I teach at a small college where we can give our students practical experience of HPLC, but we have no HPLC-MS. I need to at least give some theory and applications, since some of my students want to work in the pharmaceutical industry. I understand that in electrospray, ion formation is caused by the potential applied to a capillary, and that it works well for polar and ionic compounds. Also that electrospray is good for very large molecules (eg proteins) because multiple charged species can result, extending the mass range of some instruments. Alternatively in APCI, droplet formation is by pneumatic means only, and ionisation is produced by a corona discharge needle. It seems this technique is applicable to non-polar compounds. Let's take a drug like caffeine. At low pH this should be protonated, ie amenable to electrospray. However, I guess it would also work by APCI? Which is the best method to use in such cases? Is it trial and error when there is not an obvious answer, like the compound is neutral or has a very high molecular weight?

Most pharmaceuticals can be analysed by electrospray and ES is widely used for these applications. APCI is often empolyed when a compound exhibits a poor ES response. As well, as you point out, APCI is often a better choice when a sample contains no acidic or basic sites.

When you have a sample that can be ionized by both methods you can start to use other criteria to help you make a choice. For example, APCI is more ameiniable to higher flow rates, is less affected by mobile phase additives, and is generally more rugged than ES. On the other hand, the higher temperatures of APCI can cause compound degradation, it is not effective for high molecular weight compounds (protiens, peptides).

I think the approach that most people take today is, unless there is not an obvious reason to use APCI, try the analysis with ES and move to APCI if required.

If you contact Phenomenex (other companies may have them as well) they should be able to send you a poster with the differences between APCI and ES. It has at least one diagram on which one is more likely to work based on molecular weight and ionic strength. It sounds like you are on the right track. I am glad to hear that undergraduates are getting HPLC experience. If you haven't already, you might want to discuss the fact that LCMS requires volatile MP modifiers. It might be easy for your students to remember that APCI bridges the type of molecules analyzed by GCMS. GCMS is for the most volatile, ES is for the least (except for things like MALDI), and APCI is in between.

If you want to give them an example of compounds that work by either APCI or ES and in both, look at antimicrobials. Generally, beta-lactams work much better by APCI, sulfonamides work in either method, depending on the individual molecule, tetracyclines work better in ES but can work in APCI, and large molecules like macrolides only work in ES. Also, most drugs work better in positive mode than negative, and some, such as tetracyclines, work in both.

Michele and anonymous 2-this is really great advice and I much appreciate your help.Michele-on your last posting you said most drugs work much better in positive mode than negative. Is this because most drugs are basic and are therefore likely to be positively charged in HPLC mobile phases (which tend to be pH 2-7)? Do you generally use acid pH for HPLC-MS analysis of (basic) drugs? However, have you seen the paper by Cheng et al RCMS 2001 15 141? Here they give the anomalous example of lidocaine which is a basic analyte. However, the sensitivity in positive ion mode at pH 10.5 is about the same as at pH 2.7!!! Going back to my original question, and the examples you gave above, it does seem to me that while there are some general rules, often it is just a matter of trying both methods for a particular substance and seeing if it works-am I right?

To the first anonymous: You are right about the drugs working better because they are basic. The HPLC mobile phase can be what ever pH you want in order to give the best peak shape and response. Lowering the pH does increase the signal in positive mode and I have read papers about it increasing response in negative in what is called wrong-way electrospray. I hadn't heard about the paper by Cheng, but it sounds interesting. I have never heard of anyone raising the pH to get a better response in ES+.
As for your original question, there is some trial and error, and there are always exceptions, but with surprisingly little practice, you can learn to look at a molecular structure and tell which mode it will work best in. A quick glance at an entry in the Merck Index regarding issues such as boiling point and pKa and solubility can often clear up uncertainties.

my opinion as from practical point in pharm. analysis, unless you work with futile biomolecules like protein, peptides which will degraded at high temperatures, most chemist will choose to start with APCI, because, as already pointed out, more stable and higher ionization effciency. the ES method is usually taken as a "soft" method to treat those "soft" molecules, some researchers even don't think it is a ionization method because the molecules in LC stage usually already charged so the ES just acts as transfer method. in today's pharm. labs, with unknown property of new drug candidates,APCI is actually the one to start.

We have found many instances of quite good ES+ response at alkaline pH. I would not call it an exception any more, but it is happening rather frequently. While the sensitivity is mostly better at acidic pH, the difference that we have seen is rather small, maybe a factor of 2 to 3 only (instead of all or nothing).

To: Anonymous on Sunday, February 10, 2002 - 06:07 pm:

I would almost always choose APCI over ESI given a choice. But your comment "in today's pharm. labs, with unknown property of new drug candidates,APCI is actually the one to start", I am not so sure about. ESI probes out sell APCI probes ~ 9:1 with the pharma industry being the primary consumers. Data like these indicate that ESI is the method of choice (right or wrong).

Any other opinions?

As the person who posed the original question, I am very grateful for all these informative replies from practitioners of HPLC-MS. I could never have got such practical information from books or journals. I am much clearer on a number of issues now. I am still unsure of one issue from my original question about caffeine or say a simple acid like benzoic acid (used as a preservative-I imagine there might be people out there who actually do this analysis?). Both are ionogenic (under different mobile phase conditions!) and temperature stable. Both are volatile. Both could be analysed in simple mobile phases (no complex additives). Let's say also that I am not interested in high flow rates-I am prepared to use a small bore or capillary column. From anonymous 6.07 p.m Feb 10 I might guess that APCI will be more sensitive and that it is a "harder" ionisation method and may give more diagnostic spectra? Is this right? Anonymous 7.04 p.m 10 Feb, please tell us why you would always choose APCI over ES given a choice? Your remark is most interesting-everyone seems to agree that APCI is more rugged than ES, so why is the pharma industry mostly using ES? Many thanks again for sharing your thoughts.

For most analysis I prefer ESI, this is because it is a much softer technique than APCI and my samples frequently contain glucuronides. If you have analyte that dissociates in the source (like a glucuronide)than I would recommend ESI.

If your samples are not very clean, or are very salty (buffers) then APCI will give you less interference.

Most of the pharmaceutical compounds I analyze can be done equally well with either source, so I start with ESI since it isn't affected by injection volumes or split ratios. Also, most of my analysis have been done with minimal mobile phase modifiers (I generally use 0.1% acetic acid, formic can be too strong an acid for negative ion) for both techniques and for either positive or negative ion detection. Granted this won't work with all compounds all the time, but it's a reasonable place to start.

The first Anony,

The paper you mentioned is interesting. Could you specify the journal and title of that paper? Thank you in advance.

RCMS= Rapid Communications in Mass Spectrometry

Title of paper

Ultrafast LC/UV and LC/tandem mass spectrometric analysis.

Best wishes