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Positive Ion Electrospray LC/MS/MS Analysis of Strongly Basic Analytes with High pH Mobile Phases, “Wrong-Way-Round” Ionization
Laura Nakovich, Linge Li, Moucun Yuan, James Creegan, William R. Mylott, Bruce Hidy, and Rand Jenkins • PPD, Richmond, Virginia
INTRODUCTION
Figure 1. Acidic Mobile Phase Conditions
SMALL PEPTIDE AND METABOLITE
Figure 4. Acidic Mobile Phase Conditions
High pH mobile phases with hybrid particle or The MW of the small proprietary peptide is 1528 amu XIC of +MRM (2 pairs): 765.0/110.0 .
Max. 1.7e4 cps.
XIC of +MRM (2 pairs): 765.0/110.0 .
Max. 455.0 cps.
polymeric-based HPLC columns offer a powerful and is measured as the [M+2H]+2 with a m/z of 765. combination for basic analyte LC/MS/MS. These The MW of the metabolite is 1 amu higher than the analytes can be difficult to analyze using silica-based parent resulting in a 0.5 amu difference in m/z. The key HPLC columns, often requiring highly aqueous mobile structural difference resides with the metabolite having phases, extreme pH, and ion-pair reagents to achieve a terminal carboxylic acid and the parent having a adequate retention and peak shape. These conditions terminal amide. The chromatographic separation of the can compromise MS detection sensitivity due to parent and metabolite is paramount since the inefficient desolvation and ion suppression effects. metabolite produces the same MRM as the parent due to the resolution limits of the quadrupoles, and if not chromatographic problems by providing better resolved would lead to an overestimation of parent retention, improved peak shape, and surprisingly good concentrations for samples containing the metabolite. MS sensitivity using “wrong-way-round” ionization. Loratadine and descarboethoxyloratadine provide an example of sensitivity achieved with this ionization. The small peptide and its metabolite are isolated from A small peptide and its metabolite demonstrate the 100 µL of human plasma by protein precipitation. chromatographic selectivity gains obtained using high Figure 5. “Wrong-way-round” Conditions
XIC of +MRM (2 pairs): 765.0/110.0 .
Max. 1.4e4 cps.
XIC of +MRM (2 pairs): 765.0/110.0.
Max. 1750.0 cps.
25.0 pg/mL descarboethoxyloratadine (2.5 pg on column) LORATADINE AND
The chromatography was evaluated using a Waters DESCARBOETHOXYLORATADINE
Figure 2. “Wrong-way-round” Conditions
Atlantis dC18, 2.1x100 mm analytical column using a binary acidic aqueous ammonium formate/acetonitrile/ formic acid gradient at a flowrate of 0.4 mL/min. As can Loratadine and descarboethoxyloratadine are isolated be seen from the chromatograms the separation of the from 250 µL of human plasma by LLE with ethyl parent and metabolite using acidic mobile phase was acetate and separated via column switching using a Phenomenex Gemini C18 guard column (4x2mm), a Gemini C6 phenyl analytical column (50x2mm). Analytes are eluted with a mobile phase consisting of 80:20 methanol/10 mM ammonium bicarbonate pH 11 HPLC separation was achieved using column switching at a flow of 0.25 mL/min. The precolumn is back- with two Waters XBridge C18 columns (guard: flushed to remove phospholipids. Analysis time is 5.5 10x2.1mm; analytical: 50x2.1mm) using a binary min. Analytes are detected by Sciex API 3000 aqueous ammonium bicarbonate/acetonitrile/methanol equipped with an Ionics HSID interface (MRM mode / gradient at a flowrate of 0.4 mL/min. The precolumn is forward-flushed to remove the trapped phospholipids, and the total analysis time is 6 minutes. Analytes are CONCLUSION
detected on a Sciex API 4000 in MRM mode using The term “wrong-way-round” ionization refers to the formation of positive ions ([M+H]+) under basic pH mobile phase conditions and A loratadine and descarboethoxyloratadine assay was 25.0 pg/mL descarboethoxyloratadine (0.8 pg on column) formation of negative ions ([M-H]-) under acidic pH mobile phase validated over a concentration range of 25.0 to 10000 conditions. Historically, the formation of ions using electrospray has Figure 3. Loratadine and Descarboethoxyloratadine Assay Valve Diagram RESULTS
pg/mL with linearity demonstrated by an average been thought to occur in the liquid phase and hence to be highly correlation coefficient of 0.9990 for loratadine and The high pH mobile phase conditions significantly dependent on the mobile phase pH. Thus “wrong-way-round” 0.9984 for descarboethoxyloratadine. Inter-day QC improved the chromatographic resolution resulting in ionization conditions contradict the predicted ionization states based precision was 2.59-7.56% CV for loratadine and 2.72 – complete baseline separation. The high pH separation upon solution pH, and suggests other mechanisms of ionization are 5.46% CV for descarboethoxyloratadine. Inter-day QC can be attributed to an increase in polarity for the responsible for the sensitivities observed. Regardless of the accuracy QCs was 0.162 – 3.67%RE for loratadine and metabolite (carboxylic acid group ionized), while the mechanism, the use of “wrong-way-round” ionization has been 0.483 –3.04%RE for descarboethoxyloratadine of the polarity of the parent remains the same. demonstrated to be an effective approach to obtaining sensitivity and nominal values. LLOQ signal-to-noise was greater than improved chromatography for the compounds presented.
15:1 for both analytes. Assay ruggedness was evaluated by analyzing two 96-well blocks with total of ACKNOWLEDGMENTS
192 extracted matrix injections with no degradation of The authors would like to acknowledge Margaret L. Ware’s contribution to the

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