Drug Metabolism in Drug Design and Development Basic Concepts and Practice

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TABLE 12.10 Advantages and limitations of the common LC–NMR–MS

methods.

Mode

Setup

Advantages

Limitations

On-flow

In this mode the HPLC column is

connected directly to the NMRprobe via a DAD detector

Provides a metabolite profile of

the sample

Limited to compounds with on

column loadings

>

10

m

g

A series of 1D spectra are

collected with a predefinednumber of scans withoutstopping the chromatographicexperiment

Can be used with

1 H

and

19

F

to

monitor metabolitedegradationWith

19

F

monitoring, as most of thepeaks observed are from thecompound solvent suppressionis not needed

Limited to 1D NMR

experiments.Broadening of theNMR signal due to on-flowconditions

Solvent suppression is a challenge

due to solvent gradients usedfor LC

Stop-flow

In this mode the LC chromato-

graphy is temporarily stoppedat the peak of interest using theUV or MS (LC–NMR–MS)signal

Detailed structure determination

can be performed on the peakof interest by performing 1Dand 2D NMR

The LC peak shapes deteriorate

for the latter peaks of interest

The timing between the UV or MS

detector and the NMR flow cellis critical. Either the UV or theMS signal can be used to pausethe LC run.

>

100 ng for

1 H

experiments and

>

1

m

g for 2D

1 H



1 H

experi-

ments is required.

LC peaks of interest must be well

resolved.

>

2 min retention time

differenceCross contaminationfrom various LC fractions