Quantitative analysis of drugs in various body fluids, such as plasma, serum, and cerebrospinal fluid (CSF), is conducted in many research laboratories. SRM analysis using a triple quadrupole LC-MS/MS system has been routinely employed for quantitative analysis. Although, in most cases, SRM offers the required sensitivity and specificity, it suffers from interferences and a lack of robustness and needs expert MS users. High-resolution, accurate mass (HR/AM) mass spectrometry based on Orbitrap™ technology offers an attractive alternative with unique benefits of specificity, sensitivity and, more importantly, ease of use.
Here we describe the quantitative workflow solutions using the Thermo Scientific Exactive Plus system for the following classes of drugs:
- Immunosuppressant drugs
- Anticancer drugs
- Antifungal drugs
For additional resources, search the Orbitrap Science Library
Overview
Workflow Overview for Immunosuppressant Drugs Quantitation
Quantitative analysis of immunosuppressant drugs such as Tacrolimus, Everolimus, Sirolimus and Cyclosporin in whole blood needs high levels of accuracy and speed. Selected reaction monitoring (SRM)-based analysis is the standard technique for quantitation. The high-resolution full-scan MS or SIM offers a simpler alternative without any drawbacks.
The workflow described here uses simple full-scan MS at 100,000 FWHM resolution. The data is acquired in the Full Scan mode, and the quantitation is performed by using extracted ion chromatograms with a 3 ppm window around the m/z of interest. This application demonstrates the capability of the Thermo Scientific Exactive Plus MS coupled to a uHPLC system for quantitative analysis of the four above-mentioned drugs with a LLOQ of 1 ng/mL for all drugs except Cyclosporin. The Cyclosporin method has a LLOQ of 10 ng/mL. The dynamic range is up to at least 50 ng/mL for the first three drugs and up to 2,000 ng/mL for Cyclosporin.
Resources
Rochat B.
ASMS 2012 Presentation
Henry H, Sobhi HR, Scheibner O, Bromirski M, Nimkar SB, Rochat B.
Rapid Commun Mass Spectrom. 2012 Mar 15;26(5):499-509.
Quantitative Analysis of 4 Immunosuppressant Drugs in Blood using Exactive System
Kozak M.
Slide Presentation
Van Natta K, Kozak M, Nimkar S, Clarke W.
ASMS 2012 Poster
Sample Preparation
Sample Preparation Workflow for Immunosuppressant Drugs Quantitation
Whole blood was precipitated using the well-established ZnSO4 precipitation protocol. The precipitation solution contained Ascomycin and Cyclosporin D as internal standards.
Resources
Rochat B.
ASMS 2012 Presentation
Henry H, Sobhi HR, Scheibner O, Bromirski M, Nimkar SB, Rochat B.
Rapid Commun Mass Spectrom. 2012 Mar 15;26(5):499-509.
Quantitative Analysis of 4 Immunosuppressant Drugs in Blood using Exactive System
Kozak M.
Slide Presentation
Van Natta K, Kozak M, Nimkar S, Clarke W.
ASMS 2012 Poster
Mass Spectrometry
Mass Spectrometry Workflow for Immunosuppressant Drugs Quantitation
A Thermo Scientific Exactive Plus or Exactive mass spectrometer with an APCI source was coupled to a Thermo Scientific Accela 1250 uHPLC or Transcend system. The MS was operated SIM mode at a resolution of 70,000 FWHM during the entire LC run. The column eluent was diverted to waste after the sample injection for a specified period of time before being sent to the MS. The Exactive™ MS was calibrated at the beginning of the day and used for the entire analysis. No lock mass or internal calibration was used.
Resources
Rochat B.
ASMS 2012 Presentation
Henry H, Sobhi HR, Scheibner O, Bromirski M, Nimkar SB, Rochat B.
Rapid Commun Mass Spectrom. 2012 Mar 15;26(5):499-509.
Quantitative Analysis of 4 Immunosuppressant Drugs in Blood using Exactive System
Kozak M.
Slide Presentation
Van Natta K, Kozak M, Nimkar S, Clarke W.
ASMS 2012 Poster
Data Analysis
Data Analysis Workflow for Immunosuppressant Drugs Quantitation
Thermo Scientific TraceFinder software was used for automated processing and reporting. The quantitative processing method includes the exact m/z of fragments monitored and their retention times. The extracted ion chromatograms for molecular ions were obtained for calibrators, QCs and samples. The area under the XIC peak at the correct retention time is used for creating calibration curves and calculating values of QCs and samples. Automatic reports are created as specified in the method.
Resources
Rochat B.
ASMS 2012 Presentation
Henry H, Sobhi HR, Scheibner O, Bromirski M, Nimkar SB, Rochat B.
Rapid Commun Mass Spectrom. 2012 Mar 15;26(5):499-509.
Quantitative Analysis of 4 Immunosuppressant Drugs in Blood using Exactive System
Kozak M.
Slide Presentation
Van Natta K, Kozak M, Nimkar S, Clarke W.
ASMS 2012 Poster
Thermo Fisher Scientific
United States
