HPLC Content Determination Method Validation: A Comprehensive Guide

In HPLC method validation, analysts should always pay attention to the repeated studies of the pharmacopoeia and the standard operating procedures (SOPs) published alongside it. The importance of the former is self-evident, but the latter is often overlooked by the vast majority of analysts.

Many analysts make fundamental mistakes in experiments or become at a loss when issues arise, even though these operational errors are often described in the pharmacopoeia SOPs. Therefore, to master method development, one must repeatedly study the pharmacopoeia and its SOPs until they become second nature.

This article focuses on key points in the HPLC method validation process for content determination, serving as a reference for readers. Any omissions or inadequacies are kindly acknowledged. Please also note that procedures may differ slightly among different pharmacopoeias.

Operating Procedures: Optimize objective gradient conditions and determine the analytical wavelength:

1. Select sample solvent;
2. Test adsorption on filter membrane;
3. Under DAD detector, select the wavelength at maximum absorption of main components or at plateau;
4. Select separation conditions
5. Compare HPLC columns (according to the requirements)
6. Under DAD Detector, test water, 1 mol/L acid, base, oxygen (10% hydrogen peroxide), light exposure, and high temperature degradation, as well as separation of starting materials, intermediates, and excipients (for formulation research, blank excipient degradation should be conducted simultaneously).

Technical Points:

1. This study is necessary when no reference standard is available, or the standard method cannot be directly applied.
2. Solvent for Samples: Must dissolve the sample well, remain stable at room temperature for over 12 hours, and be miscible with the mobile phase.
3. The peak area of the filtrate should reach a stable maximum value. A volume of filtrate below 5ml is preferable; otherwise, solvent selection needs reconsideration.
4. Degradation of about 10% (5%—15%) is advisable. If the sample is not degraded after being immersed in 2mol/L acid or base at 90°C for 12 hours, it is considered stable and no further degradation is needed. Exposure to 4500lx light for 48 hours is necessary for both sample powder and solution. After degradation, both sample solution and blank solvent should be adjusted to the pH of the mobile phase before injection to prevent adverse effects on the column. If degradation exceeds 20%, the degradation conditions should be reduced.
5. Resolution must be adequate, with no interference in quantification, and each peak must meet single-peak purity requirements.
6. The gradient method must include a maximum elution capacity step to ensure that all impurities are eluted and detected.
7. The process of method exploration and optimization experiments is generally not documented but reflected in the laboratory notebook. If there is no established method basis, gradient and wavelength selection need to be documented.
8. The optimized method should be used to analyze all samples on the same day, by the same person, using the same machine for final verification to ensure comparability.
9. Method optimization experiments should be conducted using small-scale lab samples when the manufacturing process is fixed. During early raw material synthesis, broad gradient elution can be used for preliminary impurity analysis.

Operating Procedures: The purpose is to verify the method's ability to differentiate target components and to comprehensively evaluate impurity analysis.

Under DAD detector or a suitable universal detector, analyze water, 1 mol/L acid, base, oxygen (10% hydrogen peroxide), light exposure, and high-temperature degradation on starting materials, intermediates, excipients, and impurity reference standards on the same day. These can be used to support impurity profile discussions in the submission documentation.

Technical Points:

1. Prepare degraded samples according to the aforementioned procedure, including blank and negative samples.
2. Ensure good separation, with no quantification interference, and that all peaks meet single-peak purity requirements.
3. The gradient method must include a maximum elution capacity step.
4. All samples must be analyzed under the predetermined method on the same day, by the same person, and using the same instrument for comparability.
5. For regulatory documentation, data should ideally be obtained from the first production batch validation.
6. When chromatographic conditions match impurity testing methods, specificity data can be shared.
7. UV spectrum and peak purity plots of the main component must be included when preparing chromatogram documents.

Operating Procedure: The signal-to-noise ratio (S/N) method is applied — the concentration at which S/N ≥ 3 is the LOD.

Technical Points:

1. Inject a blank first, then a concentrated reference solution (1 mg/mL). Determine baseline noise within 1–2 min at the main peak’s retention time, measure peak height, and calculate the LOD as three times this height.
2. Dilute the stock solution to the approximate calculated concentration.
3. Documentation should include at least three chromatograms (blank solvent, concentrated solution, and diluted solution).

Operating Procedure: The signal-to-noise ratio (S/N) method is applied — the concentration at which S/N ≥ 10 is the LOQ.

Technical Points:

1. Calculate LOQ as ten times the baseline peak height.
2. Dilute the LOD solution to the estimated LOQ concentration and inject six times, ensuring RSD of peak area <2% (or up to 5% if necessary).
3. Documentation should include six chromatograms (diluted solutions from six injections).
4. LOD and LOQ should be determined on the same day, using the same vertical axis scale in chromatograms to aid regulatory review.

Operating Procedure: 5- or 7-point calibration curve, depending on need.

Technical Points:

1. The first point should be at the LOQ concentration, the midpoint at 100% concentration, and the last point at 200% concentration (160% or 180% is acceptable).
2. Concentration adjustments must be through dilution and not injection volume changes.
3. Correlation coefficient r > 0.999 is required.
4. Calibration should be conducted using reference standards.
5. The highest recovery test concentration must be within the linear range; otherwise, quantification is unreliable. This is a common mistake.

Operating Procedure: Inject the same sample solution six consecutive times.

Technical Points:

1. Calculate peak area RSD < 2%.
2. Documentation should include seven chromatograms (one blank, six sample injections).
3. Regulatory data should come from the first production batch validation.

Operating Procedure: Conducted alongside precision tests with well-planned time intervals. Chromatograms for stability and precision tests at the same time points can be used interchangeably to reduce work. Essential time points: 0, 4, 6, 8, 10, 12, 18, 24 hours (reducible only if stability is well-documented).

Technical Points:

1. Precision test chromatograms can be used for 2 h, 4 h, or even 6 h stability assessments.
2. Report data for six selected time points from 7–8 measured ones.
3. Stability data should confirm at least 16 h stability; the longer, the better. Overnight auto-injections can extend beyond 12 h without any human works. If the time period is too short, the data may not be enough to prove stability.
4. In special cases, alternative operating procedures like low-temperature autosamplers, special storage, or freshly prepared solutions can be processed, but they must be validated with supporting data in order to be recorded in method standards.
5. RSD of peak area across six time points should be <2%.

Operating Procedure: Analyze two reference solutions and six test solutions from the same batch by the same analyst and same method, each injected once.

Technical Points:

1. RSD of content < 2%.
2. Documentation should include nine chromatograms (one blank, two references, six test samples).
3. Regulatory data should come from the first production batch validation.

Operating Procedure: Conducted on a different day by a different analyst using a different instrument, following the same method. Analyze two reference solutions and six test solutions again. Reference materials should be re-weighed.

Technical Points:

1. To avoid instrument variation issues, perform intermediate precision test on the next day of the other procedures above.
2. All 12 content results (repeatability + intermediate precision) should be included in calculations, with RSD < 2%.
3. Documentation should include 18 chromatograms (9 from repeatability, 9 from intermediate precision).
4. Regulatory data should come from the first production batch validation.

Operating Procedure: Conduct a recovery test. For bulk drugs (APIs), direct recovery rate is applied; for formulations, spiked recovery rate is applied. Weigh the samples at 80%, 100%, and 120% proportions, each with 3 samples. Inject each sample once using the same method.

Technical Points:

1. Use the mean content from intermediate precision tests as the theoretical value.
2. Recovery range: 98%–102%, RSD < 2%.
3. Documentation should include 12 chromatograms (one blank, two references, nine samples). Regulatory data should come from the first production batch validation.
4. For spiked recovery test, mix and crush at least 20 dosage units, then spike the samples (weighed at 80%, 100%, and 120% proportions) at a 1:1 ratio.
5. If excipients adsorb or interfere, causing spiked recovery rate to fail the requirement, direct recovery can be used instead.

Column Switch

Procedure:
Use HPLC columns from three different brands to compare separation efficiency, retention time, and assay results.

• If not enough brands are available, use multiple batches from one or two brands.
• Do not use degraded or expired columns for testing.

Technical Points:

• Ideally, conduct the test on the same day using two sample solutions and two reference solutions.
• A total of 15 chromatograms from 3 columns should be included (one blank, two reference solutions, and two sample solutions per column).
• The relative standard deviation (RSD) of the assay results across six replicates (n=6) should be <2%.
• Regulatory data should come from the first production batch validation.

Mobile Phase Ratio:

Procedure:
Vary the lower component of the mobile phase by approximately ±5%.

Technical Points:

• Ideally, conduct the test on the same day using two sample solutions and two reference solutions.
• A total of 15 chromatograms from 3 ratios should be included (one blank, two reference solutions, and two sample solutions per ratio).
• The relative standard deviation (RSD) of the assay results across six replicates (n=6) should be <2%.
• Regulatory data should come from the first production batch validation.
• The chromatograms of the optimal standard condition must be redone daily to confirm column efficiency and avoid misinterpretation due to column degradation or other issues.

Flow Rate Variation:

Procedure:
Vary the flow rate by ±10%.

Technical Points:

• Ideally, conduct the test on the same day using two sample solutions and two reference solutions.
• A total of 15 chromatograms from 3 flow rates should be included (one blank, two reference solutions, and two sample solutions per flow rate).
• The relative standard deviation (RSD) of the assay results across six replicates (n=6) should be <2%.
• Regulatory data should come from the first production batch validation.
• The chromatograms of the optimal standard condition must be redone daily to confirm column efficiency and avoid misinterpretation due to column degradation or other issues.

Different pH Conditions:

Procedure:
Adjust the pH value by ±0.2.

Technical Points:

• Ideally, conduct the test on the same day using two sample solutions and two reference solutions.
• A total of 15 chromatograms from 3 pHs should be included (one blank, two reference solutions, and two sample solutions per pH).
• The relative standard deviation (RSD) of the assay results across six replicates (n=6) should be <2%.
• Regulatory data should come from the first production batch validation.
• The chromatograms of the optimal standard condition must be redone daily to confirm column efficiency and avoid misinterpretation due to column degradation or other issues.

Column Temperature:

Procedure:
Vary the column temperature by ±5°C, testing at 25°C, 30°C (or the optimal parameter), and 35°C.

Technical Points:

• Ideally, conduct the test on the same day using two sample solutions and two reference solutions.
• A total of 15 chromatograms from 3 temperatures should be included (one blank, two reference solutions, and two sample solutions per temperature).
• The relative standard deviation (RSD) of the assay results across six replicates (n=6) should be <2%.
• Regulatory data should come from the first production batch validation.
• The chromatograms of the optimal standard condition must be redone daily to confirm column efficiency and avoid misinterpretation due to column degradation or other issues.

After completing the method validation and ensuring all evaluation parameters meet the required standards, perform testing on three production verification batches and the reference product on the same day.

• This data will be included in the product inspection report for regulatory submission.
• It also serves as the Day 0 stability data.