The measurements for UPLC–MS/MS were performed using an ACQUITY Ultra Performance LC system (Waters, Milford Massachusetts), which was coupled to an API4000 tandem mass spectrometer (AB Sciex, Framingham Massachusetts). The samples were separated on a Waters ACQUITY UPLC® BEH Amide (2.1 mm × 100 mm, 1.7 μm particle size) column. The column temperature was 50 °C, with a mobile phase flow rate of 0.55 mL/min over a total run time of 2.2 min. The mobile phases consisted of (A) 0.1% formic acid in water/acetonitrile (v/v, 95/5) and (B) 0.1% formic acid in acetonitrile/water (v/v, 95/5). SAMe and SAH were eluted by the same linear gradient with 78% B at the initial time to 30% B after 1.40 min, and 0% after 1.6 min. After 2 min, it changed from 0% to 78% until the end of the experiment. The retention time was 1.24 min for SAMe, 1.24 min for D3-SAMe and 1.10 min for SAH. The sample injection volume was 2 μL. The test was repeated 3 times for each sample and the average value was calculated.
Api 4000 tandem mass spectrometer
Manufactured by AB Sciex
Sourced in United States, Germany
The API 4000 tandem mass spectrometer is a high-performance analytical instrument designed for various applications in chemical and biological analysis. It utilizes the principles of mass spectrometry to accurately identify and quantify compounds of interest within complex samples. The API 4000 is capable of performing sensitive and selective analyses, providing reliable data for researchers and scientists.
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Lab products found in correlation
Nexera hplc system, by Shimadzu (3 mentions)
Lc 30ad pump, by Shimadzu (3 mentions)
Analyst software, by AB Sciex (3 mentions)
Sil 30ac autosampler, by Shimadzu (2 mentions)
Cto 20ac column oven, by Shimadzu (2 mentions)
Cbm 20a controller, by Shimadzu (2 mentions)
Acquity uplc beh amide, by Waters Corporation (1 mentions)
Acquity ultra performance lc system, by Waters Corporation (1 mentions)
Advia 2400 clinical chemistry system, by Siemens (1 mentions)
Advia centaur xp immunoassay system, by Siemens (1 mentions)
Acquity uplc, by Waters Corporation (1 mentions)
Liquid chromatography system, by Shimadzu (1 mentions)
Analyst software version 1, by AB Sciex (1 mentions)
1200 series hplc system, by Agilent Technologies (1 mentions)
Sil 30ac, by Shimadzu (1 mentions)
Cto 20ac, by Shimadzu (1 mentions)
Cbm 20a, by Shimadzu (1 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
Synergi 4 hydro rp 80a column, by Phenomenex (1 mentions)
10 protocols using api 4000 tandem mass spectrometer
1
UPLC-MS/MS Analysis of SAMe and SAH
2
Quantifying Serum Vitamin and Homocysteine Levels
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Fasting serum levels of vitamins D2 and D3 were measured as 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, respectively, according to a previously described liquid chromatography-tandem mass spectrometry (LC-MS/MS) method21 (link),22 (link) using deuterated 25-hydroxyvitamin D2 and D3 as internal standards in an API 4000 Tandem Mass Spectrometer (AB Sciex) equipped with a Shimadzu series liquid chromatograph. The fasting level of homocysteine was also measured using an LC-MS/MS method, as described previously.23 The Analyst, version 1.5, software was used for data collection and quantitative analysis. The hepatic and renal serum profiles were recorded using an Advia 2400 Clinical Chemistry System (Siemens). The serum levels of folic acid and vitamin B12 were measured using an Advia Centaur XP Immunoassay System (Siemens). The accuracy of quality control for low, medium, and high concentrations was between 85% and 115%, and precision was within 15%. Our analysis of the levels of biochemical markers was based on the following reference ranges for healthy subjects ≥65 years of age, which had been established at Shanghai Xuhui District Central Hospital prior to the study period: Creatinine, 40 to 120 μmol/L; folic acid, >5.38 ng/mL; vitamin B12, 0.211 to 0.911 ng/mL; homocysteine, 5.0 to 15 μmol/L; vitamin D2, 2.42 to 22.4 ng/mL; vitamin D3, 10 to 55 ng/mL.
3
Quantitative Analysis of Illicit Drugs
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Intracellular accumulation was measured by HPLC-MS/MS using a Shimadzu Nexera HPLC system with a LC-30AD pump, a SIL-30AC autosampler, a CTO-20AC column oven, and a CBM-20A controller (Shimadzu, Kyoto, Japan). Separation was done on a Brownlee SPP RP-Amide column (4.6 × 100 mm inner dimension with 2.7 μm particle size) with a C18 pre-column. The aqueous mobile phase contained 0.1% (v/v) formic acid and either 3% (v/v) organic additive (acetonitrile:methanol 6:1 (v/v)) for methylecgonine, 8% for amphetamine, methylamphetamine, cathinone, cathine, (-)-ephedrine, mescaline, MDAI, and DMT, or 20% for PMA, PMMA, DOI, phentermine, MDMA, MDEA, MBDB, cocaine, and DET. Chromatography was done at a flow rate of 0.3 ml/min. For detection, an API 4000 tandem mass spectrometer (AB SCIEX, Darmstadt, Germany) was used in MRM mode. The analytes, corresponding internal standards, and detection parameters are listed in the Supplementary Table S1 . Peak integration and quantification of the analytes was done using the Analyst software (Version 1.6.2, AB SCIEX, Darmstadt, Germany) and determined by simultaneous measurement of standard curves with known concentrations.
4
Quantitative Analysis of SAM Levels
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SAM standards (Sigma-Aldrich) were utilized to create the standard curves. Standard solutions with concentrations of 20, 50, 100, 200, 500, 1000, and 2000 ng/ml SAM were contained in each batch of specimens. The UPLC-LC/mass spectrometry measurements were carried out utilizing a system of ACQUITY UPLC (Waters, Milford), which was conjugated with an API4000 tandem mass spectrometer (AB Sciex). The specimens were divided by a Waters ACQUITY UPLC1® BEH Amide (2.1 mm × 100 mm, 1.7-mm particle size) column. The temperature of the column was 40 °C, and the injection volume was 5 μl. The retention time was 1.64 ± 0.5 min. The experiment was replicated 3 times for each specimen, and the mean value was computed.
5
Liquid Chromatography-Tandem Mass Spectrometry Protocol
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Samples were analyzed using liquid chromatography-tandem mass spectroscopy (MS/MS). A liquid chromatography system (Shimadzu Corporation, Kyoto, Japan) was used to flow the sample into the MS/MS system. A mixture of acetonitrile and water (70:30, v/v) was used as the carrier solution at a flow rate of 0.14 ml/min. In total, 18 µl of the sample was added to the carrier solution; subsequently, the liquid was delivered to the ion source of the MS/MS system without column separation. The MS/MS analysis was conducted with an API 4000+ tandem mass spectrometer (AB Sciex LLC, Framingham, MA, USA) in positive ionization mode and electrospray ionization was performed with nitrogen gas at 400°C and the nebulizer pressure was 30 psi. In total, two alternating scan modes were defined in the MS setup. Neutral loss scan of m/z 102 was used for neutral amino acids (25V, 13–17 eV). All data acquisition and processing were performed with Analyst software (version 1.5.2; AB Sciex LLC).
6
Quantifying Tamoxifen and Metabolites
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Concentrations of tamoxifen, 4-OH tamoxifen, NDM, and endoxifen were measured in a total of 550 plasma specimens. Plasma concentrations of tamoxifen and its metabolites were measured by high-performance liquid chromatography-tandem mass spectrometry [42 (link), 43 (link)]. Analyses were performed on an API 4000 tandem mass spectrometer (AB Sciex, Foster City, CA, USA) equipped with an Agilent Technologies Series 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA). The column used was a Poroshell 120 EC-C18 (2.7 μm, 3 mm × 50 mm). The mobile phases A and B were water with 2 mM ammonium acetate and acetonitrile, respectively, both containing 0.1% formic acid. For simple protein precipitation, the plasma samples were mixed with acetonitrile containing an internal standard (IS, diphenhydramine) and centrifuged for 5 min. Quantitative analysis was performed in multiple reaction-monitoring mode (m/z 372.2 → 72.2 for tamoxifen, 388.2 → 72.3 for 4-OH tamoxifen, 358.2 → 58.2 for NDM, 374.3 → 58.1 for endoxifen, and 256.2 → 167.0 for IS) with a total running time of 5 min for each sample. Intra- and inter-day coefficients of variation were lower than 10%.
7
Quantification of Cellular Compound Accumulation
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The cellular accumulation of compounds for OCT1 uptake analyses were quantified by HPLC-MS/MS using a Shimadzu Nexera HPLC system (autosampler SIL-30AC, column oven CTO-20AC, pump LC-30AD pump, controller CBM-20A, all from Shimadzu, Kyoto, Japan). Separation was achieved on a Brownlee SPP RP-Amide column (4.6 × 100 mm inner dimension with 2.7 µm particle size) with a C18 pre-column. Reversed-phase chromatography was achieved with an aqueous mobile phase containing 0.1% (v/v) formic acid and organic additive (acetonitrile:methanol (6:1)) ranging von 3 to 50% (v/v) with a flow rate of 0.3 or 0.4 mL/min and a column temperature of 40 °C. Investigated compounds were detection with an API 4000 tandem mass spectrometer (AB SCIEX, Darmstadt, Germany) operating in MRM mode. Analyte peaks were integrated and quantified using the Analyst software (Version 1.6.2, AB SCIEX, Darmstadt, Germany).
8
Sensitive SCFA Quantification in Cecum
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SCFAs in the cecum and in vitro fermentation experiments were performed by LC-MS/MS. For SCFAs extraction (acetate, propionate, n-butyrate, and n-valerate) from samples, ethanol:water (3:7, v/v) was added at room temperature. After extraction, internal standard solutions, 2-nitrophenyl hydrazine, and condensation reagent were added to the tube for derivatization. Test tubes were placed in ice water for 60 min for the labeling reaction. Alkaline solution was added to stop the reaction and tubes were left in ice water for 30 min. After the reaction stopped, an acidic solution and hexane were added for liquid-liquid extraction. The hexane layer was removed, and then ether was added for liquid-liquid extraction. The ether layer was transferred to another test tube and dried under a nitrogen stream. Ammonium formate/methanol solution was added to re-fuse the residues in the tubes and an aliquot was injected into the LC-MS/MS system. Liquid chromatography was performed using an ACQUITY UPLC system (Waters, Milford, MA, USA), separated using an analytical column (AQUITY HSS T3 2.1 × 150 mm, 1.8 μm; Waters). For the detector, an API4000 tandem mass spectrometer (AB Sciex, Foster City, CA, USA) was used.
9
Quantifying Intracellular Drug Accumulation
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Intracellular drug accumulation was determined from cell lysates of the MTT assay using mass spectrometry. After MTT measurement, cell lysates were stored at −20 °C for 1–4 days prior to detection. The stability of HCQ and AZM under these conditions was confirmed for up to 7 days at −20 °C. Total of 25 µL of fresh or thawed cell lysates were diluted with 225 µL of 2 mM ammonium acetate buffer, vortexed and centrifuged for 10 min (14,000 rpm). About 10 µL of the clear supernatants were injected into the LC-MS/MS, which consists of an UltiMate3000 pump, an autosampler (Dionex, ThermoScientific, St. Louis, MO, USA) and an API 4000 Tandem mass spectrometer (ABSciex, Darmstadt, Germany) using positive electrospray ionization (ESI+; 4500 V). HCQ and AZM were determined by a Synergi 4µ HydroRP 80A column 150 mm × 3.0 mm (Phenomenex, Torrance, CA, USA) using a binary gradient with 2 mM ammonium acetate buffer and acetonitrile and a flow rate of 0.5 mL/min. The resulting retention times were 3.0 min for HCQ and 3.2 min for AZM. HCQ and AZM were measured using the multiple reaction monitoring mode with nitrogen as collision gas. The method was suitable for the quantification of HCQ and AZM in cell lysates over the range from 20 to 1000 ng/mL. Samples with higher concentrations were diluted.
10
Intracellular Drug Quantification by HPLC-MS/MS
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Intracellular drug concentrations were quantified by HPLC-MS/MS analysis using a Shimadzu Nexera HPLC system with a SIL-30AC autosampler, a CTO-20AC column oven, an LC-30AD pump, and a CBM-20A controller (Shimadzu, Kyoto, Japan). Compounds were separated on a Brownlee SPP RP-Amide column (4.6 × 100 mm inner dimension with 2.7 µm particle size) with a C18 pre-column. Reverse-phase chromatography was carried out with an aqueous mobile phase containing 0.1% (v/v) formic acid and either 3, 8, or 20% (v/v) organic additive (acetonitrile:methanol (6:1)) with a flow rate of 0.3 mL min−1 and an oven temperature of 40 °C. Detection was carried out with an API 4000 tandem mass spectrometer (AB SCIEX, Darmstadt, Germany) operating in MRM mode. Analyte peaks were integrated and quantified using the Analyst software (Version 1.6.2, AB SCIEX, Darmstadt, Germany). All analytes, corresponding internal standards, HPLC mobile phase composition, and MS detection parameters are listed in Supplementary Table S5 .
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