Methods of Impurities Calculation

 

Methods of Impurities Calculation

According to ICH guidelines impurities can be broadly classified into three categories as Organic Impurities, Inorganic Impurities and Residual Solvents. Related substances are defined as impurities derived from the drug substance and therefore not including impurities from excipients. Related substances include degradation products, synthetic impurities of drug substance and manufacturing process impurities from the drug product. Various concepts are used for the quantitation of impurities which are elaborated in this article.

 The following calculation approaches can be used:

1.      External standard approach against quantitative RS for impurities.

In this method quantitation is performed against an external standard of impurity itself. An external standard also serves as positive impurity peak identification rather than relying on relative retention times which are subject to reliability. Quantitation of impurities using this method provides accurate results.

For example: Related substances calculation for Gabapentin tablets:

Standard solution—Dissolve accurately weighed quantities of USP Gabapentin RS and USP Gabapentin Related Compound A RS in Diluent to obtain a solution having a known concentration of about 0.04 mg of each per mL.

 

Test solution—Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder,equivalent to about 500 mg of gabapentin, to a suitable volumetric flask, and dissolve the contents in Diluent with sonication, if necessary, for about 30 seconds. Dilute with Diluent to volume, and mix to obtain a final solution having a known concentration of about 20 mg per mL, based on the label claim.

 

Calculate the percentage of gabapentin related compound A in the portion of Tablets taken by the formula:

 

100(CS / CT)(rU / rS)

 

                      Area of RCA in test        0.04  

% RCA = --------------------------------- x ---------  x 100

                 Area of RCA in Standard     20        

 

in which CS is the concentration, in mg per mL, of USP Gabapentin Related Compound A RS in the Standard solution;

CT is the concentration, in mg per mL, of gabapentin in the Test solution, based on the label claim;

and rU and rS are the individual peak responses to obtain a final solution having a known concentration of about for gabapentin related compound A obtained from the Test solution and Standard solution, respectively: not more than 0.4% of gabapentin related compound A is found.

Calculate the percentage of any other unspecified degradation product relative to gabapentin content in the portion of Tablets taken using the formula:

 

100(CS / CT)(ri / rS)

               Area of imp in test          0.04

% imp = - - - - - - - - - - - - - - - - - x - - - -  x 100

               Area of standard               20

In which CS is the concentration, in mg per mL, of USP Gabapentin RS in the Standard solution;

CT is the concentration, in mg per mL, of gabapentin in the Test solution, based on the label claim;

ri is the response for each unspecified impurity in the Test solution; and rS is the peak response for gabapentin in the Standard solution: not more than 0.1% of any individual unspecified degradation product is found; and not more than 1.0% of total impurities is found.

 Total impurities = Sum of all unknown impurities and Gabapentin RCA

 

2.     External standard approach against the peak of the analyte, using RRF as needed.

The RRF of an impurity is defined as the ratio of the peak response of the impurity to that of an equal mass of the drug substance. The RRF, calculated as defined above, is placed in the denominator in the formula for calculating percent impurity. RRF values in monographs should be stated to one decimal place if the value is equal to or greater than 1.0 and to two decimal places if it is less than 1.0. The RRF values can be rounded off to 1.0 in USP–NF monographs if they are in the range 0.8–1.2.

For example: Organic impurities calculation for Montelukast chewable tablets:

Standard solution: 0.33 mg/mL of USP Montelukast Dicyclohexylamine RS in Diluent                                                                                                                               

Sensitivity solution: 0.33 mg/mL of USP Montelukast Dicyclohexylamine RS in Diluent from the Standard Solution

Sample solution (for 5-mg Chewable Tablets): Nominally 0.25 mg/mL of montelukast prepared as follows. Transfer 10 Chewable Tablets to a suitable volumetric flask, add 75% of the flask volume of Diluent, and shake vigorously for 60 min. Dilute with Diluent to volume. Pass a portion of the resulting solution through a suitable filter of 0.45-mm pore size, discarding the first mL of filtrate. Use the filtrate.

Samples: Standard solution and Sample solution

Calculate the percentage of any individual degradation product in the portion of Chewable Tablets

 Result = (rU/rS) x (CS/CU) x (Mr1/Mr2) x (1/F) x 100

 rU = peak response of any individual degradation product from the Sample solution

rS = peak response of montelukast from the Standard solution

CS = concentration of USP Montelukast Dicyclohexylamine RS in the Standard solution (mg/mL)

CU = nominal concentration of montelukast in the Sample solution (mg/mL)

M r1 = molecular weight of montelukast, 586.18

Mr2 = molecular weight of montelukast dicyclohexylamine, 767.50

F = relative response factor

                                                       

Acceptance criteria: See Table.

 Note: Disregard any peak with an area less than that of the Sensitivity solution

 

Name	RRF	Acceptance criteria (NMT %)
Sulfoxide impurity	1.0	1.5
Montelukast ketone impurity	1.7	0.2
cis-Isomer	1.0	0.2
Any other individual degradation product	1.0	0.2
Total impurities	-	2.0

 

As mentioned in above calculation method, the formula for individual known and unknown impurities will be

            Imp area       0.33       586.18       1   

%Imp=---------------x---------x-----------x-------- x 100

             Std Area       0.25       767.50     RRF

 

Total impurities = Sum of all unknown and known impurities

3.     Area normalization using the formula 100(ri/rs) in which ri is the peak response for each impurity and rs is the sum of the responses of all the peaks in sample solution.

For example: Organic impurities calculation for Metformin Hydrochloride Extended-Release Tablets:

Calculate the percentage of each impurity in the portion of Tablets taken:

 

Result = (rU/rT) × 100

 

rU = peak response for each impurity

rT = sum of all the peak responses

In this case, no need to include peak response of standard solution in calculation, but can be injected to check system suitability.

Acceptance criteria

Individual impurities: NMT 0.1%

Total impurities: NMT 0.6%

 

[NOTE—Disregard any peak less than 0.05%, and disregard any peak observed in the blank.]

4.     Quantitation against the peak of the analyte in the diluted test solutions

While this approach is being commonly used in existing Ph. Eur. monographs, USP’s preference is to use the external standard approach.

In this method, inject a solution of the substance to be examined at the concentration corresponding to disregard limit (0.05% of test solution) and note the area of principle peak. Disregard all the peaks in the chromatogram of the test solution with an area less than or equal to this peak area.

For calculation of Specified and unspecified impurities, compare the area of individual peak with (x times) the area of the peak obtained with the reference solution, as stated in the monograph.

For total impurities, sum up the areas of individual peaks and compare the figure with (y times) the area of the peak obtained with the reference solution, as stated in the monograph.

For example: Related substances calculation for Amiodarone Tablets

Solution (1) : Mix with the aid of ultrasound for 15 minutes a quantity of the powdered tablets containing 50 mg of Amiodarone Hydrochloride with 50 mL of methanol, allow to cool and filter through a 0.45-mm PTFE filter. Dilute 1 volume of the solution to 2 volumes with a mixture of equal volumes of acetonitrile and water.

Solution (2) : Dilute 1 volume of solution (1) to 50 volumes and further dilute 1 volume of the resulting solution to 10 volumes with a mixture of equal volumes of acetonitrile and water.

Solution (3) : Dilute 1 volume of solution (2) to 4 volumes with a mixture of equal volumes of acetonitrile and water.

Solution (4) : Dissolve 10 mg each of amiodarone impurity D BPCRS and amiodarone impurity E EPCRS in methanol and dilute to 50 mL with the same solvent. Dilute 1 volume of the solution to 200 volumes with a mixture of equal volumes of acetonitrile and water.

Calculation:

In the chromatogram obtained with solution (1): the area of any peak corresponding to impurity D is not greater than 2.5 times the area of the peak due to impurity D in the chromatogram obtained with solution (4) (0.5%);

The area of any other secondary peak is not greater than the peak due to amiodarone in the chromatogram obtained with solution (2) (0.2%);

The sum of the areas of all the secondary peaks is not greater than 5 times the area of the peak due to Amiodarone in the chromatogram obtained with solution (2).

Disregard any peak with an area less than the area of the peak due to amiodarone in the chromatogram obtained with solution (3) (0.05%).

Acceptance Criteria:

Impurity D – NMT 0.5%

Any other unspecified impurity - NMT 0.2%

Total impurities – NMT 1.0%

 

5.     For drug products labeled in terms of free acid/free base, a correction factor may be needed.

This concept is used above in related substances of Montelukast chewable tablets as Mr1 and Mr2. Generally this can be applied in the calculation as

X  free acid or free base formula weight / salt formula weight.

Note : While considering unspecified impurity peaks, the peaks due to blank and placebo should be disregarded. In practical calculation of impurities exactly weighed standard and sample weights, potency of standard or external standard should be used. For calculation of dosage forms, average weight, label claim should be used. For example for tablet the simplified calculation formula will be as follows: 

%Impurity =

Imp area      Std.Conc    Avg Wt.    Potency         

---------------x------------x--------------x------------- x 100 

 Std area     Spl conc.  label claim      100         

 

Conclusion :

The above mentioned methods are generally used for related substances or impurities calculation. External standard method is considered as most accurate among all the above methods. These concepts can be well understood with the practical calculation using chromatograms.

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