Mass spectrometry-based candidate reference measurement procedure for quantification of amyloid-ß in cerebrospinal fluid.

BACKGROUND: Cerebrospinal fluid (CSF) amyloid-ß (Aß42) is a well-established biomarker for Alzheimer disease. Several immunoassays for Aß42 exist but differ in absolute concentrations and may suffer from matrix interference, thereby hampering interlaboratory comparisons and the use of general cutoff levels. Together with the IFCC Working Group on CSF Proteins, we developed a candidate reference measurement procedure (RMP) for Aß42. METHODS: The antibody-independent candidate RMP was based on solid-phase extraction and isotope-dilution LC-MS/MS. The candidate RMP used 2 differently stable isotope-labeled Aß42 peptides for calibration in human CSF, an important aspect since there was no analyte-free matrix available. Because no CSF certified reference material (CRM) exists, we used a nonlabeled Aß42 standard, the concentration of which was determined by amino acid analysis. We performed measurements on a high-resolution quadrupole-Orbitrap hybrid instrument. The results were compared with a method run in a second laboratory with triple quadrupole instrumentation. RESULTS: The candidate RMP allowed quantification of CSF Aß42 from 150 to 4000 pg/mL. Validation of the method showed a recovery of 100% (15%), intraassay and interassay imprecision of 5.0% and 6.4%, respectively, and an expanded uncertainty of 15.7%. No analytical interferences or carryover were detected. CONCLUSIONS: This method will help set the value of CSF Aß42 in a CRM, which could be used to harmonize Aß42 assays and facilitate the introduction of general cutoff concentrations for CSF Aß42 in clinical trials and practice.

Quantification of vancomycin in human serum by LC-MS/MS.

BACKGROUND: The aim of our work was to develop and validate a reliable LC-MS/MS-based measurement procedure for the quantification of vancomycin in serum, to be applied in the context of efforts to standardize and harmonize therapeutic drug monitoring of this compound using routine assays. METHODS: Sample preparation was based on protein precipitation followed by ultrafiltration. In order to minimize differential modulation of ionization by matrix constituents extended chromatographic separation was applied leading to a retention time of 9.8 min for the analyte. Measurement was done by HPLC-ESI-MS/MS. For internal standardization the derivative vancomycin-glycin (ISTD) prepared by chemical synthesis was used, HPLC conditions ensured coelution of ISTD with the analyte. RESULTS: In a bi-center validation total CVs of <4% were observed for quality control material ranging from 5.3 mg/L to 79.4 mg/L; accuracy was ±4%. No relevant ion suppression was observed. Comparative measurement of aliquots from 70 samples at the two validation sites demonstrated close agreement. CONCLUSIONS: Employing a closely related homologue molecule for internal standardization and the use of MS/MS following highly efficient sample pre-fractionation by HPLC, the method described here can be considered to offer the highest level of analytical reliability realized so far for the quantification of vancomycin in human serum. Thus, the method is suitable to be used in a comprehensive reference measurement system for vancomycin.

On-line solid-phase extraction high-performance liquid chromatography-tandem mass spectrometry for the quantitative analysis of tacrolimus in whole blood hemolyzate.

Tacrolimus is an immunosuppressive drug essential for preventing organ rejection after transplantation. Since tacrolimus strongly binds to erythrocytes, therapeutic monitoring requires its quantification in whole blood lyzate, representing one of the most difficult to analyze biological fluids due to its high protein load. In this communication, we report on the successful combination of whole blood hemolysis employing ionic liquids, followed by sample preparation by means of on-line solid phase extraction (SPE) using restricted access materials (RAM), which permitted the efficient removal of hemoglobin and other large biomolecules. Among six different tested RAM columns, highest hemoglobin depletion and analyte extraction efficiency was obtained with a polymer-based, glycoprotein-coated RAM stationary phase (Biotrap 500 MS) operated at an alkaline pH of 10.7. Analyte quantification was performed by high-performance liquid chromatography-selected reaction monitoring tandem mass spectrometry (HPLC-SRM-MS/MS). The ability to quantify tacrolimus in therapeutically relevant concentrations in whole blood hemolyzates was demonstrated via external calibration with lower limits of detection and quantification of 2.00 and 7.23 ng mL(-1), respectively. Moreover, the investigation of heparin-pretreated blood samples during blood sampling led to an increase in sensitivity for the analyte, while the method appeared to be more robust with ethylenediaminetetraacetic acid as anticoagulant.