April 13, 2021, 04:15:16 AM
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Topic: Derivatization Reagents  (Read 201 times)

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Offline ss2905

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Derivatization Reagents
« on: April 02, 2021, 08:40:50 AM »
Hello,
I am setting up an LC-MS/MS analysis method for quantitative analysis of androgens in chemically castraded men (prostate cancer). Separating DHT and testosterone of such low concentrations in serum samples, seems to be quite challenging. Especially DHT, as the concentration is much lower than Testosterone. Also, DHT ionizes much more difficult. Any advice what derivatization reagent to use for DHT? One that works selectively on DHT and NOT on Testosterone. Any other advice for this analysis is also welcome.

Thanks in advance!

Offline wildfyr

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Re: Derivatization Reagents
« Reply #1 on: April 10, 2021, 05:33:31 PM »
Hmm.... testoterone is a conjugated ketone, whereas DHT is inconjugated. I wonder if their reactivity towards imine formation is different enough.

Offline Babcock_Hall

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Re: Derivatization Reagents
« Reply #2 on: April 11, 2021, 09:46:24 AM »
I don't have any specific experience in this area, but I went to PubMed and searched on "testosterone dihydrotestosterone derivatization."  54 citations came up.  Undoubtedly some will not be helpful to you, but perhaps some will be.  This paper, which I found with a slightly modified search and which mentions hypogonadism, claims baseline separation with pyridine 2,3-dicarboxylic acid:  DOI: 10.1016/j.steroids.2008.01.018

Offline ss2905

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Re: Derivatization Reagents
« Reply #3 on: Yesterday at 04:48:27 AM »
Hmm.... testoterone is a conjugated ketone, whereas DHT is inconjugated. I wonder if their reactivity towards imine formation is different enough.

Hi,
Thank you for your response. I have tried both hydroxylamine and O-tert-butyl hydroxylamine. OTB provided better results. However, I am still observing co-eluting peaks. Is this a problem with the specificity of the derivatization reagent? Which column would you recommend for the separation?
The interferences are Testosterone, EpiTestosterone, and DHEA.

Offline ss2905

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Re: Derivatization Reagents
« Reply #4 on: Yesterday at 04:49:38 AM »
I don't have any specific experience in this area, but I went to PubMed and searched on "testosterone dihydrotestosterone derivatization."  54 citations came up.  Undoubtedly some will not be helpful to you, but perhaps some will be.  This paper, which I found with a slightly modified search and which mentions hypogonadism, claims baseline separation with pyridine 2,3-dicarboxylic acid:  DOI: 10.1016/j.steroids.2008.01.018

I will check it out. Thank you so much!

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