Sensitive, High Throughput HPLC-MS/MS Method With On-line Sample Clean-up for Everolimus Measurement

Abstract

A new HPLC-MS/MS method for everolimus measurement was developed that includes the following features: small sample volume, short run time, fast, simple and cost-efficient sample preparation, assessment of performance of two internal standards (IS), SDZ RAD 223-756 and ascomycin and comparison of the method with an HPLC-MS/MS reference method. The authors established a multiple reaction monitoring positive ion HPLC-MS/MS method with on-line extraction and sample cleanup. This procedure includes: an API 2000 triple quadrupole mass spectrometer with turbo-ion spray, built-in Valco switching valve, an HPLC system; guard column; a Nova-Pak C18 analytical column; washing solution, methanol:30 mM ammonium acetate pH 5.1 (80:20); eluting solution, methanol:30 mM ammonium acetate pH 5.1 (97:3); flow rate 0.8 mL/min; and a run time of 2.8 minutes. The first and third quadrupoles were set to detect the ammonium adduct ion and a high mass fragment of everolimus (m/z 975.5-->908.5), and two ISs: SDZ RAD 223-756 (m/z 989.8-->922.8) and ascomycin (m/z 809.5-->756.5). The LLOQ was 1.0 microg/L for everolimus using either IS. Between day precision ranged from 3.1% to 5.7% for SDZ RAD 223-756 and 6.0% to 8.6% for ascomycin using spiked blood with everolimus concentrations 2.0 to 25.0 microg/L. Absolute recoveries using spiked samples over the range of 2.5 to 25 mug/L averaged 77.3% (SDZ RAD 223-756) and 76.8% (ascomycin). No matrix effect on everolimus was demonstrated based on the mean observed signal detection of 98.6% (SDZ RAD 223-756) and 105% (ascomycin). Comparison of everolimus concentrations obtained using this method with two internal standards with a reference laboratory demonstrated that the mean everolimus concentration obtained with ascomycin was statistically different (lower) than results with the reference method and the method that used SDZ RAD 223-756 as the internal standard gave equivalent results compared with the reference method.

Comparison of blood sirolimus, tacrolimus and everolimus concentrations measured by LC-MS/MS, HPLC-UV and immunoassay methods

“…Several LC-MS/MS methods have been published for the quantitation of individual [16][17][18][19][20] or multiple immunosuppressants [21][22][23] in whole blood. Our laboratory initially introduced a previously published LC-MS/MS method for the analysis of sirolimus [20], which has subsequently been modified to also allow the additional simultaneous quantitation of tacrolimus and everolimus.…”

Antineoplastic drugs and their analysis: a state of the art review

“…589 The sensitivity and selectivity provided by MS helped to ease sample preparation in clinical chemistry laboratories. Column-switching systems coupling the extraction support to the chromatographic column before MS detection were also used for the analysis of sirolimus [598][599][600][601][602][603][604][605][606][607][608][609][610][611] and everolimus 598,599,602,603,[605][606][607]609,[612][613][614] in whole blood. LLE 615 or SPE 616 on 96-well plates constitutes another approach for sample preparation automatisation.…”

Sensitive quantification of sirolimus and everolimus by LC–MS/MS with online sample cleanup

“…One transition per analyte was regarded to be sufficient, since only few substances can be ionized in negative mode using APCI and since the masses of the analytes are quite high, therefore interferences with other compounds are unlikely. Also in most other methods published, only one transition per analyte is monitored [10,11,16].…”

“…doi:10.1016/j.jchromb.2010.02.029 [9][10][11]. Within the last 10 years, several online extraction procedures have been published [12][13][14][15][16][17][18][19][20][21][22][23]. Most of them use either online solid-phase extraction (SPE) or an additional chromatographic column for extraction.…”