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Dear All,
I am trying to develop a method for a steroid and the lower limit required for the assay is in pg/ml. Now i have noticed while tunning the compound that at very high response is observed during Q1 scan but while in Q3 the response after breakage is too less.?
I want to know that what are possible ways for increasing the response?
Please reply
Jacob
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Dear Jacob,
Try MRM that may give you good response.
Dr Zafar
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The following message was posted to: PharmPK
you may try APCi
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Dear Jacob,
Sensitivity of a molecule is directly dependant on ionosation
efficiency. Q1 response is not real indicator of ionization efficiency
as there will be contribution from chemical noise.
Steriods generally have low response.One way to address is to look for
some adducts like Sodium or Ammonium.
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Dear Jacob,
It's always been a challenge to develop steroid molecules in LC-MS/MS. In your case, the molecule seems to be very fragile. May be most of the part breaks as neutral lose. Ways:
1. Consider all the major fragments and prepare a quantification method (Using sum option)
2. Try to develop derivitization method. 3. You can also have a look of IS and CE voltage parameters.
Regards,
Sudipta Basu
Senior Research Scientist
Sai Advantium Pharma Ltd, Pune
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Dear all
you can try by changing the mobile phase. So try to tune the
compound in presence of
acetic acid in the ratio of 50:50 and see. Check that in column
compatible with acetic acid like
Sax column.
with regards
karthik
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Jacob,
Steroids do not light up nicely in any of the API sources (ESI, APCI, APPCI), which is not a surprise given their chemical structure.
If the issue is that you are getting adequate response using Q1, but then loosing that in Q3, this could be due to:
1-Noise contribution to the Q1 signal
2-Fragmentation in the fly through Q2
3-Tuning/contamination issues with Q3
We have managed to boost the signal for many endogenous steroids by considering multiple product ions. Some product ions (fragments) are not picked up by autotune functions because they have low absolute signal, but their signal/noise turned about to be way better than the ones from high absolute signal.
When we failed to obtain fragments of a decent mass, we used MIM (same mass on Q1 and Q3, using very low CE).
Finally, insource fragmentation is a big problem with some of these steroids. You can either use a low DP, or in some cases we ended up using an in-source fragment rather than the parent molecule.
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I was wondering how steroidal analysis has been this much difficult in LC-MS.
We had done before some couple of analyses of the plant sterols in nanoscale which went very fine usinf APCI source. I assume it is quite within the arena of polarity and ionizability covered by APCI. Yes, if they are metabolites or the degradation products formed of steroids which have lost their ionizable part and render more hydrocarbon in nature, then you need to think of alternatives (derivetization, more precisely).
In triple-Quad, the analysis is more straight forward if you have sample of right form. If your noise are annoying then you can use, IS-CID (in source collision induced dissociation of your defined noise) prior to its entry in Q1. You can find some invaluable articles from M. Thevesis etal on the mass spectrometric analysis of steriods at the forensic level!!!
Thanks and Best Regards,
Amrit
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Yes you can use APCI for steroid analysis as most of them are non polar. Though I have worked on several steroid molecules (more than 20), I faced lot of problem while developing. I faced problems like the molecules are not stable at Q1, no Q3 frags, response are not reproducible and also selectivity issues. As they are non-polar their chromatography are very easy and also UV absorbance. The molecules I worked were big molecules and some are indigenous and we have to achieve pg/mL level. In such case derivitization is a good and alternative option. Generic method developments for steroids are easy but we need to think differently for method development of difficult steroid molecules.
Regards,
Sudipta Basu
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Dear Amrut,
General literature observation is Steriods are difficult molecules for
analysis. May be with newer instrument geometry and column chemistry,
sensitivity is improved. Could you give specific examples from your
experience with sensitivity achieved? Also, please give detailed
reference of article cited.
Thanks,
Vinayak Nadiger
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Dear Vinayak,
First of I want to correct you in my name its, Amrit (I am Nepalese FYI) instead of Amrut.
Regarding mass spectrometric analysis of steroid, plenty literatures are available. If you excurs through some standard journals (RCM, JMS; JASMS, JPBA and Planta medica) you can find them quite a lot for endogenous steroids, conventional steroidal drugs, plant sterols and upto lately advanced marine steroids candidates. The common of them is the structural skeleton and biosynthesis (most are from shikkemic acid pathway).
Now for analyses, you can even nice MRM methods developed for the regio isomeric steroids. I cannot recall them (literatures) by heart as I am no more in that field but i do kon they exist.
The analytical condition for the steroids for precursor ion scanning is crucial where you have to play more with the depolarization potential of the ionization source. But indeed there is no more the rule of thumb other than experientially learning their structure and behavior towards ionization.
Even, if the signals in Q1 and Q3 both are subtle then, you can run the neutral loss scans as the such losses in case of steroidal molecules are exculsive and reproducible.
I remember one of the challanges in Innocentive for a development of LC-MS method for analysis of steroidal samples upto pcMolar level and my collegues won the award.
So, understanding EI-fragmentation behavior of steroids is crucial to correlate its ionization potential with the common instrumental variables of MS.
Thanks and Best Regards,
Amrit
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Dear Amrit,
Indeed analysis of seroids and specific hormones presents a very complex picture for the bioanalytical chemist. To achieve the selectivity by applying the normal acceptance criteria does not always work due to the presence of endogeneous matter in the matrix. I guess, the most logical approach would be to calculate the base level in a few plasma lots and then go for a LOQ which would be more than five times of the base level reading. Other approaches like charcoal stripping, etc could also be employed depending upon the type of submission.
Regards, Noel
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Dear Amrit,
All your points are correct. I am trying to emphasize that Steroid
analysis is not as easy as you are portraying it. May be you were lucky
to have a sensitive molecule to achieve great sensitivity. As Noel put
it, it is a challenge for bioanalytical scientist. I have not worked on
plant sterols and do not know about it. I am asking for specific example
you worked on and the sensitivity achieved.
Vinayak
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