Lc 20ad hplc

Media and lysate samples were stored at −80 °C prior to analysis. 2 volumes of ice cold MeOH containing deuterated internal standards (d₄-LTB₄, d₈-5S-HETE, d₄-PGE₂, d₅-LXA₄ and d₅-RvD2, 500 pg each) were added to the samples. These were then kept at −20 °C for 45 minutes to allow for protein precipitation and subjected to solid phase extraction as per previous publication^{26 (link)}. Methyl formate fractions were then brought to dryness using a TurboVap LP (Biotage) and products suspended in water-methanol (50:50 vol:vol) for LC-MS-MS. A Shimadzu LC-20AD HPLC and a Shimadzu SIL-20AC autoinjector (Shimadzu, Kyoto, Japan), paired with a QTrap 5500 (ABSciex, Warrington, UK) were utilised and operated as described^{26 (link)}. To monitor each lipid mediator and respective pathways, a Multiple Reaction Monitoring (MRM) method was developed with diagnostic ion fragments and identification using recently published criteria^{26 (link)}, including matching retention time (RT) to synthetic and authentic materials and at least six diagnostic ions for each lipid mediator. Calibration curves were obtained for each using authentic compound mixtures and deuterium labeled lipid mediator at 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, and 200 pg. Linear calibration curves were obtained for each lipid mediator, which gave r² values of 0.98–0.99.

Blister exudates were placed in 2 volumes of ice-cold methanol containing deuterium-labeled standards (Cayman Chemicals). These were then kept at –20°C for 45 minutes to allow for protein precipitation and lipid mediators were extracted using C-18–based solid phase extraction as previously described (24 (link)). Methyl formate fractions were brought to dryness using a TurboVap LP (Biotage) and products suspended in water/methanol (50:50 vol/vol) for LC-MS/MS–based profiling. A Shimadzu LC-20AD HPLC and a Shimadzu SIL-20AC autoinjector, paired with a QTrap 5500 (ABSciex) were utilized and operated as previously described (24 (link)). To monitor each lipid mediator and deuterium-labeled internal standard, a multiple reaction monitoring (MRM) method was developed using parent ions and characteristic diagnostic ion fragments as previously described (24 (link)). This was coupled to an information-dependent acquisition and an enhanced product ion scan. Identification criteria included matching retention time to synthetic standards and at least 6 diagnostic ions in the MS/MS spectrum for each molecule. Calibration curves were obtained for each molecule using authentic compound mixtures and deuterium-labeled lipid mediator at 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, and 200 pg. Linear calibration curves were obtained for each lipid mediator, which gave r² values of 0.98–0.99.

Maceration and fractionation of the mangosteen pericarp were carried out using a procedure similar to that reported by Mulia et al. [15 ]. Dried mangosteen pericarp powder was mixed with 96% ethanol at a ratio of 1:3 (w/v) and macerated for 14 days with continuous stirring. The fractionation of extracts obtained from maceration was performed using a mixture of ethyl acetate and water (volume ratio of 1:1) in a separating funnel. The ethyl acetate fraction was separated and solvent was evaporated using a rotary evaporator.
The uv-spectrophotometry and high-performance liquid chromatography (HPLC) were used to determine the total mangostin and the α-mangostin content, respectively. The total mangostin in the ethyl acetate fraction was quantified as α-mangostin equivalent, based on the absorbance curve of α-mangostin standard solutions measured at a wavelength of 316 nm. The α-mangostin content was determined using a Shimadzu LC 20AD HPLC equipped with a reversed-phase C18 column (250nm×4.6nm, 5μm) at maintained at 30°C, and, a UV detector set at a wavelength of 244 nm. The mobile phase consisted of 95% acetonitrile and 5% buffer (0.1% H₃PO₄) was used with a flow rate of 1 mL/min and elution time of 8 min [16 ].