Chinese Pharmacopoeia, US Pharmacopoeia on the adjustable range of chromatographic conditions

The so-called pharmacopoeia method, as the name implies, the method contained in the pharmacopoeia monograph. In the process of chromatographic analysis and method development with reference to the Pharmacopoeia method, due to different laboratory conditions, it is impossible to perform a reliable analysis in accordance with the Pharmacopoeia method. We should understand that the pharmacopoeia method has a certain adjustable range considering the differences of each laboratory.

Let’s discuss this issue in detail!

Chinese Pharmacopoeia 2020 Regulations

0512 High performance liquid chromatography is specified as follows:

The chromatographic conditions (parameters) specified under the category text should not be changed except for the type of filler, mobile phase components, and detector type, column temperature, injection volume, detector sensitivity, etc. can be appropriately changed to meet the requirements of the system suitability test.

When adjusting the proportion of mobile phase components:

When the percentage X of the small proportion component is less than 33%, the allowable change range is 0.7X~1.3X.

When X is greater than 33%, the allowable change range is X-10%~X+10%.

It can be seen from the above that the Chinese Pharmacopoeia monograph method should not be changed except for the type of filler, mobile phase components (the ratio is adjusted within the specified range), and the type of detector, and other conditions have changed, and it is still considered as a pharmacopoeia method.

However, the Chinese Pharmacopoeia does not stipulate method validation. Therefore, even for Chinese Pharmacopoeia methods, a full set of method validation should be carried out in accordance with the relevant provisions for analytical method validation.

US Pharmacopeia Regulations

USP43(621) CHROMATOGRAPHY specifies the following:

Operating conditions adjusted to meet system suitability requirements are permitted unless otherwise specified under the monograph. The following is the maximum adjustable range listed, and a full set of method validation should be carried out in accordance with the relevant provisions for analytical method validation. Adjustment of multiple conditions will have a cumulative impact on system performance, which needs to be carefully considered before implementation.

1. The pH of the mobile phase buffer (HPLC): Adjust within ±0.2 units or within the specified range, suitable for gradient and isocratic.
2. Mobile phase buffer salt concentration (HPLC): ±10% (pH allows), suitable for gradient and isocratic.
3. The proportion of components in the mobile phase (HPLC): components with a component proportion of ≤50% can be adjusted by ±30%, but the adjustment of any component ratio should not exceed ±10% (relative to the total mobile phase) , the adjustment of the mobile phase containing three components can be carried out in groups, and the adjustment of the mobile phase containing two components can be carried out in the smallest component.

Two-component adjustment example: such as mobile phase 50:50, 30% of 50% of one component is 15%, but it exceeds the regulation of ±10% of the total proportion, so the adjustment range of this mobile phase ratio should be ± It is limited to 10% and can be adjusted within the range of 40:60~60:40. For another example, the mobile phase ratio is 2:98, in which 30% of the 2% of the small component is 0.6%. This value does not exceed the regulation of ±10% of the overall proportion. The ratio of this mobile phase can be no more than 1.4:98.6~2.6: Adjustable between the range of 97.4.

Three-component adjustment example: mobile phase 60:35:5, 30% of its subcomponent 35% is 10.5%, but it exceeds the regulation of ±10% of the overall proportion, so the subgroup in this mobile phase ratio is 10.5%. The adjustment range of the points should be limited to ±10%. 30% for the minimum component 5% is 1.5%. It can be adjusted within the range of no more than 50:45:5~70:25:5 or 58.5:35:6.5~61.5:35:3.5.

4. Ultraviolet detector wavelength (HPLC): The detection wavelength specified in the monograph is not allowed to be changed. The detector supplier’s program or another verification program indicates that the wavelength detection error should not exceed ±3nm at most.
5. Stationary phase:
   Column length (GC): Adjustable within ±70%.
   Column Length (HPLC): See under Particle Size.
   Column ID (HPLC): Can be adjusted if the linear velocity is kept constant. See HPLC flow rate.
   Column Inner Diameter (GC): Adjustable up to ±50%.
   Film thickness (capillary column GC): Adjustable from −50% to 100% maximum.
6. Particle size (HPLC): Isocratic elution: Both particle size and column length can be modified, but the ratio (L/dp) of column length (L) to particle size (dp) is constant or specified in the column length and particle size. within the range of -25%~+50% of the ratio. Alternatively (for particle size adjustment applied to superficially porous particles), columns of other column length (L) and particle size (dp) may be used, but the number of theoretical plates (N) should be within -25% to +50% of the specified column within the range. Caution should be exercised if the adjustment results in a higher number of theoretical plates, which will result in smaller peak volumes, and should be adjusted by reducing factors for additional extra-column peak broadening, such as instrument tubing, detection cell volume, sampling rate, and input sample volume. When particle size is not specified in the monograph, the ratio shall be calculated using the maximum particle size of the specified column.

Particle size (GC): The particle size can be changed from large to small or from small to large if the chromatogram meets the system suitability requirements and the particle size range ratio is the same, defined as the largest particle size diameter divided by the smallest particle size diameter.

7. Flow rate (GC): The flow rate can be adjusted up to ±50%.

Flow rate (HPLC): When the particle size is changed, the flow rate may need to be adjusted, because to achieve the same performance, the small particle size column requires higher linear velocity (measured through less tray height), flow rate, column ID, particle size through the following Formula conversion.

F2=F1×[（dc2²×dp1）/（dc1²×dp2）]

For isocratic elution, if the particle size changes from ≥3μm to <3μm, the linear velocity will be increased (by adjusting the flow rate) so that the column efficiency does not drop by more than 20%, similarly, if the particle size changes from <3μm to ≥3μm, Linear velocity (flow rate) should be reduced to avoid a reduction in column efficiency of more than 20%. During gradient elution, flow rate, column diameter, and particle size will not be allowed to be adjusted. In addition, the flow rate can be adjusted at ±50% (only for isocratic elution).