Run Zhang Shi (Presenter)
Stanford University School of Medicine
Bio: Run-Zhang Shi is a Clinical Assistant Professor at Stanford University School of Medicine.
Authorship: Run-Zhang Shi
Stanford University School of Medicine
| Short Abstract Paper spray (PS) is open atmosphere ionization for direct and rapid mass spectrometry analysis. It is performed by simultaneously extracting and ionizing dried sample deposited on paper matrix. A PS-MS/MS method for TDM of cyclosporin, sirolimus, and tacrolimus was developed and evaluated. The method uses PS disposable cartridges and automated sample extraction and ionization interface (Prosolia Inc.) and TSQ Vantage tandem mass spectrometry (Thermo Scientific). Assay sensitivity, specificity, linearity, imprecision, and accuracy met set criteria. PS-MS/MS is a viable alternative to immunoassays or LC-MS/MS methods for immunosuppressive drug TDM. |
Long Abstract
Introduction
Paper spray (PS) is an open atmosphere ionization method for direct and rapid mass spectrometry analysis. It is performed by simultaneously extracting and ionizing dried sample deposited on paper matrix. PS-MS/MS can be attractive to a busy clinical lab with limited staff and LC-MS/MS expertise, since it is a simpler alternative to conventional LC-MS/MS with the removal of chromatography separation step.
Therapeutic drug monitoring (TDM) of immunosuppressive drugs such as cyclosporin, sirolimus, and tacrolimus is mandatory for organ transplant patient care especially in pediatric patients. Physicians rely on faster turnaround time (TAT) of TDM results for prompt dosage adjustment to improve pediatric organ transplant outcome. PS-MS/MS is an attractive alternative TDM method because of its simplicity while maintaining accuracy and sensitivity of mass spectrometry. In addition analysis time is shortened, and with much reduced volume of blood sample. Since multiplexing is an inherent advantage of MS analysis, simultaneous quantitation of cyclosporin, sirolimus, and tacrolimus by PS-MS/MS may be readily achievable.
Methods
We developed and evaluated such a PS-MS/MS method for TDM of cyclosporin, sirolimus, and tacrolimus in our clinical diagnostic lab. Briefly blood sample (50-200 µL) was premixed (proportionally) with a solution containing stable isotopically labeled internal standard (SIL-IS) analogs of each analyte. The above mixture of 10-12 µL was spotted on paper contained in a disposable cartridge (Prosolia Inc.). After a brief time drying, the cartridge was placed in front of TSQ Vantage tandem mass spectrometry (Thermo Scientific) by an automated PS sample extraction and ionization interface (Prosolia Inc.). Each drug was detected as singly charged sodium adduct ion. Sodium was chosen over ammonium because sodium adduct ions gave more selective fragmentation during MS/MS, due to a desirable larger neutral losses.
Results
The lower limits of detection (LOD) were 0.2, 5 and 0.5 ng/mL for tacrolimus, cyclosporin and sirolimus, respectively, determined by means of sample blank plus three standard deviation (sd). The lower limits of quantitation (LLOQ) were 1, 35, and 2 ng/mL for tacrolimus, cyclosporin, and sirolimus, respectively, at which drug concentration the method imprecision (CV) reached a 20% limit. Inter-day imprecision (CV) was less than 20% at low, medium, and high therapeutic concentrations for all three analytes. The assay was linear within the analytical measurement range. Accuracy was evaluated by (1) analyzing CAP proficiency samples, in which most of the PS-MS/MS determinations were within 1 sd of LC-MS/MS peer group mean values; (2) analysis of spiked blood samples, in which the determined concentrations were routinely within 15% of the expected values; (3) correlation study comparing concentrations determined by PS-MS/MS to those of Dimension RXL immunoassays (Siemens). Passing-Bablok regression analysis yielded the following relationship: PS-MS/MS=0.9*RXL+0.5, r2=0.90 (n=75) for tacrolimus; PS-MS/MS=1.2*RXL-21, r2=0.95 (n=45) for cyclosporin; and PS-MS/MS=1.1*RXL-1, r2=0.81 (n=44) for sirolimus.
Matrix effects were evaluated by comparing signal for standards prepared in methanol versus in whole blood. For each drug, the signal was about 3 times lower in blood. The decrease in signal arises from both ion suppression and lower recovery from dried blood as previously reported. Accurate and robust quantitation was obtained despite the presence of matrix effects because matrix matched whole blood calibrators and SIL-IS were used to account for variation arising during extraction and ionization, and results normalized. The effect of hemolysis, lipemia, icterus, and high concentrations of 50 steroids, vitamins, diuretics, and other immunosuppressive compounds was evaluated. There was no effect on low or medium level of tacrolimus, cyclosporin and sirolimus concentrations when compared to blood samples without these factors.
Conclusions
This study demonstrates that PS-MS/MS is a viable alternative to immunoassays or LC-MS/MS methods for immunosuppressive TDM, having lower ongoing costs than immunoassays and being suitable for a clinical lab with limited expertise in the maintenance and trouble shooting of LC-MS/MS. Despite current limitations of PS-MS/MS which remain to be resolved, we see several advantages of PS-MS/MS over traditional LC-MS/MS methods for clinical laboratories. First, TAT could be decreased due to removal of sample preparation, and the simpler method is more suited to random access analysis in a clinical lab. Second, the required person-hours and expertise are lower compared to LC-MS/MS due to the simplification of the method and the removal of a chromatography system. Third, solvent consumption was much lower, and on average less than 1 mL per day of solvent waste was generated. Finally, the potential risk of carry-over is eliminated because the entire fluid path that interacts with the sample, including extraction and ionization, is contained within a disposable cartridge.
References & Acknowledgements:
We thank Prosolia Inc. for the loan of PS automated sample extraction and ionization interface.
We thank Thermo Scientific for the load of TSQ Vantage tandem mass spectrometry.
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