title: Determination of Three Corticosteroids in the Biologic Matrix of Vitreous Humor by HPLC-tandem Mass Spectrometry: Method Development and Validation.
abstract: To develop a simple, specific, and rapid method to determine corticosteroid concentrations in vitreous humor.
  
  
  
  An analytical method based on high-pressure liquid chromatography-tandem mass spectrometry (HPLC-MS) with a simple extraction procedure was developed. New Zealand albino rabbits (n = 54) received a single (0.1 mL) intravitreal injection of dexamethasone (DXM, 0.1 mg), methylprednisolone (MP, 2 mg), or triamcinolone acetonide (TA, 10 mg). Eyes were enucleated and mean vitreous steroid levels were quantified at 12 h and 1, 2, 3, 7, and 14 days.
  
  
  
  Corticosteroids were extracted from the vitreous with acetonitrile, and TA was extracted with ethyl acetate, yielding high protein precipitation and clean solution samples. Vitreous samples were analyzed by isocratic HPLC-MS with mobile phase comprising acetonitrile and 2 mM ammonium formate buffer in water, pH 3.5. The linear range was 50-100,000 ng/g with a lower quantification limit of 45 ng/g for DXM and MP, and 50 ng/g for TA. Vitreous levels of DXM and MP were not detectable 14 days post-administration. Vitreous levels of TA were positive and stable throughout the study in both injected and control eyes.
  
  
  
  The HPLC-MS analytical method is an alternative to HPLC-MS/MS methods, sensitive enough for identifying and quantifying steroids in vitreous humor at a therapeutic dosage scale.
authors.affiliation: a Ophthalmology Department , Hospital Universitario Miguel Servet , Zaragoza , Spain.|b Aragón Institute for Health Research (IIS-Aragón) , Zaragoza , Spain.
authors.forename: Esther
authors.initials: E
authors.lastname: Prieto
authors.affiliation: c Laboratorio de Cromatografía y Espectroscopia, Instituto de Síntesis Química y Catálisis Homogénea (ISQCH), Chemical Sciences School , University of Zaragoza-CSIC , Zaragoza , Spain.
authors.forename: Eugenio
authors.initials: E
authors.lastname: Vispe
authors.affiliation: a Ophthalmology Department , Hospital Universitario Miguel Servet , Zaragoza , Spain.|b Aragón Institute for Health Research (IIS-Aragón) , Zaragoza , Spain.
authors.forename: Sofía
authors.initials: S
authors.lastname: Otín-Mallada
authors.affiliation: a Ophthalmology Department , Hospital Universitario Miguel Servet , Zaragoza , Spain.|b Aragón Institute for Health Research (IIS-Aragón) , Zaragoza , Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: Garcia-Martin
authors.affiliation: a Ophthalmology Department , Hospital Universitario Miguel Servet , Zaragoza , Spain.|b Aragón Institute for Health Research (IIS-Aragón) , Zaragoza , Spain.
authors.forename: Vicente
authors.initials: V
authors.lastname: Polo-Llorens
authors.affiliation: d Instituto de Síntesis Química y Catálisis Homogénea (ISQCH), Chemical Sciences School , University of Zaragoza-CSIC , Zaragoza , Spain.
authors.forename: José M
authors.initials: JM
authors.lastname: Fraile
authors.affiliation: a Ophthalmology Department , Hospital Universitario Miguel Servet , Zaragoza , Spain.|b Aragón Institute for Health Research (IIS-Aragón) , Zaragoza , Spain.
authors.forename: Luis E
authors.initials: LE
authors.lastname: Pablo
authors.affiliation: d Instituto de Síntesis Química y Catálisis Homogénea (ISQCH), Chemical Sciences School , University of Zaragoza-CSIC , Zaragoza , Spain.
authors.forename: José A
authors.initials: JA
authors.lastname: Mayoral
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003907:Dexamethasone / Q000008:administration & dosage / Q000493:pharmacokinetics*; D004195:Disease Models, Animal; D005260:Female; D005938:Glucocorticoids / Q000378:metabolism / Q000493:pharmacokinetics; D058449:Intravitreal Injections; D008775:Methylprednisolone / Q000008:administration & dosage / Q000493:pharmacokinetics*; D011817:Rabbits; D012164:Retinal Diseases / Q000175:diagnosis / Q000188:drug therapy* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014222:Triamcinolone Acetonide / Q000008:administration & dosage / Q000493:pharmacokinetics*; D014822:Vitreous Body / Q000378:metabolism*
chemical_list: D005938:Glucocorticoids; D003907:Dexamethasone; D014222:Triamcinolone Acetonide; D008775:Methylprednisolone
keywords: Corticosteroids; intravitreal injection; liquid chromatography-tandem mass spectrometry; rabbits; vitreous humor