title: Development and validation of a method for the analysis of hydroxyzine hydrochloride in extracellular solution used in in vitro preclinical safety studies.
abstract: In the process of drug development, preclinical safety studies are to be performed that require the analysis of the compound at very low concentrations with high demands on the performance of the analytical methods. In the current study, a UPLC-MS/MS method was developed and validated to quantify hydroxyzine hydrochloride in an extracellular solution used in a hERG assay in concentrations ranging from 0.01 to 10μM (4.5ng/ml-4.5μg/ml). Chromatographic separation was achieved isocratically on an Acquity BEH C18 analytical column. The assay was validated at concentrations of 0.11-1.1ng/ml in end solution for hydroxyzine hydrochloride. Linearity was demonstrated over the range of concentrations of 0.06-0.17ng/ml and over the range of concentrations of 0.6-1.7ng/ml in end solution with the coefficient of correlation r>0.99. Accuracy of the achieved concentration, intra-run, and inter-run precision of the method were well within the acceptance criteria (being mean recovery of 80-120% and relative standard deviation ≤10.0%). The limit of quantification in extracellular solution was 0.09ng/ml. Hydroxyzine hydrochloride in extracellular solution proved to be stable when stored in the fridge at 4-8°C for at least 37 days, at room temperature for at least 16 days and at +35°C for at least 16 days. The analytical method was successfully applied in hERG assay.
authors.affiliation: UCB Pharma S.A., Chemin du Foriest, 1420 Braine L'Alleud, Belgium. Electronic address: willy.brione@ucb.com.
authors.forename: Willy
authors.initials: W
authors.lastname: Briône
authors.affiliation: WIL Research Europe B.V., Hambakenwetering 7, 5231 DD 's-Hertogenbosch, The Netherlands.
authors.forename: Mari
authors.initials: M
authors.lastname: Brekelmans
authors.affiliation: WIL Research Europe B.V., Hambakenwetering 7, 5231 DD 's-Hertogenbosch, The Netherlands.
authors.forename: Freek van
authors.initials: Fv
authors.lastname: Eijndhoven
authors.affiliation: UCB Pharma S.A., Chemin du Foriest, 1420 Braine L'Alleud, Belgium.
authors.forename: Eric
authors.initials: E
authors.lastname: Schenkel
authors.affiliation: WIL Research Europe B.V., Hambakenwetering 7, 5231 DD 's-Hertogenbosch, The Netherlands.
authors.forename: Theo
authors.initials: T
authors.lastname: Noij
mesh_terms: D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004353:Drug Evaluation, Preclinical; D004355:Drug Stability; D004356:Drug Storage; D051638:Ether-A-Go-Go Potassium Channels / Q000235:genetics; D045604:Extracellular Fluid / Q000737:chemistry*; D006801:Humans; D006919:Hydroxyzine / Q000032:analysis*; D057230:Limit of Detection; D016014:Linear Models; D012015:Reference Standards; D015203:Reproducibility of Results; D012996:Solutions; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D051638:Ether-A-Go-Go Potassium Channels; D012996:Solutions; D006919:Hydroxyzine
keywords: Human ether-a-go-go–related gene (hERG); Hydroxyzine; MS; UPLC–MS/