doubt about using the mobile phases

[indianhplc]

hello group,

i do work presently in HPLC method development of an anticancer drug,its molecular weight is 449 and its pkas are 5.4 and 7.2, dear group member kindly help me by giving me suggestion about certain mobile phases that could i try.
the drug is a free base.

the work that i have still done is mentioned below....

at first the mobile phase tried was 50:50 methanol water..it didnt gave any peak,then drug stock was prepared in methanol and the final dilution was also done in methanol.

in the second step, i tried out one of the reported LC-MS method mobile phase of 30:70 acetonitrile water with 0.1% formic acid and the flow rate was maintained at 0.5 ml/min and 1.0ml/min, the peak werwe obtained at 6.1min and 3.1 min respectively .herre i got a negative peak too but this was removed while using the drug stock in acetonitrile and diluting with mobile phase.

in the third step while looking the pH of the above mentioned mobile phase was around 2.7 so to protect the column the 0.1% formic acid was made to 0.05%formic acid and the same combination was tried.but the result was same and now 1 ml flow was treied and the retention was 2.7 min.

as to increase the retention time as the retention time was around dead volume the pH was adjusted with orthophosporic acid to 4.0 ,it was suprising that the same retention time was obtained.

i did also try mobile phases like 30:70 acetonitrile water and 40 :60 acn water........without any pH adjustment. but i didnt get anyresult.

dear group i am in a real dilemma please help me out.

thanking you

vivin

[Rob Burgess]

If I understand you correctly your compound is a base, yes? Therefore to gain greater retention in a reversed phase mode HPLC you could trying going to a higher pH (say around 9) which would be two units greater than your pKa's. In that way your compound should be chromatographed as neutral solute (rather than being ionised) and should (in theory) yield greater retention.

[JA]

Hello & welcome to the forum.

There really are a great number of things one could comment on regarding the problem you have posted. Ordinarily when seeking advice on the board you may find people will reply asking for "a few more details, please" where some helpful tidbits have been omitted. This could be the instrumentation you are using, specifically the detection method you are employing, or want to employ, as the mobile phases might have to be tailored to have low UV cutoff or be volatile for example.

For your molecule some confirmation of its functionality would be useful. It's a free base so I guess it has at least one amino type function (the pKa is therefore strictly that of the conjugate acid) but is it dibasic or is there some acid character there? This is useful to know because for ionisable species one controls retention by controlling the ionisation state. If the compound is a diamino then operating with formic or orthophosphoric acids certainly has them both protonated and ionised, giving less retention by reversed phase. Even with your peak at 3.1 mins in 30% ACN with formic your work leaves room open to investigate - you could continue to lower percentages of acetonitrile down to 20% and 10%. If your column can handle it (aqueous specific or polar embedded, for example) you could try a run without organic modifier.

With acidic mobile phase (the compound ionised) one can look to employ other means of retention by ion pairing or ion exchange. For the former you could use a combination of pairing anion, typically an alkanesulfonate at 5-10 mM, with a "regular" C18. There can be a lot to say about the many different reagent types which may be used here. There are specialty columns available where you do not need this reagent. For ion exchange you might even find you have a capable C18 with sufficient silanolic character (perhaps not fully endcapped) to gain retention in mildly acidic conditions. A silica column could be directly employed here.

Sticking with the silica column and the compound in the charged state you can look for retention by a HILIC mechanism using the mixture of water, acetonitrile and formic acid. Start at 50:50 v/v and decrease the water content run-by-run in steps of 10%. Alternatively employ a gradient from 5-50% water to save on experimentation time. HILIC can be performed on numerous bonded phases as well.

You can eliminate what will and won't be possible from my suggestions above based on the hardware you have to hand.

If you've still no luck or want to mix up the work you can try, as Rob suggested, to neutralise the compound and look for retention at high pH. This ordinarily requires a compatible column so check the manufacturer's recommended limits.

Probably on the weighting of my reply you can tell I don't work too often at high pH

Good luck. Let us know how you get on.

[Uwe Neue]

I think the first thing to do is to buy a book that tells how the different things in reversed-phase chromatography affect the retention of compounds.

While the book is on order, let us improve your method. You got retention with 30% acetonitrile and 70% water with 0.1% formic acid. This is good, the retention was not enough. Therefore I suggest to increase the water content. Maybe 25% acetonitrile and 75% water with 0.1% formic acid (pH 2.7 is not a problem). The retention will increase, and you may need to wait for a while. If the retention is way too much, use solvent composition in between, say 17.5% acetonitrile.

Problem solved?

[JA]

I wish I had been more concise like that

[Uwe Neue]

Oops .... I was typing too quickly.

For the last intermediate composition I meant to suggest to use 27.5% acetonitrile...

[indianhplc]

Dear group,

I am grateful to Rob Burgess, JA, Uwe Neue for sending me valuable suggestions.
A mentioned by all of you, I would like to present some more details about my work. My primary aim is to develop specific and repeatable stability indicating method of this drug.
As mentioned by you all I am including the further dtails about the drug.
It has the molecular formula C22H24ClFN4O3,( 4-Quinazolinamine, N-(3-chloro-4- fluorophenyl) -7-methoxy-6- [3-4-morpholin) propoxy]) a relative molecular mass of 446.9 sparingly soluble at pH 1, but is practically insoluble above pH 7, with the solubility dropping sharply between pH 4 and pH 6. In non-aqueous solvents, the drug is freely soluble in glacial acetic acid and dimethylsulphoxide, soluble in pyridine, sparingly soluble in tetrahydrofuran, and slightly soluble in methanol, ethanol (99.5%), ethyl acetate, propan-2-ol and acetonitrile.

I work with UV detection, and the drug has two peak absorbances at 250 and 332 for HPTLC I performed my work using methanol as my solvent and scanned at 332nm
Respected sir, while performing stability indicating method will there be some problems regarding detection of the degraded products as if I reduce my content of acetonitrile ,from 30% .

Sir I would also like to know whether if I perform my method in a lower flow rate ,will there be a problem in publishing my work in the future.

Once again I would like to thank all those who replied me taking there valuable time.
And I do expect further help from them.

Thanking you
vivin

[DR]

Flow rate changes are not going to help you (have you ordered the book yet ). You have to change K or alpha. Pending the arrival of "Practical HPLC Method Development", I would suggest that you search the literature and column vendor web sites for separations of similar molecules. Anything with similar ring structures and functional groups may lead you to a good starting point. Working between the pKs may lend some solubility relief, but you will have to be careful that you stay between them through the course of any gradients you come up with. It may be worth investigating the use of zwitterionic additives to 1) establish a buffer system to maintain a desired pH, 2) enhance solubility.

[Uwe Neue]

OK we are getting closer to what we need to do.

You want to do a stability-indicating assay. The most important thing about a stability indicating assay is to make sure that none of the degradants interfere with the detection of the parent compound. With a classical UV detector, you needed to separate the parent from all degradants. Since you are using LC/MS, I am not sure how much separation the regulatory agencies require, but I would not be comfortable doing this with peaks overlapping with the parent, even with MS. With UV, the classical way to look for peak overlap was with the peak purity algorithms that came with the PDA. Maybe somebody else can comment on what the situation is with MS detection.

From the standpoint of chromatography, you want to get your peak into a middle range of an isocratic chromatogram, say around k = 5. This is why you may want to play a bit with the organic content of your mobile phase.

The next thing to worry about is what to do, if indeed you have overlap of your parent with degradants. The usual approach is to change the mobile phase from acetonitrile to methanol, or play with the pH, or use a column with a much different selectivity.

[Uwe Neue]

Sorry, I needed to reread your original message. You are not using LC/MS, but LC/UV.

In this case, you need to do the estimation of peak purity with the software tools that come with your PDA.

All your peaks are moving around as you change the organic composition. In general, you get better separation from neighboring peaks as you increase the retention of the parent. But you also do not want the retention to be too large, otherwise your assay will take too long.

You can also play with pH. Considering the pK of your compound, your selectivity will change as you change the pH from acidic to neutral with a phosphate buffer. But I would do any selectivity manipulations only if I have evidence that there is peak overlap under the original conditions.

[indianhplc]

dear group,

from all u r suggestions, i am planning to try some more mobile phases.

please tell me whethr these trails would be worthwhile or should i try some other mobile phases.

my trails include,

acetonitrile:water(25:75) at ph 4.0

metahanol :water(40:60) at ph 4.0

dear group please giv me suggestion for this.

and should i go or lowr flow rate with 30:70 ACN:WATER at ph 4.0

thanking you

vivin

[anilray1155]

friend,
you will tryed some other mobile phase given below

(1)1-pentane sulphonic acid sodium salt buffer:acetonitrile
or 1-octane sulphonic acid sodium salt buffer:acetonitrile for ion pair chromatography using ph range 3.0 to 5.0and the suggested ratio for buffer and organic are 25:75(approximate)
you try different ratio
(2)you try phosphate buffer .
i am suggested here you should use buffered mobile phase .
if you will work with ion pair chrmatography it is necessary to adjust the pH of buffer accurately.

[Uwe Neue]

Anilray:
Why do you want them to ruin their column with an ion-pair reagent? It is difficult to completely remove ion-pair reagents from the column, and the general recommendation is to dedicate a column to ion-pair chromatography, once it has been used for this.