Hi everybody,
I am setting up a method for analysis of colistin sulphate by HPLC-UV. I tried different previously-published methods but the results were not acceptable (long run time, bad resolution, tailing, broadening and ...). Finally, I tried the European Pharmacopoeia method for identification of colistin sulphate. The parameters of HPLC method were as follows:
C18 column (15*4.6), detection at 215 nm, oven tempearture 30 oC, flow 1 ml/min, mobile phase (4.46 g anhydrous sodium sulphate in 1 liter water, pH adjustment to 2.4
[I used phosphoric acid] and acetonitrile with ratio of 78:22), injection volume 20 microliter, sample concentration 1000 microgram/milliliter of water.
I saw two major peaks at Rt of 8 and 16 min related to colistin b and colistin a, respectively.
Now, my problems:
1- According to EP, the AUC of the second peak must be greater but in my chromatogram, the first one is greater, why and how to solve it?
2- Each of major peaks have a subsequent minor impurity-related peak. The resolution between major and impurity peaks was good at first. I even draw a good calibration curve (100-1000 microgram/milliliter, R2=0.999). Then, I tried a different gradient method on my column (water:ACN from 10% to 65% in 20 min, pH was 2.2 with phosphoric acid, temp 45 oC) and after that, everything went wrong. When I used the EP method again, the peaks were in bad shape and resolution. I changed the guard and the first major peak became better but the second one was again broaden with tailing and without separation from its impurity. Finally, I changed the column but the result was like previous. I don't know how to fix the problem?
Mrs/Miss Kahkeshani,
There is currently only one method, which provide exact analytical quantitative and 3D molecular structural information about the analytes and this method is mass spectrometry (MS.) However, within the framework of our innovative stochastic dynamic theory (mine and my author's one) and new model formulas applicable to account exactly (or absolutely) for the experimentally MS measurable variables.
Please, consider detail on our theory and method [1,2].
So far, our approach has been tested at pg.(mL)-1 and ng.(L)-1 concentration levels of the analytes. The obtained coefficients of linear correlations between our theory and experimentare /r/ = 0.99997 - 1. The method is validated chromatographycally, as well.
[1] Steroids, 164 (2020) 108750
Stochastic dynamic mass spectrometric quantification of steroids in mixture — Part II; Bojidarka Ivanova, Michael Spiteller
[2] B.Ivanova, M. Spiteller, Stochastic dynamic mass spectrometric approach to quantify reserpine in solution, (2020) in press.
You should take into consideration, the fact that according to the European Union Directives, the classical protocols approved, therein, operate at lower capability of the mass spectrometric method (at about 98 %.) As can be seen, from the results shown above; however, according to our new method the full capability of the mass spectrometry as an absolute method for analytical quantification is completely revealed.
Why there is unable to you to reproduce exactly the protocol according to the EU? Due to, the complexity of the 3D molecular structure of your analyte. The peptides, are characterized with great polyproton accepting capability; flexibility of the molecular structure and both inter- and intramolecular protron transfer effects, which determins their chemistry in solution as a very complex. Very small changes of the 3D molecular structure and positional isomers obtained in solution, due to inter- and intramolecular protron transfer processes are out of the capability of the chromatography (even using UPLC) or UV-VIS spectroscopy. They are unable to be determined precisely even qualitatively by means of these methods. As I have written above, such analyses can be carried our precisely, accurately, selectively and sensitively only by mass spectrometry within the framework of our new method [1,2].
You could pay attention to our reference [3] dealing namely with these complex processes of the chemistry of peptides and the application of our innovative mass spectrometric method to study peptides in solution, as well.
[3] B. Ivanova, M. Spiteller; EXPERIMENTAL MASS SPECTROMETRIC AND THEORETICAL TREATMENT OF THE EFFECT OF PROTONATION ON THE 3D MOLECULAR AND ELECTRONIC STRUCTURES OF LOW MOLECULAR WEIGHT ORGANICS AND METAL–ORGANICS OF SILVER(I) ION; pp. 1-182; in Book ''Protonation: Properties, Applications and Effect,'' A. Germogen (Ed.) NOVA Science Publishers, New York, (2019); ISBN: 978-1-5361-4886-2.
In other words, there is needed comprehensive theoretical and experimental knowledge of the chemistry of your analyte in solution with respect to the experimental conditions, in order to, reproduce exactly the EP protocol.
Please look at the following below links which may help you in your analysis:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC153303/pdf/0327.pdf
https://downloads.hindawi.com/journals/jchem/2015/624316.pdf
Thanks
Mrs/Miss Kahkeshani,
In the context of the recent posting by Mr. Elgailani regarding work [ Article Use of High-Performance Liquid Chromatography To Study the P...
], I should underline that there is a deviation from the linearity range shown in Figure 2 (page 1768.)Unfortunately, there is a lack of chemometric data from a linear fitting; however, even without them it is obvious that within the range of time (T) T = 0 - 60 mins, there is a deviation from the linear dependence.
One of the advantages of our method, shown in my posting (reference [2],) among many other ones, is that it is applicable despite the experimental conditions.
An additional comments on: Reference ANTIMICROBIAL AGENTS AND CHEMOTHERAPY (2003) 1766-1770 lacks of detail comment on these data in Figure 2.
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