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Jim Vrbanac, Matthew Marchewka; Determination of Dexamethasone To 13 pg/g (mL) in Ocular Fluids and Tissues Following Unilateral, Topical Administration. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3252.
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Quantitative analysis of drug in ocular tissues is often challenging due to the low concentrations and small sample weights. This paper describes a method for determination of the concentration of dexamethasone in ocular tissues with a LLOQ of 13 pg/g(mL) in TM.
Calibration curve and QC samples consisted of 0.5 mL of control plasma, or control retina, choroid, and vitreous humor, or AH and 0.25 mL of methanol containing 2.50 ng/mL of D4-Dexamethasone. Reversed phase LC (C-18); gradient conditions; APCI; Thermo TSQ Ultra; Resolution, 0.7 daltons at half peak height; centroid; SRM, dexamethasone ([2H4]dexamethasone): m/z = 393 (397) CAD to 373 (377), loss of HF (-10 volts) (-HF).
After comparison of +/- ESI and +/- APCI, +APCI was chosen as the method. The base peak in the +APCI mass spectrum was observed at m/z 393 (MH+). Losses of HF, m/z 373, and [HF + H2O], m/z 355, were observed at approximately 40% and 10% of the base peak, as was the acetonitrile adduct ion (< 10%). At 10 volts collision energy, the product ion mass spectrum of MH+ exhibited loss of HF, m/z 373, and loss of [HF + H2O], m/z 355, with two more successive losses of water, m/z 337 and 319. A retention time of 4.9 minutes was observed using conditions to be outlined. The method was observed to be rugged and reproducible and met CDER guidelines. Back calculated concentrations for 13, 27, 53 and 106 pg/mL were -12, 2.6, -9.6 and -1.6%, respectively. A LLOQ of 13 pg/mL (g) was observed using these conditions. The concentration of dexamethasone in anterior retina samples varied from 0.6 to 2.4 ng/g and 0.9 to 14 ng/mL in the untreated and treated eyes, respectively. The concentration of dexamethasone in posterior retina samples varied from < LLOQ to 1.7 ng/g and < LLOQ to 4.8 ng/mL in the untreated and treated eyes, respectively. The concentration of dexamethasone in anterior choroid samples varied from 1.1 to 6.2 ng/g and 5.0 to 46 ng/mL in the untreated and treated eyes, respectively. The concentration of dexamethasone in posterior choroid samples varied from < LLOQ to 5.2 ng/g and < LLOQ to 4.7 ng/mL in the untreated and treated eyes, respectively.
A method was described with an approximate 1-2 order of magnitude lower LLOQ for dexamethasone in TM relative to literature methods.
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