INJECTION SEQUENCE FOR CHROMATOGRAPHY

I.    PURPOSE :

    To have a procedure to follow the injection sequence for HPLC and GC analysis.

II.    SCOPE:

    This SOP is applicable to the HPLC and GC analysis in Quality Control Department.

III.    RESPONSIBILITY:
        It is the responsibility of  Quality Control personnel to follow this SOP.

            
IV.    DOCUMENT REFERENCE :
              SOP    :  Nil.
    Form    :  Nil.
V          PROCEDURE:
•    Ensure that the chromatographic conditions are as per the Method / SOP pertaining to the particular product.
•    Inject the mobile phase or diluent in which the sample is to be dissolved as blank run. [Ensure that the base line is steady and there is no drifting].
•    Perform the system suitability. [For finished/bulk product where it is mentioned in method of analysis]
•    Following the injection sequences mentioned if not any variation specifically mentioned in the relevant STP.
•    Follow the injections sequence in analysis for assay by HPLC of Bulk or finished product as follows: 

•    The system is suitable only if the similarity factor is between 0.98 and 1.02.  Calculate the similarity factor using the following formula:

Peak area of standard solution (1) x Wt. (mg) of standard solution (2)
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Peak area of standard solution (2) x Wt. (mg) of standard solution (1)

If it is so proceed with the following sequence.

•    Calculate the average area of the five injections/As mentioned in the MOA of Working standard preparation (2) and use it in Assay calculation.
•    Assay difference between two preparations should not be more than 1.0 %.
•    The % RSD should be calculated for the Initial five injections of working standard preparation (2).
•    The % RSD should be calculated for the last five injections of working standard preparation (2) whenever a bracketing standard is injected including that injection.
•    Not more than 15 batches shall be analyzed with same set of standard preparations.
•    If %RSD /System suitability fails at a point of time analysis should not be carried forward and the injections after that are invalid.
•    Continue injections for 4th batch if present as follows

• And so on for up to 15 batches if present with same set of standard preparations.

• Follow the injections sequence in analysis for purity by HPLC as follows:

  S. No	Injection	Replicate	Requirement (System suitability – Theoretical plates, Tailing factor and resolution)
  1.	Diluent blank	1	------
  2.	Resolution – If required as per MOA	1	As per MOA
  3.	Impurity low load injections for Quantification If required as per MOA	3	As per MOA
  4.	Main compound low load  If required as per MOA	1	As per MOA
  5.	Test sample preparation Batch 1	1	As per MOA
  6.	Test sample preparation Batch 2 (If present)	1	As per MOA
  7.	Test sample preparation Batch 3 (If present)	1	As per MOA
  8.	Resolution – If required as per MOA / Main compound low load For bracketing	1	As per MOA

• Calculate the purity or impurity as per method of analysis.
• Not more than 15 batches shall be analyzed with same set of standard preparations.

•  

   

• If system suitability fails at a point of time analysis should not be carried forward and the injections after that are invalid.
• Continue injections for 4^th batch if present as follows

• S. No	Injection	Replicate	Requirement (System suitability – Theoretical plates, Tailing factor and resolution)
  1.	Test solution preparation Batch 4 (If present)	1	As per MOA
  2.	Test solution preparation Batch 5 (If present)	1	As per MOA
  3.	Test solution preparation Batch 6 (If present)	1	As per MOA
  4.	Resolution – If required as per MOA	1	As per MOA

• And so on for up to 15 batches if present with same set of standard preparation

• Follow the injections sequence in analysis for OVI / RS by GC for Bulk and finished products as follows.

• S. No	Injection	Replicate	Requirement (System suitability – Resolution, %RSD for replicate injections)
  1.	Diluent blank	1	------
  2.	Resolution – If required as per MOA	1	As per MOA
  3.	STD solution	6	As per MOA
  4.	Test sample preparation Batch 1	1	As per MOA
  5.	Test sample preparation Batch 2 (If present)	1	As per MOA
  6.	Test sample preparation Batch 3 (If present)	1	As per MOA
  7.	STD solution Bracketing standard	1	As per MOA

• Calculate the levels of solvents in the test solution using the average areas of first six standard injections.
• Assay difference between two preparations should not be more than 1.0 %.

•  

• The % RSD should be calculated for the last five injections of standard preparation whenever a bracketing standard is injected including that injection.
• Not more than 9 batches shall be analyzed with a standard preparation.
• If %RSD / System suitability fails at a point of time analysis should not be carried forward and the injections after that are invalid.
• Continue injections for 4th batch if present as follows

• S. No	Injection	Replicate	Requirement (System suitability – Resolution, %RSD for replicate injections)
  1.	Test solution preparation Batch 4 (If present)	1	As per MOA
  2.	Test solution preparation Batch 5 (If present)	1	As per MOA
  3.	Test solution preparation Batch 6 (If present)	1	As per MOA
  4.	Resolution – If required as per MOA	1	As per MOA
  5.	STD solution Bracketing standard	1	As per MOA

• And so on for up to 9 batches if present with a standard preparation
• Follow the injections sequence in analysis of Purity by GC / HPLC for raw materials, in process and intermediate samples as follows: 

• S. No	Injection	Replicate	Requirement (System suitability – Theoretical plates, Tailing factor)
  1.0	Diluent blank	1	--
  2.0	Resolution /System Suitability Injection (If required as per MOA)	As per MOA	As per MOA
  3.0	Working standard	1	As per MOA
  4.0	Test sample Batch 1 (If present)	1	As per MOA
  5.0	Test sample Batch 2 (If present)	1	As per MOA
  6.0	Test sample Batch 3 (If present)	1	As per MOA
  7.0	Resolution /System Suitability Injection (If required as per MOA) OR Working standard	1	As per MOA

• Report results as per method of analysis.
• Not more than 15 batches shall be analyzed with a standard preparation.

•  

• If  System suitability fails at a point of time analysis should not be carried forward and the injections after that are invalid.
• Continue injections for 4th batch if present as follows

• S. No	Injection	Replicate	Requirement (System suitability – Theoretical plates, Tailing factor)
  1.0	Test sample Batch 4 (If present)	1	As per MOA
  2.0	Test sample Batch 5 (If present)	1	As per MOA
  3.0	Test sample Batch 6 (If present)	1	As per MOA
  4.0	Resolution /System Suitability Injection (If required as per MOA) OR Working standard	1	As per MOA

• And so on for up to 9 batches if present with a standard preparation

Injection, sequence, chromatography,