CPSA Digest 2003

Day 3

WeOB3

Bioanalytical Applications of Hydrophilic and Ion Exchange LC-MS/MS

Premise
Hydrophilic and ion exchange chromatography are under-utilized for bioanalytical LC-MS/MS applications

• Mobile phase (80/20 ACN/buffer solution) is compatible with MS ionization interface for maximum sensitivity.
• Allows for high throughput.
• Straightforward optimization of LC conditions.
• Robust and reproducible.

Flavoxate and metabolite MFA (3-Methylflavone-8-Carboxylic Acid) Case Study

• Flavoxate is a basic compound, but MFA metabolite is acidic,
• Deuterated internal standards were used.
• Extraction scheme was simply protein precipitation using acetonitrile.
• Flavoxate: LC column, Thermo Hypersil silica, 50x3mm, 3um; Mobile phase, 80/20 acetonitrile/10mM ammonium formate, 1mL/min.
• MFA: LC column, Thermo Hypersil BioBasic AX, 10x3mm, 5um; Mobile phase, 80/20 acetonitrile/10mM ammonium formate, 1mL/min.
• Interface: ESI positive ion mode.
• Retention time was 0.9 min with a runtime of 1.5 min.

Stable Label Separation Observed
A rare case of LC separation of deuterium labeled internal standard from its corresponding compound was observed. The mechanism of the separation has been determined as an ionic interaction between ionized silanol groups (LC column) and basic analytes. From the chemical structures of these compounds, the deuterium was labeled on -NCH3 for d3-oxymorphone and on -OCH3 for both d3-oxycodone and d3-noroxycodone. It is possible that the isotope effect indirectly changed the electron distribution of tertiary amine, therefore affecting the ionic interaction between the tertiary amine and silanols, causing the separation of oxymorphone and d3-oxymorphone. When the pH of the mobile phase was lowered, the amount of ionized silanol groups was reduced and no separation was observed.

NNAL Case Study
LC-MS/MS Bioanalysis of Total 4-Methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) in Human Urine

SCX vs. Silica Chromatography for NNAL

• SCX showed very clean chromatogram; no matrix effect.
• SCX showed very good chromatographic resolution; showed chiral separation (split peak) with k' ~4, which is not desired.
• Changed to silica chromatography to eliminate chiral separation (single peak), but gained a matrix effect and an interference peak.
• Retention time on silica column was doubled to separate analyte from matrix effect and interference peak.
• LC column, Thermo Hypersil silica, 50x3mm, 3μm; Mobile phase, 70/30/0.7 acetonitrile/water/formic acid.

Conclusions

• Hydrophilic and ion exchange chromatography are popular for small molecule LC-MS/MS bioanalytical methods in our lab (> 90% of methods developed).
• A stable labeled internal standard is the best choice.
• Don't ignore the matrix effects!
• Lengthy chromatographic separation is not the only way to overcome matrix effects; try different interfaces or post-column infusion.

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