SSA was administered to C57B6 mice of both males and females via oral garage as a single bolus dose. Mice were sacrificed at predetermined time points and plasma and brain tissue samples were harvested for analysis by PPL labs (Redwood City, CA). SSA and its metabolite SA were measured simultaneously using oxopropanoyloxy benzoic acid as an internal standard solution59 (link). To extract SSA/SA from brain, 200 μl of 50% acetonitrile in 0.1% formic acid was added to 30–50 mg of cortical tissue, followed by homogenization and vortexing. After addition of 200 μl of the internal standard solution, the samples were centrifuged and the supernatants were transferred for injection. To measure SSA/SA in the plasma, 10 μl of plasma samples were mixed with 90 μl H2O and 100 μl of Internal Standard Solution, followed by centrifugation and injection. The HPLC was performed in C18 column, following a gradient program (10–90% acetonitrile in 6 min) with a flow rate of 0.4 ml/min. The injection volume is ~ 5 μl. The mass spectrometry analyses were perform on 3200 Q TRAP system (AB SCIEX, Framingham, MA) using MQL algorithm.
3200 q trap system
Manufactured by AB Sciex
Sourced in United States
The 3200 Q TRAP system is a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform designed for sensitive and accurate quantitative analysis. The system combines a triple quadrupole mass analyzer with a linear ion trap, providing enhanced selectivity and sensitivity for targeted and discovery-based applications. The 3200 Q TRAP system is capable of performing multiple reaction monitoring (MRM) and information-dependent acquisition (IDA) analyses.
Automatically generated - may contain errors
Lab products found in correlation
Prominence uflc system, by Shimadzu (3 mentions)
C17 0 sm, by Avanti Polar Lipids (2 mentions)
C17 0 ceramide cer, by Avanti Polar Lipids (2 mentions)
C17 0 lactosylceramide, by Avanti Polar Lipids (2 mentions)
C17 0 glucosylceramide, by Avanti Polar Lipids (2 mentions)
C17 0 gb3, by Avanti Polar Lipids (2 mentions)
D18 1 d5 c18 0 gm3, by Avanti Polar Lipids (2 mentions)
Acetonitrile hplc grade, by Samchun (1 mentions)
1260 infinity 2, by Agilent Technologies (1 mentions)
Shim pack gis c18, by Shimadzu (1 mentions)
Trifluoroacetic acid, by Samchun (1 mentions)
Capcell pak mgii column, by Shiseido (1 mentions)
Sequant zic hilic column, by Merck Group (1 mentions)
7 protocols using 3200 q trap system
1
Quantification of SSA and Metabolite SA
2
Quantification of SSA and Metabolite SA
3
HPLC Characterization of Biomolecular Compounds
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Chromatography experiments were performed using an HPLC system (1260 Infinity II, Agilent, Santa Clara, CA, USA). Water and acetonitrile (HPLC grade) were purchased from Samchun Pure Chemicals (Pyeongtaek, Republic of Korea), and trifluoroacetic acid of Alfa Aesar (Schiltigheim, France) was used. For the identification of each individual peak appearing in the HPLC chromatogram, authentic compounds (assay ≥ 98%) of adenosine (#A9251), guanosine (#G6752), phenylalanine (#P2126), tryptophan (# T0254), tyrosine (#T3754), and uridine (#U3750) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The analytical column was a Shim-pack GIS C18 (5 μm, 4.6 × 250 mm, Shimadzu, Kyoto, Japan), and the mobile phase was a mixture of eluent A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). Elution was performed with a linear gradient of eluent A and B under the following conditions: 0→5 min, 0→0% B; 5→75 min, 0→75% B; 75→77 min, 75→95% B; 77→82 min, 95→95% B; 82→84 min, 95→0% B and 84→95 min 0→0% B. The flow rate was 1.0 mL/min, the injection volume was 20 μL, and detection was performed at 210 nm. All samples were filtered with a 0.45 μm syringe filter (PTFE, Advantec, Tokyo, Japan) before injection. To identify peaks from the HPLC results, we conducted MS/MS using the 3200 QTRAP system (AB Sciex, Framingham, MA, USA), as described previously [29 ].
4
Toxicological Analysis of Overdose Deaths
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In all 23 autopsy cases, qualitative and quantitative analysis of blood collected from the external iliac vein using liquid chromatography (LC)-tandem mass spectrometry (MS/MS) determined that death was caused by intoxication due to drug overdose. Every case demonstrated lethal, supra-therapeutic drug levels. LC was performed with a P r o m i n e n c e L C s y s t e m ( S h i m a d z u , K y o t o , J a p a n ) . Chromatographic separation was achieved on a CAPCELL-PAK MG II column (35.0 × 2.0 mm id, 5 lm; Shiseido, Tokyo, Japan). MS/MS detection was performed with a 3200 QTRAP system equipped with an electrospray ionization probe (AB Sciex, Foster City, CA). All analyses were performed by the forensic toxicologist (K.U.) in charge of laboratory sample testing at our institution.
5
Lipidomic Analysis Using HILIC-MS
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Lipidomic analysis of these extracted lipids was performed as previously described (Nakao et al., 2019 ; Watanabe et al., 2022 (link)). The extracted lipids were dissolved in equal volumes of methanol and acetonitrile, and subjected to the liquid chromatography-mass spectrometry system consisted of a Prominence UFLC system (Shi-madzu, Kyoto, Japan) equipped with a SeQuant ZIC-HILIC column (5 μm, 2.1 mm × 150 mm, Merck Millipore) coupled to a 3200 QTRAP System (Sciex, Redwood, CA, USA). The optimal conditions for the ionization and fragmentation of each lipid were determined as previously described (Watanabe et al., 2022 (link)).
6
Lipidomic Analysis of Cultured Cells
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Cells were seeded in a 6-cm dish at a density of 0.5 × 106 cells/dish in 5 ml of culture medium. After 24 h, cells were washed twice with PBS and harvested. A total of 1 nmol each of the internal standards C17:0 SM, C17:0 ceramide (Cer), C17:0 lactosylceramide, C17:0 glucosylceramide, C17:0 Gb3, and d18:1-d5-C18:0 GM3 (Avanti Polar Lipids) were then added for LC-MS/MS analysis. The amount of protein was determined and lipids were extracted from the cells. Sphingolipids were analyzed by an LC-MS system that consisted of a Prominence UFLC system (Shimadzu) coupled to a 3200 QTRAP System (SCIEX) as described previously (27 ).
7
Sphingolipid Quantification by LC-MS/MS
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Cells were seeded in a 6-cm dish at a density of 0.5 × 106 cells/dish in 5 mL of culture medium. After 24 h, cells were washed twice with PBS and harvested. The amount of protein was determined and lipids were extracted from the cells. A total of 1 nmol each of the internal standards C17:0 SM, C17:0 ceramide (Cer), C17:0 lactosylceramide, C17:0 glucosylceramide, C17:0 Gb3, and d18:1-d5-C18:0 GM3 (Avanti Polar Lipids, Inc) were then added for quantification. Sphingolipids were analyzed by an LC-MS/MS system that consisted of a Prominence UFLC system (Shimadzu Corporation) coupled to a 3200 QTRAP System (SCIEX) as described previously (Nakao et al., 2019 (link)).
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