Chromatography Forum

Hellow all,

I have to develop a LC method for determination of NSAIDs (non steroidal antiinflamatory drugs) in foods of animal origin. The number of analytes is 9 + IS. We separate them in 15 minutes on conventional sized Eclipse Plus PhenylHexyl column using a gradient program and the separation characteristics are quite good. But...
The problem is that all the analytes have an UV absorption maxima between 200 and 300nm and the intensity is not sufficiently high. Something else - it is hard to believe that we could purify the extracts from meat, milk etc. with sufficient degree to overcome the interferences from the matrix absorbing at these wavelenghts. With other words - the interval of detection wavelenghts between 200 and 300-350nm is not appropriate for analysis of residues in biological samples because there are a number of interfering compounds that also absorbs strongly.
Probably you will say - LC/MS is the right alternative and this will be true. We will make the method with MS detection and I don't expect big problems because as I saw the structures the ESI ionization will be easy at proper conditions. But we want to develop the LC/MS method as a confirmatory method because our LC/MS systems are quite busy. It will be more convenient for our routine work to have a LC screening method and only the positive samples will be confirmed on LC/MS.
So I noted that all the analyte's molecules have a carboxylic group. This group can be derivatized with fluorescent reagent to give a derivative with fluorescence properties. I read in "Modern Derivatization Methods for Separation Sciences" from Toyo'oka many possible derivatization reagents but it is hard to orientate which of them is most suitable - in terms of stability, high fluorescence intesity, simple reaction conditions. It is also important for the sutable reagent to be commersially available. The options are: bromomethyl reagents (coumarins, Br-DMEQ, Br-MMEQ etc.), diazomethyl reagents, amine reagents, hydrazine reagents and so on.
Please give me your opinion on the problem, is someone have an experience with derivatization of carboxilic acids for fluorescence detection? Thanks to all in advance...

Come on...I can't believe that no one from here with an opinion on the problem...or experience

Come on...I can't believe that no one from here with an opinion on the problem...or experience

I can.

Pre-column derivatization is rarely done these days for the very reason you point out: MS is a better way to go.

Yes, Tom, you're correct but...What about costs with LC/MS per hour (for example triple quad which is the most convenient for residue analysis of foods) compared to traditional HPLC for example with fluorescence detection. You can imagine how much geater is the initial investment and servise with LC/MS. And this will have an impact on the analysis price per sample. In some cases it is more reasonable to use cheaper HPLC...for example sulphonamide residue analysis in honey (the one can obtain quite satisfactory results with HPLC/FlD through simple derivatization of the analytes with fluorescamine).
But this is all about lab managment. Simply I want to understand is there some useful derivatization reagent for carboxylic acids to obtain without great complications fluorescent derivatives.? I found some compounds but it is very hard to found a supplier...

try this site/supplier

link describing Coupling Hydrazines, Hydroxylamines and Amines to Carboxylic Acids

http://probes.invitrogen.com/handbook/s ... /0303.html

link that lists their avaliable Derivatization Reagents for Carboxylic Acids and Glutamine

http://probes.invitrogen.com/servlets/p ... t?id=32085

good luck

Another list of reagents is also available at:
http://www.sigmaaldrich.com/catalog/sea ... ge/8659001.
There are some online resources at that site as well.

I don't have any direct experience with derivatizing acids, but there are numerous reagents available and it's just a matter of checking out the reaction conditions. But if your analytes have amines, they would be easier to deal with.

Regarding your initial list, I would stay away from the diazomethyl reagents, mostly because of the safety, reactivity, and toxicity issues. After that look at reaction conditions that aren't too extreme. I don't know what equipment you have, but with several of the newer autosampler designs, it is possible to do an automated pre-column derivatization as part of the injection cycle. Once set up, it is much more efficient. Agilent, for example, has a nice application for amino acids that does it all in the autosampler; all you do is load in the reagents. I have done methods using this approach and it does work.

And I agree with you that MS does not have to be the only answer. There is a lot of good chemistry out there that can do a similar job for a lot less money, if someone wants to spend a little time getting it set up.

I have worked with NSAIDs residues since few years and I understand your problems with developing good multi-residue method. I used LC-MS/MS for screening and confirmation of this drugs,and in my opinion it is most reasonable (but of course very expensive) solution.
Use of fluorescence detection without derivatisation is possible for naproxen and ketoprofen. Use of derivatisation is theoretically possible but I did not find any publication for NSAIDs.
This idea is very interesting but some important drugs do not have -COOH group, like phenylbutazone.
What can be interesting for you, P.Gallo (Italy) presented poster during last Euroresidue VI conference describing HPLC-FLD method for NSAIDs "The confirmatory analysis of non steroidal anti inflammatory drugs by HPLC with fluorescence detection". He did not use any derivatisation (I can not understand how, I did not repeat his work ). Please send me an e-mail, I will send you back abstract of this work (jedziCANCEL_IT@piwet.pulawy.pl)

Here's my rationale which I'm happy for somebody to pick apart:

The problem is that all the analytes have an UV absorption maxima between 200 and 300nm and the intensity is not sufficiently high. Something else - it is hard to believe that we could purify the extracts from meat, milk etc. with sufficient degree to overcome the interferences from the matrix absorbing at these wavelenghts.
...
Probably you will say - LC/MS is the right alternative and this will be true.

I disagree with the choice to use MS detection as, if I understand correctly, you have co-elution of one or a few of your target analytes with background signals from the matrices. The impact on quantitation due to the resulting signal supression is the shortcoming of this approach.

In the same sense I reason that analyses utilising UV or fluorescence would be detrimentally affected if the matrix components can cause a baseline deflection, not necessarily an additive positive signal, at the monitoring wavelength used. In other words, I would expect difficulties not resulting solely from the fact your analytes have weak extinction coefficients in their native, underivatised form.

Since I have never personally used, or been demonstrated the operation of, a fluorescence detector I write predominantly with reference to variable UV. Presumably the aim is now to derivatise the target analytes selectively from the matrix components, or at least those which may co-elute with a compound of interest, to yield either fluorescence or a bathochromic shift in the absorption spectrum which will necessitate observation at a wavelength which affords specificity. The detection wavelength now discriminates against any change in response that background interferences could produce. In this case I reason that quantitation should be possible even in the case of co-elution. This is before taking into account the change in retention behaviour, and thus chromatographic selectivity, which will result from derivatisation - maybe the problem of overlapping signals is removed or made easier to control as a result of the characteristics imparted by the newly-coupled moiety.

Anyway I thought I would just write my feelings with regard to the chromatographic aspects of the problem because, as I inferred earlier, I have no experience with fluorescence derivatisation procedures. I guess I'm wondering why Fl detection is so widely applied when, if my reasons are sound, one could attain the same specificity by creating a 'more highly chromaphoric' derivative observable with a VWD.

JA:
You are correct in many respects. Yes, derivatization to form a product that absorbs at a longer wavelength is a possibility. In addition, many of these derivatives will also have stronger absorptivities, which further helps sensitivity. Of course, the selectivity advantage is a plus as well.

Fluorescence offers the same advantages, but with a few twists. You can now select an excitation wavelength and emission wavelength, with some (but not much) flexibility. But the biggest advantage comes from the fact that not all compounds fluoresce, especially at the specific wavelengths involved with some of these deriviatives. The improvement in selectivity can be dramatic, depending on the sample.

Finally, unlike absorbance, the intensity of fluorescence is directly proportional to the intensitiy of the exciting light. The resulting improvement in sensitivity can be 100 - 1000X.

I would guess that a modern fluorescence detector would generate better precision than an MS, at a fraction of the cost. It is not a perfect detector, but for many analytes it is a very good option.

Fluorescence offers the same advantages, but with a few twists. You can now select an excitation wavelength and emission wavelength, with some (but not much) flexibility. But the biggest advantage comes from the fact that not all compounds fluoresce, especially at the specific wavelengths involved with some of these deriviatives. The improvement in selectivity can be dramatic, depending on the sample.

I second mbicking's opinion. The fact that only small number of molecules fluoresce naturally and the possibility to add a fluorophore with a derivatization step for those molecules which do not fluoresce naturally, speaks for the specifity and selectivity of the fluorescence derivatization and fluorescent detection. With such derivatization the benefit is not only the fluorescent tagging that could be made for selective determination of nonfluorescent analytes, but also the derivates have different chemistry and speaking with the chromatographic language, have different partition properties and will behave different in a chromatographic column. That said, previously (before derivatization) coeluting "natural" analytes could be beautifully separated after derivatization, which I hope you'll agree that this is the desired "picture" for every chromatographer.

Regards

At this point of this discussion it appears to be worthwhile to explain why I had decided, initially, to stay mute, even though I had some experience with some of the derivatives mentioned by cyanogen78. The experimentation was done a long time ago, I understood cyanogen´s call for help to be for specifics. It is just too tedious for me to look up my old notes. Also, having been away from this for so long I found the description of cyanogen78´s reagents somewhat confusing.
A few things stuck in memory, allowing some nonspecific opinion: Coumarins produced at least 4 prominent peaks for each carboxylic acid. Not acceptable for me. Hydrazines produced osazones with ascorbic acid (a vinylog of the carboxyl group), with two isomers interconverting within a few hours. The reaction in con H2SO4 proved very messy. We tried hard but had to give it up (there are sundry colorimetric methods in the very old literature, they are guesses, to be kind...). We did extensive work (published ~1982) on diazomethane derivatization of fatty acids, I became a great fan of this reaction. It can easily be handled safely (I published something on this as well, don´t have the ref. here). Solid diazo compounds are even less of a health hazard. If one uses them, one has to remember that they react also with phenols or other similarily active alcohols, or with water. In the case of FA and diazomethane, conditions can be chosen to eliminate all deleterious side reactions. One can also easily eliminate carbene reactions.
Thus: In my opinion, derivatization for fluorescence detection is a highly specialty type of method, not a general one.
The only use of fluorescence that I can enthusiastically recommend from my experience is that of native protein (antibodies) fluorescence to identify proteins using a UV-Fl dual detector setup.
The problems mentioned here are probably the basis for what Tom pointed out.

HW:
I agree with most of your comments. While we have been discussing the advantages of derivatization reactions, every procedure has its disadvantages. Among them is the fact that the reagent is present in a large (100X) excess, and thus any impurities in the reagent can become major peaks. Finding multiple peaks for one analyte can also occur due to formation of different isomers. The carboxylic acids appear to be problematic for LC derivatization because the reaction chemistry is not as efficient.

However, I have done adehydes and ketones with no problem, even in an automated procedure using the autosampler. And the method for amino acids works quite well; many labs use it routinely.

So, you are correct in pointing out there are some issues with derivatization, especially for acids, where you have some experience. But I think there are many other success stories also, so the technique is worth considering for those labs that don't have, or can't afford, an LC-MS system.

I'm happy that my question give rise to such detailed discussion, thanks to all for the responsiveness. Last 3 days I look up extensively in the published literature...mainly in Toyo'oka's "Modern derivatization methods for separation sciences" 1999 & in one review article named "Ultraviolet and Fluorescence Derivatization Reagents for Carboxylic Acids Suitable for High Performance Liquid Chromatography: a Review", Mukherjee&Karnes in BIOMEDICAL CHROMATOGRAPHY, VOL. 10, 193-204 (1 996). There is few options to derivatize a carboxilic group to obtain a fluorescent derivate but most of them don't like me because of complicated conditions for reaction, stability of reagents/products or comersial unavailability...Reading the available information and of course all your posts (I see that there very, very good chemists here) I pick out 2-3 reactions:

1. The first choise is derivatization with 6,7-Dimethoxyl-l-methyl-2(1H)-quinoxalinone-3-propionyl carboxylic acid hydrazide (DMEQ-hydrazide). This tends to be highly sensitive fluorescence label and the reaction seems to be not so complex. The fatty acids reacted in the presence of pyridine and EDC (Iwata, T., lnoue, K., Nakamura, M. and Yamaguchi, M. Biomed. Chromatogr. 6, 120 (1992).


Unfortunately I can't find any supplier of DMEQ-hydrazide...

2. Derivatization with 9-(Hydroxymethy1)anthracene;HMA
This reagent was used also for Ibuprofen. Probably I will try this because of commersial availability from Sigma and low cost (EDC, l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride is also needed for reaction and is available from Sigma at low price). The reaction is:

The excitation wavelenght reported is around 350-360nm and the emission is at 430nm. This looks suitable for me regarding to selectivity.

3. The 3-rd possible reagent could be 1-Pyrenyldiazomethane (PDAM) which reported to be more stable than ADAM (9-Anthryldiazomethane - very unstable even at -10degrees C). But 25mg of PDAM cost 386Euro from the Sigma's website...Its expensive but probably the bigger problem for me is that I can't assess how more stable is this reagent compared to very unstable ADAM. What I will have to do in case of low stability - to loose around 400Euro for such small amount of reagent? No, thanks. The reaction of PDAM with carboxylic acid is:



Finally I will try the second type of derivatization...Please comment such decission. My analytes are: Ibuprofen, Vedaprofen, Ketoprofen, Carprofen, Flunixin, Tolfenamic acid, Diclofenac and Methamizole. All these have a carboxylic group but not all possess a native fluorescence. And have to be derivatized if one wants to work with FlD.

To dr_Pyrex:
Thank you for the information I'm in contact with dr. Gallo from Italy since 2-3months and we discuss this method. I have an abstract of his presentation at EuroResidue but for now he and his colleagues have studied the native fluorescence of NSAIDs only.

To HW:
Your memories are valued for me and suport my feeling that with most of reagents I will have big problems. Hope that my choise will be right.

To mbicking: thanks for the optimism, we all have to be realists for the moment with optimistic look at the future.

there should also be some other dyes with hydrazide or cadaverine (from Invitrogen or even Sigma-aldrich).
In addition there is http://www.dyomics.com who have dyes in all colors with primary amino groups. (they are water soluble and also available via Thermo/Pierce if I remember right)