Chromatography Forum

Dear Friends

I am working on diclofenac analysis in plasma sample using API 2000.
During the optimization phase ( I infused 1 ng/ul of a diclofenac solution in acetonitrile-amonium acetate 1mM 1:1) , I noticed that a had a better signal in the positive mode, but a lot of noise. In the negative mode the noise is much lower.
Most of the papers published about diclofenac and LC-MS, use positive mode.
What is the importance of this noise when I start with the chromatography part, in other words, should I say that if I use positive mode (higher signal but lot of noise) I will have problems in the chromatography ?

Thanks
Maristela

Maristela,

Both the signal (S) and the background (chemical) noise (N) that are observed in LC/MS are important. However, the detectability (and therefore any quantitation) is determined by the Signal-to-Noise ratio (S/N). This is why LC/MS/MS methods are usually more sensitive, i.e. lower limit of detection, than LC/MS. With MS/MS, the chemical noise is very reduced compared to MS, and so the S/N ratio is increased. You have noted that the noise is much lower in the -ve ion mode vs. the +ve mode, and this is a general observation.

You should move to diclofenac spiked at a reasonably "high" level into plasma, develop the chromatography to resolve it from other components in the plasma (that may cause suppression of ionization) and take some measurements of S/N in both -ve and +ve modes with decreasing levels. This will help to establish the best set of MS conditions for your analysis.

Generally speaking, if there are published methods for a particular analyte that meet your detectability needs in a given matrix with instrumentation available to you, then do not waste time by re-inventing the wheel.

JMB

Maristela,

Both the signal (S) and the background (chemical) noise (N) that are observed in LC/MS are important. However, the detectability (and therefore any quantitation) is determined by the Signal-to-Noise ratio (S/N). This is why LC/MS/MS methods are usually more sensitive, i.e. lower limit of detection, than LC/MS. With MS/MS, the chemical noise is very reduced compared to MS, and so the S/N ratio is increased. You have noted that the noise is much lower in the -ve ion mode vs. the +ve mode, and this is a general observation.

You should move to diclofenac spiked at a reasonably "high" level into plasma, develop the chromatography to resolve it from other components in the plasma (that may cause suppression of ionization) and take some measurements of S/N in both -ve and +ve modes with decreasing levels. This will help to establish the best set of MS conditions for your analysis.

Generally speaking, if there are published methods for a particular analyte that meet your detectability needs in a given matrix with instrumentation available to you, then do not waste time by re-inventing the wheel.

JMB

OK.

I will make the tests you suggest, but I would like to know if there is any specific reason (stability of the positive compound or...) to choose the positive instead of negative.
Thank you anyway.
Maristela

Maristela,

Diclofenac is essentially a diphenylamine, substituted with a -CH2COOH on one phenyl ring. You can either protonate the secondary amine, or deprotonate the carboxylic acid group. I suspect that previous workers have used a low pH to protonate the -NH and keep the COO- also protonated (as the neutral -COOH) for good, sharp chromatography and better S/N than if the Aryl-COO- was tailing under high pH conditions.

JMB

The Aryl-COO- won't tail under high pH conditions. For this compound, you are completely free to choose the pH that gives you the best separation from the matrix, followed by the selection of the +ve or -ve ionization mode that is best for the chosen pH.