low recoverys for metanephrine , normethanephrine

[TheProphet]

Hi everybody,

Let me introduce my problem to you,

I`m trying to set up a routine method to detect metanephrine and normethanephrine in plasma via LC-MS-MS. We are using a triple quad 6410 from agilent technologies.

The method consist of 3 parts.

First the manuel sample preparation via SPE.

For the extraction i`m using waters MCX columns 1 CC because in literature they are always having the highest recoverys , about 70 - 80 %.
The problem is i`m only getting 20 %.

The protocol for the SPE looks like this

condition : 1 mL MeOH
equilibrate: 1 mL Water
Load: 1 mL sample ( just pure MN in water, i`m first testing this because i want to exclude protein problems from the plasma)
Washstep1 : 1 mL HCL 0.1 N
Wasstep 2 : 1 mL MeOH
Elute: 1 mL NH4OH 5% in MeOH

After the Elution it gets dryed @ 45 C and under a low stream of N2. afterwards it gets reconstituted in 100 uL 95% (99.9 % ACN + 0.1% F.A.) and 5 % (ammoniumformate 100 mM). 75 uL gets injected into the HPLC. I need to inject so much else i don`t get my LOD.

For the HPLC separation

I`m using a Atlantis HILIC column from Waters.

Gradient with 2 mobile fases
A: 99.9% ACN + 0.1% F.A.
B 100 mM Ammoniumformate

Gradient starting At 95 % A and 5 % B
In time going to 80 % A and 20 % B
And then back to 95 % A and 5 % B

Separation is always good and no problems occur.

For the MS

We are measuring in MRM mode via elektrospray ionisation for 3 ions of each molecule.
The only thing that worries me is the high level of ACN. Somebody told me it is not good for the ionisation? can somebody explain this for me ?

So in the end i`m only getting 20% recoverys. already took the loadstep washsteps and analysed them , no losses in here...

Hope you experts can help me on this one

Sry for my bad english

Grtz TheProphet

[yangz00g]

Based on my experience with those compounds, your problem may be related to the wash and elute steps of your SPE. You need optimize those steps yourself to get desirable recovery.

[Uwe Neue]

You need to acidify your sample before loading it onto the SPE.

Yangz00g: methods developed in my lab have been verified over and over again and proven to function well. Your comment is very inappropriate.

[yangz00g]

Yangz00g: methods developed in my lab have been verified over and over again and proven to function well. Your comment is very inappropriate.

Sorry if my comments offended you (deleted), but it's exactly what I thought of literature. If all of them are reliable and trustworthy, we won't see so many scandals in the research society, and we won’t need any "experienced" scientist or this forum except those working for instrument companies, because simply following a published procedure or application note can get the job done perfectly.

Again, IMO.

In my lab, we use exactly the same procdure and same instrument developed by a major instrument company, but coluldn't get even close to the results published in an application note. I sincerely hope my experience is a wrong imagination. All the application notes should work well as they say, any question to it is not acceptable. If I cannot follow the procedure to "make" it work, I am simply too stupid.

[TheProphet]

You need to acidify your sample before loading it onto the SPE.

Yangz00g: methods developed in my lab have been verified over and over again and proven to function well. Your comment is very inappropriate.

Thx for the reply, acidify with what ? what do you recommend ? F.A. ? and in wich concentration ?


If i have to be honest most of the time i have to make little changes to the published protocols for getting the results that I want. Sometimes ennoying but it keeps me of the streets ;o)

Grttzzz TheProphet

[Uwe Neue]

we typically use phosphoric acid, but considering the pKa of your analyte, you should be able to do equally well with formic acid. Mix your analytes with 0.5 M acid 1:1.

[TheProphet]

Hi Neue thx again for the response ,

my stock solutions are already made in 0.1N HCl when i load my monster on the SPE columns, so i guess it`s acid enough... but still the same problem

do you know if the high ACN concentration has a bad influence on the ionisation ? i know this has nothing to do with my recovery but if the recovery is reproducible and i could get higher responses via better ionisation then i could counter my LOD problem. It would still be a weak step in the method but then my boss can decides what he wants..

Thx in advance

TheProphet

[Uwe Neue]

No, the responses in a high acetonitrile content have been reported to be better than in a high water content. So I expect the opposite.

What we have not tested is a possible loss of analyte in the containers after the SPE. Basic analytes often adsorb to glass. Maybe the loss is in the drying step, or in the sample vial.

[carls]

Below is a procedure using weak cation exchange for sample clean-up.

Elution with 5 v/v% formic acid gives quantitative recovery.

Its been a while since I ran this analysis so I dont recall if stability at high pH was a problem but I do remember this was not a particularly challenging application.

HILIC is the best separation mode for MS detection of these analytes:

http://www.phenomenex.com/cms400min/lit ... n_0018.pdf

[Uwe Neue]

When I wrote that "we have not tested the loss of ananlyte in the containers after SPE", I meant you and me. Any results?

(I am very well aware of loss of bases onto glass surfaces... Nothing new. Been there, done that..)