Acceptance Criteria:
Minimum 4400 IU/mg (dried substance)

ALSO READ: Steps for Analytical Methods Validation

METHOD VALIDATION PARAMETERS

System Suitability Test

The system suitability of an analytical method is the degree of repeatability of the result in a series of experiments run during a single session operator with identical reagents & equipment.

Standard Solution:

Weigh accurately 20 mg of Nystatin working standard and transfer into 50 mL volumetric flask add 30mL mobile phase, sonicate for 5 minutes, make up the volume with mobile phase & mix. Filter through 0.45µ filter.

Procedure:

Inject separately 10µL standard preparation in 6 replicates of standard preparation as shown in the following sequence

I. 1 × injects the diluent (Blank run)

II. 6 × Inject the standard preparations.

Accuracy

The accuracy is the degree of agreement of test results with the true value, or the closeness of the results obtained by the procedure to the true value. It is normally established on samples of the material to be examined that have been prepared to quantitative accuracy. Accuracy should be established across the specified range of the analytical procedure.

Set the instrument operating conditions as given under the analytical method.

Standard Stock Solution:

Weigh accurately 40 mg of Nystatin working standard and transfer into a 50mL volumetric flask add 30mL mobile phase, sonicate for 5 minutes, make up the volume with mobile phase & mix.

Standard Solution:

Transfer 2.5 mL of Standard stock solution to 20mL volumetric flask make up the volume with mobile phase & mix. Filter through 0.45 µ filter.

Carry out three experiments for each level as follows.

Level I (80%): Weigh accurately placebo powder about 40 mg to 20 mL volumetric flask, add 10 mL mobile phase & 2 mL standard stock solution, shake for 5 minutes, add mobile phase to produce 20 mL & filter.

Level II (100%): Weigh accurately placebo powder about 40 mg to 20 mL volumetric flask, add 10 mL mobile phase & 2.5 mL standard stock solution, shake for 5 minutes, add mobile phase to produce 20 mL & filter.

Level III (120%): Weigh accurately placebo powder about 40 mg to 20 mL volumetric flask, add 10 mL mobile phase & 3 mL standard stock solution, shake for 5 minutes, add mobile phase to produce 20 mL & filter.

Procedure: Refer to ANALYTICAL METHOD FOR NYSTATIN

Calculation Formula:

Precision

Precision is the degree of agreement among individual results. The complete procedure should be applied repeatedly to separate, identical samples drawn from the same homogeneous batch of material. It should be measured by the scatter of individual results from the mean (good grouping) and expressed as the relative standard deviation (RSD).

Precision may be performed at three different levels: repeatability, intermediate precision, and reproducibility.

Repeatability: should be assessed using a minimum of nine determinations covering the specified range for the procedure, e.g. three concentrations/three replicates each, or a minimum of six determinations at 100% of the test concentration.

Procedure : Refer to ANALYTICAL METHOD FOR NYSTATIN

Minimum of six determinations at 100% of the test concentration.

Intermediate precision (Ruggedness): expresses within laboratory variations (usually on different days, different analysts and different equipment). If reproducibility is assessed, a measure of intermediate precision is not required.

Procedure: Refer to ANALYTICAL METHOD FOR NYSTATIN

Minimum of six determinations at 100% of the test concentration.

Specificity

Specificity (selectivity) is the ability to measure unequivocally the desired analyte in the presence of components such as excipients and impurities that may also be expected to be present. An investigation of specificity should be conducted during the verification of identification tests, the determination of impurities and the assay.

Standard Solution:

Weigh accurately 20 mg of Nystatin working standard and transfer into 50 mL volumetric flask add 30mL mobile phase, sonicate for 5 minutes, make up the volume with mobile phase & mix. Filter through 0.45µ filter.

ALSO READ: Checklist for Analytical Method Validation

Sample Preparation:

Weigh accurately about 20 mg of sample and transferred in to 50 mL volumetric flask, add 30 mL mobile phase & sonicate for 5 minutes, dilute with mobile phase to volume & mix. Filter through 0.45µ filter.

Preparation of placebo:

Weigh accurately about 20 mg of placebo powder and transferred in to a 50mL volumetric flask, add 20mL mobile phase & sonicate for 5 minutes, dilute with mobile phase to volume & mix.

Standard solution spiked with placebo:

Weigh accurately about 20mg of placebo powder and transferred in to 50mL volumetric flask, add 30mL mobile phase & 10mL standard solution, sonicate for 5 minutes, dilute with mobile phase to volume & mix. Filter with 0.45 µ filter.

Observation: 

Inject a placebo & diluent solution as per assay procedure.

Conclusion: 

No peaks were obtained while running the solution containing placebo ingredients which proves that the method is specific.

Linearity & Range

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.

Standard Stock solution:

Weigh accurately 25mg of Nystatin working standard and transfer into 25mL volumetric flask add 10mL mobile phase, sonicate for 5 minutes, make up the volume with mobile phase & mix. Filter through 0.45µ filter. (Concentration of this solution 1000 µg/mL).

Solution A (50%): 0.5 mL of Standard Stock Solution --- 10 mL (50 µg/ mL)

Solution B (75%): 0.75 mL of Standard Stock Solution -- 10 mL (75 µg/ mL)

Solution C (100%): 1.0 mL of Standard Stock Solution --  10 mL (100 µg/ mL)

Solution D (125%): 1.25 mL of Standard Stock Solution -- 10 mL (125 µg/ mL)

Solution E (150%): 1.5 mL of Standard Stock Solution --- 10 mL (150 µg/ mL)

Determination:

Linearity is calculated by statistical method of linear regression analysis with five standard solutions in the range of 50% to 150% concentration. 

The correlation coefficient (R2), Y-intercept & slope of the regression line is submitted.

Robustness

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters & provides an indication of its reliability during normal usage.

Set the instrument operating conditions as given under analytical method.

Actual Parameter

Standard Solution:

Weigh accurately 20 mg of Nystatin working standard and transfer into 50 mL volumetric flask add 30mL mobile phase, sonicate for 5 minutes, make up the volume with mobile phase & mix. Filter through 0.45µ filter.

Sample Preparation:

Weigh accurately about 20 mg of sample and transferred in to 50 mL volumetric flask, add 30 mL mobile phase & sonicate for 5 minutes, dilute with mobile phase to volume & mix. Filter through 0.45µ filter.

Follow the procedure as per assay of Nystatin BP.

Change in flow rate ( ± 0.2 mL)

Procedure: For initial result refer actual parameter.

For flow rate of 0.8 mL: Record the chromatogram as per actual parameter.

For flow rate of 1.2 mL: Record the chromatogram as per actual parameter.

Calculate % assay from formula given in analytical method.

Change in mobile phase composition ( ± 2 volume)

Procedure: For initial result refer actual parameter.

For mobile phase composition of Water : Methanol : Dimethylformamide (14 : 72 : 14, v/v ) : Record the chromatogram as per actual parameter.

For mobile phase composition of Water : Methanol : Dimethylformamide (16 : 68 : 16, v/v ): Record the chromatogram as per actual parameter.

Calculate % assay from formula given in analytical method.

Change in column (different lots and/or suppliers)

Procedure: For initial result refer actual parameter.

For change in column (different lots and/or suppliers) : Record the chromatogram as per actual parameter.

Calculate % assay from formula given in analytical method.

STABILITY OF ANALYTICAL SOLUTIONS

Stability of analytical solution is a measure of its capacity to remain unaffected storage.

Develop chromatogram using assay preparation and inject it for following intervals.

12 hours

18 hours

24 hours

CONCLUSION

The conclusion should state whether the outcome of the activity was successful or not.

It should also include validation recommendations (if any) for any improvements.

REVALIDATION

Re-validation shall be necessary under the following circumstances:

1. Change in the synthesis of drug substances.
2. Change in the composition of the finished product.
3. Change in the analytical procedure.

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