The process flow of preparing mouse serum samples is illustrated in Figure 1 . The detailed protocols can be found in the previous report18 (link). In brief, an ammonium formate solution (1% in methanol) was added to the mouse serum sample and mixed thoroughly. The precipitation was pelleted down by centrifugation at 7500 × g for 5 min, and the precipitated proteins were removed. The supernatant was then transferred into a HybridSPE-Phospholipid cartridge (Sigma, St. Louis, MO) to get rid of phospholipids. The eluate was reconstituted with water and then loaded into a DSC-18 SPE cartridge (Sigma, St. Louis, MO) that had been conditioned with methanol and water. After washing with water, organic molecules on the cartridge column were eluted out using methanol. The final eluate in methanol was dried under nitrogen stream and reconstituted in the sample matrix solution designed for CSEI-sweeping-MEKC analysis.
Hybridspe phospholipid cartridge
Manufactured by Merck Group
Sourced in United States
The HybridSPE-Phospholipid cartridge is a laboratory equipment product designed for sample preparation. It is used for the selective and efficient removal of phospholipids from biological samples prior to analysis.
Automatically generated - may contain errors
Lab products found in correlation
5 protocols using hybridspe phospholipid cartridge
1
Mouse Serum Preparation for CSEI-sweeping-MEKC
2
Plasma Metabolome Analysis by CE-TOFMS and LC-TOFMS
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Metabolite extraction and metabolome analysis were conducted at Human Metabolome Technologies (HMT), Japan. For CE-TOFMS analysis, 50 μl of plasma was added to 450 μl of methanol containing internal standards (H3304–1002, HMT) on ice. The solution was then mixed thoroughly with 500 μl chloroform and 200 μl Milli-Q water and centrifuged at 2,300 × g for 5 min at 4 °C. The upper aqueous layer was centrifugally filtered through Millipore 5 kDa cut-off filter (UltrafreeMC-PLHCC, HMT) at 9,100 × g for 120 min at 4 °C to remove macromolecules. The filtrate was then centrifugally concentrated and reconstituted in 25 μl Milli-Q water prior to CE-TOFMS analysis.
For LC-TOFMS analysis, 500 μl plasma samples were added to 1,500 μl acetonitrile with 1% formic acid containing internal standard solution (H3304-1002, HMT) on ice. The solution was then mixed thoroughly and centrifuged at 2,300 × g for 5 min at 4 °C. The supernatant was applied to a Hybrid SPE phospholipid cartridge (55261-U, Sigma-Aldrich). The filtrate was dried by nitrogen gas and reconstituted in 200 μl of 50% isopropanol prior to LC-TOFMS analysis.
For LC-TOFMS analysis, 500 μl plasma samples were added to 1,500 μl acetonitrile with 1% formic acid containing internal standard solution (H3304-1002, HMT) on ice. The solution was then mixed thoroughly and centrifuged at 2,300 × g for 5 min at 4 °C. The supernatant was applied to a Hybrid SPE phospholipid cartridge (55261-U, Sigma-Aldrich). The filtrate was dried by nitrogen gas and reconstituted in 200 μl of 50% isopropanol prior to LC-TOFMS analysis.
3
Quantitative Analysis of CLP-m and TCP in Biological Matrices
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For the analysis of CLP-m and TCP content, approximately 0.2 mL of bile or 0.5 g (wet weight) of liver tissue were transferred to an Eppendorf tube. After adding 0.8 mL (1 mL for liver analysis) of acetonitrile:acetone (80:20, v/v) containing 1% formic acid, the tube was stirred in a vortex for 1 min. The extract was then passed through a hybridSPEphospholipid cartridge (Sigma-Aldrich) (in the case of liver, 0.4 g magnesium sulphate were previously added to the mix and stirred in a vortex for 30 s). The eluate was diluted with HPLC water (two-and five-fold for bile and liver, respectively). After adding the internal-labelled internal standard mix (composed by CLP-m-D6 and TCP-13C3), 2 µL were finally injected in the liquid chromatography-tandem mass spectrometry (LC-MS/MS) system, equipped with a triple quadrupole analyser (TQS, Waters). For further details, please see supplementary material (Appendix B).
4
Sample Pretreatment and Cleanup for TBBPA
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The sample pretreatment method was performed as described in a previous study with slight modifications (Yu et al. 2016 (link)). In brief, the cell pellet (approximately cells) of each sample was mixed with of hydrochloric acid ( ) and of dimethylcarbinol. Next, of a mixture of hexane and methyl tertiary butyl ether (1:1, vol/vol) was added to extract the chemicals from the cells by applying an ultrasonic cell disrupter for . The supernatant was collected after centrifugation at 4,000 rpm for 15 min. The above ultrasonic extraction process was repeated twice, and the supernatants were collected and mixed together. Next, of 1% KCl and of water were added to the collected extraction solution to precipitate the proteins and impurities. Anhydrous sodium sulfate was added to remove water. The extraction solution was then removed gently with a rotary evaporator and nitrogen blowing. The target compounds were then reconstituted in of methanol and introduced to a HybridSPE-Phospholipid cartridge ( , ; Supelco) for further purification. Finally, of Deuterated-labeled TBBPA ( ) was spiked as an internal standard and redissolved in of methanol before analysis.
5
Quantitative Analysis of Amphetamine and Methamphetamine
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The reference compounds AP and MA were purchased from Cerilliant (Round Rock, TX, USA) at a concentration of 1000 µg/mL in methanol. Methanolic solutions of the deuterated IS AP-d8 and MA-d11 in each vial at a concentration of 100 µg/mL were also purchased from Cerilliant. HPLC-grade acetone, ethyl acetate, and methanol were supplied by J. T. Baker (Phillipsburg, NJ, USA). Methanolic HCl (HCl, 1.25 M) was obtained from Fluka (St. Gallen, Switzerland). PFPA was acquired from Acros Organics (Geel, Belgium). HybridSPE phospholipid cartridge (30 mg/1 mL) was purchased from Supelco (Bellefonte, PA, USA). Strata-X 33 µm polymeric reversed phase cartridge (60 mg/3 mL) was purchased from Phenomenex (Torrance, CA, USA), while Oasis HLB extraction cartridge (60 mg/3 mL) was from Waters (Milford, MA, USA). All other chemicals were of analytical grade or higher. Polypropylene tubes (2 mL) were obtained from Eppendorf (Safe-Lock tube, Hamburg, Germany). Water was purified with a Millipore AFS-16 water purification system (Molsheim, France).
Stock solutions of the target compounds were mixed and diluted in methanol to prepare final mixed standard solutions at 10 µg/mL of AP and 20 µg/mL of MA. A working solution of IS (AP-d8 and MA-d11) at 0.3 µg/mL was also prepared in methanol. All these solutions were stored at −20 °C before use.
Stock solutions of the target compounds were mixed and diluted in methanol to prepare final mixed standard solutions at 10 µg/mL of AP and 20 µg/mL of MA. A working solution of IS (AP-d8 and MA-d11) at 0.3 µg/mL was also prepared in methanol. All these solutions were stored at −20 °C before use.
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