bstfa not reacted!!

[che313]

i'm working with extraction of carboxy thc from urine.
as the carboxilic acid present dervatization is must.
bstfa is widley used in researches but for me it not works
I check also mstfa, mtbstfa and bstfa with 1%TMS no reaction at all.

I use agilent 6890N and msd 5973 with hp-5ms 30m, 0.25mm, 0.25um
100C for 3 min then raising to 300C @ 20C/min and hold 6 min. with He flow is 1 ml/min and injection is 250C, source 230C and quadropol 150C and inonization @ 7o mev.

[Bruce Hamilton]

I think we need much more details about your sample preparation, perhaps also your chromatography.

My suspicion is that you have too much water somewhere - either in your reagents or procedure. . But please provide full sample preparation details, or point to the detailed published method.

Bruce Hamilton

[che313]

the sample is prepared by hydrolyz urine by KOH and then adjust the ph to 2 and extracting carboxy thc by organic solvent. after evaporate the solvent to dryness under nitrogene its dervatized by bstfa @ 60C for 20 min.
I did TLC also and the it gives very clear red spots when spray with fast blue.
I try mstfa, bstfa with 1%tms, mtbstfa at 60C for 20min and no carboxy-tms in chromaotogram but there is another drugs reacted like ibuprofene and acids!!
When try @ 90C for 15 min no peaks at all same as blank!!

[krickos]

Hmmm

Should work, are you doing exactly as in this application with regard so sample preparation?
http://www.innovatek.com.co/pdf/ANALISI ... %20DSQ.pdf

Are you sure you use an excess of reagent?

TLC shows that extraction worked or do mean that dervatization worked?

Edit:
Also it seems like the acid you look for gives a high boiling product even after dervatization with reagents used in the above application or similar. So you might need to increase temperatures in injector/source as well and consider what injection type you do (split/splitless?).

[Consumer Products Guy]

I've used BSTFA for decades (with 1% TMCS). Any issues I've had can ALWAYS be traced to not enough excess reagent. Sometimes adding trimethylsilyl group(s) decreases the retention times by reducing polarity even though additional mass is added, sometimes retention time increases (like when BHT is derivatized). I'd say start with your standard; derivatize and optimize your chromatography on that, then move on to samples.

[che313]

Hmmm

Should work, are you doing exactly as in this application with regard so sample preparation?
http://www.innovatek.com.co/pdf/ANALISI ... %20DSQ.pdf

Are you sure you use an excess of reagent?

TLC shows that extraction worked or do mean that dervatization worked?

Edit:
Also it seems like the acid you look for gives a high boiling product even after dervatization with reagents used in the above application or similar. So you might need to increase temperatures in injector/source as well and consider what injection type you do (split/splitless?).

- No this application is utlizing solid phase extraction while I'm liquid liquid extraction
- I use 0.1, 0.2, 0.5 & 1.0 mL of BSTFA

-TLC shows extarction is worked very well

- Injector is splitless and its temp is 250 where source is 230

-thanks for your comment I'll try with incresing injector temp

[kishorebits]

I have used BSTFA and MSTFA quiet a lot for the analysis of endogenous metabolites. I noticed that even slightest amount of moisture in the sample will make BSTFA inactive. Therefore, subsequent to drying extracted samples under nitrogen, I spike 100 micro L of toulene (dried on sodium sulphate or using molecular sieve) vortex and dry one more time. In my case, if I donot use additional step of drying using toulene, I see irregular patterns of BSTFA reaction. Toulene helps in removing residual amount of moisture which may be present after nitrogen drying.

[Bruce Hamilton]

I'm with kishorebits, any water needs to be eliminated before derivatisation, as well as ensuring you are adding excess reagent - which also often has the benefit of helping keep your injector less active.

As CPG suggests, work with your standards first, then look at spiked sample
recoveries.

Bruce Hamilton

[che313]

Can the pH affecting the reaction and gc sepration? I assume the pH in my case is <2

[Consumer Products Guy]

There are publications which described using DMF to which HCl had been added, then derivatizing, so I'd say that your suspicion of low pH affecting this is unfounded.

[krickos]

hi

There are some other pitfalls.

Moist seems not the issue as other drugs react but watch that factor in any case.

For one your TMS product might NOT be soluble in the reagent while ibuprofene is
, then you might have to use a solvent as well like dry toluene or likewise.

Dugged around in "ye old" lab notes and recalled a similar compund (sadly patent restricted) but I used MSTFA + 1% TMCS with toluene and a hint of pyridine without any issues.

The link below gives some good hints for MSTFA:

http://www.piercenet.com/files/0519TB-1.PDF

And as mentioned earlier start with standard then extractions, good luck

[che313]

Thank you for everybody shared with me his/her knowledge.
Finally did it but with thiese conditions, the pH for extraction is 4, and I use toluene to dry the sample before add mstfa or bstfa.

4 samples are tested, two with mstfa and two with bstfa
one of mstfa shows very good result although the abundance is not high but the ions quality was 96.
the other one shows very very large peak of ibuprfene-tms!!
for bstfa one not react with carboxy thc and the the other react and gives both ibuprofen-tms high quality 95+ and only 58 for carboxy thc.

[Bigbear]

I'm with the too much moisture crowd. In the past we replace the air in the reaction tube with dry nitrogen prior to derivatization.

[Don_Hilton]

Che313,

Dry a small quantity of dry pyridine in the derivitization. To keep the pyridine dry put some KOH pellets into the bottle. You may have to play with the volume a bit, but you can start with something like half the volume of BSTFA you add. As long as you add the BSTFA in excess, you should derivatize any thing that can be derivatized.

[che313]

Che313,

Dry a small quantity of dry pyridine in the derivitization. To keep the pyridine dry put some KOH pellets into the bottle. You may have to play with the volume a bit, but you can start with something like half the volume of BSTFA you add. As long as you add the BSTFA in excess, you should derivatize any thing that can be derivatized.

U mean I have to add dry pyridine to my bstfa bottle? And to get dry pyridin I have to add some KOH pellets in its bottle?
Is that what u want to say?