Analytical Method Development for New Products: Assay and Related Substances

Following are the list of attributes to be followed;

1. Objective:

The purpose of the study is to develop an analytical method for the determination of Assay & Related substances in a new formulation product by HPLC using a UV-Visible detector.

2. Introduction:

This document will help analysts to develop robust Analytical methods for routine testing.

3. Scope:

This guideline will provide information about analytical method development to be carried out as per ICH Guidelines.

4. Physico-Chemical details of API:

Following items to be checked viz;

IUPAC name, Molecular formula, Chemical structure, Molecular weight, Solubility, Appearance, Melting Point, pH solubility, pKa, LogP, BCS Classification, Polymorphism, Isomerism, Density, Hygroscopicity, Impurities listed as per process either in DMF or official monograph

5. Details of formulation product:

Following items to be checked viz;

Detailed Name, Molecular formula, Chemical structure, Molecular weight, Official monograph status in USP/BP/EP, Dosage form, Dosage strength, Maximum daily dose, Reporting and Identification thresholds, Reference listed drug / Innovator information, Solubility, pKa, impurities information, Information on potential metabolites.

• Literature review
• Impurities information from both API and finished product
• Route of synthesis of API to understand impurity profile
• Potential impurities with relevant information- process related and degradant impurities
• Impurities structure, IUPAC name, molecular weight

6. Chromatographic conditions:

• Selection of detector:

The detector can be selected based on the structure of analyte and impurities, at times different detectors can be used.

• Wavelength selection:

This should be done based on the spectra of analyte or impurities.

• Buffer selection:

Buffer should be chosen based on the pH of solution/ pKa and final pH of buffer should be maintained at +/- 0.2 unit of target pH, this will ensure no distortion in peak shape during elution is found.

• Column selection:

Column can be selected based on polarity of Analyte and impurities.

• Mode of elution:

Initial assessment can be done using different combination of gradient technique to elute all possible impurities and then final fine tuning should be done.

• Samples to be used for method development:

Initial 1 batch of formulation and API can be used for assessment, further at least 3 different batches should be checked for getting consistent impurity profile.

• Sample extraction:

Appropriate diluent and technique to prepare sample should be evaluated and further finalized.

Sample Assay after extraction should be in the range of 95% to 98% with appropriate % of total impurities.

Test concentration and injection volume should be selected such that impurities LOQ are less than reporting threshold.

• Solution stability:

Solution stability of standard and sample should be evaluated for at least 12 hours.

• Matrix interference study:

Relevant mobile phase blank, diluent blank, placebo blank and interference from syringe filter used for sample preparation should be evaluated.

7. Finalization of method:

Method should be finalised after evaluating data for different robustness parameters such as flow rate of mobile phase, temperature of column, evaluating different make and series of columns, pH of mobile phase, composition of mobile phase, system suitability parameters.

8. Forced degradation:

Both API and finished product should be treated for different stress conditions such as;

1. Thermal: 105^o C for 12 to 24 hours
2. Water: Reflux for 12 to 24 hours
3. Acid: Dilute acid treatment either immediately or after exposure for different time intervals
4. Alkali: Dilute alkali treatment either immediately or after exposure for different time intervals
5. Oxidation: Sample exposure to dilute oxidising agent for different time intervals
6. Photo stability: Light exposure using 200 watt to achieve 1.2 million lux hour intensity
7. Humidity: 60%RH for 7 days

After treatment of samples, they should be immediately analysed, if required further dilution can be done to check Assay using different method of short runtime than related substances method and check for peak purity.

All data; Assay and impurity profile should be tabulated based on RRT.

Mass balance should be calculated against a standard solution of API of same concentration which is not treated.

Alternatively samples can be evaluated via LC-MS technique to gain information on major degradant impurities.

9. Linearity and Accuracy studies:

Linearity studies is done to establish RRf (relative response factor) and Accuracy is done to ensure on spiking of impurities in related substance method shows recovery in the range of at least 98%, additionally Assay method accuracy study is done to check recovery of drug from matrix.

10. Comparison of Pharmacopeial and in-house developed methods:

Side by side comparison of both methods should be done and establish equivalency studies.

11. System Suitability criteria:

Based on different studies carried out during method development and partial validation System suitability criteria such theoretical plates, tailing factor, resolution, % RSD of standard, S/N ratio, Capacity factor should be set based on data.

12. Conclusion / Method development report:

Document conclusions on stability indicating performance of method

Based on robustness studies, clarify on sensitivity of the method

Special precaution to be taken during using method for routine testing

Specific handling instructions of standards, samples and chemicals

Justification of specifications based on literature reference and tentative setting of specifications based on evaluation of Innovator product.

Draft method of analysis, to be further verified by another analyst to ensure workability/ reproducibility of method.

All relevant chromatograms and documents can be attached for reference.

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