Lc 10ad hplc

The analysis of media glucose content by HPLC (high performance liquid chromatography) was performed as follows: after filtration with a 0.22-μm membrane, medium supernatants were applied to a Bio-Rad Aminex HPX-42A carbohydrate column and analyzed with an LC-10AD HPLC (Shimadzu, Japan) equipped with a RID-10A refractive index detector. The column was maintained at 78 °C and eluted with double-distilled water at a flow rate of 0.4 ml/min.

Metabolic stability was determined as described in reference 14 (link). CD101 (1 μM) was incubated with liver microsomes and hepatocytes from Sprague-Dawley rats, cynomolgus monkeys, and humans or hepatocytes only from dogs with appropriate cofactors for up to 2 h at 37°C. Following incubation, samples were quenched with ice-cold methanol or acetonitrile (ACN), containing an appropriate internal standard and centrifuged to remove precipitated protein, and the supernatants were analyzed to quantitate the remaining parent. Samples from in vitro experiments were analyzed either by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Agilent 6410 mass spectrometer coupled with an Agilent 1200 high-performance liquid chromatography (HPLC) device and a CTC PAL autosampler (microsomes) or an Applied Biosystems API4000 mass spectrometer coupled with a Shimadzu LC10 AD HPLC and a CTC PAL autosampler (hepatocytes). Two control agents (warfarin and verapamil) were analyzed under similar conditions. Data were converted to the percentage remaining by dividing by the time zero concentration value. Data were fit to a first-order decay model to determine half-life. Intrinsic clearance was calculated from the half-life and the protein concentrations.

The concentration of XOS was analyzed using a LC-10AD HPLC (Shimadzu, Japan) equipped with a Shodex Sugar KS-802 packed column (8 mm ID ×300 mm, F6378020) and a RID-10A according to Wu et al. [26 (link)]. The column was maintained at 65 °C and eluted with deionized water at a flow rate of 0.8 mL/min. The concentration of glucose, xylose, and arabinose was determined by HPLC system equipped with Aminex HPX-87H column (Bio-Rad, Hercules, USA). The column temperature was kept at 65 °C and the mobile phase was 5 mmol/L H₂SO₄ at a flow rate of 0.6 mL/min.

where P(X) is the XOS, xylose, glucose, or arabinose yield, C(X) is the XOS (DP = 2–5), xylose, glucose, or arabinose concentration in the reaction mixture, V is the volume of LF (L), and W is the hemicellulose or total of cellulose and starch weight in the test sample (g).

The GABA content in the brains of the control and ACEA groups was determined using a Shimadzu (LC-10 AD) HPLC with an injection circuit attached to an LC-20AT pump, analytical Shimadzu C18 column, and fluorescence detector (RF-10AXL). The mobile phase (phase A) consisted of a solution containing 50 mM of sodium acetate, 5% methanol, and 2-propanol. Phase B contained 70% methanol. Immediately after the behavior task, the animals were cryoanesthetized individually and their brains were dissected as soon as possible. The samples were then transferred to culture dishes containing Na⁺—Hanks buffer (128 mM NaCl; 4 mM KCl; 1 mM MgCl₂; 2 mM CaCl₂, 12 mM glucose, and 20 mM HEPES, pH. 7.2) and maintained for 20 min in a CO₂ stove at 37°C. The analysis matrix was prepared by adding 1% TCA, followed by centrifugation for 10 min at 5,000 rpm. The matrix was made by mixing 60 μl of each sample in 10 μl of methanolic o-phthaldialdehyde and pH 9.5 borate buffer. The final volume of the samples was vortexed and injected into the system for analysis after 5 min. The GABA levels in each sample were determined using a standard GABA curve, with the values being expressed as a percentage of the control (telencephalon = 0.14 ± 0.002 μM/mg ptn and mesencephalon = 0.02 ± 0.006 μM/mg ptn). The protein content was determined by the Bradford method.

The fermentation broth was collected via centrifugation, and the aliquots of the supernatant were diluted to measure enzyme activity. To examine β-glucosidase activity, pNPG and cellobiose (Sigma, USA) were used as substrates. We conducted the enzyme reaction in acetate buffer (pH 4.8) at 50 °C for a total of 30 min, after which we added 10 % Na₂CO₃ to stop the reaction. pNP release was measured and the absorbance was read at 405 nm. The glucose level was measured with the Biosensor. One enzyme activity unit represents the amount of enzyme required to either produce one µmol glucose, or pNP per minute under the above condition. To measure the FPase activity, the enzyme reaction was conducted in 0.2 mol/L acetate buffer (pH 4.8) at 50 °C for a total of 60 min with 0.05 g Whatman No. 1 paper as the substrate. DNS method was used to quantify the released reducing sugars. A protein concentration assay was performed using a Bradford kit (Sangon Biotech, Shanghai, China). Three biological triplicates were performed throughout all described experiments. Both equal quality and volume of culture supernatants were performed for SDS-PAGE analysis, and the predicted β-glucosidase bands were excised for MALDI-TOF–MS identification. We measured the released sugars within the broth supernatants via LC-10 AD HPLC (Shimadzu, Japan) by a BioRad Aminex HPX-42A carbohydrate column (Bio-Rad, USA).