Cve 2000

Aliquots (40 μl) of purified Gb3 (10 μg/ml) were mixed with 0.8 ml of CHCl₃/MeOH (2:1 (v/v)) for lipid extraction, and aqueous lipids were separated by the addition of 0.2 ml of 1.5% FA in H₂O and centrifugation at 6500 × g using a personal centrifuge (Gyrogen, Seoul, Korea) for 5 min. After removal of the upper layer, 40 μl of the lower layer was transferred to another tube and dried. Samples were suspended in 40 μl of enzyme mixture (5 μg/ml of SCDase in 25 mm sodium acetate buffer, pH 5.5, containing 0.5% Triton X-100, 5 mm CaCl₂, and 5% DMSO) and incubated at 37 °C for 1 h. The reaction was stopped by the addition of 0.8 ml of CHCl₃/MeOH (2:1 (v/v)). The reaction products were mixed with 20 μl of 100 ng/ml glycine derivative of lyso-Gb3 (Gly-lyso-Gb3 as an internal standard (37 (link))) and 0.2 ml of 1.5% FA in H₂O and extracted by centrifugation at 6500 × g for 5 min. Then 150 μl of the clear upper layer was transferred to another tube and dried using a centrifugal evaporator CVE-2000 (EYELA, Tokyo, Japan). Samples were redissolved in 50 μl of H₂O and extracted with the same volume of H₂O-saturated 1-butanol. The extracts were diluted with MeOH and analyzed by UPLC-MS/MS at GlycoPharma Corp.

The sample preparation was described in a previous publication [20 (link)]. In brief, firstly the fillets were freeze-dried and powdered in a mill. Into each 50 mg of powdered sample, mixed solutions of methanol/ultrapure water/chloroform (2.5/1/1 v/v/v, 1 mL) and ribitol (internal reference standard, 0.2 mg/mL, 60 µL) were added. After stirring for 5 min, the mixture was centrifuged (16,000× g, 0 °C, 5 min). Ultrapure water (400 µL) was added to 800 µL of the supernatant, followed by stirring for 1 min and then centrifugation (16,000× g, 0 °C, 5 min). A 400-µL portion of the supernatant was concentrated for 1 hour using a centrifugal evaporator (CVE-2000, Eyela, Japan), and then freeze-dried overnight. Methoxyamine hydrochloride solubilised with pyridine (20 mg/mL, 50 µL) was added to the freeze-dried sample, and oxime formation was carried out by reacting at 30 °C for 90 min. N-methyl-N-(trimethylsilyl) trifluoroacetamide with a volume of 100 µL (MSTFA, GL Sciences, Japan) was further added, and trimethylsilylation was carried out by reaction at 37 °C for 30 min. The derivatised samples were submitted to GC-MS analysis.

Sample preparation was conducted as described in previous studies [8 (link),9 (link)]. Briefly, fish fillets were freeze-dried and powdered in a mill. Mixed solutions of methanol/ultrapure water/chloroform (2.5/1/1 v/v/v, 1 mL) and ribitol (internal reference standard, 0.2 mg/mL, 60 µL) were added to 50 mg of the powdered sample. After stirring for 5 min, the mixture was centrifuged (16,000× g, 0, 5 min). Ultrapure water (400 µL) was then added to 800 µL of the supernatant, followed by stirring for 1 min and then centrifugation (16,000× g, 0 °C, 5 min). A 400-µL aliquot of the supernatant was concentrated for 1 h using a centrifugal evaporator (CVE-2000, Eyela, Japan), before being freeze-dried overnight. Methoxyamine hydrochloride solubilized with pyridine (20 mg/mL, 50 µL) was added to the freeze-dried sample, and oxime was formed in a reaction at 30 °C for 90 min. Subsequently, 100 µL of MSTFA was added, and trimethylsilylation was conducted by reaction at 37 °C for 30 min. The derivatized samples were then subjected to GC-MS analysis.