Methods

Sample preparation: Rat plasma was protein precipitated and loaded onto the mixed mode SPE cartridge. The eluents were diluted 1:1 (v/v) by water and served as the processed biological matrix. Human ANP, human BNP and rat ANP were spiked into the processed rat plasma, endogenous rat ANP was used as the internal standard. Serial dilution with processed plasma was performed to create the calibration curves for analysis.

LC-MS conditions: Samples were analyzed in triplicate by a SCIEX Triple Quad 7500 LC-MS/MS System – QTRAP Ready, coupled with an ExionLC™ system. The method details are summarized in Table 1 and 2. The same sample set was also analyzed using a SCIEX Triple Quad 6500+ LC-MS/MS System, coupled with the same HPLC system, to characterize the performance difference between the two mass spectrometers. All MRM parameters were optimized on both mass spectrometers for accurate performance comparison.

Data processing: Data was processed using the Analytics function in SCIEX OS Software 2.0.

Cyclic peptide quantification results 

The SCIEX Triple Quad 7500 LC-MS/MS System – QTRAP Ready has multiple novel hardware features to improve instrument sensitivity.³ The OptiFlow Pro Ion Source with E Lens Technology has a universal geometry that supports flow rates from 1 µL/min to 3000 µL/min without positional adjustment, using exchangeable probes for micro and high-flow applications. ³ The E Lens Technology creates a stronger field at the ESI probe leading to more efficient release of ions from the droplet and deflection of ions towards the orifice for improved sensitivity.³ The D Jet Ion Guide behind the orifice plate efficiently captures and transmits the ions in the higher vacuum region. Its tapered dodecapole geometry, efficiently focuses the ions into the second stage QJet® Ion Guide.³

Combining these technologies together, greater sensitivity is achieved through gains in the ion generation and ion focusing. To investigate the sensitivity improvement, the same sample set was analyzed on both the SCIEX Triple Quad 7500 LC-MS/MS System – QTRAP Ready and the Triple Quad 6500+ LC-MS/MS System. A >10-fold increase in peak area and a 5-fold improvement in S/N was observed (Figure 2).

As shown in Figures 3, 4 and 5, both ANP and BNP are robustly quantified at 0.05 ng/mL in rat plasma, with tight %CV (<11%) and high accuracies (90-114%) across the entire linear dynamic range (0.05-100 ng/mL). 100 ng/mL is the highest concentration prepared in the sample set, the assay ULOQ would potentially be higher than 100 ng/mL as saturation was not yet reached. 

Conclusion
 

• An ultra-sensitive MRM based cyclic peptide quantification workflow using SCIEX Triple Quad 7500 LC-MS/MS System – QTRAP Ready has been presented 
  
  
• Human ANP and BNP were quantified at 0.05 ng/mL in rat plasma with outstanding reproducibility, accuracy and linearity
  
  
• Combining the OptiFlow Pro Ion Source with E Lens Technology and D Jet Ion Guide, significant sensitivity improvements were observed compared to a previously published method ²
  
  
• A greater than 10-fold increase in peak area and a 5-fold improvement in S/N was observed versus the SCIEX 6500+ System

References
 

1. Das BB, Solinger R (2009) Cardiovasc. Hematol. Agents Med. Chem. 7(1), 29-42.
   
   
2. Ciccimaro E., et al, (2014) Anal. Chem, 86, 11523−11527.
   
   
3. Enabling new levels of quantification. SCIEX technical note RUO-MKT-02-11886-A.