A rapid and sensitive LC/MS/MS assay for the quantitation of brimonidine in ocular fluids and tissues

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 877; no. 3; pp. 107 - 114
• Main Authors: Jiang, Sherwin, Chappa, Arvind K., Proksch, Joel W.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15.01.2009; Elsevier
• Subjects: Adrenergic alpha-Agonists - analysis; Analysis; Analytical, structural and metabolic biochemistry; Animals; Aqueous Humor - chemistry; Bioanalysis; Biological and medical sciences; Brimonidine; Brimonidine Tartrate; Chromatography, Liquid - methods; Ciliary Body - chemistry; Drug Stability; Eye - chemistry; Fundamental and applied biological sciences. Psychology; General pharmacology; Internal standard; Iris - chemistry; LC/MS/MS; Linear Models; Medical sciences; Ocular tissues; Pharmacokinetics; Pharmacology. Drug treatments; Quinoxalines - analysis; Rabbits; Reference Standards; Reproducibility of Results; Retina - chemistry; Sensitivity and Specificity; Tandem Mass Spectrometry - methods; Vitreous Body - chemistry; Brimonidine; Pharmacokinetics; LC/MS/MS; Bioanalysis; Internal standard; Ocular tissues; α2-Adrenergic receptor; Antiglaucomatous agent; Imidazole derivatives; Quinoxaline derivatives; Agonist; HPLC chromatography; Tissue; Mass spectrometry MS/MS; α-Adrenergic receptor agonist; Quantitative analysis
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2008.11.009

We report here the development and validation of an LC/MS/MS method for the rapid and accurate quantitation of brimonidine in ocular tissues and fluids using brimonidine- d 4 as an internal standard (IS). Brimonidine was extracted from retina, iris/ciliary body, and vitreous humor samples with an acetonitrile:water (1:1) solution followed by sonication and vortexing. Aliquots of aqueous humor, iris/ciliary body, retina, and vitreous humor samples were diluted with acetonitrile containing IS and were separated on a reverse-phase HPLC column under isocratic conditions. Brimonidine ( m/ z transition: 292 → 212) and the internal standard ( m/ z transition: 296 → 216) were analyzed via multiple-reaction monitoring (MRM) in the positive electrospray mode on a 4000 Q TRAP ® instrument. The total analysis time for each sample was less than 2.0 min. The calibration curves for brimonidine (1–1000 ng/mL) were constructed using a linear regression with 1/ x 2 weighing. The lower limit of quantitation for brimonidine was 1.0 ng/mL for aqueous humor, 10 ng/g for iris/ciliary body, 12.5 ng/g for retina, and 1.6 ng/g for vitreous humor. Intra-day and inter-day estimates of accuracy and precision were within 15% of their nominal values indicating that the method is reliable for quantitation of brimonidine in ocular tissues and fluids.