Davide Vecchietti (Presenter)
Shimadzu Italy
Bio: Davide Vecchietti P.hD, LCMS&Life Sciences product specialist
Authorship: Davide Vecchietti (1), Daisuke Kawakami (2), Taku Tsukamoto (2), Maura Brambilla (3)
(1)Shimadzu Italy, Milan, Italy, (2) Shimadzu Corporation, Kyoto, Japan, (3) Desio Hospital, Toxicology and Mass Spectrometry Unit, Desio, Italy
| Short Abstract Therapeutic drug monitoring of immunosuppressant agents needs to be accomplished by extremely accurate techniques. Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) shows higher sensitivity and superior specificity compared to immunoassay-based approaches; however, LC-MS/MS approaches lack in standardization since they require sample preparation procedures that usually involves complex offline extraction methods. These procedures are time-consuming and are more vulnerable to variability due to errors in manual preparation. To increase the data quality, safety, and throughput of LC-MS/MS quantitation of immunosuppressant drugs, a fully automated platform for the quantitation, has been introduced. |
Long Abstract
Introduction
Immunosuppressant medications plays a critical role in transplantation of allografts by decreasing patient morbidity and mortality. However, overdosing with these critical dose drugs can cause serious toxicity and long-term morbidity, while rejection can occur in case of underdosing. For that reason therapeutic drug monitoring need to be accomplished by extremely accurate techniques. Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) show higher sensitivity and superior specificity compared to immunoassay-based approaches. Nevertheless, LC-MS/MS approaches lacks in standardization and the necessary throughput for the application in routine analysis. We report a fully automated platform for the quantitation of four major immunosuppressant (cyclosporine A, tacrolimus, sirolimus and everolimus) in whole blood samples with high throughput and without operator sample preparation.
Method
The analysis of immunosuppressant was performed using a fully automatic LCMS preparation Unit (CLAM-2000, “For Research Use Only. Not for use in clinical diagnostics.” Shimadzu) online with HPLC-LCMS (NexeraX2-LCMS8050, Shimadzu) starting from Standard Blood Collection Tubes using “MassTox®” kit (Chromsystems,93000). Whole blood samples were firstly frozen o/n at -20°C and subsequently incubated at RT for 45 min under gently mixing before the analysis. Samples (EDTA whole blood), calibrators, Extraction Buffer, Precipitation Reagent, and Internal Standards (Chromsystems,93046) were loaded on CLAM-2000. The fully automatic preparation/analysis procedure consist in: I) 25 ul of sample were dispensed in filtration-collection vial; II) 50 ul Extraction buffer added (liquid-liquid extraction); III) 12,5 ul IS added; IV) stirring and incubation (2 min); V) 125 ul Precipitation reagent added; VI) stirring and filtration (deproteinization); the sample is then transported using an arm from the CLAM-2000 to the HPLC without human intervention for LC-MS/MS analysis.
The treated samples were trapped using Trap Column (Chromsystem, 93122) and then separated by Analytical column (Chromsystem, 93100) at 65°C with isocratic gradient system at a flow rate of 0.3 ml/min in 2 min.
Results
The quantitation of four major immunosuppressant (cyclosporine A, tacrolimus, sirolimus and everolimus) in EDTA whole blood samples was performed by LC-MS/MS approach. Usually LC-MS/MS analysis of whole blood samples require some manual preparation steps for extraction and protein precipitation before the injection. With the aim to reduce the operator involvement, to increase the throughput and the data quality, we completely eliminated the manual sample preparation procedure by the use of a novel automatic preparation unit (CLAM-2000, Shimadzu). We tested firstly if the LOQ and linearity of our method were compatible with suggested therapeutic intervals for immunosuppressant (100-350 ug/L cyclospirin A, 3-8 ug/L everolimus, 3-20 ug/L sirolimus, tacrolimus 4-20 ug/L). We used a wide range of quantification (6 levels) for all the compounds (22.7-930 ug/l for cyclosporin A, and 2.2-45 ug/l for everolimus,sirolimus,tacrolimus), and we obtained a good linearity over the entire range (r2>0,997). We tested the intra-day precision (repeatability) of the method, by analyzing 4 reference samples (Chromsystems MassCheck® whole blood control, 0081) spanning from low concentration to high concentration levels and the automated sample preparation resulted efficient in providing CV% values under CLSI reference intervals. Also the inter-day precision (reproducibility) was in accordance with CLSI guidelines, with CV% <15% over all the 4 levels of concentration. The accuracy of the method resulted for all the compound close to the reference material (Bias%<15%). Finally results on whole blood samples showed accordance with immunoassay.
Conclusion
We completed Immunosuppressant analysis using the automated sample preparation system coupled to LC-MS/MS. The results shows the capability of the system for large sample set analyses with improved accuracy and precision by eliminating human error associated with manual sample handling. The procedure resulted more safe for operators by reducing risk of contamination.
Novel Aspect
The completely automated quantification method for Immunosuppressant allow routine analysis with high data quality/precision, reduced time, increased throughput and safety.
References & Acknowledgements:
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