Evgueni Fedorov (Presenter)
Biotrial Bioanalytical Services
Authorship: Evgueni Fedorov
Biotrial Bioanalytical Services Inc., Laval, QC, Canada
| Short Abstract Some bioactive peptides and proteins exhibit their activity at very low concentrations. Selective and sensitive assays are also required for biomarkers. Although signature peptides are often used to increase the sensitivity in LC-MS/MS determination of large molecules, there is a risk that isobaric interferences might be accounted for the target compound. The use of Differential Ion Mobility Spectrometry (DMS) as an additional separation dimension helps to increase selectivity and specificity. The SelexION® option with 6500 triple quadrupole mass spectrometer from Sciex permitted to remove endogenous interferences and achieve an LLOQ of 5 pg/mL in LC-MS/MS method for Exenatide, a GLP-1 receptor agonist. |
Long Abstract
• Introduction:
Some bioactive peptides and proteins exhibit their activity at very low concentrations. Selective and sensitive assays are also required for biomarkers. LC-MS/MS with a triple quadrupole mass-spectrometer is considered to be state-of-the-art technique for the analysis of small molecules in biological fluids. For large peptides and proteins, however, the sensitivity is adversely affected by the charge distribution between multiple charged ions formed during electrospray ionization.
It was demonstrated earlier that even for relatively large peptides, like Exenatide, a potent GLP-1 receptor agonist, an approach used in quantification of proteins, involving trypsin digestion, might lead to a significant improvement in sensitivity and allows achieving an LLOQ of 10 pg/mL [1].
However, the use of a more sensitive triple quadrupole instrument to attain a greater sensitivity was not entirely successful on its own. Its effectiveness in this particular case was lessened by the relatively large volume of plasma used, which gave rise to a high level of chemical noise produced undoubtedly by endogenous species still present after sample processing, and by the use of a signature peptide consisting of only seven amino acid, which increases the chances of obtaining isobaric products from endogenous proteins and peptides. While the use of a high-resolution mass spectrometer could likely improve the situation by resolving almost isobaric interferences, there is a less expensive alternative.
• Methods:
In this work, a Differential Ion Mobility Spectrometry (SelexION® option from Sciex) was used as an additional dimension, orthogonal to LC and MS/MS separations.
Sample Preparation:
Human plasma samples were thawed and aliquoted (0.5 mL), the IS solution of custom-synthesised Exenatide d5 labeled with deuterium on second Phenylalanine unit was added.
A solid phase extraction on Strata-XC (Phenomenex) cation-exchange cartridges was performed.
After evaporation, the samples were reconstituted with a bicarbonate buffer and a solution of trypsin was added.
After incubation of 1 hr at 50°C, the samples were applied to a conditioned SOLAµ HRP microextraction plate (Thermo Scientific).
After elution in 50 mcL of eluent, the samples were diluted with 150 mcL of water for the compatibility with the HPLC conditions.
Chromatography:
Pump A: Shimadzu LC-20AD
Pumps B and C: Shimadzu LC-10AD
Autosampler: PAL CTC with DLW option
Analytical Column: Supelco, Ascentis Express Peptide ES-C18, 50 x 2.1 mm, 2.7 µm with matching Guard Column, heated at 60°C
Trapping Column: Supelco, Ascentis Express Peptide ES-CN, 5 x 2.1 mm, 2.7 µm, at ambient temperature
Injection volume: 40 µL
Flow rate in the Analytical Column: 0.5 mL/min
Mobile Phase A: 15/85/0.2/0.1/0.1 v/v/v/v/v Acetonitrile/Water/Formic/Hexfluoroisopropanol/3-Nitrobenzyl alcohol
Mobile Phase B: 95/5/0.1 v/v/v Acetonitrile/Water/Formic acid
A trap-elute technique with a switch valve was used, then a linear gradient HPLC with a washing step was applied.
Mass Spectrometry:
Instrument: AbSciex QTrap 6500 with SelexION® option
Ionization source: ESI
Modifier: none
Monitoring of ions: Positive mode MRM
Resolution Q1: Unit
Resolution Q3: Unit
Dwell time: 50 msec analyte and IS
CUR: 30.0
GS1: 55.0
GS2: 70.0
IS: 5500
TEM: 450°C
CAD: 12.00
CE: 21.0
DP: 85.0
EP: 10.00
CXP: 12.00
DT: High
DR: Open
COV: 28.80
DMO: -3.00
SV: 3800.00
• Results:
With the SelexION® option installed and operating, the linearity was achieved over the range from 5 to 2000 pg/mL, with precision and accuracy meeting FDA Guidance criteria for LC-MS/MS methods. Thirteen different lots of human K2 and K3 EDTA plasma blanks were tested for potential interferences both with and without differential ion mobility spectrometry (SelexION®). All the other settings were exactly the same.
As seen from the results, in most lots of plasma analyzed without SelexION® there are interferences, which, after integration, are attributable to peaks pertaining to the signature peptide. In some lots, this contribution can be even much higher than the signal from some other plasma spiked at the LLOQ concentration. Clearly, this does not meet the established criteria for non-endogenous analytes. Moreover, automatic integration could not be applied to all these samples.
On the other hand, when SelexION® was used, the highest interference observed from a blank sample accounted for only approximately 16% of the LLOQ, well within the acceptance criteria, and automatic integration was possible for all blanks.
• Conclusions:
The use of Differential Ion Mobility spectroscopy as an additional dimension in LC-MS/MS quantification using a signature peptide showed the following advantages:
The chemical noise and contributing signal from matrix impurities were significantly decreased.
A lower LOQ was achieved in compliance with regulatory acceptance criteria.
The need of manual integration was eliminated.
These benefits were obtained at lower cost, compared to a high resolution instrument.
The module is easy to tune and operate, robust and does not require any calibration.
References & Acknowledgements:
1. J.-N. Mess, D. Villeneuve and F. Garofolo. Bioanalysis of Exenatide: Intact Versus Signature Peptide Approach to Reach Optimal Sensitivity in Large Molecule Quantification by LC-MS. 61th Conference of ASMS, 2013, Minneapolis, USA.
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