SOP for Good Chromatography Practices

The use of chromatography methods such as High-Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC) in quality control laboratory analysis has increased significantly in recent years. 

Observations during inspections have shown that there was a need for a specific good practices document. HPLC and GC methods are used in, for example, the identification of materials and products, for determination of assay and related substances in materials and products, as well as in validation such as process validation and cleaning validation. 

Due to the criticality of the results obtained through chromatography, it must be ensured that the data acquired meet ALCOA+ principles (i.e. attributable, legible, contemporaneous, original and accurate).

What are Good Chromatography Practices?

Chromatographic methods should be suitable for their intended use. Appropriate values should be specified for parameters such as slope sensitivity, noise threshold, peak width, area threshold, bunching factor and skim ratio.

What is a Chromatographic System?

• The Chromatographic systems should meet regulatory requirements and expectations for GLP. This should include, for example, ensuring that data are acquired, processed, and stored by national legislation and ALCOA+ principles.
• Vendor qualification should ensure that hardware and software are suitable for their intended application.
• Valid agreements should specify the respective responsibilities between the purchaser and supplier and include arrangements for after-sales services.
• Chromatographic systems selected and installed should be appropriate for their intended use.

What is the Chromatographic Method?

• Chromatography, a group of methods for separating very small quantities of complex mixtures, with very high resolution, is one of the most important techniques in environmental analysis. The ability of the modern analytical chemist to detect specific compounds at ng/g or lower levels in such complex matrices as natural waters or animal tissues is due in large part to the development of chromatographic methods.
• The science of chromatography began early in the twentieth century, with the use of columns packed with calcium carbonate to separate plant pigments. The method was developed rapidly in the years and began to be applied to environmental problems with the invention of the electron capture detector (ECD) in 1960 by James Lovelock. This detector, with its specificity and very high sensitivity toward halogenated organic compounds, was just what was needed to determine traces of pesticides in soils, food and water and halocarbon gases in the atmosphere. This happened at exactly the time when the effect of anthropogenic chemicals on many environmental systems was becoming an issue of public concern. Within a year, it was being applied to pesticide analysis. The pernicious effects of long lived, bioaccumulating pesticides, such as DDT, would have been very difficult to detect without the use of the ECD. The effect of this information on public policy has been far-reaching.
• The basis of all types of chromatography is the partition of the sample compounds between a stationary phase and a mobile phase which flows over or through the stationary phase. Different combinations of gaseous or liquid phases give rise to the types of chromatography used in analysis, namely gas chromatography (GC), liquid chromatography (LC), thin layer chromatography (TLC), and supercritical fluid chromatography (SFC).
• Chromatography has increased the utility of several types of spectroscopy, by delivering separate components of a complex sample, one at a time, to the spectrometer. This combination of the separating power of chromatography with the identification and quantitation of spectroscopy has been most influential in environmental analysis. It has enabled analysts to cope with tremendously complex and extremely dilute samples.

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Good Chromatography Practices

OBJECTIVE

To lay down a procedure for good chromatography practices.

SCOPE

This SOP shall be applicable for HPLC & GC analysis performed in the quality control department.

RESPONSIBILITY

QC officer / QC chemist

ACCOUNTABILITY

HOD QC

PROCEDURE

What is the System Suitability Test (SST)?

• System suitability test (SST) is a test to determine the suitability and effectiveness of chromatographic systems prior to use. The performance of any chromatographic system may continuously change during its regular use, which can affect the reliability of the analytical results. 
• The operation parameters of the whole chromatographic system can be checked with properly selected SST mixtures. These mixtures are used to establish characteristic chromatographic parameters, such as the number of effective theoretical plates, resolution, asymmetry, detection limit, and selectivity. 
• The system is then only declared suitable if the responses are within given limits.

System Suitability

• Consider peak area/peak height as a measure of response from the detector as specified in the method of analysis. While entering the system suitability data in the notebook, only average area and %RSD obtained from the summary report shall be entered.
• When system suitability requirements are specified in the method of analysis, only those parameters will be considered.

• When limit for %RSD of replicate injections of a standard solution is specified as 2.0% or less, then five consecutive injections of reference solution will be made, unless otherwise specified.
• When limit for %RSD of replicate injections of a standard solution is specified as more than 2.0%, then six consecutive injections of the standard solution will be made, unless otherwise specified.
• For calculation of %RSD consecutive 5 or 6 sequential standards shall be considered.
• Inject bracketing standard as specified in the method of analysis and calculate %RSD with respect to original system suitability. For the bracketing standard, the limit or % RSD will be 1.5 times than the original limit.

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System suitability parameters are not specified, then following parameters shall be considered.

• For tests of assay, dissolution, content uniformity; five replicate injections of reference/standard solution shall be made & RSD limit will be not more than 2.0%.
• For the tests of related substances (other than British Pharmacopeia) where %RSD limit of replicate injections is not specified, only a single injection of the reference / standard solution will be made.
• For the tests of related substances (of the British Pharmacopeia) where %RSD limit of replicate injections is not specified, minimum three replicate injections of the reference / standard solution will be made & %RSD limit will be not more than 5.0%.
• For the tests of the residual solvents %RSD limit of six replicate injections will be 15% whenever not specified in the specifications.

In the event of any interruption maximum up to six hours during HPLC run due to 

1. Electric interruption
2. Computer interruption
3. The time gap between system suitability injection and sample injection is due to a delay in sample receipt.
4. The time gap between the two sets of samples

• Bracketing standards shall be injected before proceeding with further next sample injection.
• The above shall be applicable only in case of the system is in the continuous state of equilibration.
• The deviation between retention times of standard or sample injections should not be more than 15% from the specified retention time in the method of analysis. For related substances test, the retention time of the sample shall be compared with the specified retention time.
• In the test of related substances, run the chromatogram for at least 1.5 times the retention time of the principal peak unless otherwise specified.
• Adjustment allowed for Liquid chromatography conditions.

1. Following mentioned adjustment to the chromatographic conditions can be made to meet system suitability criteria, Prior permission of QC Head/Designee should be taken before proceeding with any change in the chromatographic conditions.
2. Composition of the Mobile Phase: the amount of the minor solvent component may be adjusted by ± 10% relative or ± 2% absolute, whichever is the larger. No other component is altered by more than 10% absolute
3. pH of the aqueous component of the mobile phase: ± 0.05 pH unless otherwise stated in the monograph, or ± 1.0 pH when neutral substances are to be examined. The salts concentration in a mobile phase's buffer component: ±10%.
4. Detector Wavelength: no adjustment permitted
5. Stationary Phase: Column Length: ± 70%, Column Internal Diameter: ± 25%
6. Particle Size: maximal reduction of 50%, no increase permitted.
7. Flow Rate: ± 50%. When in a monograph the retention time of the principle peak is indicated, the flow rate has to be adjusted if the column internal diameter has been changed. No decrease of flow rate is permitted if the monograph uses an apparent number of theoretical plates in the qualification section.
8. Temperature: ± 10%, to a maximum of 60°C
9. Injection Volume: may be decreased, provided detection and repeatability of the peak (s) to be determined are satisfactory.
10. Gradient Elution: the configuration of the equipment employed may significantly alter the resolution, retention time and relative retentions described in the method. Should this occur, it may be due to excessive dwell volume which is the volume between the point at which the 2 eluents meet and the top of the column.

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Adjustment allowed for Gas chromatography

• Stationary phase: NO
• Column length: ± 70%,
• Column internal diameter: ± 50%
• Particle size: maximal reduction of 50%, no increase permitted,
• Film thickness: -50% to + 100%
• Flow rate: ± 50%.
• Temperature: ± 10%
• Injection volume: may be decreased, providing detection and repeatability are satisfactory.

Data Generation

• Follow the method of analysis & respective standard operating procedures to create appropriate method files, data files, and sequence files.
• Method Files

1. Once the method file is created check it for compliance as per the requirement of the method of analysis & save it.
2. When exact integration parameters or instrument parameters are not known then an atrial run can be taken to identify the integration & or instrument parameters. After a trial run, the integration & or instrument parameters can be fixed by saving the method.
3. For related substances / chromatographic purity test, peak up to void volume is to be inhibited, All data must be integrated using the same integration parameters throughout the chromatographic analysis.
4. The integration parameters must be optimized for the entire analysis before processing to obtain between the representation of chromatography, While saving the changed method, always enter the reason in the software.
5. After the analysis is over, take prints of method parameters & sign

Data Files

• Prior to any sample injection single or sequence, feed the required data file name with the required data path as per software requirement. Select the data file name, that is not previously used.
• If data reprocessing is required with or without parameter change, then all the related data files like blank, standard, samples generated during that particular analysis will be reprocessed at the same time.
• During or after the sequence run, the data file print will be taken & signed. If printouts are being taken online & after completion of injection but before completion of the entire sequence, ensure that same processing method is being used for all the injections.
• During or after sequence run, data file print will be taken & signed. If printouts are being taken online & after completion of injection but before completion of the entire sequence, ensure that the same processing method is being used for all the injections.
• Data description should be present on the data file whether printed or written manually on the printed data.

Sequence Files

• While preparing any sequence always ensure that data file names given are not being repeated. The product information like stage, batch number/ A.R. number, test name product name strength, stability conditions etc can be entered in the report through the sequence.
• Once the required sequence file is created & saved, load it to run the test.
• A sequence can be amended or altered before running any particular analysis and Printouts of sequence parameters are taken & signed after testing.

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Log-Book Entries

• Data files, which are generated during analysis & not considered due to failure injections or some other reason, will be attached with the same set of data.
• All such extra data will be signed & justification will be written on the printed data about their non-consideration & same shall be approved by QC.
• The analyst shall sign the printed data.
• The results like dissolution, dissolution profile, content uniformity and K factor obtained using excel sheet can directly be pasted in the notebook as final results & processing methods(s) and same shall be reviewed by QC.

Column Care

• All HPLC & GC columns are to be handled with due care.
• Always keep columns in their respective casing or box.
• Avoid all types of jerks or falls of columns
• Always flush columns with miscible solvents if used one after other.
• Always replace end fittings of the respective columns after use.
• Check the direction of the column before attaching it
• Always check for any leakage after attaching the column, & then only close the lid of column compartment

Precautions

• Always ensure that no air bubbles are entrapped in the solvent or mobile phase path.
• Prior to injection, use syringe filters or centrifugation of solutions as applicable.
• Always use filtered (through 0.45μ or less porosity filters) & sonicated mobile phase only
• Before running any sequence, ensure that all the respective solutions (in vials), are located properly in the auto-sampler tray (s) as per the sequence.
• Always check mobile phase or solutions for the absence of turbidity or opalescence, before & during use
• During GC analysis always ensure that the flow of carrier gas, as well as other gases, is as per the requirement.
• Inject blank solution intermittently to nullify the effect of carryover if required.

ABBREVIATIONS

QA: Quality Assurance

QC: Quality Control

RSD: Relative Standard Deviation

No. : Number

SOP: Standard Operating Procedure.

HOD: Head of Department

HPLC: High-Pressure Liquid Chromatography

GC: Gas Chromatography

REVISION HISTORY

Nil

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