Determination of Three Corticosteroids in the Biologic Matrix of Vitreous Humor by HPLC-tandem Mass Spectrometry: Method Development and Validation. (1st February 2017)

• Record Type: Journal Article
• Authors: Prieto, Esther; Vispe, Eugenio; Otín-Mallada, Sofía; Garcia-Martin, Elena; Polo-Llorens, Vicente; Fraile, José M.; Pablo, Luis E.; Mayoral, José A.
• Is Part Of: Current eye research. Volume 42:Number 2(2017)
• Page Start: 244
• Page End: 251
• Publication Date: 2017-02-01
• Subjects: Corticosteroids -- intravitreal injection -- liquid chromatography-tandem mass spectrometry -- rabbits -- vitreous humor; Ophthalmology -- Periodicals; Eye -- Diseases -- Periodicals; Ophthalmology -- Periodicals; 573.88
• DOI: 10.1080/02713683.2016.1183795
• Languages: English
• ISSNs: 0271-3683

Abstract

ABSTRACT: Purpose : To develop a simple, specific, and rapid method to determine corticosteroid concentrations in vitreous humor. Methods : 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. Results : 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. Conclusions : 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.