title: 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.
authors.affiliation: Department Pathology and Laboratory Medicine, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Korecka
authors.forename: Sandra G
authors.initials: SG
authors.lastname: Solari
authors.forename: Leslie M
authors.initials: LM
authors.lastname: Shaw
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D016903:Drug Monitoring / Q000379:methods; D000068338:Everolimus; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood / Q000737:chemistry; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D020123:Sirolimus / Q000031:analogs & derivatives* / Q000032:analysis / Q000097:blood; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000032:analysis; D013997:Time Factors
chemical_list: D007166:Immunosuppressive Agents; C514512:SDZ RAD 223-756; D000068338:Everolimus; C058028:immunomycin; D020123:Sirolimus; D016559:Tacrolimus