Superseded Protocol No. Nil
Effective Date  


Prepared By:

Functional Area Name Designation Signature/ Date
Quality Control      

Reviewed By:

Functional Area Name Designation Signature/Date
Quality Assurance      
Head Quality Control      

Approved By:

Functional Area Name Designation Signature/Date
Head QA      

Authorized By:

Functional Area Name Designation Signature/Date
Head Quality      

Table of contents :

Sr. No. Subject
  Responsibility of Validation Team
  Product Profile
  Revision History
  1. Objective:                                                                                                                                                     The objective of this validation is to validate the method validation of residual solvent in Citalopram Hydrobromide by considering thefollowing parameters:
  2. Specificity
  3. Precision
  4. System Precision
  5. Method Precision
  6. Intermediate Precision (Ruggedness)
  7. Limit of Detection and Limit of Quantification(LOD & LOQ)
  8. Linearity and Range
  9. Accuracy
  10. Stability of Analytical Solutions
  11. Robustness  

Following parameters will be seen during system suitability:

Sr. No. Parameters Limit
1. % RSD for peak areas of each solvent obtain by 6 replicate injections of standard solution. Not more than 15.0

When the above parameters meet the method, the actual experiment shall be started.

  1. Scope :

This protocol is based on SOP and applicable for the method validation of residual solvent in Citalopram Hydrobromide.

  • Responsibility of Validation Team:
Departments Responsibilities
QC Preparation & Review of Protocol.
Analysis of samples and recording of data.
Compilation and checking of data
Preparation of Summary Report.
QA Review and approval of protocol.
Co-ordination with QC to carryout Validation.
Review of data and summary report.
Head Quality Authorization of protocol.
  • Product Profile:
Category Residual solvent
Reason for Validation 1st Validation
Methodology  In-House
Method Reference In-House
Specification Limits (ppm) Acetone                : NMT 3000 ppm
Tetrahydrofuran    : NMT 720 ppm
Methanol               : NMT 3000ppm
Isopropyl alcohol   : NMT 5000ppm      
Toluene                  : NMT 890ppm
  • Methodology:

Residual solvents by GC

Instrument: A gas chromatograph system is equipped with flame ionization detector and


GC Conditions

Column                               : DB-FFAP or equivalent

Length                                : 30.0 meters

Film thickness                    : 1.00 µm

ID                                        : 0.53 mm

Carrier gas                          : Nitrogen

Flow rate of nitrogen          : 3.0 ml/min

Injector port temperature    : 220°C

Detector port  temperature  : 220°C

Split ratio                             : 1:10

Injection volume                  : 1.0 µl

Sample Run time                : 15 minutes

Column temperature          : Set and hold at 40°C for 3 minutes, raise to 220°C at a rate of 30°C

per minute, then hold for 6 minutes, Let the instrument gets cooled of

 its own to come to the normal temperature.

Solvent A Pre wash      : 3                                           Solvent B Pre wash     : 3       

Sample wash                : 3                                           Sample pump               : 3

Solvent A Post wash    : 3                                          Solvent B Post wash     : 3

Viscosity Delay : 0 Sec   

Preparation of stock solution:

  • Transfer 112.5 mg of Acetone,  27mg of Tetrahydrofuran, 112.5 mg of Methanol, 187.5 mg of Isopropyl alcohol  and 33.40 of Toluene in 25 ml volumetric flask containing 10.0 ml of Dimethyl Sulfoxide. Make up to volume with Dimethyl Sulfoxide.

Preparation of standard solution:

  • Transfer 1.0ml of stock solution in to a 100 ml volumetric flask containing 20 ml of Dimethyl Sulfoxide. Make up to the volume with Dimethyl Sulfoxide.

Preparation of sample solution:

  • Transfer about 150.0 mg of test sample into a 10 ml volumetric flask.
  • Dissolve and makeup with Dimethyl Sulfoxide. (15 mg/ml concentration).

System suitability:

  • Inject standard solution six times into the chromatographic system.
  • Record the chromatograms obtained with standard solution and measure the peak responses.
  • The relative standard deviation of the individual peak responses from six replicate injections should be less than 15.0 %.


  • Inject DMSO as blank solution and record the chromatographic system.
  • Record the chromatogram obtained with blank solution and measure the peak responses.
  • Inject standard solution six times into the chromatographic system.
  • Record the chromatograms obtained with standard solution and measure the peak responses.
  • Inject test sample solution two times  into the chromatographic system.
  • Record the chromatogram obtained with sample solution and measure the peak responses.
  • Identify the peaks in the chromatograms of the sample preparation, based on the retention time of corresponding peaks present in the standard preparation.
  • Calculate and note down the peak responses of individual components from the sample and standard preparations.
  • Apply the blank correction in case of any interference from the solvent used as diluent.
  • Calculate the amount of residual solvents present in the sample by the formula given below.


AT – AB           WS           1           10

————- x ————x———x ———- x P x10000

AS – AB            25          100        WT


AT = Peak area of each solvent observed in test sample chromatogram.

AB = Peak area of each solvent observed in blank chromatogram.

AS = Average area of each solvent observed in standard chromatogram.

Ws = Weight of the respective standard in standard solution in mg test.

Wt = Weight of the test sample taken.

P = Potency of respective standard.

  • Specificity:

Specificity of test method shall be established by injecting all solvents to be examined simultaneously. Prepare blank solution, standard solutions individually, mixes standard and spiked test sample as per method of analysis. Retention time observed for each solvent shall be recorded

Acceptance criteria:

Retention times of each solvent should match in, individual, mixed and spiked solution and  there should no interference at the retention time of the solvents.

  • Precision:System Precision:

Prepare and inject separately Blank and Standard solution at target level. Calculate the % RSD of response for six replicate injections.

Analysis sequence

Blank – 1 injection

Standard solution – 6 injections

Acceptance criteria

The relative standard deviation for peak area counts is not more than 15.0%.

  • Method Precision:

Prepare test sample six times from the same batch and analyze as per the method and report the content of each solvent    

Acceptance criteria

% RSD of content of each solvent in six sample preparations should be NMT 10.0  

  • Intermediate Precision ( Ruggedness ):

The analysis of the same batch will be done in six replicate analyses by using different columns by different analyst, by different system on different day. The mean, standard deviation and relative standard deviation will be calculated.


Calculate the mean, standard deviation, and % RSD for the operators and instruments and record on data sheet.

         Acceptance criteria:

    % RSD of content of each solvent in six sample preparations should be NMT 10.0

  • Determination of LOD and LOQ:

LOD and LOQ will be estimated based on the signal to noise ratio values .Determine the LOD and LOQ of each solvent theoretically having S/N ratio more than 3 and 10 respectively. These value shall be calculated from the signal to noise ratio of 100% specification level of standard.                                                       

Confirmation of LOD:                                                                                                                                                                                     

Prepare the LOD  solution of mixed standard as determined from the above and confirm by injecting solution in six times.

         Acceptance criteria:

The % RSD for the peak area should be NMT 30.0 and S/N ratio of the peak should be NLT 3.0.

Precision at LOQ:

Prepare a solution containing mixed standard at LOQ concentration and analyze six  times.                      

Determine the %RSD of peak area.

         Acceptance criteria:

The % RSD for the peak area should be NMT 10.0 and the S/N ratio should be NLT 10.0

  • Linearity and range:

    The linearity of an analytical procedure is its ability (within a given range) to obtained test results which are directly proportional to the concentration levels shall be prepared.

    Determine the linearity by preparing and inject the standard solution in the range of LOQ to 120% of concentration level and calculate the correlation coefficient “r”.

Test Procedure:

Prepare the standard solutions at five concentrations, typically LOQ, 50%, 80%, 100% and 120% of target concentration following finished product testing procedure. Duplicate replicates at each concentration will be analyzed.


Record results on a datasheet. Calculate the mean, standard deviation, and Relative Standard Deviation (RSD) for each concentration. Plot concentration (x-axis) versus mean response (y-axis) for each concentration. Calculate the correlation coefficient (r). Record these calculations on the datasheet.

Acceptance criteria

The correlation coefficient (r) shall be not less than 0.99.  

  • Accuracy:

The accuracy of an analytical procedure express the closeness of agreement between the value which accepted either as a conventional true value or an accepted reference value and the value found. The accuracy shall be established across the specified range of the analytical procedure.

Test Procedure:

Accuracy can be accessed by spiking the standard solution of each solvent into test sample at levels 50%, 100% and 150% with respect to target levels. Analyze the solutions in three sample preparation at each level. Calculate the % RSD for recovery obtained at each level separately and overall %RSD.

Acceptance criteria

The mean recovery at each level shall be between 85% to 115 % .

  • Solution stability:

    It is essential when validating an analytical method to confirm that the analyte has adequate stability in both the standard and sample solution during analytical measurement stages of the testing.

    Test Procedure:

    Inject blank, six replicates standard solution and one injection of the sample solution.

Acceptance criteria

    % RSD of peak areas observed in standard and sample solution injected at different time  intervals should not be more than 15%.                                                                                       

     Note: If the solvent are not detected in the sample inject spike sample solution.

  • Robustness:

The method shall show reliability of an analysis with respect to deliberate variation in method parameters.

Following deliberate variations shall be done in method parameters:

  • By changing the flow rate by ±5%
  • By changing the Oven temperature by ±2°C                                                                                                                                                                                                                                                                                                                                   

Test Procedure:

Inject blank, six replicate injections of standard solution and two injection of spiked sample solutions.

Acceptance criteria

The system suitability and specificity should meet.

  •   Revision History:
Revision No.   Details of changes Reason
00   New Document Nil

Bhanu Pratap Singh


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