Division of Pharmaceutical Sciences, School of Pharmacy, 5258 Health Science Building, University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, MO 64108-2718, USA.
J Pharm Biomed Anal. 2010 Aug 1;52(4):525-33. doi: 10.1016/j.jpba.2010.01.015. Epub 2010 Jan 18.
A sensitive, selective, accurate and robust LC-MS/MS method was developed and validated for the quantitative determination of glucocorticoids in rabbit ocular tissues. Samples were processed by a simple liquid-liquid extraction procedure. Chromatographic separation was performed on Phenomenex reversed phase C18 gemini column (50mmx4.6mm i.d.,) with an isocratic mobile phase composed of 30% of acetonitrile in water containing 0.1% of formic acid, at a flow rate 0.2mL/min. Dexamethasone (DEX), prednisolone (PD) and hydrocortisone (HD) were detected with proton adducts at m/z 393.20-->355.30, 361.30-->147.20 and 363.20-->121.0 in multiple reaction monitoring (MRM) positive mode respectively. Finally, 50microL of 0.1% novel DEX mixed micellar formulation was topically administered to a rabbit eye and concentrations were measured. The method was validated over a linear concentration range of 2.7-617.6ng/mL. Lower limit of quantitation (LLOQ) of DEX and PD was measured in the concentration range of 2.7 and 11.0ng/mL respectively. The resulting method demonstrated intra and inter-day precision within 13.3% and 11.1% and accuracy within 19.3% and 12.5% for DEX and PD, respectively. Both analytes were found to be stable throughout freeze-thaw cycles and during bench top and postoperative stability studies (r(2)>0.999). DEX concentrations in various ocular tissue samples i.e., aqueous humor, cornea, iris ciliary body, sclera and retina choroid were found to be 344.0, 1050.07, 529.6, 103.9 and 48.5ng/mg protein respectively. Absorption of DEX after topical administration from a novel aqueous mixed micellar formulation achieved therapeutic concentration levels in posterior segment of the rabbit eye.
建立并验证了一种用于兔眼组织中糖皮质激素定量测定的灵敏、选择性、准确且稳健的 LC-MS/MS 方法。样品经简单的液液萃取处理。色谱分离在 Phenomenex 反相 Gemini 柱（50mmx4.6mm id.）上进行，采用由水中含 0.1%甲酸的 30%乙腈组成的等度流动相，流速为 0.2mL/min。用质子加合物在正离子多反应监测（MRM）模式下检测地塞米松（DEX）、泼尼松龙（PD）和氢化可的松（HD），m/z 分别为 393.20--->355.30、361.30--->147.20 和 363.20--->121.0。最后，将 50μL 0.1%新型 DEX 混合胶束制剂局部给予兔眼，并测量浓度。该方法在 2.7-617.6ng/mL 的线性浓度范围内进行验证。DEX 和 PD 的定量下限（LLOQ）分别在 2.7 和 11.0ng/mL 的浓度范围内进行测量。该方法的日内和日间精密度分别为 13.3%和 11.1%，准确度分别为 19.3%和 12.5%。两种分析物在冻融循环以及在台式和术后稳定性研究中均表现出稳定性（r(2)>0.999）。在各种眼组织样品（如房水、角膜、虹膜睫状体、巩膜和脉络膜视网膜）中均检测到 DEX 浓度分别为 344.0、1050.07、529.6、103.9 和 48.5ng/mg 蛋白。从新型水性混合胶束制剂局部给药后，DEX 在兔眼后节中达到治疗浓度水平。
