Lc 20ab pump

Mycolactones A/B were purified from M. ulcerans extracts as previously described [6 (link),127 (link)]. Briefly, S4018, an African strain of Mycobacterium ulcerans obtained from a patient in Benin, was grown in Middlebrook 7H10 agar supplemented with oleic albumin dextrose catalase growth supplement. The bacteria were resuspended in chloroform-methanol (2:1, v/v) and cell debris was removed by centrifugation. Folch extraction was performed by adding 0.2 volumes of water. The organic phase was dried and phospholipids were precipitated with ice-cold acetone. The acetone-soluble lipids were loaded onto a thin layer chromatography plate and eluted with chloroform-methanol-water (90:10:1, v/v/v) as the mobile phase. The yellow band with a retention factor of 0.23 was scraped off the plate, filtered, evaporated, resuspended in absolute ethanol and then stored in amber glass tubes in the dark. Its concentration was determined by measuring absorbance (λ_max  =  362 nm, log ε  =  4.29), and its purity (>98%) was evaluated with a Shimadzu Ultra-Fast Liquid Chromatograph (UFLC XR system with a CBM-20A controller, a CTO-10AS Prominence column oven, LC-20AB pumps, an SPD M20A diode array detector (Shimadzu, Japan)) and a reverse C18 column (Zorbax 23 Eclipse XDB-C18, 9.4×250mm, Particle Size: 5 μm (Agilent, USA)).

The concentration of rosuvastatin in cells was determined by LC-MS/MS system consisted of Shimadzu LC-20AB pumps (Shimadzu Corporation, Kyoto, Japan) and an AB SCIEX API 4000 mass spectrometer (Applied Biosystems/SCIEX, Foster, CA, USA). Data acquisition was performed using Analyst 1.6.1 software (AB SCIEX). Chromatographic separation was achieved on a Luna C18 column (50 × 2.0 mm i.d., 5 µm; Phenomenex Technologies). The mobile phase consisted of 10-mM ammonium formate (A) and acetonitrile (B) using a gradient elution of 40-90% B at 0.0–1.0 min, 90%–90% B at 1.0–2.5 min, and 40%–40% B at 2.51–3.5 min. The flow rate was 0.4 ml/min, the operating temperature was 25°C.
Samples were ionized utilizing an electrospray-ionization probe in the positive-ion mode, and quantification was performed using the multiple-reaction monitoring (MRM) method, with the precursor-to-product transition being m/z 482.3→258.2 for rosuvastatin and m/z 559.2→440.0 for atorvastatin (IS). Nitrogen was used as the curtain and auxiliary gas, and air was used as the nebulizer gas under the following conditions: curtain gas, 40 psi; ion-spray voltage, 5500 V; nebulizer gas, 50 psi; auxiliary gas, 50 psi; and turbo temperature, 500°C. The collision energy (CE) was 45 V for rosuvastatin and 28V for atorvastatin, and the declustering potential (DP) was 118 V for rosuvastatin and 100 V for atorvastatin.

The ascorbic acid content was determined according to the method described by Wojdyło^{25 (link)} with some modifications. In this analysis, 2 to 3 g of sliced fresh fruit was used. The sample was added to 50 mL of 2% oxalic acid solution and homogenized for 10 min using a magnetic stirrer. The homogenized suspension was centrifuged at 3,000 rpm for 15 min, and the supernatant was collected and filtered with a 0.22 μm syringe filter for chromatographic analysis. High-performance liquid chromatography was performed using an LC-20A system (Shimadzu, Kyoto, Japan) equipped with two LC-20AB pumps, an SIL-20A injector, and an SPD-M20A detector. A C18 column (I.D. 250 × 4.6 mm; Dikma Ltd, Tianjin, China) was used to separate the compounds. A 10 μL aliquot of the sample was injected into the system where the column oven temperature was adjusted to 30 °C. The samples were eluted using 0.5% formic acid at a flow rate of 0.6 mL/min. Absorbance was monitored at 243 nm, where L-ascorbic acid was used as the standard. The AA concentration was expressed as g/kg FW.