A rapid and sensitive LC/MS/MS assay for the quantitation of brimonidine in ocular fluids and tissues.
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.
Jiang
Sherwin
S
Global Preclinical Development, Bausch & Lomb Inc., Rochester, NY 14609, USA.
Chappa
Arvind K
AK
Proksch
Joel W
JW
D000316:Adrenergic alpha-Agonists / Q000032:analysis*; D000818:Animals; D001082:Aqueous Humor / Q000737:chemistry; D000068438:Brimonidine Tartrate; D002853:Chromatography, Liquid / Q000379:methods*; D002924:Ciliary Body / Q000737:chemistry; D004355:Drug Stability; D005123:Eye / Q000737:chemistry*; D007498:Iris / Q000737:chemistry; D016014:Linear Models; D011810:Quinoxalines / Q000032:analysis*; D011817:Rabbits; D012015:Reference Standards; D015203:Reproducibility of Results; D012160:Retina / Q000737:chemistry; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014822:Vitreous Body / Q000737:chemistry
D000316:Adrenergic alpha-Agonists; D011810:Quinoxalines; D000068438:Brimonidine Tartrate