06 June 2017 12 1K Report

I have a new molecule for which I have initially developed an isocratic LC-MS/MS method (Synergi Fusion column, H2O/ACN 20/80 (+0.1% HCOOH)). Analysis of this method gives limited sensitivity because of broad peak shape (but it was sufficient for the time being), and with no traces of carry-over.

Later on I needed a more sensitive method, so I switched to a gradient method to sharpen up the peak. Now I have great sensitivity but a carry-over of 50%!

Gradient:

A: H2O+0.1% HCOOH

B: ACN + 0.1% HCOOH

t        %A      %B

0       90        10

3       90        10

3.1    20        80

5       20        80

5.1    10        90

8       10        90

Rinse solvent: ACN

Injection of a standard solution followed by a series of blanks showed that the peaks decrease somewhat evenly by about half, after each injection. It will be impractical to therefore injection 10 blanks after each standard solution and sample.

Injection of a completely fresh blank vial (injected as a 'sample) after injection of a standard gave a peak, suggesting that the analyte does not stick to the outside of the needle. Use of an external wash vial showed no improvement in the carry-over. So the problem must be somewhere in the system.

I tried a number of things already, none of which had any effect:

- Tried a different column (Synergi Polar instead of Synergi Fusion)

- raise the gradient to 100% ACN and keep it for an extra minute

- rinse solvent: ACN/H2O 50/50, and MeOH/H2O 50/50 + 1% acetic acid

- used an external wash vial with ACN/THF 90/10

I then went back to using the isocratic method, and no carry-over was observed. But it is not sensitive enough, so I really need this gradient method to work.

What other rinse solution combinations could work? Perhaps something more similar to the mobile phase?

- H2O/ACN 20/80 + 0.1% HCOOH ??

- H2O: ACN: MeOH: Isopropanol (25: 25: 25: 25 V/V) ??

The LC-MS/MS system is an Agilent Series 1290 Pump and Autosampler, with an API 5500 MS

Any help would be appreciated!

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