Chromatography Forum

Dear all,

I am trying to develop a new HPLC-MS/MS method for the analysis of tetracyclines using a synergi Polar-RP column. The peaks looks pretty good, however, I don't know what is is going wrong!!! I am not able to achieve linearity. The standards are prepared in methanol and diluted in 100 % of water.
I will really appreciate any comment or suggestion,

Sincerely,

Carolina

What is your mobile phase at time of injection?

Does it match the 100% water of your standard?

Do you inject the same volume for each standard? (I know some methods vary the injection volume to make a calibration curve)

Can you give the calibration levels you are using?

ESI can be very sensitive to analyte concentration and the ionization can fall off with higher concentrations, if the curve is bending low at the top end this could be what is happening.

Thanks for the reply,
The gradient has initially 100 % of water 0.1% of formic acid,
- the injection volume is always 10 uL,
- the concentrations I am injecting is 100, 250, 500 and 1000 ng/ml.
The curve does not make sence at all!!! My R2 is 0.89 I have deleted the last point but even though!!

Thanks in advance! :

what instrument are you using? Electrospray is never as linear as PDA, and on some instruments it is truly horribly curvy.

Hi again,

I know, but with a sunfire column I get linearity for these compounds but back pressure problems.

In a situation like this, there are several possible sources of error....

1) Standards are off
2) Injection volumes are off
3) Some non-reversible binding of analytes to different column
4) Transfer of analytes to ion source is off
5) Ionization/detection are off

1), 2) & 3) can be checked by using measurements from the UV detector--is calibration linear ??

Your top std (1000 ng/mL) delivers only 10 ng per analyte---hardly enough to saturate the ionization/detection. I would expect the 250/500/1000 ng stds. to give linearity, and the 100 ng std. to be below the calibn. curve.
Do they ?? If not, do any 3 of 4 stds give linearity ?

Since the Sunfire column does give linearity (R2 = ???), it would seem that 1), 2), 4) & 5) are all OK; fault then lies with 3), which is the only change to the system.