HPLC analysis and system suitability check procedure

HPLC analysis and system suitability; Purpose:

The purpose of this SOP is to describe the procedure for HPLC analysis and checking of system suitability.

HPLC analysis and system suitability; Scope

This procedure is applicable to all HPLC analysis that will be carried out in quality control laboratory of XX Pharmaceuticals Limited.

Definitions/Abbreviation

• Standard Operating Procedure (SOP)
• Standard Operating Procedure. A written authorized procedure, which gives instructions for performing operations.
• HPLC: High performance liquid chromatography
• QC: Quality Control
• RSD: Relative standard deviation

Responsibilities

The roles and responsibility is as follows:

Officer/Executive/ Senior Executive, Quality Control

• To ensure that the instructions of this procedure are correctly followed.
• To maintain the records properly as per SOP

Assistant Manager, Quality Control

• To confirm that this procedure is kept up to date.
• To check that the SOP reflects the required working practices.
• To organize training on the SOP to all concerned personnel & to ensure implementation of the SOP after training.

Head of Quality Assurance

• Take initiative to approval of the SOP.
• To confirm the overall implementation of the SOP.

Procedure:

General Precaution(s):

• Ensure that the mobile phase is filtered and degassed properly by sonication before use.
• Suggested injection procedure should be run in one single sequence.

Analytical procedure:

• Prepare desired mobile phase, filtered by 0.22 or 0.45 µm membrane filter & degassed for at least 5 minutes with the help of sonication.
• Equilibrate the column for at least 30 minutes or more until baseline gets stabilized with desired mobile phase & check the baseline to ensure the system is ready for injection.
• Prepare standard solution & assay sample and keep record of weights.
• At first inject blank to confirm the absence of unknown peak at the retention time of Principal peak & then inject standard solution to check retention time of the principal peak of interest. Run chromatogram minimum 2 minutes extra after principal peak elution is over and peak is properly integrated.
• Unless otherwise specified in the individual method, data from five replicate injections of standard are used to calculate RSD, if the requirement is 2.0% or less; data from six replicate injections are used if the RSD requirement is more than 2.0%. Tailing factor of principal peak of interest should be between 0.8 and 1.5 unless otherwise stated in individual method.
• Inject standard & assay preparation solution into the HPLC system as per suggested injection procedure previously mentioned.
• Following are the suggested injection procedure to be followed unless otherwise specified in respective method.

Injection procedure for test homogeneity of Blend/Granules sample

Sample ID with No. of Injection

• Blank: 1 Time
• Resolution or System Suitability solution (If applicable) : 1 Time
• Standard: 5 Times
• Sample-1: 1 Time
• Sample-2: 1 Time
• Sample-3: 1 Time
• Sample-4: 1 Time
• Sample-5: 1 Time
• Sample-6: 1 Time
• End Standard: 1 Time

Injection procedure for Assay

• Blank: 1 Time
• Resolution or System Suitability solution (If applicable) : 1 Time
• Standard: 5 Times
• Sample: 1 Time
• End Standard: 1 Time

Injection procedure for dissolution

• Blank: 1 Time
• Resolution or System Suitability solution (If applicable) : 1 Time
• Standard: 5 Times
• Sample-1: 1 Time
• Sample-2: 1 Time
• Sample-3: 1 Time
• Sample-4: 1 Time
• Sample-5: 1 Time
• Sample-6: 1 Time
• End Standard: 1 Time

Injection procedure for Content Uniformity

• Blank: 1 Time
• Resolution or System Suitability solution (If applicable) : 1 Time
• Standard: 5 Times
• Sample-1: 1 Time
• Sample-2: 1 Time
• Sample-3: 1 Time
• Sample-4: 1 Time
• Sample-5: 1 Time
• Sample-6: 1 Time
• Sample-7: 1 Time
• Sample-8: 1 Time
• Sample-9: 1 Time
• Sample-10: 1 Time
• End Standard: 1 Time
• For injection of more than 12 samples, run end standard after every 12th sample.
• Calculate the result comparing the area of sample solution with the average area of 5th standard (6th standard if six replicate injections required) and respective end standard.

Chromatogram review and documentation:

The custom report covers the following information and can be modified as per the specific need.

• Acquired by
• Sample Name
• Sample ID
• Tray No.
• Vial No.
• Injection Volume
• Data File
• Method File
• Batch File
• Date Acquired

The peak table in custom report for standard covers the following data, however select other data as per requirement.

• Title
• Sample Name
• Sample ID
• Ret. Time
• Area
• Theoretical Plate
• Tailing Factor
• Resolution

The peak table in custom report for sample covers the following data, however select other data as per requirement.

• Title
• Sample Name
• Sample ID
• Ret. Time
• Area
• Result
• Unit

General guideline:

• In the test for Assay, run all Standard & Sample chromatograms minimum 2 minutes extra after the principal peak elution is over & peak is properly integrated or as per the method.
• In Chromatographic Purity/Degradation/Related Substances, run the chromatogram 2.5 times the retention time of principal peak or as specified in individual method. In case of specific impurity analysis, run the chromatogram minimum 2 minutes extra after the principal peak elution is over and peak is properly integrated.

Allowable modification in chromatographic system:

Following are general criteria, which provide extent of allowable variation to get system suitability. The adjustments are allowed only to improve quality of chromatography unless otherwise directed in the respective method/pharmacopoeial monograph.

pH of mobile phase:

pH of aqueous buffer used in mobile phase preparation can be adjusted to within ± 0.2 units of the value or range specified. (Example: If specified pH is 7.0 then allowable limit for adjustment is 6.80 – 7.20)

Concentration of salts in buffer:

Concentration of salts used in the preparation of aqueous buffer employed in mobile phase can be adjusted within ± 10% if the permitted pH variation is met. (Example: If specified concentration is 1.0% then allowable limit for adjustment is 0.90%–1.10%)

Ratio of components in the mobile phase

Following adjustment limits apply to minor components of the mobile phase (specified at 50% or less). The amount(s) of these component(s) can be adjusted + 30% relative. However the change in any component cannot exceed + 10% of absolute (i.e. in relation to the total mobile phase). Adjustment can be made to one minor component in ternary mixture. Examples of adjustments for binary and ternary mixture are given below.

Binary mixtures

Specified ratio of 50:50: 30% of 50 is 15% absolute, but this exceeds the maximum permitted change of + 10% absolute in either component. Therefore, the mobile phase ratio may be adjusted only within the range of 40:60 to 60:40.

Specified ratio of 2:98: 30%of 2 is 0.6% absolute. Therefore the maximum allowed adjustment is within the range of 1.4:98.6 to 2.6:97.4.

Ternary Mixtures

Specified ratio of 60:35:5: For the second component, 30% of 35 is 10.5% absolute, which exceeds the maximum permitted change of + 10% absolute in any component. Therefore the second component may be adjusted only within the range of 25% to 45% absolute. For the third component, 30% of 5 is 1.5% absolute. In all cases, a sufficient quantity of the first component is used to give a total of 100%. Therefore, mixture range of 50:45:5 to 70:25:5 or 58.5:35:6.5 to 61.5:35:3.5 would meet the requirement.

Detector wavelength

• Deviations from the wavelengths specified in the method are not permitted.
• Stationary phase:
• Column length: ±70%.
• Column internal diameter: ±25%.
• Particle size: Maximum reduction of 50%, No Increase    permitted.
• Flow rate: When the particle size is changed, the flow rate may require adjustment, because smaller particle columns will require higher linear velocities for the same performance (as measured by reduced plate height). Flow rate changes for both a change in column diameter and particle size can be made by:

F₂=F₁x [(dc₂2xdp₁)/( dc₁2xdp₂)]

Where F1 and F2 are the flow rates for the original and modified conditions, respectively; dc₁ and dc₂ are the respective column diameters; and dp₁ and dp₂ are the particle sizes.

When column dimensions are changed, the flow rate may be adjusted using the following equation:

F₂=F₁x [(l₂d₂2/( l₁d₁2)]

Where F₁ and F₂ are the flow rates for the original and modified conditions, respectively; l₁ and l₂ are the respective column lengths; and d₁ and d₂ are the diameters.

• Injection volume: Injection volume can be reduced as far as consistent with acceptance precision and detection limits; no increase is permitted.

Column temperature: ±10°C.      F₂=F₁x [(l₂d₂2/( l₁d₁2)]

This is all about

HPLC analysis and system suitability.