Calculations in Analytical Methods(HPLC and GC)

I want to write this topic for giving an idea about how to calculate the Assay, related substances and Residual solvents.

Calculations for Assay Method (Tirtimetry)

Titire value(Sample - Blank) x M x Factor x 100 x 100

--------------------------------------------------------

                            Ws x (100-LOD)

M= Molarity of the volumetric solution 

Ws= Weight of the sample 

Factor = Factor for Drug substance 

LOD is Loss on drying or MC is water content 

Calculations for Assay Substances Method (HPLC)

             

                    At x Ws x P x (100-WC of std)

Assay % = ----------------------------------

                    As x Wt x (100-WC of sample)

At = Area due to sample Preparation 

As= Area due to working standard solution 

Wt= Weight of the sample in mg

Ws= Weight of the working standard

P=Potency  or Assay of the standard 

WC=% water content of the sample or standard 

Note: WC may be replaced by LOD based on the drug substance 

Calculations for Related Substances Method (HPLC)

                                          ri

% of Known Impurity = ---x 100 X RF

                                         rs

                                              ri

% of unknown Impurity = ---x 100

                                              rs

Total Impurities = Sum of all known and unknown impurities 

ri =Area of each impurity Peak in the chromatogram of the sample solution preparation

rs=Sum of areas of Main drug and all impurity Peaks in the chromatogram of the sample solution preparation

RF= Response factor 

Calculations for Residual solvents Method (GC)

     

    At        Volume of solvent taken x density     0.1 ml       dilution of sample(ml)

= ------x -------------------------------------------x ---------x-------------------------------x 10 ^ 6

    As                     10 ml                                                                   10 ml        Weight of the sample

At= Area of the solvent in the sample - Blank

As= Area of the solvents in the standard -blank 

Note: Volume of the solvents taken should be mentioned in microliters 

As we are multiplying with 10 power 6 we can get the results in PPM 

Note: Refer Merck index for solvent densities

Types of Analytical Procedures

Analytical procedures are used to evaluate characteristics of the drug substance. Analytical procedures are classified into three types. There are

1. Regulatory analytical procedures
2. Alternative Analytical procedures
3. Stability Indicating Methods
 

Regulatory Analytical Procedures or Compendial Methods
The analytical procedures which are defined in the USP/EP/EP/JP/IP etc.. are termed as regulatory analytical procedure for a particular Drug substance. If the manufacturer claims that his/her material is EPgrade, then the manufacturer must analyse the drug substances as per the procedures given in the EP and the material must meet the specifications given by EP. 

Eg: Famotidine, Ciprofloxacin Hydrochloride etc..

Alternative Analytical Procedures or In-House procedures:
There are situations were the regulatory analytical procedures may not be sufficient to analyze the drug substance, in such cases applicant can come up with own analytical methods. These methods are called as alternative analytical procedures. The method should be same or better than the regulatory method and fully validated and also comparative to the regulatory analytical procedures.
 

Stability Indicating Methods(SIM):
A SIM is a quantitative analytical procedure used to detect a decrease in the amount of the active pharmaceutical ingredient (API) present due to degradation.

According to FDA guidelines, a SIM is defined as a validated analytical procedure that accurately and precisely measures active ingredients (drug substance or drug product) free from potential interferences like degradation products, process impurities, excipients, or other potential impurities.

All the assay procedures for stability studies be stability indicating.