Immunosuppressant drugs hinder the immune system from rejecting foreign tissue and are applied in organ transplant patients. With highly variable pharmacokinetics and narrow therapeutic indices, these drugs need to be monitored to improve the scientific understanding of dosage requirements for organ transplant patients. Until recently, immunoassays have been the main mehtod used for therapeutic monitoring of these drugs.
Liquid chromatography-mass spectrometry (LC-MS/MS) is now increasingly used for the identification of immunosuppressant drugs. The LC-MS/MS technique provides unique benefits of selectivity, sensitivity, and accuracy by eliminating the cross-reactivity with metabolites in immunoassay. It can make up for initial high costs of LC-MS/MS instrumentation and an experienced analytical chemist if used in high-throughput processes.
This article discusses the application of the EVOQ Elite LC-MS/MS system from Bruker to simultaneously analyze everolimus, sirolimus, tacrolimus, and cyclosporine A in human plasma.
EVOQ Elite LC-MS/MS system
The EVOQ triple quadrupole for liquid chromatography (LC-TQ) (Figure 1) was intended for a singular purpose - to perform reliable quantification of many real samples at the fastest sample-to-report time possible.
It offers unprecedented sensitivity, linearity, accuracy, precision and an extensive dynamic range for multiple reaction monitoring (MRM) assays. Advances in atmospheric pressure ionization (API) technology and software make it a revolutionary technique for routine high-sensitivity, quantitative analysis.
Figure 1. The EVOQ Elite LC-MS/MS system.
With the lowest system delay-volume possible, the Advance LC pump technology provides unprecedented ballistic gradient reproducibility at analytical flow rates. This design-for-purpose feature reduces gradient delay and ascertains highly precise retention times, resulting in shorter run-to-run cycle times with the reproducibility essential for quantitative LC-MS/MS analyses.
The LC-TQ performance is extended by the OLE module by facilitating method-driven, on-line clean-up or sample pre-concentration. The inclusion of a third pump in the same module saves bench space and simplifies configuration under full software control, while delivering the flexibility of quick method development. The following are the key benefits of the EVOQ Elite LC-MS/MS system:
- Ultra-high sensitivity for biomolecules and tiny molecules can be easily obtained due to the novel interlaced quadrupole (IQ) dual ion funnel.
- Reliably run matrix-rich samples on the powerful orifice plate based API interface.
- Thermally labile molecules can be efficiently analyzed at high flow rates with the innovative vacuum insulated probe (VIP) heated electrospray probe.
- Exception-based data-review software saves time by conveniently highlighting chromatograms that do not satisfy preset method criteria.
The sample preparation involved diluting standard solutions of everolimus, sirolimus, tacrolimus, and cyclosporine A in MeOH to make primary stock solutions, followed by spiking them in matrix blank to make calibration solutions. The preparation of the plasma matrix blank involved mixing 9ml of cold Acetonitrile into 3ml of plasma (3:1 v/v).
The solution obtained was then vortexed for 30s and allowed to settle for 15 minutes at temperature followed by centrifugation at 15,000rpm for 15 minutes. The supernatant was separated as matrix blank to make the matrix spiked calibration solutions.
Chromatography (Advance UHPLC)
- Column: YMC Triart C18 (50mm x 2.1mm x 1.9µm)
- Column temperature: 80°C
- Flow rate: 0.5mL/min
- Mobile phase A: Water with 0.1% Formic acid + 10mM Ammonium Format
- Mobile phase B: Acetonitrile with 0.1% Formic Acid
- Gradient conditions:
- Injection volume: 5µL
Mass Spectrometer (EVOQ Elite)
- HESI: +4500V
- Probe Temperature: 450°C
- Probe gas: 45 units
- Nebulizer gas: 40 units
- Cone gas temp: 350°C
- Cone gas: 20 units
- Active exhaust: On
- Collision gas: Argon 1.25mTorr
Optimized MRM transitions for each compound are listed in Table 1. The selectivity and sensitivity of the EVOQ Elite LC-MS/MS system simplifies the sample preparation with only protein precipitation, thereby enabling the direct feeding of the supernatant for subsequent analysis. The LC-MS/MS system determines the concentration levels of 0.5ng/ml for everolimus, sirolimus and tacrolimus and 10ng/ml for cyclosporine A in human plasma.
Table 1. Optimized MRM transitions.
||Collision Energy (V)
|Cyclosporine D (IS)
The representative chromatograms at these levels in plasma are delineated in Figure 2. The Bruker MSWS software that controls the EVOQ LC-MS/MS system was equipped with the optional PACER™ software, which allows for ‘exception based data-review’ to drastically minimize the error rate of peak integration for quantitative analysis.
Figure 2. MRM Chromatograms of Four Imminosuppressents in Plasma Matrix.
Calibration curves of four imminosuppressents in plasma matrix processed by PACER™ are depicted in Figure 3. The curves shown are from 0.5ng/mL to 50ng/mL for everolimus, sirolimus, and tacrolimus with linearity of R2 >0.997, and from 10ng/mL to 1000ng/ml for cyclosporine A with linearity of R2 >0.995.
A series of seven level calibrations (each three replicates) and three level QC standards in matrix (each six replicates) were repeatedly performed for five times to corroborate the robustness of the EVOQ LC-MS/MS system. The R2 of the calibration curves were observed to be >0.994 and RSD for each QC level variation is typically 3-8% across over 200 injections of matrix samples.
In this analysis, the EVOQ Elite LC-MS/MS system was used to analyze four human plasma samples subjected to immunosuppressant drugs subsequent to a simple protein precipitation of the plasma sample. The method was proved to be rapid, sensitive and powerful with improved limit of quantitation, easily satisfying the requirement for quantification of these drugs in human plasma.
This information has been sourced, reviewed and adapted from materials provided by Bruker - Chemical And Applied Markets.
For more information on this source, please visit Bruker Daltonics.