Methods

Samples and reagents: The antibody trastuzumab emtansine was purchased from Myonex, and SILUmab (Sigma) was used as internal standard. For digestion, a trypsin/lys-C mixture (Promega) was used.

Sample preparation: For this study, the sample was similar to that of an immunoaffinity extracted antibody, which typically is free of interference and can be readily analyzed by microflow LC-MS/MS after digestion² . A 150 µL aliquot of a 20 µg/mL solution of either trastuzumab emtansine or SILUmab in 500 mM NH₄HCO₃/1 mM CaCl was first denatured at 90°C for 10 minutes, and then digested with 1 µg of trypsin/lys-C for 1 hour at 50°C. The digestion was stopped by adding 3 µL formic acid. Samples were prepared from the stock solutions by dilution with the same mixture of digestion buffer and formic acid as was used for the digestion. Finally, 2% acetonitrile and 5% acetic acid was added for increased sample stability. For the robustness test, the concentration of both the antibody trastuzumab emtansine and the internal standard (IS), SILUmab, was 100 ng/mL. The final concentration of the IS for the standard curves was also 100 ng/mL.

Chromatography: A Waters ACQUITY UPLC M-Class system, consisting of a micro binary solvent manager (µBSM), a micro sample manager with fixed loop (µSM-FL), an auxiliary solvent manager (ASM), and a trap valve manager (TVM) was used in trap-and-elute mode. Figure 2 shows the setup for the trap-andelute mode. 

Gradient and other chromatographic conditions and columns are summarized in Tables 1 and 2. A volume of 10 µL of sample was loaded onto the trap column for 1 minute with a loading flow rate of 35 µL/min. The analytical flow rate was 5 µL/min, and a 4 min gradient from 3 to 50 %B was used for the separation.

Mass spectrometry: A SCIEX 7500 system with an OptiFlow Pro ion source with E Lens probe and a micro (low) electrode was used in MRM mode. All source and MS parameters were optimized (Tables 3 and 4). The MS method included multiple MRM transitions of signature tryptic peptides for both trastuzumab emtansine and the IS.

Data processing: MRM data was processed with SCIEX OS software 2.0 using the MQ4 integration algorithm. Linear regression with a 1/x weighting was used.

Results and discussion

Software integration
 

Once the driver for the ACQUITY UPLC M-Class system is installed on the SCIEX 7500 system acquisition computer, the system can be added in SCIEX OS software configuration as an “Integrated System” (Figure 3). LC methods are generated in the “LC Method” section of SCIEX OS software, using the tabs for the various modules of the M-class system. As an example, Figure 4 shows the tab where the gradient method is entered. After the LC method is completed and saved, it can be used in the batch with the corresponding MS method.

Robustness test

One thousand consecutive injections were performed to demonstrate the robustness of the combined system. For every 200 injections, 10 sample vials were prepared from frozen aliquots of the sample prepared as described in the methods section. Figure 1 demonstrates the excellent stability of the entire system. The RSD based on peak area for the trastuzumab emtansine signature peptide used for quantification was 2.5% over the one thousand injections. The RSD based on the area ratio with the IS was 3.5%. Figure 5 shows the XICs for the signature peptides and IS peptide for the first and last injections. No significant changes in peak widths or tailing were observed. The RSD on retention time for the signature peptide used for quantification was 0.5% over all injections. Figure 6 shows an overlay of the analytical column pressure traces of the 1^st and 1,000^th injection; the pressure trace was comparable throughout all runs. The trap column backpressure only increased by about 3% from 1,450 to 1,500 psi.

Dynamic range and LLOQ

A standard calibration curve was measured from 1.0 ng/mL to 10,000 ng/mL, with 5 replicates for each concentration. Excellent accuracy and precision were achieved for all standards within this data set (Table 5). LLOQs and linear calibration curve range were determined based on the requirements that the %CV of the calculated mean of the concentration is below 20% at the LLOQ and below 15% for all higher concentrations, while the accuracy of the calculated mean is between 80% and 120% at the LLOQ and between 85% and 115% for the higher concentrations. The LLOQ was 1.0 ng/mL, and the LDR was 3.3 orders of magnitude. The precision was 7.8% at the LLOQ, and less than 3.6% at the higher concentrations. Accuracy was 90% at the LLOQ and between 93% and 102% above the LLOQ. Figure 7 shows the calibration curve, and Figure 8 the XIC at the LLOQ and blank.

Carryover was determined to be around 0.5% in the first blank after injecting of the 10,000 ng/mL sample. This will likely be lower after injection of the upper limit of quantification (ULOQ) of 3,000 ng/mL and could be further reduced by adding extended column and trap washes. Alternatively, a lower ULOQ can be declared.

Conclusion
 

• Reliable integration of the ACQUITY UPLC M-Class system with the SCIEX OS software was demonstrated; the system ran without any issue for one thousand injections consecutively, plus another 100 for the calibration curve
  
  
• A low LLOQ of 1.0 ng/mL was achieved for a signature peptide of the antibody trastuzumab emtansine with an LDR of 3.3 orders of magnitude
  
  
• Trap-and-elute capability allows for large sample volume analysis without increasing method run time, resulting in high sample throughput
  
  
• Easily switch between analytical and microflow setup with no need for electrode position optimization, using the OptiFlow Pro ion source with E Lens probe
  
  
• SCIEX OS software increases productivity by providing a user-friendly interface and one single and compliance-ready platform for data acquisition, processing and management 

References
 

1. Sub-picogram level quantitation of desmopressin in small volumes of human plasma using a trap-elute microLC-MS system. SCIEX technical note, RUO-MKT-02-8508-A.
   
   
2. Ultra-sensitive quantification of trastuzumab emtansine in mouse plasma using trap-elute microLC MS method. SCIEX technical note, RUO-MKT-02-8288-A.
   
   
3. Achieving best in class sensitivity for antisense oligonucleotides in plasma using trap-and-elute microflow LC. SCIEX technical note, RUO-MKT-02-13206-A