question about steroid LC-MS analysis

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

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Hi All,

I was trying to use LC-MS to analyze a couple of steorids. Several compounds are just fine, but I got some difficulty with DHEA, DHEAS and Estradiol. I tried both methanol and acetonitrile as mobile phase B, water is mobile phase A. No acids were added. These three steroids just show no signals even at a relatively high conc. I checked a few references on website and don't see strange comments for them, so I feel very strange why they are not detected in my exp. I was using positive ion mode with Nanospray. Is it because I didn't have formic acid in the mobile phase? Other similar steroids such as cortisol, Corticosterone, etc were giving nice signals
In my work, Estradiol in Standard +ESI was fine by using Ammonium Acetate in 0.1% FA/Water as MP A and 0.1%FA/ACN as MP B. I have not analyze DHEA and DHEAS before, but I think it can be ionize in -ESI as well.
Jetjamnong wrote:
In my work, Estradiol in Standard +ESI was fine by using Ammonium Acetate in 0.1% FA/Water as MP A and 0.1%FA/ACN as MP B. I have not analyze DHEA and DHEAS before, but I think it can be ionize in -ESI as well.

Yep, ammonium acetate. Also ammonium hydroxide will work too.

See EPA method 539
I cannot remember which instrument I had a problem seeing DHEA on. I tried a different instrument and I saw the parent peak.
Cannot remember the solution make-up, probably meoh/0.1% formic acid.
I tried on an API -III and and API-3000.
One saw the compound, one did not (even after trying to optimize declustering potential and the like).
I was rather amazed that such a strong parent peak was not seen on the other instrument. Was something wrong with the instrument or was everything fine? ... I had no reason to suspect the instruments were not working at the time.

So, no I am not surprised you are having difficulty. At least try adding some formic acid.
I seem to recall seeing a steroid method begin run in negative APCI on a triple Quad.. Sorry this is all the information i can remember..
Kind regards

At our lab we are also working on those compounds and we use 2 mM ammoniumacetaat +0.1% F.A. and ACN +0.1% F.A. The problem with those steroids are that they don"t have much or none (cant remember atm) double bonds in their structure so they are hard to ionize in ESI. For our LOD en LOQ we are making picolinic acid derivates so that they ionize better and you can measure at lower concentrations.

How difficult is cholesterol to see with ESI or APCI? Would the methods described above work on this compound?
Try a basic media in mobile fase(water + NH3 0.02%) and (MeOH + NH3 0.02%)

I also see nothing with acidic mobile fase, and with NH3 0.02% works OK

Sorry for my english
DHEA and DHEAS are a pain in the ass in our streroid profile. for some reson ionisation improves when ammoniumfluoride is added to mobile phase .....
You should be aware of the use of low level FA for steroids.

Search the forum for "Formic acid and steroids."
This returns 6 matches.
Scroll down to 4th match, by JMB.
This gives a lit. ref. to the use of VERY low formic acid concns (20-100 ppm) for steroid LC/MS analysis.
The authors are H. Marwah et al,in I believe J. Pharm Biomed Anal., about 2005
and this was the first use of ppm FA.

A second ref. is this Applications note by Thermo from about 2015 ? ... 333-EN.pdf

The original ref. for very low concn of acis for steroids is,

Journal of Chromatography A
Volume 964, Issues 1–2, 26 July 2002, Pages 137-151

Analysis of ergosteroids: VIII: Enhancement of signal response of neutral steroidal compounds in liquid chromatographic–electrospray ionization mass spectrometric analysis by mobile phase additives

Ashok Marwah, Padma Marwah, Henry Lardy

The signal response of moderately polar to nonpolar neutral steroidal compounds in positive ion mode was significantly improved in electrospray ionization mode by addition of volatile organic acids (trifluoroacetic acid, acetic and formic) at concentrations much lower than those normally employed for high-performance liquid chromatographic separations of ionic compounds. Each of the three acids enhanced the sensitivity, the order being: formic acid (∼50–200 ppm, v/v)>acetic acid (100–500 ppm)>trifluoroacetic acid (5–20 ppm). Higher concentrations caused decrease in the sensitivity. The extent of increase in the sensitivity was compound specific and also depended on the nature of organic modifier present in the mobile phase. Acetic acid was the acid of choice for the ‘wrong-way-round’ ionization of sulfate conjugates. The postcolumn addition of silver nitrate produced highly stable (M+Ag)+ adducts with concomitant increase in signal response and reduction in baseline noise.

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