After animal sacrifice, the blood was harvested by cardiac puncture with a disposable syringe, immediately transferred to blood collection tubes (Covidien, Mansfield, MA), and centrifuged (1,500 g, 10 min, 4 °C) to yield plasma. The plasma was stored at −80 °C until analysis. To extract lipid metabolites from plasma, deuterated internal standards (d11-14,15-DHET, d11-11,12-EET, d4-9-HODE, d8-5-HETE, d4-6-keto PGF1a, d4-LTB4, d4-PGE2, d4-TXB2, 500 nM) were added into ~200 μL plasma. The mixture was then loaded onto pre-washed Waters® Oasis solid phase extraction (SPE) cartridges, and washed with 5% methanol containing 0.1% acetic acid, and the analytes were eluted with methanol and ethyl acetate, dried by a centrifugal vacuum evaporator, and then dispersed in methanol for LC-MS/MS analysis. The LC-MS/MS analysis was performed on an Agilent 1200SL HPLC coupled with a 4000 QTRAP MS/MS (AB Sciex) as previously14 (link). The peaks were identified based on retention time and specific multiple reaction monitoring (MRM) transitions of the standards of lipid metabolite. The concentrations of the lipid metabolites were calculated according to calibration curves of standards.
Oasis solid phase extraction cartridges
Manufactured by Waters Corporation
Sourced in United States
Oasis solid phase extraction (SPE) cartridges are laboratory equipment designed for sample preparation. They are used to isolate and purify analytes from complex matrices prior to analysis.
Automatically generated - may contain errors
Lab products found in correlation
4000 qtrap ms ms, by AB Sciex (4 mentions)
Agilent 1200sl hplc system, by Agilent Technologies (3 mentions)
Blood collection tubes, by Medtronic (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Hplc grade acetonitrile, by Thermo Fisher Scientific (1 mentions)
Sodium carboxymethyl cellulose cmc na, by Sinopharm (1 mentions)
Milli q water system, by Merck Group (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Formic acid, by Sinopharm (1 mentions)
Pierce trypsin protease, by Thermo Fisher Scientific (1 mentions)
Savant iss110, by Thermo Fisher Scientific (1 mentions)
Peg4000, by Merck Group (1 mentions)
Direct q 5 water purification system, by Merck Group (1 mentions)
Hydroxypropyl methylcellulose hpmc, by Merck Group (1 mentions)
Glycerine, by Avantor (1 mentions)
Pvp k90, by Merck Group (1 mentions)
Peg 10 000, by Merck Group (1 mentions)
8 protocols using oasis solid phase extraction cartridges
1
Plasma Lipid Metabolite Extraction and Quantification
2
Isolation and Characterization of Capilliposide Compounds
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Capilliposide B (LC-B) and LC-C were isolated from Lysimachia capillipes Hemsl (Primulaceae) [2 (link)]. The purity of both compounds was higher than 98%, measured by an HPLC-ELSD method [9 (link)]. LC-A was hydrolyzed from LC-C with a purity of >98%, as described in a previous report [12 (link)]. Dioscin (98% purity, Jiangxi University of Traditional Chinese Medicine, Nangchang, China) was used as internal standard (IS). Midazolam and nomifensine (IS) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Nicotinamide adenine dinucleotide phosphate (NADPH) was purchased from Dingguochangsheng Biotechnology Co., Ltd. (Beijing, China). Pooled liver microsomes originating from different species such as rats, mice, and humans were purchased from BD Gentest (Woburn, MA, USA). Acetonitrile and methanol were HPLC grade and provided by Fisher Scientific (Pittsburgh, PA, USA). Formic acid (≥96%) was purchased from Sinopharm Chemistry Reagent Co., Ltd. (Shanghai, China). The OASIS solid phase extraction (SPE) cartridges were obtained from Waters Corp. (Milford, MA, USA).
3
HPLC-Based Quantification of Alisol Compounds
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HPLC-grade acetonitrile and methanol were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Formic acid (≥ 96%) and carboxymethylcellulose sodium (CMC-Na) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ultra-pure water was produced using a Milli-Q water system (Millipore, Bedford, MA, USA). Oasis solid phase extraction (SPE) cartridges were purchased from Waters Corp. (Milford, MA, USA). All the other solvents and chemicals were of reagent grade or better.
Dried AR materials were obtained from Sichuan Province, China, and identified and authenticated by Professor Keli Chen of Hubei University of Chinese Medicine, China. The reference standards included alisol A-24 acetate, alisol A-23 acetate, 24-deacetyl alisol O, alisol G and 25-O-methylalisol A, with a purity of ≥98%, and they were isolated in house from the dried AR. In addition, reference standards of alisol B-23 acetate, alisol C-23 acetate, 16-oxo-alisol A and alisol F with a purity of ≥98% were obtained from Shanghai Yuanye Bio-Technology Co., Ltd. Shanghai, China.
Dried AR materials were obtained from Sichuan Province, China, and identified and authenticated by Professor Keli Chen of Hubei University of Chinese Medicine, China. The reference standards included alisol A-24 acetate, alisol A-23 acetate, 24-deacetyl alisol O, alisol G and 25-O-methylalisol A, with a purity of ≥98%, and they were isolated in house from the dried AR. In addition, reference standards of alisol B-23 acetate, alisol C-23 acetate, 16-oxo-alisol A and alisol F with a purity of ≥98% were obtained from Shanghai Yuanye Bio-Technology Co., Ltd. Shanghai, China.
4
Lipid Metabolite Extraction from Colon Tissues
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To extract lipid metabolites from colon tissues, ~100 mg tissues were mixed with an antioxidant solution (0.2 mg/mL butylated hydroxytoluene and 0.2 mg/mL triphenylphosphine in methanol), deuterated internal standards, and 400 μL extract solution (0.1% acetic acid with 0.2 mg/mL butylated hydroxytoluene in a methanol solution), and then homogenized; the resulting homogenates were kept in −80 °C overnight. After centrifugation of the homogenates, the pellets were washed with methanol (containing 0.1% butylated hydroxytoluene and 0.1% acetic acid) and then combined with the supernatant. The combined solutions were loaded onto pre-washed Waters® Oasis solid phase extraction (SPE) cartridges, washed with a solution of 95:5 water/methanol with 0.1% acetic acid, the analytes were eluted with methanol and ethyl acetate, dried using a centrifugal vacuum evaporator, then reconstituted in methanol for LC-MS/MS analysis. The LC-MS/MS analysis was carried out using an Agilent 1200SL HPLC system (Agilent, Santa Clara, CA) coupled to a 4000 QTRAP MS/MS (AB Sciex, Foster City, CA) as described in our previous report [7 ].
5
Protein Extraction and Digestion Protocol
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Protein pellets were resuspended using 8 M of urea in 50 mM of triethylammonium bicarbonate (TEAB), pH 8.5, sonicated for 20 min at 37°C, and centrifuged at 20,627 × g for 2 min. Protein contents were measured using the BCA assay (see section “Induced Changes of the Root Proteome”). Protein of 1 mg/cm3 was reduced with tris(2-carboxyethyl)phosphineı (TCEP; 10 mM of final concentration) at 37°C for 45 min and alkylated with iodoacetamide (IAA; 55 mM of final concentration) at 37°C for 45 min in the dark. Solution was diluted with 25 mM of TEAB at pH 8.5 to 1 M of urea. Trypsin (Pierce Trypsin Protease, mass spectrometry grade, Thermo Fisher Scientific) in 25 mM of TEAB was added to the samples (1:40) and shaken overnight at 37°C. TFA was added to 1% final volume. Peptides were loaded in Oasis solid-phase extraction (SPE) cartridges (Waters Co., United States), washed with 0.1% TFA, and eluted out using 80% acetonitrile (ACN) with 0.1% TFA. Peptides were dried in a vacuum concentrator (Savant ISS110, Thermo Fisher Scientific) and a freeze-dryer (Alpha 3-4 LSCbasic, Christ).
6
Adipose Tissue Lipid Metabolite Extraction and Analysis
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To extract lipid metabolites from adipose tissues, ~100 mg tissues were mixed with an antioxidant solution (0.2 mg/mL butylated hydroxytoluene and 0.2 mg/mL triphenylphosphine in methanol), 10 µL of deuterated internal standards (500 nM of d4-6-keto PGF1a, d4-TXB2, d4-PGE2, d4-LTB4, d11-14,15-DHET, d4-9-HODE, d8-5-HETE, d11-11,12-EET), and 400 µL extract solution (0.1% acetic acid with 0.2 mg/mL butylated hydroxytoluene in methanol), and then homogenized; the resulting homogenates were kept in −80 °C overnight. After centrifugation of the homogenates, the pellets were washed with methanol (containing 0.1% butylated hydroxytoluene and 0.1% acetic acid) and then combined with the supernatant. The lipid metabolites in the combined solutions were loaded onto pre-washed Waters® Oasis solid phase extraction (SPE) cartridges, washed with 95:5 v/v water/methanol with 0.1% acetic acid, the analytes were eluted with methanol and ethyl acetate, dried using a centrifugal vacuum evaporator, then reconstituted in methanol for LC-MS/MS analysis. The LC-MS/MS analysis was carried out on an Agilent 1200SL HPLC system (Agilent, Santa Clara, CA) coupled to a 4000 QTRAP MS/MS (AB Sciex, Foster City, CA) as described in our previous report (24 (link)). The lipid mediators whose levels were above the detection limit of LC-MS/MS were reported.
7
PVA-PEG-PVP Hydrophilic Polymer Formulation
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Poly (vinyl alcohol) (PVA, Mw = 85 -124 kDa, 87 -89% hydrolysed), poly (ethylene glycol) (PEG 4000, Mw = 400 and (PEG 10,000, Mw 10,000 Da), poly (vinyl pyrrolidone) K90 (PVP K90, Mw = 360 kDa) and hydroxypropyl methylcellulose (HPMC) with a viscosity of 40 -60 centipoise, 2% in H 2 O (20 °C) were purchased from Sigma-Aldrich (Dorset, UK).
Citric acid (CA) was supplied by BDH Laboratory Supplies (Poole, UK). PVP K29-32 (Mw = 58 kDa) was kindly donated by Ashland (Surrey, UK). Methotrexate disodium (MTX) (99.35% purity) was purchased from Haihang Industry Co., Ltd (Shandong, China).
Methotrexate polyglutamates (MTX-PG 1 -5 ) standards were purchased from Schircks Laboratories (Jona, Switzerland). Guthrie cards (Schleicher & Schuell 903) were purchased from Aston Ltd. (Oldham, England). Oasis solid-phase extraction (SPE) cartridges were obtained from Waters (Dublin, Ireland). Glycerine (99.5% purity) was purchased from VWR International (Lutterworth, England). HPLC grade water was produced using a Millipore Direct-Q™ 5 water purification system from Millipore (Watford, England). All other chemicals and materials were of analytical reagent grade supplied by Sigma-Aldrich (Dorset, UK) and Fisher Scientific (Loughborough, UK).
Citric acid (CA) was supplied by BDH Laboratory Supplies (Poole, UK). PVP K29-32 (Mw = 58 kDa) was kindly donated by Ashland (Surrey, UK). Methotrexate disodium (MTX) (99.35% purity) was purchased from Haihang Industry Co., Ltd (Shandong, China).
Methotrexate polyglutamates (MTX-PG 1 -5 ) standards were purchased from Schircks Laboratories (Jona, Switzerland). Guthrie cards (Schleicher & Schuell 903) were purchased from Aston Ltd. (Oldham, England). Oasis solid-phase extraction (SPE) cartridges were obtained from Waters (Dublin, Ireland). Glycerine (99.5% purity) was purchased from VWR International (Lutterworth, England). HPLC grade water was produced using a Millipore Direct-Q™ 5 water purification system from Millipore (Watford, England). All other chemicals and materials were of analytical reagent grade supplied by Sigma-Aldrich (Dorset, UK) and Fisher Scientific (Loughborough, UK).
8
Adipose Tissue Lipid Metabolite Extraction and Analysis
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