Analysis of Micronutrient of Premixes By HPLC method

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sarika sawant
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Analysis of Micronutrient of Premixes By HPLC method

I have a problem in Calculation of Assay ,which is analyse by HPLC method .Retension Area of standard as well as sample is much more vary , it indicate unequal concentration.But due to back calculation ,i am not getting expected result . I have a Question that i) The sample which i analyse have lower label claim or ii) Is there any factor is to be added while calculating % Assay .or iii) The instrument is not giving proper reading. Can anybody will help me out ? Looking forward of anyone's reply.....

Lynn E.
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Additional information would

Additional information would be very helpful.

Is the problem with all or only some of the analytes?

What is the sample preparation- could it change speciation/retention time?

Have you tried running one of the problem analytes alone (in the sample matrix) to determine if the problem is with the sample, or a column problem?

Have you spiked one of the problem analytes into a sample to see if you get the expected peak and recovery?

Could you have ghost peaks interfereing (because something in the sample elutes past the run time of your assay?

How many injections have been analyzed with the column?  (They do not last forever_.

What is your eluent?  Is it stable (Would daily preparation help?)

Is the elution by gradient?  Could the precision of the gradient be inconsistent?  (leads to all kinds of problems, including changing the order of elution in some cases.  If you have a set of peaks that gives you a characteristic chromatogram, multiple replicates of a standard solution containing these can help you visualize this problem)    Another issue that can occur with a gradient pump happens when the gradient you have programmed exceeds the ability of the pump.

sarika sawant
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Sir, Thanks a lot for your reply.
I have recently tried this method , basically its from (USP 24) on Agilent 1120 compact system (i.e of Isocratic mode) . I got fabulous result from this method .
As from my view its very important to know the exact quantity of active ingredient in your sample.
As you discuss of comparing Blank , standard and sample simultaneously.
Assay :-
i)-iv)]   For Vitamin B1, Vitamin B2, Vitamin B3 and Folic acid.

 
Chromatographic conditions :-
Mobile phase: - Transfer 0.2 ml of triethylamine, 7.5 ml of glacial acetic acid and 175 ml of methanol to a 1000 ml volumetric flask and dilute with 0.008 M (1.505 gm to 1000 ml of water) of sodium hexane sulphonate to volume mix it, degassed.  adjust pH to 3.2 with GAA
 
Column                        :-  C18 ,25 cm x 4.6 mm , 5µ
 
Flow rate                      : - 2.0 ml per min.
 
Injection volume          :-  20 µl
 
Wavelength                  :-  270 nm
 
Run time                      : - 32 min.
 
Retention Time            :-    For  Niacin : About 2.0 min
                                              For Thiamine mononitrate : About 13 min
                                              For  Folic acid                   : About 16 min
                                              For Riboflavin                   : About 27 min.
 
Standard stock solution: -   Dissolve 24 mg of Vitamin B1, 8 mg of Vitamin B2, 150 mg of Vitamin B3 and 8 mg of Folic acid in 100 ml of reagent solution.
 
Standard preparation: - Transfer 5 ml of stock solution, Add 10 ml mix. of Methanol and GAA (9 : 1), 30 ml of methanol and ethylene glycol (1: 1). Shake for 15 min in water bath 600 and cool. Filter discards few ml.
 
Assay Preparation: - Weigh equivalent to about 1.2 mg of thiamine, 1.2 mg of riboflavin, 20 mg of Niacin and 0.205mg of folic acid. (i.e.  200 mg of premix ) Add 10 ml mix. of Methanol and GAA  (9 : 1), 30 ml of methanol and ethylene glycol (1: 1). Shake for 15 min in water bath 600 and cool .Filter discard few ml.
 
Procedure: - Separately inject equal volume of the blank, standard preparation and sample preparation into the chromatograph, record the chromatograms.
 
Observation: - Observe the retention time of major peak in the chromatogram of sample preparation to that in standard preparation.

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                  Calculation  :-
         = Area (spl)  x wt of std (mg) x  5_____x    Average wt_x  100
              Area (std)     100                     wt of spl      label claim
 

 

sarika sawant
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                  Calculation  :-
                 = Area (spl)  x wt of std (mg) x  5_____x    Average wt_x  100
                    Area (std)     100                     wt of spl      label claim
 

Lynn E.
Lynn E.'s picture
This site seems to have

This site seems to have information on this separation:

http://www.urmediazone.com/signup?sf=search&ref=4851781&q=HPLC%20Analysis%20of%20Vitamins%20in%20Tablets%20using%20HPLC.pdf

(You do have to create a free account)

This pdf relates to food (not sure what your matrix is initially but it couls have something helpful):
http://a2zproject.org/pdf/Manual_Foods.pdf

I hope you find some information that helps.

Do you have an injection valve that is automatic, or one that you load and turn to inject manually?  That is another place things can go awry.  In either case, you need to be sure that enough sample goes through the valve to rinse it (like you would a test tube with sample prior to pouring an aliquot for analysis) and enough to completely fill the loop.

Lynn E.
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http://www.phenomenex.com

http://www.phenomenex.com/Application/Detail/14235?returnURL=/Application/Search

Phenomenex has good technical support.  Here is a column with some of yur analytes in 12 minutes or so-

sarika sawant
sarika sawant's picture
Thank you sir ,

Thank you sir ,

Its really been so Informative link.

Have a good day.