Důležité změny chromatografických metod z pohledu lékopisu EP a USP (Rainer Turek, Waters VIZE 2022)

Unites States Pharmacopoeia

• Changes in <621> Chromatography
• <1220> Analytical Procedure Life Cycle

European Pharmacopoeia

• Changes in 2.2.46 Chromatographic Separation Techniques
• Addition of 5.26 Implementation of Pharmacopeial Procedures

Pozvánka: Waters seminář VIZE 2024

Termín: 19. listopad 2024 (09:00-14:00)
Místo: Hotel International Brno

• REGISTRACE - prezenčně
• REGISTRACE - on-line stream

USP <621> & EP 2.2.46 | Harmonization

Parameter / Currently effective / Effective soon¹

CHANGE METHODS

• Changing HPLC methods to UHPLC condition / Permitted for isocratic Not allowed for gradients / Permitted for isocratic and gradients

CALCULATIONS

• Theoretical Plates / Tangent width reporting as plate count / Half-Height width reporting as plate number
• Resolution & Relative Resolution / Tangent width / Half-Height width 
• Tailing factor / No formula change but renamed, harmonized, and now reported as Symmetry Factor
• S/N Calculation / 5 times the peak width at half height / 20 times the peak width at half height

⁽¹⁾ Effective by 01/DEC/2022 in USP and 01/JAN/2023 in EP

Waters Corporation: USP 621 & EP 2.2.46 Change HPLC methods to UHPLC condition

USP <621> & EP 2.2.46 | Change HPLC methods to UHPLC condition

Stationary Phase

… no change of the identity of the substituent…

Column dimension and particle

… the particle size and/or length of the column may be modified provided that the ratio of the column length (L) to the particle size (dp) remains constant or in the range − 25 per cent to + 50 per cent of the prescribed L/dp ratio…

Column Internal diameter

… In absence of a change in particle size and/or length, the internal diameter of the column may be adjusted. Caution is necessary when the adjustment results in smaller peak volumes due to a smaller particle size or smaller internal column diameter, a situation that may require adjustments to minimize extra-column band broadening by factors such as instrument connections, detector cell volume and sampling rate, and injection volume.

When the particle size is changed, the flow rate requires adjustment, because smaller-particle columns will require higher linear velocities for the same performance (as measured by reduced plate height). The flow rate is adjusted for both the change in column diameter and particle size using the following equation…

Flow rate

Isocratic: … in the absence of a change in column dimensions, an adjustment of the flow rate by ± 50 per cent is permitted…

Gradient: … Flow rate is adjusted for changes in column diameter and particle size using the following equation …

Gradient time points

… A change in column dimensions, and thus in column volume, impacts the gradient volume, which controls selectivity. Gradients are adjusted to the column volume by changing the gradient volume in proportion to the column volume. This applies to every gradient segment volume…

Temperature

Isocratic: ± 10 °C, where the operating temperature is specified, unless otherwise prescribed

Gradient: Column temperature: ± 5 °C, where the operating temperature is specified, unless otherwise prescribed

Wavelength (nm)

No adjustment permitted

Injection Volume

... When the column dimensions are changed, the following equation may be used for adjusting the injection volume: …

Dwell volume

… include an isocratic step before the start of the gradient program so that an adaptation can be made to the gradient time points to take account of differences in dwell volume between the system used for analytical procedure development and that actually used. It is the user’s responsibility to adapt the length of the isocratic step to the analytical equipment used…

pH of the aqueous component in the mobile phase

±0.2 pH units, unless otherwise prescribed

Concentration of salts in the buffer component of a mobile phase

±10 %

1. Recommendation

Determine the System Dwell volumes for all your instruments

Waters can help you to setup custom fields (13 fields) to automatically calculate the dwell volume, following the guidelines (USP).

Validation service is also available.

2. Adjust the column length and particle size according to L/dp.

• The column must match the description of the pharmacopoeia.
• The column must be available in the desired particle size and dimensions.

Waters Corporation: USP 621 & EP 2.2.46 Changing HPLC methods to UHPLC condition

The tools located here help you to identify the right column, to do the maths (L/dp) with the column calculator, and much more…

Use the column calculator for the maths (also embedded in Empower) & visit the web-shop

Waters Corporation: USP 621 & EP 2.2.46 Changing HPLC methods to UHPLC condition

Sometimes the pharmacopeia publishes reference chromatograms

Waters Corporation: USP 621 & EP 2.2.46 Sometimes the pharmacopeia publishes reference chromatograms

When peaks are close together (8-11 min), you may be best served with L/dp ≥ the original column’s L/dp – trading speed for resolution.

3. Adjust the flow rate for changes in particle size and column diameter. Adjust the gradient time of each segment for changes in column length, diameter, and flow rate.

The tool also provide information for the isocratic hold step to compensate dwell volume differences, computes pressure, etc.

Waters Corporation: USP 621 & EP 2.2.46 Changing HPLC methods to UHPLC condition

Everything is exactly explained

Waters Corporation: USP 621 & EP 2.2.46 - running the right column on the right system will give best results

…running the right column on the right system will give best results…

USP <621> & EP 2.2.46 | Calculations within Empower

“The system suitability and acceptance criteria in monographs have been set using parameters as defined below. With some equipment, certain parameters, such as the signal-to-noise ratio and resolution, can be calculated using software provided by the manufacturer. It is the responsibility of the user to ensure that the calculation methods used in the software are equivalent to the requirements of the US Pharmacopeia and to make any necessary corrections if this is not the case.” 

USP <621>

Formula used by Empower is impacted by the pharmacopeia selection

Chinese pharmacopoeia supported in Empower version 3.7 or later

• I. Press [F1] within Empower to open the help menu, preferably inside a processing method.
• II. Lookup for “Verifying System Performance” to display the formula used by Empower for your selection (US, EP, JP, ChP, All) and to what field it is reported.

Resolution (Rs)

The resolution between peaks of two components (Figure 1) may be calculated using the following
equation:

Waters Corporation: Calculations within Empower Resolution (Rs)

• t_R1, t_R2 = retention times of the peaks
• W_h1, W_h2 = peak widths at half-height

Empower uses H-H width calculation in the field “USP Resolution (HH)” and the field Resolution (for EP and JP).

Empower uses the tangent width in the field “USP Resolution” an should therefore not be used any
longer.

When using all pharmacopoeia or the filed “Relative Resolution” additional measures are needed.

Relative Resolution (R_s) field & All Pharmacopoeia selection

ALL or ChP will use the tangent approach

EP, JP and USP will use the H-H approach

If you must use the ALL or ChP selection the HH approach is not determined

• -> use of a custom field required
• -> to obtain a result using H-H calculations

Plate Count / Plate Number

Empower fields: EP Plate Count and JP Plate Count

USP Plate Count uses the tangent approach.

In the Empower report method, consider to rename the field as “Plate number”

Symmetry Factor / Tailing Factor

No change to formula

Empower fields: USP Tailing, Symmetry Factor (for EP and JP), ChP Tailing Factor

Signal-to-Noise

Waters Corporation: Calculations within Empower - Signal-to-Noise

H = height of the peak (Figure 6) corresponding to the component concerned, in the chromatogram obtained with the prescribed reference solution, measured from the maximum of the peak to the extrapolated baseline of the signal observed over a distance equal to 20 times the width at half-height

h = range of the noise in a chromatogram obtained after injection of a blank (Figure 7), observed over a distance equal to 20 times the width at half-height of the peak in the chromatogram obtained with the prescribed reference solution and, if possible, situated equally around the place where this peak would be found Figure 6 Figure 7

If a baseline of 20 times the width at half-height is not obtainable because of peaks due to the solvents or reagents, or arising from the mobile phase or the sample matrix, or due to the gas chromatographic temperature program, a baseline of at least 5 times the width at half-height is permitted.

Summary - Calculations

Waters Corporation: Summary - Calculations

Summary - Calculations

Probably these require an update:

• Processing Methods
• Report Methods
• Displayed Views and View Filters
• Custom Fields

Waters will not change built-in formula in Empower due to data integrity.

• All existing system suitability fields will remain and will still be calculated using the same formulas as in Empower 3.7.0. This provides backwards compatibility with legacy data and custom fields.
  • New fields are proposed for new calculations for future Empower versions.
  • Default multiplier (for S/N) will be set to 20.

European Pharmacopoeia | Addition of chapter 5.26 Implementation of Pharmacopeial Procedures

This general chapter is published for information. It provides guidance on setting up an approach for the implementation of analytical procedures given in monographs of the Ph. Eur. (or ‘pharmacopoeial procedures’hereinafter).

The approach set out below is valid only when used in accordance with the principles laid down in the General Notices (including a suitable quality system).

The term “implementation” is used to describe the overall activities performed, whereas “verification” is used exclusively to refer to the experimental activities.

As the first step of the implementation process, an assessment is performed prior to the first use of the pharmacopoeial procedure in the implementing laboratory. The purpose of this assessment is not to evaluate the intrinsic capability of the procedure, but to determine whether there are any factors associated with the complexity of the procedure and the actual conditions of its use in the implementing laboratory that may affect the performance of the procedure.

If such factors are identified, an experimental verification is the second step to evaluate the analytical procedure performance characteristics (APPCs), such as accuracy and precision, that are considered relevant.

The publication of a revised monograph requires re-evaluation of the implementation of the concerned analytical procedure.

The aim of the pharmacopoeial procedure implementation assessment is to identify any critical factors related to the actual conditions of use in the implementing laboratory that may affect the performance of the procedure.

Such factors may include, but are not limited to, the following:

• the composition of the article under test;
• the complexity of the sample preparation;
• the reagents required to run the procedure;
• the laboratory equipment required to run the procedure;
• the laboratory environment.

Addition of EP chapter 5.26 plus USP <1220>

USP <1220> Analytical Procedure Life Cycle

also advises to conduct a similar assessment, during stage 1 (Procedure Design), for CQAs, to verify/validate them (Procedure Performance Qualification) and continuous monitoring (Ongoing Procedure Performance Verification).

Waters Corporation: USP 1220 Analytical Procedure Life Cycle

Waters Corporation: Addition of EP chapter 5.26 plus USP 1220

Addition of chapter 5.26 Implementation of Pharmacopeial Procedures

USP <1220> Analytical Procedure Life Cycle

heat map capturing a qualitative risk assessment related to sample preparation and HPLC setup steps from ananalytical procedure. Impact levels are strong (red), medium (amber), and minor (green).

Waters Corporation: USP 1220 Analytical Procedure Life Cycle

Assessment Outcome

• if no factors are identified as critical, the procedure may be used in the implementing laboratory without any specific verification experiments to demonstrate its suitability under the actual conditions of use
• if factors are identified as critical, the procedure may be used in the implementing laboratory provided a set of verification experiments evaluating the impact of identified critical factors on selected APPCs is performed to demonstrate the suitability of the analytical procedure under the actual conditions of use

In some cases, the tests prescribed for the purpose of verifying the suitability of analytical procedures in an individual monograph and/or relevant general chapter can be used as a partial or full verification of the corresponding APPCs.