5
For high throughput assays, automated sample preparation platforms are now used. One such example is the Clinical Laboratory Automated sample preparation Module (CLAM-2000) integrated with LC-MS/MS (LCMS-8060; Figure 1). The CLAM-2000/LCMS- 8060 platform is the fi rst of its kind in bringing an automated solution to routine clinical pathology accelerating the concept of patient sample to result. The CLAM-2000 supports integrated calibrators and quality controls throughout the batch analysis, parallel analysis and sample preparation and can be fully adapted to a range of sample preparation protocols including reagent aliquoting, ISTD addition and extraction for automated LC-MS/ MS analysis.
The system is designed as an open architecture for method development and routine sample analysis enabling the validation of LDT’s to increase sample throughput, reduce the risk of human errors, minimise user contact with biological samples and simplify operation.
Its application to immunosuppressant’s and anticonvulsant’s is highlighted below. 4. Immunosuppressant drugs
Immunosuppressant drugs are used to reduce the activity of the immune system and prevent transplant rejection. The major drugs used are calcineurin inhibitors, cyclosporine and tacrolimus, the mTOR inhibitors, sirolimus and everolimus. Circulating concentrations of these compounds should remain within a narrow therapeutic range, as overdosing can cause serious toxicity and long-term morbidity, and underdosing can cause rejection [3]. As immunosuppressant drugs result in a high pharmacokinetic variability between individual patients, TDM is now an established approach to mitigate the risks associated with organ transplantation.
Several commercial immunoassays are available for the TDM of immunosuppressant’s, however, all immunoassays show a signifi cant positive bias compared to LC-MS/MS methods [18]. Despite the availability of automated immunoassays each test is restricted to one analyte for each test when in many clinical settings multiple immunosuppressants are used in one individual patient [1, 19]. In this example, an automated LC-MS/MS method is described for the routine TDM analysis of immunosuppressants.
4.1. Materials and Methods
The quantitative analysis of immunosuppressant drugs was performed using reagents provided in a Dosimmune®
kit (Asachim, France) [20]. A UHPLC-MS/MS system (Nexera X2
and LCMS-8050, Shimadzu Corporation, Kyoto) within online SPE was used for sample analysis (see Figure 2). Automatic sample preparation was performed using the CLAM- 2000 module (Shimadzu Corporaiton, Kyoto). Sample preparation was performed using the extraction buffer and internal standard set provided in the kit.
Figure 4: Calibration curves for (a) Everolimus, (b) Sirolimus, (c) Tacrolimus and (d) Cyclosporine A.
everolimus, sirolimus and tacrolimus, and from 5 to 1500 ng/mL for cyclosporin A. Four QC levels were processed in 8 individual replicates.
4.2. Linearity
Linearity of the method was assessed by calculating the relative deviation of calibration standards against the calculated linear regression model. In all cases, deviation was within ±15%, meeting the acceptance criteria. Typical calibration curves are showed in Figure 4.
4.3. Accuracy and Precision
Accuracy and precision of the QC samples were calculated across the 8 replicates per concentration level. Results are reported in Table 3. A typical chromatogram of target compounds is shown in Figure 5. All QC analyses were within the acceptance criteria for accuracy and precision.
Figure 2: Flow diagram of online SPE-LC-MS/MS system for immunosuppressants.
25 µL of whole blood (calibrators, quality control samples (QC) or sample) were mixed with 12.5 µL of SIL-ISTD solution and 175 µL of extraction buffer (mixture of zinc sulphate 0.1M, methanol and acetonitrile 5/3/2 v/v). After 30 seconds of vortex, the samples were fi ltered for 1 min with the CLAM-2000 device. The resulting extract was then automatically transferred into the LC-MS/MS autosampler for analysis. Then, 20µL of extract was injected onto the online SPE column (Ascentis C8 5µm 30x4.6mm, Supelco, USA). After 0.15 min, the SPE column was backfl ushed and the resulting analytes transferred to the LC column (Ascentis C18 5µm 50x2.1mm, Supelco, USA), maintained at 65°C, for separation and detection. The SPE mobile phase (formate buffer/methanol 9/1 v/v) and LC mobile phase (formate buffer/methanol 1/9 v/v) were pumped at 2 mL/min and 0.8 mL/min, respectively. An overview of the analytical process is shown in Figure 3. Using sample preparation overlapped with analysis, a result was generated every 4 minutes. Mass spectrometry parameters are described in Table 2. As it is common for these compounds, the monitored transitions used ammonium adducts [M+NH4]+
as the precursor ion.
Figure 5: Middle level QC chromatogram for (a) Everolimus, (b) Sirolimus, (c) Tacrolimus and (d) Cyclosporine A. 4.4. Comparison with immunoassay
Patient samples were assayed in parallel by immunoassays and LC-MS/MS (see Figure 6, data not published, courtesy of Shimadzu Italy). For each drug compound, there was good agreement between both techniques with a correlation coeffi cient r>0.9.
Figure 3: Sample processing overview for immunosuppressants.
Calibration standards and QC samples prepared in whole blood provided with the kit were used to assess data quality. Calibration (6 levels) ranged from 0.5 to 40 ng/mL for
Figure 6: Correlation data between immunoassay and LC-MS/MS for immunosuppressants.
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