LOD LOQ

[nageshwar]

Hi to all,

Recently I have been asked to develop LOD and LOQ for n-methylmethanamine is an impuriy F in Metformin HCl as per EUROPEAN PHARMACOPOEIA 5.0 method (HPLC-UV detection at 218nm).

As per the method at 218nm impuriy F (n-methylmethanamine) peak shape is dispersing, merging with other known impurity and have very low response. I spiked the metformin Hcl and impurity F in 10:0.5 ratio (injection concentraion 5mg/ml) but there is no expected result. System suitability parameters are meeting the monograph requirements.

As the Ph. Eur. methods are validated, how i can proceed further?
Please guide me.

Thanks in advance

T.Nageshwar

[tom jupille]

Can you provide more detail regarding what you mean by "no expected result"?

[nageshwar]

Dear TomJupille,
Thanks for your response.
The details are as follows:
Related substances. Liquid chromatography (2.2.29).
Test solution. Dissolve 0.50 g of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase.
Reference solution (a). Dissolve 20.0 mg of cyanoguanidine R in water R and dilute to 100.0 ml with the same solvent. Dilute 1.0 ml to 200.0 ml with the mobile phase.
Reference solution (b). Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. Dilute 1.0 ml of this solution to 20.0 ml with the mobile phase.
Reference solution (c). Dissolve 10.0 mg of melamine R in about 90 ml of water R. Add 5.0 ml of the test solution and dilute to 100.0 ml with water R. Dilute 1.0 ml of this solution to 50.0 ml with the mobile phase.
Column:
— size: l = 0.25 m, Ø = 4.6 mm,
— stationary phase: irregular, porous silica gel to which benzenesulphonic acid groups have been chemically bonded (10 µm),
or
— size: l = 0.11 m, Ø = 4.7 mm,
— stationary phase: regular, porous silica gel to which benzenesulphonic acid groups have been chemically bonded (5 µm).
Mobile phase: 17 g/l solution of ammonium dihydrogen phosphate R adjusted to pH 3.0 with phosphoric acid R.
Flow rate: 1ml/min.
Detection: spectrophotometer at 218 nm.
Injection: 20 µl.
Run time: twice the retention time of metformin hydrochloride.
System suitability : reference solution (c) :
— resolution: minimum of 10 between the peaks due to melamine and to metformin hydrochloride.
Limits :
— impurity A: not more than the area of the corresponding peak in the chromatogram obtained with reference solution (a) (0.02 per cent),
— any other impurity: not more than the area of the principal peak in the chromatogram obtained with
reference solution (b) (0.1 per cent).
IMPURITIES
Specified impurities : A.
Other detectable impurities : B, C, D, E, F.
A. cyanoguanidine,
B. R = NH-C(=NH)-NH2 : (4,6-diamino-1,3,5-triazin-2-yl)guanidine,
C. R = N(CH3)2 : N,N-dimethyl-1,3,5-triazine-2,4,6-triamine,
D. R = NH2 : 1,3,5-triazine-2,4,6-triamine (melamine),
E. 1-methylbiguanide,
F. CH3-NH-CH3 : N-methylmethanamine.

I have spiked with impurity F in metformin with various concentrations, but with 0.1% spike there is no any impurity peak observed, with 0.5% spike observed a small peak merged with impurity A which is around 0.1%. With 5% spike there is about 1% area but peak shape is not good and dispersion/split.

I am expected about 5% impurityF and 95% Metformin in the resultant chromatogram(5% spike solution injection).

Thank you again.
T.NAGESHWAR

[Bruce Hamilton]

You should be using EP 6.x, however the method appears unchanged.
According to the EP Knowledgebase ( freely available at the EP site ), the column used was Whatman Partispher SCX 5, so you should first ensure your column is equivalent - or even purchase that column.

Secondly, you can contact the EP and ask them, as the monograph is listed as currently under revision, and they may be aware of the problem and have a solution.

Please keep having fun,

Bruce Hamilton

[nageshwar]

Dear Bruce Hamilton

Thanks for your prompt response.
Yes I am using EP6.0 version.
I am using the Column:
Make: Macherey Nagel
Brand name: Nucleosil-100SA-10micron
Size: l = 0.25 m, Ø = 4.6 mm,
Stationary phase: irregular, porous silica gel to which benzenesulphonic acid groups have been chemically bonded.

Thanks again
T.Nageshwar

[tom jupille]

I would agree with Bruce's suggestion that your column does not have the same selectivity as the original. The problem with compendial methods is that they try to be "generic" in describing the columns used.

[nageshwar]

Thanks to all for the input

T.Nageshwar

[vishnu]

Dear all,
As per my experience, Impurity F is not detectable in HPLC system.
I tried many more and never got good results.

But it is giving good results in the TLC. You may use Ninhydrin or Iodine as the visualizing agents.

[nageshwar]

Dear all,
As per my experience, Impurity F is not detectable in HPLC system.
I tried many more and never got good results.

But it is giving good results in the TLC. You may use Ninhydrin or Iodine as the visualizing agents.

Dear Vishnu
Let me know the TLC method please
Regards
T.Nageshwar

[vishnu]

Just use the same method specified for metformin in EP.

Instead of metformin, you can use the sample and standard.
But for LOD - LOQ, i dont know how it gives the result.