Acyclovir on LC/MS/MS

[smkh]

Dear All,

Iwant to work abioequivalence study of acyclovir (human plasma) on the LC/MS/MS , and I'm now in the development phase ,any one have agood experience on that ? Ihave aproplem of Extraction procedure ,first I use Aprecipetation of perchloric acid which was agood approach but it makes many problems to the instrument ,then i change it to precipetation of acetonitrile but Iget abad peak shape and anot reproducible results , Ineed any advices about this but not SPE ..

thanks

[Uwe Neue]

Honestly, I am not at all surprised that the methods that you tried are creating problems for you with this compound. A good SPE method would solve the problem, but you specifically say that you do not want any advice on this...

[smkh]

Idont know if ican optimize my method using the same procedure by precipetation of acetonitrile ,is the problem in the drug or in the precipetation although acetonitrile precipetation is common use on LC/MS/MS.
May SPE is agood solution but ihope it not the only solution because ihave alot of samples but ihavent agood equibment.

[Noser222]

What equipment are you lacking for SPE? In my experience, if I have a whole lot of samples, I set all the SPE tubes up at the same time and let them flow by gravity.
You can try acetonitrile in the meantime, but that still won't get your sample as clean as SPE would.

[fishin' addict]

What is the instrument type?
What Type of column?
What was the mobile phase?
What were the LC Conditions i.e. Gradient (give a rough descriptio) or iso?
How much sample was crashed and what's the ratio (ACN:Matrix)?
What was the reconstitution solvent?
How much sample did you inject?
Were the curves divergent or just scattered error aka .. did the QC's calibrate low or high?

FA

[smkh]

Instrument type : LC/MS/MS API 5000 (ABI)
Column:Gimini C18 (50*3.00 mm) 5u
Mobile phase : isocratic 90% H2o+10%Acetonitile+0.1% formic acid
0.50 ml plasma with ratio (2:1)
Direct injection after precipetation without evaporation
20 ul injected
qc's give high result by time

thanks

[Bryan Evans]

You can try direct injection without protein precipitation.
If you contact me - I tell you more about how.

[HW Mueller]

Bryan, most likely there are a bunch of people, like me, who would like to be informed on what you will suggest.
A restricted access type column?
If so, have you solved the problem some proteins pose, especially of some patients, by accumulating in places where one does not want them? (I finally had to add a partial precipitation step by adding some Na2SO4 when using Pinkerton columns, plugging and occasionally huge protein peaks were requiring that).
For me an academic question as there is no LC-MS here: How compatible is your suggestion with MS?
Do you have any examples in which your method was used on patients or test persons for extendet routine analyses?

[Bryan Evans]

Hi Hans - thank you for your interest.

Imtakt has developed a new column, Cadenza HS-C18. This column can be used for the direct injection of plasma and serum. Large proteins elute quickly through the column - and the drug compound is retained on the C18 ligand. It is LC-MS compatible (all of our phases are) - meaning there is very low column bleed.

The proteins will not coagulate in the column under gradient elution - here are some examples:

http://www.imtakt.com/TecInfo/TI302E.pdf
http://www.imtakt.com/TecInfo/TI296E.pdf

Acyclovir is challenging because it is not well retained on ODS phase. But we do have an application with it on our instruction manual. That I would have to email to you.

[smkh]

Hello Bryan

Thanks for your interest
Ihope your suggestions can help me , can you send them to me on this
E-mail
sewar@pru.com.jo

[HW Mueller]

Bryan, the pdf links don´t open on my PC, because of a Japanese language problem.
Anyway, I was not wondering about column bleed but mobile phases which are compatible with protein as well as MS.
My main concern was with routine injection of patients plasma/serum which will inevitably include some with enough weird proteins to give you a peak width of one hour (in favorable cases) or gunk up the whole system (if you have the luck which I always had). (As a matter of fact, even normal plasma/serum appears to leave enough traces to produce a problem in routine applications).