Polarity switching speeds metabolic stability studies using cassette analysis and UHPLC-QQQ

By Anabel S. Fandiño
Agilent R&D LC/MS Application Scientist

Metabolic stability is an important assay in drug discovery, where higher throughput is necessary to screen more compounds in less time. The Agilent 1290 Infinity LC System delivers the rapid LC separations that you need for this application, but the resulting subsecond peak widths require a triple quadrupole MS that can acquire data fast enough to provide good quantitation. The Agilent 6460 Triple Quadrupole LC/MS system meets this demand, even while switching polarity within the run.

Cassette analysis with polarity switching increases throughput

In vitro assays like metabolic stability play an important role in the early understanding of in vivo pharmacokinetic characteristics, and help to eliminate compounds that would fail in later stages of development. Typically, researchers assess metabolic stability using the cassette approach and fast LC/MS/MS methods. In the cassette approach, investigators create a cocktail of substrates post-incubation, which reduces the number of samples to analyze by LC/MS/MS (Figure 1). Fast LC methods, which use high flow rates and short sub-2-µm columns, reduce the analysis time and further increase sample throughput.[1, 2] Detection is usually performed using a triple quadrupole (QQQ) mass spectrometer operated in multiple reaction monitoring (MRM) mode.

Figure 1. This example shows how cassette analysis combines samples from metabolic stability assays, to decrease the sample load for UHPLC/MS/MS. (Click here to see this image larger.)

With cassette analysis, each cocktail typically ionizes in either positive or negative mode. Although it would be advantageous to pool substrates that ionize in both modes, that requires the MS to alternate between the two polarities within a run, which slows the data acquisition. For good quantitation, the MS must collect at least nine to ten data points across the extremely narrow peaks generated using fast chromatography, which is impossible if positive/negative switching is too slow.

With 30-millisecond polarity switching and 5-millisecond MRM dwell times, the Agilent 6460 Triple Quadrupole LC/MS system meets the challenge of high-speed data acquisition. To demonstrate, we performed metabolic stability studies with four drugs (buspirone, verapamil, dextromethorphan, and diclofenac) and two internal standards (dextrorphan-d₃ and diclofenac-d₄). At each incubation time (0, 5, 10, 15, 25 and 35 minutes), we took aliquots of each of the four samples and combined them. We then analyzed the samples using the Agilent 1290 Infinity LC System coupled to the Agilent 6460 Triple Quadrupole. A recent Application Note (5990-4469EN) provides details on the sample preparation and instrument conditions.

Figure 2. Even with polarity switching, the Agilent 6460 Triple Quadrupole provided reliable quantitation with these narrow UHPLC peaks. (Click here to see this image larger.)

Figure 3. The Agilent 6460 Triple Quadrupole gave excellent results both with polarity switching (left) and with single-polarity analyses (right). (Click here to see this image larger.)

Figure 4. The straight line indicates that the results with polarity switching correlated well with the results that used conventional analysis, all while delivering higher throughput. (Click here to see this image larger.)

High speed and high data quality using fast polarity switching

Operating the UHPLC system at high flow rates and pressures up to 1100 bar enabled a run time of only 1.5 minutes. The chromatographic peak widths were typically only 0.4 to 1.0 second at half height, but the 6460 Triple Quadrupole collected between nine and 35 data points across each peak, ensuring high-precision quantitation. Response relative standard deviations (RSDs) were 10% or less. Figure 2 compares the MRM chromatograms, data quality, and analysis times achieved using flow rates of 1.5 and 1.0 mL/min.

Excellent RSDs from both switched and non-switched analyses

Average area responses were similar using switched analysis (fast polarity switching) and non-switched analysis (positive-only or negative-only). Relative area RSD values were less than 10% using polarity switching and less than 9% using non-switched analysis. Figure 3 compares the data obtained for the pooled incubates with and without polarity switching, analyzed using the UHPLC method at 1.0 mL/min.

Similar metabolic stability results with and without polarity switching

We determined the percent parent drug remaining by comparing the average relative areas of the parent compound measured in the 5-, 10-, 15-, 25- and 35-min samples to that in the 0-min sample. Figure 4 shows the excellent correlation of results obtained using fast polarity switching and non-switched analysis, with correlation coefficients (r² > 0.9848).

More samples per day, with outstanding data quality

In summary, the combination of cassette analysis and fast polarity switching in the Agilent UHPLC/MS/MS system provided:

Highest sample throughput
Precise and accurate quantitation
Excellent chromatographic resolution

The fast data acquisition rate provided by the Agilent 6460 Triple Quadrupole LC/MS system makes it perfectly compatible with the Agilent 1290 Infinity LC, for excellent quantitation of peaks with subsecond peak widths at half height. To read all the details about this study, download the full Application Note (5990-4469EN).

References

T. J. Carlson and M. B. Fisher, “Recent advances in high throughput screening for ADME properties,” Combinatorial Chemistry and High Throughput Screening, 11(3), 258–264, 2008.
W. A. Korfmacher, “Principles and applications of LC-MS in drug discovery,” Drug Discovery Today, 10(20), 1357–1367, 2005.

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