Ultrasensitive bioanalytical quantification with minimal method development time

Methods

Sample preparation: Monomethyl auristatin E (MMAE) was obtained from Achemblock (cat# 10353), and the internal standard, dolastatin-15 was obtained from Sigma (product # D5566). Stock solutions were prepared to 1 mg/mL for each in DMSO and stored at -20 ºC until use. Working standards were prepared by serial dilution to 10x the final concentrations. Standards were prepared by adding 10 µL of working standard to 100 µL of K2EDTA rat plasma (Bioreclamation IVT).

Standards and blanks were prepared for analysis by adding 300 µL of acetonitrile, containing 10 ng/mL of the internal standard, to 100 µL of sample to precipitate plasma proteins. The samples were vortexed for 1 min, then centrifuged at 14k rpm for 15 min. The supernatant was transferred to a clean Eppendorf tube and evaporated to near dryness under N₂. The samples were reconstituted with 100 µL 5% methanol in water and transferred to insert lined HPLC vials for analysis.

Chromatography: The nanoLC™ 425 System (SCIEX) with a 1-10 µL/min flow module and a 10 µL injection loop, operated in direct injection mode was used for separation. Mobile phase A was water with 0.1% formic acid by volume and mobile phase B was acetonitrile with 0.1% formic acid by volume. The column was a Phenomenex 0.3 x 50 mm Luna C18 (Phenomenex part # 00B-4251-AC). A simple 5-95% B gradient was used at a flow rate of 5 mL/min, with a total run time of 9 minutes which included the sample load time and column re-equilibration.

Mass spectrometry: A SCIEX 7500 System, operated in positive ESI MRM mode was used for data acquisition. The MRM transitions monitored are listed in Table 1, and the source conditions are listed in Table 2. 

Data processing: MS data acquisition was performed using SCIEX OS Software. Data processing was performed using the Analytics module in SCIEX OS Software. The calibration curve was run in triplicate with good linearity from 2.5 pg/mL to 10 ng/mL (Figure 2) with good accuracy and precision for all concentrations, shown in Table 3.

Requirement for very high sensitivity detection

Monomethyl auristatin E (MMAE) is a highly cytotoxic compound that is a synthetic analogue of the naturally occurring dolastatin class of compounds. It works by inhibiting the formation of tubulin during cellular division, and while it is far too toxic to be used independently as a cancer therapeutic, it is used as the toxic component of some antibody drug conjugates (Adcetris, Padcev). Monitoring of the unconjugated toxin is required as part of ADC clinical trials to ensure the drug linker, which binds the toxic payload to the antibody, does not release the toxic compound prior to entering the targeted cancer cells. Levels of unconjugated toxin are expected to be very low, and because they are so acutely toxic in their free state, very low detection limits are required to accurately and relevantly monitor for their presence.

Combining the well characterized sensitivity improvements of microflow HPLC with its ability to improve ionization efficiency, along with a highly sensitive triple quadrupole mass spectrometer, provided a very effective platform for routine, highly sensitive bioanalytical assays. Here, complex sample preparation and chromatography development time was minimized, so less time was spent on development, allowing results to be generated more quickly.

Conclusions

The method and data presented here illustrate the utility of using the SCIEX 7500 System in conjunction with microflow  chromatography as an analytical platform for routine, highly sensitive bioanalytical sample analysis. The 2.5 pg/mL LLOQ that was observed for MMAE in rat plasma was achieved using a generic, unoptimized protein precipitation sample preparation, and a generic HPLC gradient with standard stationary phase and mobile phases. The ability to routinely detect ultra-low analyte levels, with minimal method development time, will allow bioanalytical groups to deliver high quality data with rapid turnaround times.

References

1. Enabling new levels of quantification. SCIEX technical note RUO-MKT-02-11886-A.
2. Microflow chromatography: Bridging the sensitivity gap for quantitation of a pyrrolobenzodiazepine dimer. SCIEX technical note RUO-MKT-02-9512-A.
3. Pharmacologically relevant measurement of high potency drug candidates with improved sensitivity in in-vivo transporter assays. SCIEX technical note RUO-MKT-02-11960-A.