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Authors: Magdalena Rajska (1), Prochazkova Petra (1), Karolina Kotalova (1), Natalie Kinclova (1), Martin Radina (1)
Our Antiepileptics method was validated with in-house spiked calibrators. Recently, the calibration curve stopped meeting acceptance criteria, which necessitated the purchase of a commercial calibrator set (ClinCal®-Antiepileptics 5 calibrators, Recipe). When evaluated with the commercial calibrators, topiramate (TOPI) controls failed the acceptance criteria, while the remaining 6 antiepileptics passed. After ruling out errors in acquisition and quantitation methods and confirming that ClinCal®-Calibrator concentrations were assigned correctly, further troubleshooting steps revealed that ESI+ transitions in use for TOPI and TOPI-d12 were affected by ClinCal®-Calibrator matrix. When TOPI was analyzed in negative ESI mode, the controls met the acceptance requirements.
Our Antiepileptics in Serum by LC-MS/MS assay measures 7 antiepileptic drugs – gabapentin, lacosamide, levetiracetam (LEVE), pregabaline, topiramate (TOPI), vigabatrine, zonisamide. When we changed calibrators from in-house prepared to commercially available ClinCal®-Antiepileptics 5 calibrators (Recipe), the measured concentration for TOPI was nearly twice as high as the reported mean concentrations in the ClinCheck®-Antiepileptics 2 Controls Level I and II in current use. The control values for all other analytes were within the acceptance criteria.
There are two methods for antiepileptics in current use in our laboratory: Antiepileptics in Serum assay (AED assay), using a 7-min LC gradient and measuring all analytes in positive ESI mode, and an abbreviated antiepileptics method for LEVE and TOPI only (LEVE TOPI assay), using a 4-min LC gradient, with TOPI being measured in negative ESI mode. All other parameters (below) are common to both methods.
- 25ul sample (calibrator, control, serum) was precipitated with 125 ul protein precipitation solution (containing internal standards in acetonitrile, c=1mg/l)
- Agilent 1290 Infinity LC System
- Agilent 6460 Triple Quadrupole LC/MS System
- MP-A: 0.1% FA in water
- MP-B: ACN
- Flow rate: 0.35 ml/min
- Column: Kinetex®-2.6um PFP 100Å LC Column 50x3 mm
- Column oven temperature: 35 ˚C
- Injection volume: 1.5 ul
- Quantitative MRM acquisition
As the first troubleshooting step we investigated whether there were any accidental changes or errors made in either the acquisition or the quantitation methods. We did not find any. Secondly, we considered that the commercial calibrator concentrations may not have been assigned correctly. When inquiring with a Recipe representative in the Czech Republic, we were told that no other laboratory using ClinCal®-Antiepileptics 5 calibrators reported any issue.
Next, we tested the ClinCal®-Antiepileptics 5 calibrators with our abbreviated antiepileptics LEVE TOPI assay (negative ESI mode for TOPI) and found that TOPI concentrations in ClinCheck®-Antiepileptics 2 controls were in range. Therefore, we considered that the problem could be matrix related.
As further troubleshooting steps, we ran a comparison of a series of samples (10 patient samples, 2 QC samples, 2 EQAS samples) under different conditions (5 assays which differed in gradient and ESI mode, see below) and evaluated TOPI results using ClinCal®-Antiepileptics 5 calibrators as well as an older lot of in-house calibrators prepared before the calibration curve stopped meeting acceptance criteria.
I LEVE TOPI assay (in current use) ESI- for TOPI
II AED assay 0.3 ml/min ESI+
III AED assay 0.35 ml/min (in current use) ESI+
IV AED assay pos neg 0.3 ml/min ESI-
V AED assay pos neg 0.35 ml/min ESI-
Our troubleshooting revealed that TOPI signal in our 7-analyte Antiepileptics in Serum assay was affected by the ClinCal®-Antiepileptics 5 calibrators matrix. When in-house calibrators were used, results for TOPI in ClinCheck®-Antiepileptics 2 controls were in range and consistent for all five assay variations. With ClinCal®-Antiepileptics 5 calibrators, we observed significantly higher TOPI concentrations only when using Assay III, the current 7-analyte Antiepileptics in Serum assay measuring TOPI in ESI+ mode with a flowrate of 0.35 ml/min. For patient and EQAS samples the same trend was observed.
To solve the problem with TOPI quantification, we changed the MRM transitions for TOPI in the Antiepileptics in Serum assay. TOPI is now measured in negative ESI mode and there is no difference observed in results when evaluated with spiked or commercial calibrators.
References & Acknowledgements:
IP Royalty: no
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