HPLC Method Development Steps For Pharmaceuticals: How To Make

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Introduction and outcome

HPLC method development requires analytical chemistry knowledge, organic chemistry, skills and experience. Integrating these components and creating a development strategy is key to the HPLC method development process. However, it is a challenging task for any chromatographers. That’s why I decided to share my 30 years of experience in 15 minutes in this blog. In this article, you will learn the HPLC method development step by step, optimizations of separation, detection and method finalisation with case studies and FAQs.

HPLC Method Development Strategy

The process of making a vision-based plan for developing an HPLC method keeping in mind the cost, reliability, practicality and validity of the method is called method development strategy.

HPLC Method Development steps

Follow the following steps for effective HPLC method development:

1. To Know about the Goal of the Method
2. To Know Details about the sample
3. HPLC Mode Selection
4. Column Selection
5. Mobile Phase Selection
6. Detector Selection
7. Elution Mode Selection
8. SST Selection
9. Sample preparation procedure
10. Mode of Calculation
11. Sample preparation procedure
12. Flow Rate Optimisation
13. Column Temperature Optimization
14. Method Verification/Mini Validation
15. Documentation and Reporting
16. Routine Monitoring and Maintenance

To Know about the Goal of the Method

In this step find out the goal of the method such as whether you are going to develop a method for identity testing, purity testing, assay testing, related substance testing, chiral purity or limit testing. What will be the specification? What will be the method of calculation?

Types of test

• Qualitative test
  • Identification test
  • Monitoring the reaction mixture
  • Purity test
  • Impurity profile test or Related substances test
• Quantitative test
  • Assay test
  • Impurity profile test or Related substances test
  • Content test

To Know Details about the sample

Get the following sample details in this step:

1. Route of Synthesis (ROS)
2. Possible impurities and degradants (sample matrix)
3. Literature reported impurities
4. Stability of the sample and impurities
5. Structures of main analyte and impurities
6. pKa value
7. Hydrophilicity/hydrophobicity of main analyte and impurities
8. Total impurities (in number)
9. Standards availability
10. Sample and impurities solubility

Route of Synthesis (ROS)

Collect ROS of all purchasing materials like starting materials, key starting materials, reagents, chemicals and final API (Active Pharmaceutical Ingredients).

Possible impurities and degradants (sample matrix)

Collect information about possible impurities and degradation products with the help of ROS and literature reports.

Literature reported impurities

Collect all the published data and make the literature report. This report contains a pices of information related to impurities, chromatographic conditions, pH, pKa, stability etc.

Stability of the Sample and Impurities

Collect all the information related to the stability of the main analyte and impurities using ROS and literature report. It is very helpful during diluent selection

Structures of main analyte and impurities

In this step write down the structure, molecular weight and polarity nature of the molecule in the tabular form. It is very helpful during column selection and mobile phase selection during method development.

pKa value

Collect information related to pK value for concerned impurities and main analyte with the help of literature. If not available then find out the same using titration or UV method. It is very helpful in pH selection of the mobile phase.

Hydrophilicity/hydrophobicity of main analyte and impurities

Collect information related to Hydrophilicity and hydrophobicity for concerned impurities and main analyte with the help of literature. It is very helpful in the pH selection of the mobile phase and column selection.

Total impurities (in number)

Find out all concerned impurities related to a method with the help of literature and write it down in tabular form. It is required to prove the selectivity and specificity of the HPLC method which you are going to develop.

Standards availability

Collect all the parameters of related impurities and main analysis as without this method development is impossible. If the standard is not available then a marker or contaminated sample (containing relevant impurities) can be used.

Sample and Impurities Solubility

Collect information related to the solubility of the sample and impurities with the help of chemical R&D scientists and literature. For any new compound where solubility information is not available, conduct a solubility study. This is very helpful in Diluent selection during sample preparation

HPLC Mode Selection

In this step select the HPLC mode like whether you are going to develop a method reverse phase chromatographic (RPC) mode or normal phase chromatographic (NPC) mode or any other mode. The mode is best decided on the basis of polarity, solubility and nature of impurities and the main analyte.

Select the most appropriate chromatography mode based on the sample and their impurities properties:

• Normal Phase HPLC: For polar analytes.
• Reverse Phase HPLC (RP-HPLC): For non-polar or moderately polar analytes.
• Ion-Exchange HPLC: For charged analytes (typically used for peptides, proteins, or inorganic ions).
• Size-Exclusion HPLC: For large molecules like proteins or polymers.
• Affinity Chromatography: For specific binding interactions (e.g., antibodies or specific ligands).

Check out this article for more details: NPC & RPC

Column Selection

It doesn’t matter how sophisticated and expensive your HPLC system is you can’t perform method development if you haven’t chosen the right column. The general rule is to use a polar column for a polar molecule and a non-polar column for a non-polar molecule.

• Column Chemistry: Select an appropriate column based on the mode chosen (e.g., C18, C8 for RP-HPLC, silica-based for normal phase, or ion-exchange columns for ionic compounds).
• Particle Size: Smaller particle sizes (e.g., 1.8-5 µm) improve resolution but can increase backpressure.
• Column Dimensions: The column length and inner diameter affect separation efficiency. Common sizes are 4.6 mm × 150 mm or 4.6 mm × 250 mm.

Check out this article for more details: HPLC Column: How to Select

Mobile Phase Selection

Mobile Phase Selection plays a vital role in HPLC method development. The general rule is to use an acidic mobile phase for an acidic molecule, a basic mobile phase for a basic molecule and a neutral mobile phase for a non-polar molecule.

• Solvent System: The mobile phase needs to match the polarity of the analytes. In RPC, a gradient or isocratic system using water and organic solvents like methanol or acetonitrile is common.
• Buffer Selection: If working with ionic compounds or adjusting pH, use an appropriate buffer (e.g., phosphate, acetate) to maintain consistent pH.
• pH Control: For stability and reproducibility, choose a mobile phase with a stable pH. For example, in RP-HPLC, the pH is usually maintained between 2-8.

Detector Selection

The commonly used detectors in pharmaceutical industries are:

• Ultra-violet/UV detecto
• Mass spectrometer(MS) detector and
• Refractive Index (RI) detector

Among the above detectors, UV detector is widely used in industries. Mass detectors are extremely expensive and are used for structure elucidation and quantification at very low levels.RI detector is used for pharmaceuticals which do not have any reaction like glucose, sucrose etc.

Check out this article for more details: HPLC Detectors

Elution Mode Selection

The following elution modes are widely used in HPLC analysis:

• Isocratic mode and
• Gradient Mode

If your sample has components of almost similar polarity and the number of components is 3 to 5 then you can use isocratic mode. If your sample has multiple components of different polarity you can use gradient mode.

Method Optimisation

• Retention Time: Adjust the mobile phase composition, flow rate, or column temperature to fine-tune the retention times and ensure good separation of analytes.
• Peak Resolution: Aim for a resolution (Rs) of ≥1.5 for baseline separation. Modify parameters such as the gradient program, mobile phase pH, or ion-pairing agents to improve peak separation.
• Sensitivity: Adjust the detection wavelength or method parameters (e.g., UV wavelength, flow rate) to improve sensitivity if needed.

SST (System Suitability Test) acceptance Criteria Selection

The selection of SST acceptance criteria plays an important role in HPLC method development as it tells about the performance of the HPLC column and the HPL system for the intended analyte. Wrong SST selection may result in failure of the pharmaceuticals. The Commonly used SST parameters are:

Precision

Precision is mostly used for assay tests, content tests and related substances tests by area normalization/ % area methods.

General rule keeping precision in SST:

• Precision should be part of SST for related substances and assay tests when the external standard method is used to calculate the impurities and assay test
• It should not be part of related substances test if calculation is performed by area normalisation method
• RSD of 6 injections should be kept in the SST and it should be based on the specification

QL (Quantification Limit)

QL is part of SST for Related substances test/Impurity profile test/content test (at low level) but not assay test. SST evaluation criteria S/N≥ 10

Resolution (R)

When peaks are eluting close to each other then R is kept as one of the SST acceptance criteria. The General rule for keeping the R in SST:

• Must be kept between two close eluting peaks
• R can be kept between analyte peak vs impurity peak
• R can be kept between Impurity peak vs Analyte peak
• R can be kept between Impurity peak vs Impurity peak
• R≥ 2 (with baseline separation between the adjacent peaks
• Can be kept less than 2 with scientific justification

Theoretical plate or Column Efficiency (N)

The following are the conditions for keeping the N in SST:

• When peaks are eluting far away then N is kept as one of the SST acceptance criteria
• The limit must be decided based on trend data
• N≥ 5000 for HPLC and N ≥ 10000 for GC
• A lower N limit can be considered with scientific justification
• Generally, N of the main peak is kept as a SST acceptance criteria

Tailing Factor(T)

The following are the conditions for keeping the N in SST:

• When the peak is unsymmetrical then Tailing Factor (T) is kept as one of the SST acceptance criteria
• The limit must be decided based on trend data
• T≤ 1.5
• Higher T limit can be kept with scientific justification

Check out this article for more details: How To Decide SST?

Sample preparation procedure

Sample preparation involves:

• Selection of solvent to use as a diluent: Select the solvent in which the sample and its impurities are soluble. Organic solvents such as acetonitrile, methanol, ethanol, isopropyl alcohol or their mixtures or their mixtures with aqueous can be used.
• Compatibility of diluent with mobile phase and wavelength: Diluent must be compatible with mobile phase and should not be reactive with the column or sample components
• Concertation: The sample concentration should be adjusted so that the column does not become oversaturated. Also ensure that the analyte concentration falls within the linear dynamic range of the detector.
• Stirring or Sonication: Stirring or sonication time and temperature should be properly evaluated to avoid any degradation of impurities or the sample
• Sample Clean-up: If the sample matrix is complex, it may require filtration, solid-phase extraction (SPE), or protein precipitation to minimize matrix interference.
• Injection Volume: A typical volume for HPLC injections is between 5 µL and 20 µL, but this depends on the detector and column
• Filtration: Filter the sample solution to remove undissolved matter. Filtration is generally performed for dosages from samples which contain excipients such as tablets, capsules syrups etc. Filter effects must be evaluated during development.

Flow Rate Optimisation

• Flow Rate: A typical starting flow rate for a 4.6 mm column is 1 mL/min. This can be adjusted depending on the system’s backpressure and the separation needs.
• Column Efficiency: Higher flow rates reduce resolution, while lower flow rates might improve resolution but also increase analysis time.

Column Temperature Optimization

Most of the HPLC columns are stable up to 80⁰C. But try to optimise the HPLC column temperature between 10 to 35⁰C because at higher temperatures life of the column decreases.

Mode of Calculation

In this step mode of calculation is selected. In HPLC the following modes of calculation are widely used:

• Area % or Area normalisation method and
• External standard Method

External standard is used for quantitative tests such as assay, related substances and content tests whereas area normalisation is used for qualitative analysis such as purity tests, reaction monitoring tests and impurity profile tests.

Method Verification/Mini Validation

Perform method validation or mini-validation so that the method can be validated when needed and any surprises can be avoided. The verification parameters can be selected based on the sensitivity of the method which may include:

• Specificity: Ability to separate analytes from matrix components.
• Linearity: The method’s ability to produce a straight-line response over the concentration range.
• Accuracy: The closeness of measured values to the true value.
• Precision: Repeatability and reproducibility of results.
• Detection Limit (DL) and Quantification Limit (QL).
• Robustness: The method’s reliability under small variations in conditions (e.g., slight changes in temperature or mobile phase composition).

Check out this article for more details: Analytical Method Mini-Validation

Documentation and Reporting

• Method Description: Document all parameters used, such as column type, mobile phase, flow rate, detection conditions, and sample preparation procedures.
• Standard Operating Procedures (SOPs): Write SOPs to ensure the method is reproducible by different operators or in different laboratories.

Check out this article for more details: STP: How to Decide

Routine Monitoring and Maintenance

This step includes:

• Routine Monitoring and Maintenance
• Regularly monitor system performance to ensure stability.
• Perform routine maintenance on the HPLC system (e.g., cleaning, replacing filters, and inspecting pumps).

Case Study-1

How to develop a Related substance/ Impurity profile test method for N-(4-Hydroxyphenyl)acetamide (Paracetamol) containing the following ROS:

Method Development Steps

1. Type of Test: Related Substances/Impurity profile test
2. To Know about the sample
   • ROS: Available
   • Possible impurities: Phenol, 4-Nitrophenol, 4-aminophenol
   • Maine analyte: N-(4-Hydroxyphenyl)acetamide
   • Main analyte and impurities: Both main analyte and impurities are soluble in a mixture of water and acetonitrile
3. HPLC Mode Selection: Reverse phase mode
4. Column Selection: Both the main analyte and impurities are separated on the C18 and C8 columns. But C18 selected for routine analysis.
5. Mobile Phase Selection: All analytes are well-separated in acidic pH (pH 3 to 4) in the mobile phase contains 0.02M KH₂PO₄ and acetonitrile
6. Detector Selection: Since all analytes can give good absorbance therefore a UV detector is the best choice. The PDA detector suggests a 230 nm operating wavelength as all analytes have good absorption at this wavelength.
7. Elution Mode Selection: Since compounds have different polarities and hence linear gradient mode was selected for this test.

*Typical gradient design (not actual)

1. Method Optimisation: The method has been optimized using adjustments to column temperature, flow rate, and sample concentration.
2. SST (System Suitability Test) acceptance Criteria Selection: Amino phenol and phenol are the nearest eluting peaks and hence R is kept between these two as the first SST acceptance criteria. The Tailing factor has been kept as the second SST acceptance criterion
3. Sample preparation procedure: Analytes found soluble in the mixture of water and acetonitrile (60:40)
4. Mode of Calculation: The area normalisation method using relative response factor(RRF) has been selected as a mode of calculation
5. Method Verification/Mini Validation: Mini validation confirms the suitability of this method

The following is the elution pattern of the final optimised related substances method:

1. 4-aminophenol
2. Phenol
3. N-(4-Hydroxyphenyl)acetamide
4. 4-Nitrophenol

Case Study-1: Separation of Nonpolar compounds: Check out this article

Case Study-2: Separation of Acidic compounds: Check out this article

Case Study-2: Separation of Basic compounds: Check out this article

Conclusion

HPLC method development is an innovative analytical skill that involves fine-tuning various parameters to optimize separation, sensitivity, and reproducibility for specific analytes. By carefully considering each step – starting with the goal of the analysis and ending with validation and documentation. I hope this article has helped you understand HPLC Method Development Strategy.

You may also want to check out other articles on my blog, such as:

• Difference between HPLC and GC
• How to develop a method using GC-MS?
• Need of Chromatographic Method in Drug Development
• What is the Chiral Gas Chromatography?
• How to decide system suitability test (SST)?
• Allowable GC Method Adjustment
• How to develop the HPLC method for basic compounds?
• How to develop the HPLC method for acidic compounds?
• How to develop the HPLC method for non-polar compounds?
• What should be the Analytical method development approach?
• GLP, GDP and OOS-OOT.

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FAQs

How are HPLC methods developed?

Using proper column selection, mobile phase selection and sample preparation procedure HPLC can be developed

How to do HPLC step by step?

HPLC step-by-step procedure involves HPLC mode selection column selection, mobile phase selection, detector selection and elution mode selection, SST selection and sample preparation procedure

What are the principles of HPLC method development?

The process of making a vision-based plan for developing an HPLC method keeping in mind the cost, reliability, practicality and validity of the method is called method development strategy.

References

• Practical High-Performance Liquid Chromatography: VERONIKA R.MEYER
• ScienceDirect.COM
• ICH Q14

Abbreviations

• SST: System suitability test
• HPLC: High pressure liquid chromatography
• R & D: Research and Development

Disclaimer

To explain the topic, case studies have been prepared based on chromatography theory and practical experience.