sop for Chromatographic practices and system suitability

1.0. OBJECTIVE:
To lay down a procedure for Chromatographic practices and system suitability.

2.0. SCOPE:

This SOP is applicable for procedure of Chromatographic practices and system suitability in quality control department

3.0. RESPONSIBILITY:
Executive /Officer QC

4.0 ACCOUNTABILITY:
Head- Quality Control

5.0 DEFINITION:
Chromatography is a Laboratory technique for the identification and separation of drugs mixture. Where it is used on a large

scale to purify the intermediates and products in various synthesis in Pharmaceuticals industries etc.

PROCEDURE
Standard Practices:
Chromatographic conditions are set as per the method specified in respective standard testing

Procedure.

Analyst should not change any system parameters without prior information to Head-QC. If any change in system

parameters is performed then it should be acknowledged and properly documented.
Prepare mobile phase, according to STP or Pharmacopoeia and filter with 0.45 µ filter.
Mobile phase bottles and all solvents bottles in use should be labeled.
Mobile phase should be freshly prepared for analysis and solvents should not be used for more than 72 hours.
Check the solvent quantity in solvent bottles before start the instrument.; no any air bubbles should be observed in line. Solvent bottles should be covered.
Check no any blockage in wastage line and outlet of wastage line not touch with surface of waste bottles.
Waste bottles wastage should be discarded when it will be fill.
Hot water should be run once in 07 days for about 1 hour for all HPLC instrument. This removes any particulate matter that may cause blockages, Pump plungers, seals
Buffers should be prepared freshly on the day required. and solvents
must be filtered through 0.45 micron filter paper.

Freshly prepared mobile phase should be thoroughly degassed to remove all dissolved gases with sonicator.
Ensure that the buffer is soluble in the proposed wash or storage phase. If it is not, first flush the system with a solvent mix that is highly aqueous to remove the buffer from the system and column , then change to the proposed wash or storage solvent mix.
integration procedure for assay

Integrate all samples and standards both with chromatograms within a sequence using the same integration events.
Check the each chromatogram within a sequence to ensure that integration is correct.
integration procedure for related substances

Integrate the standard chromatograms within a sequence using the same integration events.
If required same integration method for Blank, Placebo, system suitability injections can be prepared & in test injection, auto integration can be performed, if required.
Integrate the sample chromatograms within a sequence using the integration events and disregard the blank and placebo peaks. Integrate the known and unknown peaks

in sample chromatograms with using following types of integrations.

Check each chromatogram to ensure correct integration obtained for all relevant peaks. After peak integration, save the processing methods for Blank, Placebo, Standard, System suitability and sample injections.
baseline to baseline integration

integration baseline is a continuation of the existing baseline. Begin integration when the signal first begins to rise above the baseline and end when the signal returns to the original baseline.
types of integration practices

Pictorial representation of peak area, peak height and Gaussian peak shape are as follows:

Base to Base: The peak should be symmetrically shaped and exhibits no indication of co-elution. The baseline is stable

and returns to the same level. Peaks of this nature are usually appropriately integrated baseline-to-baseline integration.
Below mentioned is an example of improperly integrated peak. The tailing facet of the peak has important area unit that

has been eliminated that shall be enclosed within the peak and another one is an example for improperly elevated baseline.

This clearly excludes an outsized space of peak: during this case a base-to-base integration is correct apply.

When peak eluted on falling of baseline, excessive baseline area need to be eliminated as below manner:

When excessive peak tailing is observed peak shall be integrated covering its complete area

base-to-base with dropline:
The drop methodology involves addition of a vertical line from the valley between the peaks to the horizontal baseline

that is drawn between the beginning and stop points of the peak group.

VALLEY TO VALLEY: This timed events sets the baseline from the peak starts and where it ends in a less resolved peak Integrate each peak separately.

.System Suitability :

During HPLC analysis, Analyst injects the blank and standard preparation as per the test procedure and checks the chromatogram

for peak shape and system suitability parameters like Theoretical plates, Tailing factor and resolution, etc. if applicable.
System Suitability is established as per the procedure and shall be determined by the first chromatogram of replicate injections of standard solution except resolution.
Following system suitability criteria shall be consider if not mentioned in pharmacopoeia for Theoretical

plates NLT 1500, tailing factor NMT 2 and RSD of three replicate injection NMT 2.0%.
During analysis of Bulk and In-process sample 3 standards shall be injected.

Sequence:

Sequence of blank, standard and sample injections are followed as – Blank, replicate standard solution, duplicate

injection of sample solution for assay and one Bracketing standard solution or as specified in respective standard test procedure.

After injections of sample Solution (i.e. in assay) inject bracketing standard solution or as specified in the respective standard test procedure.
In case of content uniformity (CU), after 5 injections inject bracketing standard solution or as specified in the respective standard test procedure.
RSD shall be calculated with Bracketing solution and standard solution or specified in respective standard test procedure.
Assay calculation should be done using the mean area of replicate injections of standard solution.
An acceptance criteria of relative standard deviation (RSD) with bracketing standard is NMT 2.0%.
If the acceptance criteria for RSD not met for any bracketing standard the analysis in between of those run shall be

canceled with proper justification and authorization of Head-QC.
In case of Content uniformity and related substances single injection of sample solution shall be injected, rest

of the injection sequence shall remains the same as specified under assay.
At the end of sequence analyst shall add the column washing method.

Procedure for Column washing

Column washing should be performed with appropriate solvent.

Column should be stored in organic solvent.

After each batch table column washing should be added.

Column washing should be performed for at least 30 minutes.

Columns washing should be done by using ratio of Solvent and water in 30:70.

general:

Section In charge shall prepare the instrument method.

Before run the sequence, Instrument method shall be checked as per STP by Section In charge / designee.
documentation

System suitability chromatogram, sequence table, and chromatographic method (Instrument method) print shall be attach with sample report.
Every chromatogram and page which is compile with report shall be duly signed and checked.

7.0 ABBREVIATION:

SOP Standard Operating Procedure

QC Quality Control

STP Standard Testing Procedure

CU Content Uniformity

RSD Relative Standard Deviation

8.0.
ANNEXURES :
NA

9.0
DISTRIBUTION:
Controlled Copy Quality Control Department
Master Copy Quality Assurance Department

10.0
REFERENCES:
In-House

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