Drug Metabolism in Drug Design and Development Basic Concepts and Practice

TABLE 12.9 Advantages and limitations of the most common hyphenated NMR methods.Technique

Setup

Advantages

Limitations

LC–NMR

HPLC system connected to the

NMR flow probe (30–120

m

l)

via a UV detector. Experimentscan be performed in on-flow,stop-flow, and loop collectionmodes

Eliminates the need for sample

isolation and purificationassociated with regular tubeNMR

Useful for analysing

compounds that may degradeduring isolation

Use of deuterated solvents for

LCSample limited to columnsize, NMR flow cell andchromatographic resolution

Solvent suppression techniques

are necessary even when bothsolvents are deuterated

DAD UV detector used for peak

detectionRegular reversed-phase C18 (4.6

150 mm) columns

CH

CN or CH 3

OH:H 3

O with 2

trifloroacetic acid or formicacid as a modifier.

At least one of the solvents

is deuterated.

Isocratic and gradient HPLC

methods can be used

Need for NMR friendly solvent

modifiers

Disconnect between UV and

MS retention time due to theuse of two separate instruments

LC–NMR–MS

20 : 1 flow splitter added after the

LC column resulting in 5% ofthe flow going into the MSsource and 95% going into theNMR flow cell

Disconnect between the UV and

MS retention times are addressed

MS and MS/MS data can be

collected during analysis

Need for MS friendly solvent

modifier (i.e., formic acid)

Complex setup. Maintenance

of the hardware istime consuming

All three modes can be performed

Can be used to determine number

of exchangeable protons in themetabolite

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