In vitro release of LNG from effervescent MN patches was measured by placing an MN patch into 1 liter of PBST with varying concentrations of ethanol as the release media in a glass vessel incubated in a shaker water bath at 37°C and shaken at 80 rpm. PBST solution contained 137 mM NaCl, 2.68 mM KCl, 10.14 mM Na2HPO4, 1.76 mM KH2PO4, and 0.02% (w/v) Tween 80. Ethanol was added to PBST to final concentrations of 0, 15, 20, or 25% (v/v) ethanol. One milliliter of release medium was collected at predetermined time points (0, 1, 3, 6, 12, 18, 24, 30, 36, 42, 48, 54, and 60 days) and replaced with the same amount of fresh medium.

Sample analysis by ultraperformance liquid chromatography (UPLC)–mass spectrometer (Waters, Milford, MA) was used to quantify LNG concentration. LNG was separated on an Acquity UPLC Ethylene-Bridged Hybrids (BEH) C18 column (100 mm by 2.1 mm; inner diameter, 1.7-μm particle size) at 50°C. A mixture of acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid (8:2 ratio, v/v) comprised the mobile phase. The injection volume was 10 μl, with a flow rate of 0.3 ml/min. LNG detection was performed by electrospray ionization mass spectrometry in the positive ion mode. Quantification was based on the target analyte of LNG [M + H+; m/z (mass/charge ratio) = 313.4].

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