HPLC Content Determination Method Validation: A Comprehensive Guide

Research Content

Operational Process

Optimization of Objective Gradient Conditions to Determine Analytical Wavelengths:

1. Selection of Solvent for Sample
2. Adsorption Test on Filter Membrane
3. Under DAD Detector, Selection of Wavelength at Maximum Absorption of Main Components or at Plateau
4. Selection of Separation Conditions
5. Comparison of Chromatographic Columns (Adjust according to the situation)
6. Under DAD Detector, separation of components such as water, 1 mol/L acid, base, oxygen (10% hydrogen peroxide), light exposure, and high temperature degradation, including starting materials, intermediates, excipients (for formulation research, blank excipient degradation should be conducted simultaneously).

1. When standard methods with no reference or direct applicability are unavailable, this research content is necessary, such as for Class 3 drugs.

2. Solvent for Samples: It should have excellent solvency for the sample, be stable when left undisturbed in the laboratory for over 12 hours, and exhibit good mutual solubility 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 remains stable after being immersed in 2mol/L acid or base at 90°C for 12 hours, degradation is considered acceptable. Exposure to 4500lx light for 48 hours is necessary for both sample powder and solution. After degradation, both sample solution and blank solvent need adjustment to the pH of the mobile phase before injection to prevent adverse effects on the chromatographic column. If degradation exceeds 20%, the degradation conditions need to be reduced.

5. Good separation with no interference in the determination, and purity of each peak meets the requirements for single peaks.

6. The gradient should have a process with the highest elution capacity 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, unless there is no method basis, such as in the case of Class 3 drug applications, where gradient and wavelength selection need to be documented.

8. After method optimization, all samples should be analyzed on the same day, by the same person, using the same machine to ensure comparability.

9. Method exploration and optimization experiments use laboratory-scale trials and laboratory-scale samples with established processes. In the initial stage of material synthesis sample coordination testing, a wide range of gradient elutions can be used for fuzzy detection analysis.

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

Under DAD detector or suitable universal detectors, analysis of water, 1 mol/L acid, base, oxygen (10% hydrogen peroxide), light exposure, and high-temperature degradation, starting materials, intermediates, excipients, and impurity reference standards are conducted on the same day. These can be discussed as part of the impurity spectrum in the application documentation.

1. Follow the above process for preparing degraded samples, ensuring both blanks and negative samples are included.

2. Ensure good separation, interference-free measurements, and peak purity of each peak meeting single peak requirements.

3. The gradient should include a process with the highest elution capacity.

4. Analyze and determine all samples using the same machine, by the same person, on the same day, under previously established analysis conditions and methods to ensure comparability.

5. Data from this section should be included in the documentation, preferably using the first batch of samples validated on the production line.

When chromatographic conditions match those of relevant substance examination methods, experimental data of specialty can be shared.

UV spectra of main component peaks and peak purity plots should be included when plotting graphs.

Detection limit

The signal-to-noise ratio method defines the quantitation limit as the concentration when the signal-to-noise ratio (S/N) is equal to or greater than 10.

• The first point is at the quantitation limit concentration, the middle point is at 100% concentration, and the last point is at 200% concentration (160% or 180%). Dilution method must be used, adjustment of injection volume is not allowed. r>0.999.

• Control materials are used.

• The linear range must include the highest concentration tested for recovery experiments. Otherwise, if the recovery concentration exceeds the linear range, there will be a lack of quantitative basis, which is a very common mistake.

1. Calculate the peak area with a relative standard deviation (RSD) of less than 2%. Include seven images in the documentation (1 blank and 6 sample solutions).

2. Graphs included in the documentation must use the first batch of samples validated on the production line.

The stability of the solution is tested on the same day, interleaved with precision testing. Prior to the experiment, a proper sorting setting is established to minimize workload by sharing graphs where possible. The following time points are mandatory (unless stability is already known, reducing the number of time points):

0, 4, 6, 8, 10, 12, 18, 24 hours.

• During the experiment, graphs from precision testing can be used for 2 hours, 4 hours, or even 6 hours.

• Perform tests at 7 to 8 time points, but report only 6 data points in the final application documentation.

• Solution stability data should demonstrate stability for at least 16 hours (ideally longer, as automatic injection systems typically operate overnight, ensuring at least 12 hours of stability). In extremely rare cases, special handling methods such as low-temperature injectors or unique storage methods may be employed, but supporting data must be provided to justify inclusion in the standard.

• Calculate the RSD of peak areas for the 6 data points, ensuring it is less than 2%.

The same batch of samples are taken by the same operator using the same method. Two portions of control material and six portions of test sample solution are prepared, each injected once.

• Calculate the content with a relative standard deviation (RSD) of less than 2%. Include nine images in the documentation (1 blank, 2 control, and 6 sample solutions).

• Graphs included in the documentation must use the first batch of samples validated on the production line.

• The mean content from intermediate precision is used as the theoretical content to calculate recovery rate. The acceptable range for recovery rate is 98% to 102%, with a relative standard deviation (RSD) of less than 2%. Include 12 images in the documentation (1 blank, 2 control, and 9 sample solutions). Graphs included in the documentation must use the first batch of samples validated on the production line.

• For active pharmaceutical ingredients, direct recovery rate is used.

• For formulations, recovery rate determination is conducted through spiking recovery. For this, 20 units of the final product are mixed and ground, and samples are spiked at 80%, 100%, and 120% proportions. If the recovery rate does not meet standards (due to significant adsorption or interference from excipients), direct recovery rate can be used. The acceptable range for recovery rate is 98% to 102%, with a relative standard deviation (RSD) of less than 2%. Include 12 images in the documentation (1 blank, 2 control, and 9 sample solutions). Graphs included in the documentation must use the first batch of samples validated on the production line.

Comparing different brands of chromatographic columns, a total of 3 brands will be evaluated. The comparison will involve assessing separation efficiency, retention times, and content determination results.

If there are fewer than 3 brands available, multiple batch numbers or columns from different brands can be used. However, using discarded columns for experimentation is not permissible.

1. It's preferable to conduct experiments on two sets of sample solutions and two sets of control solutions on the same day.

2. A total of 15 images will be included for the 3 brands of chromatographic columns (1 blank for each column, 2 controls for each column, and 1 sample for each column). The content data (n=6) for each condition should have a relative standard deviation (RSD) of less than 2%. Graphs included in the documentation must use the first batch of samples validated on the production line.

1. It's preferable to conduct experiments on two sets of sample solutions and two sets of control solutions on the same day.

2. For the 3 different flow rates of the mobile phase, a total of 15 images will be included (1 blank for each flow rate, 2 controls for each flow rate, and 1 sample for each flow rate). The content data (n=6) for each condition should have a relative standard deviation (RSD) of less than 2%. Graphs included in the documentation must use the first batch of samples validated on the production line. Graphs for the optimal standard conditions should be redone daily to avoid any issues arising from decreased chromatographic column efficiency or other problems.

• It's preferable to conduct experiments on two sets of sample solutions and two sets of control solutions on the same day.

• For the 3 different pH values of the mobile phase, a total of 15 images will be included (1 blank for each pH value, 2 controls for each pH value, and 1 sample for each pH value). The content data (n=6) for each condition should have a relative standard deviation (RSD) of less than 2%. Graphs included in the documentation must use the first batch of samples validated on the production line. Graphs for the optimal standard conditions should be redone daily to avoid any issues arising from decreased chromatographic column efficiency or other problems.

Column temperature

Column temperature varies by ±5°C.

25°C, 30°C (or optimal parameters), 35°C

1. It's preferable to conduct experiments on two sets of sample solutions and one set of control solutions on the same day.

2. For the 3 different temperatures, a total of 15 images will be included (1 blank for each temperature, 2 controls for each temperature, and 1 sample for each temperature). The content data (n=6) for each condition should have a relative standard deviation (RSD) of less than 2%. Graphs included in the documentation must use the first batch of samples validated on the production line. Graphs for the optimal standard conditions should be redone daily to avoid any issues arising from decreased chromatographic column efficiency or other problems.

Testing of 0-day samples