Supernatant from the inflammatory lesions were placed in ice cold methanol containing deuterated internal standards (d8-5S-hydroxyeicosatetraenoic acid (HETE), d4-leukotriene (LT) B4, d4-prostaglandin (PG)E2 and d5-lipoxin (LX) A4; 500pg each) and homogenized using a PTFE dounce (Kimble Chase). Proteins were allowed to precipitate (4°C), and lipid mediators were extracted using C18 solid-phase cartridges and a Biotage RapidTrace®. Measurement of lipid mediators was carried out by liquid chromatography-tandem mass spectrometry using a QTrap 5500 (ABSciex, Framingham, MA) equipped with a Shimadzu LC-20AD HPLC and a Shimadzu SIL-20AC autoinjector (Shimadzu, Kyoto, Japan). An Agilent Eclipse Plus C18 column (100mm × 4.6 mm × 1.8 μm) maintained at 50°C was used with a gradient of methanol/water/acetic acid of 55:45:0.01 (v/v/v) to 100:0:0.01 at 0.4 ml/min flow rate. Multiple reactions monitoring (MRM) was used to monitor lipid mediator profiles with more than 60 bioactive products from specific biosynthetic pathway including their pathway markers. Identification was carried out with signature ion fragments for each target lipid mediator (pro-inflammatory mediators PG, LT as well as SPM) using a minimum of six diagnostic ions. Quantification was achieved using calibration curves (Colas et al., 2014 (link)).
Sil 20ac autoinjector
Manufactured by Shimadzu
Sourced in Japan, United States
The SIL-20AC autoinjector is a laboratory instrument designed for automated sample injection and handling. It provides precise and consistent sample introduction into analytical instruments, such as chromatography systems, for accurate and reliable data collection. The SIL-20AC autoinjector is a core component of Shimadzu's analytical instrumentation solutions.
Automatically generated - may contain errors
Lab products found in correlation
Lc 20ad hplc, by Shimadzu (6 mentions)
Qtrap 5500, by AB Sciex (5 mentions)
Qtrap 6500, by AB Sciex (5 mentions)
Eclipse plus c18 column, by Agilent Technologies (4 mentions)
Lc 20ad binary pump, by Shimadzu (4 mentions)
Lc 20at pump, by Shimadzu (2 mentions)
Spd m20a diode array detector, by Shimadzu (2 mentions)
Thermasphere ts 130, by Phenomenex (2 mentions)
Poroshell 120 ec column, by Agilent Technologies (2 mentions)
Cto 20a column oven, by Shimadzu (2 mentions)
Synergi hydro rp column, by Phenomenex (2 mentions)
Ptfe dounce, by DWK Life Sciences (1 mentions)
Rapidtrace, by Biotage (1 mentions)
Turbovap lp, by Biotage (1 mentions)
C r3a integrator, by Shimadzu (1 mentions)
Cto 20ac column oven, by Shimadzu (1 mentions)
Cbm 20a communication bus module, by Shimadzu (1 mentions)
Hplc system, by Shimadzu (1 mentions)
C18 column, by Shiseido (1 mentions)
Lc solution software, by Shimadzu (1 mentions)
15 protocols using sil 20ac autoinjector
1
Quantitative Lipid Mediator Profiling
2
Quantification of Lipid Mediators by LC-MS/MS
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Media and lysate samples were stored at −80 °C prior to analysis. 2 volumes of ice cold MeOH containing deuterated internal standards (d4-LTB4, d8-5S-HETE, d4-PGE2, d5-LXA4 and d5-RvD2, 500 pg each) were added to the samples. These were then kept at −20 °C for 45 minutes to allow for protein precipitation and subjected to solid phase extraction as per previous publication26 (link). Methyl formate fractions were then brought to dryness using a TurboVap LP (Biotage) and products suspended in water-methanol (50:50 vol:vol) for LC-MS-MS. A Shimadzu LC-20AD HPLC and a Shimadzu SIL-20AC autoinjector (Shimadzu, Kyoto, Japan), paired with a QTrap 5500 (ABSciex, Warrington, UK) were utilised and operated as described26 (link). To monitor each lipid mediator and respective pathways, a Multiple Reaction Monitoring (MRM) method was developed with diagnostic ion fragments and identification using recently published criteria26 (link), including matching retention time (RT) to synthetic and authentic materials and at least six diagnostic ions for each lipid mediator. Calibration curves were obtained for each using authentic compound mixtures and deuterium labeled lipid mediator at 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, and 200 pg. Linear calibration curves were obtained for each lipid mediator, which gave r2 values of 0.98–0.99.
3
Quantification of FTC in Pharmaceutical Samples
4
Quantification and Profiling of Biomolecules by LC-MS/MS
5
HPLC Quantification of Cefdinir Drug
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Sample analysis was performed using a Shimadzu HPLC system consisting of LC-20AT pump, SIL-20AC autoinjector, CBM-20A communication bus module, CTO-20A column oven, and SPD-M20A diode array detector (Shimadzu, Kyoto, Japan). The separation of cefdinir was performed on a reverse phase C18 column (250 mm × 4.6 mm, 5 μm; Shiseido, Tokyo, Japan). The mobile phase consisted of 1 M tetramethylammonium hydroxide solution, water, 0.1 M ethylenediaminetetraacetic acid, and methanol (1.40:92.40:0.04:6.16, v/v/v/v) (pH 5, adjusted with diluted phosphoric acid), at a flow rate of 1.0 mL/min by modifying method in United States Pharmacopoeia (USP) (2010) [23 ]. The column and autosampler tray were maintained at 40 °C and 4 °C, respectively. The analytical run time was 10 min. UV detection was monitored at 254 nm and injection volume of sample was 10 μL. Data acquisition and processing were carried out using the Shimadzu LC Solution software.
6
Quantitative Lipid Mediator Profiling by LC-MS/MS
7
Extraction and LC-MS/MS Profiling of Metabolites
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Samples were placed in one volume of ice-cold MeOH containing deuterated internal standards (5 μg of 13C2-citrate, 5 μg of 13C2-fumarate, and 10 μg of 13C6-glucose) and homogenized. One volume of ice-cold water was added, followed by the addition of two volumes of chloroform. Samples were then centrifuged for 10 min at 4000g. Aqueous phase was collected and brought to dryness under a gentle nitrogen stream using TurboVap LV before suspension in H2O for LC-MS/MS profiling.
Extracted samples were analyzed using an LC-MS/MS system. Here, a QTrap 6500+ (Sciex) was equipped with a Shimadzu SIL-20 AC autoinjector and LC-20 AD binary pump (Shimadzu Corp.). Separation was conducted with a method adapted from (40 ). Briefly, a Synergi Hydro-RP column (250 mm by 4.6 mm by 4 μm, Phenomenex), maintained at 30°C, was used with a gradient of methanol/water/acetic acid of 0:100:0.5 (v/v/v) that was ramped to 100:0:0.5 (v/v/v) over 16 min. The flow rate was maintained at 0.5 ml/min. To monitor and quantify the levels of analytes, a multiple reaction monitoring method was used as in (41 (link)). Table S3 reports the transitions used for each of the molecules. Calibration curves were obtained using a mixture of standards for each of the molecules of interest with an r2 of 0.98.
Extracted samples were analyzed using an LC-MS/MS system. Here, a QTrap 6500+ (Sciex) was equipped with a Shimadzu SIL-20 AC autoinjector and LC-20 AD binary pump (Shimadzu Corp.). Separation was conducted with a method adapted from (40 ). Briefly, a Synergi Hydro-RP column (250 mm by 4.6 mm by 4 μm, Phenomenex), maintained at 30°C, was used with a gradient of methanol/water/acetic acid of 0:100:0.5 (v/v/v) that was ramped to 100:0:0.5 (v/v/v) over 16 min. The flow rate was maintained at 0.5 ml/min. To monitor and quantify the levels of analytes, a multiple reaction monitoring method was used as in (41 (link)). Table S3 reports the transitions used for each of the molecules. Calibration curves were obtained using a mixture of standards for each of the molecules of interest with an r2 of 0.98.
8
HPLC Quantification of Tulearine
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TUL was measured using an HPLC method. A Shimadzu LC-20AT system equipped with an SIL-20AC auto-injector and a CTO-20 A column oven (Shimadzu Corp.) was used to measure the TUL content. TUL was dissolved in methanol containing internal standard (1 μg/mL methyl p-oxybenzoate), and 10 μL was injected into the HPLC. The column used was an Inertsil® ODS-3 column (3 μm, column size: 2.1 mm × 50 mm; GL Science Co., Inc., Tokyo, Japan), and 0.02 M potassium dihydrogen phosphate:acetonitrile (87:13, v/v) was utilized as the mobile phase at 0.25 mL/min and 35 °C. TUL was detected at 211 nm.
9
Lipidomic Analysis of Spinal Cord Samples
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For LC-MS/MS analysis, 2ml ice-cold methanol was added to each spinal cord and the samples were profiled using metabololipidomics as described previously (Colas et al., 2014 (link)). In short, for lipid mediator profiling deuterated internal standards d8–5S-HETE, d4-LTB4, d5-LXA4, d4-PGE2 and d5-RvD2 (500 pg each) representing each chromatographic region of identified lipid mediators were added to samples to perform quantification and assessment of sample recovery. Proteins were allowed to precipitate at ‒20°C overnight prior to centrifugation (1200 g at 4°C for 10 min) and then supernatants were taken to solid-phase extraction on C18 columns. Products were eluted using methyl formate, brought to dryness under nitrogen and suspended in methanol-water (50:50) for lipid mediators (Colas et al., 2014 (link)). The system consisted of a Qtrap 6500 (AB Sciex, Framingham, MA, USA) equipped with a Shimadzu SIL-20AC autoinjector, LC-20AD binary pump (Shimadzu, Kyoto, Japan), and an Agilent Eclipse Plus C18 column (100 mm x 4.6 mm x 1,8 μm). To identify and quantify lipid mediators, multiple reaction monitoring (MRM) was used with signature ion pairs Q1 (parent ion) to Q3 (characteristic daughter ion) for each molecule (Table 1 ). Identification was conducted by using published criteria with six diagnostic ions and retention time matching of standards (Colas et al., 2014 (link)).
10
HDL Particle Size Analysis
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To investigate changes in HDL particle size, patient HDL fractions were analyzed on an HPLC system equipped with a LC-20ADVP pump, a DGU-20A degassing unit, a CTO-20A column oven, an SPD-20A ultraviolet detector, and an SIL-20AC autoinjector (Shimadzu Corporation, Kyoto, Japan). Each sample (40 μL) was injected into serially connected size exclusion columns (PROTEIN KW-803 and KW-804; 300 mm × 8.0 mm i.d., Shodex, Tokyo, Japan), eluted with PBS at a flow rate of 1 mL/min, and monitored by absorbance at 280 nm.
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