Methods

Immunocapture: A streptavidin coated immunoaffinity magnetic bead slurry was aliquoted and washed with PBS buffer (1x) three times. Biotinylated goat anti-human IgG antibody was added to the beads and incubated at room temperature for 1 hour with shaking. The conjugated beads were washed three times and resuspended in PBS buffer. Calibration standard samples were prepared by spiking trastuzumab-emtansine and SILuMab (internal standard) into rat plasma and performing serial dilution. The concentrations of trastuzumab-emtansine in plasma ranged from 0.5 - 20,000 ng/mL. To each calibration standard sample, 250 µL of PBS Buffer (1×) and 50 µL conjugated bead slurry were added and the mixtures were incubated at room temperature for 1 hour with shaking. The beads were accumulated by magnetic stand and washed sequentially with PBS buffer and 10 mM ammonium bicarbonate. The target proteins were eluted by incubating the beads with 0.1% TFA in water and vortexing for 10 min.  

Protease digestion of immuno-enriched eluents: The eluents were neutralized with 500 mM ammonium bicarbonate in water and incubated at 95 °C for 10 mins with shaking. The samples were cooled to room temperature and incubated with 1 µg of trypsin/Lys-C mix per sample overnight at 37 °C. The digestion was aborted by adding formic acid. The samples were centrifuged at 15,000×g for 5 min. The supernatants were collected and subjected to LC-MS/MS analysis.

LC-MS/MS conditions: Samples were analyzed in triplicate by a SCIEX Triple Quad 7500 System, coupled with an ExionLC System. The method details are summarized in Tables 1, 2 and 3. The data were processed using the Analytics module in SCIEX OS Software 2.0.

Quantification results

The SCIEX Triple Quad 7500 System integrates innovations that provide improvements in both ion generation and ion sampling. The OptiFlow Pro Ion Source with E Lens Technology provides improvement in ion generation and the D Jet Ion Guide efficiently captures and transmits the ions in the high gas flow behind the orifice plate. ¹

With the optimized method, the presented immunocapture-LCMRM assay demonstrated ultra-high sensitivity to quantify trastuzumab-emtansine in rat plasma, with the LLOQ at 1 ng/mL and the LOD at 0.5 ng/mL (Figure 1). As summarized in Figure 2, the assay accuracy is 86-106% and %CVs are below 10% for all tested samples. The calibration curve covered 4.5 orders of magnitude (1-20000 ng/mL) (Figure 3) and displayed a regression coefficient (r) of 0.996 using a weighting of 1/x².

Conclusion
 

• A highly sensitive immunoaffinity-LC-MRM workflow for quantifying trastuzumab emtansine in rat plasma was demonstrated
  
  
• By using the SCIEX Triple Quad 7500 System coupled with analytical flow HPLC, trastuzumab-emtansine was solidly quantified at 1 ng/mL level
  
  
• Good reproducibility, accuracy and wide linear dynamic range as 4.5 orders of magnitude were achieved at the same time

References
 

1. Enabling new levels of quantification. SCIEX technical note RUO-MKT-02-11886-A.
   
   
2. Quantification of trastuzumab in rat plasma using an improved immunoaffinity-LC-MS/MS method. SCIEX technical note RUO-MKT-02-7597-A.