Api 4000 mass spectrometer

Manufactured by Thermo Fisher Scientific

Sourced in United States, Canada, Japan

The API-4000 is a triple quadrupole mass spectrometer designed for precise and sensitive quantitative analysis. It features a high-performance ion source, efficient ion optics, and a triple quadrupole mass analyzer. The API-4000 is capable of performing selective and sensitive detection of target analytes in complex matrices.

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Lab products found in correlation

Transwell type filters, by Corning (6 mentions) Keratanase 2, by Seikagaku (3 mentions) Analyst 1, by AB Sciex (2 mentions) Hypercarb column, by Thermo Fisher Scientific (2 mentions) Hp1100 system, by Agilent Technologies (2 mentions) δdi 4s, by Seikagaku (2 mentions) δdi 6s, by Seikagaku (2 mentions) δdihs ns, by Seikagaku (2 mentions) Chondroitinase b, by Seikagaku (2 mentions) Heparitinase, by Seikagaku (2 mentions) Analyst 1, by Thermo Fisher Scientific (1 mentions) Methanol, by Merck Group (1 mentions) Venusil mp c18 column, by Agela Technologies (1 mentions) High performance liquid chromatography system, by Shiseido (1 mentions) Turboionspray esi interface, by Thermo Fisher Scientific (1 mentions) 1100 binary pump, by Agilent Technologies (1 mentions) δdihs 0s, by Seikagaku (1 mentions) Ultrafast liquid chromatography system, by Shimadzu (1 mentions) Kinetex c18 column, by Phenomenex (1 mentions)

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26 protocols using api 4000 mass spectrometer

Quantification of Urinary and Plasma GAGs

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Concentrations of urinary and plasma GAGs and KS were measured by tandem mass spectroscopy as described elsewhere (16 (link)). Briefly, the chromatographic system consists of an HP1100 system (Agilent Technologies) and a Hypercarb column (Thermo Electron). The API-4000 mass spectrometer (Applied Biosystems) was equipped with a turbo ionspray ion source. Disaccharides of keratan, heparan, dermatan sulfate were used as standards [Galß1,4GlcNAc(6S), ΔDiHS-0S, ΔDiHS-NS, ΔDi-4S, ΔDi-6S (Seikagaku). Chondrosine was used as internal standard (Glycosyn). Urine or plasma was centrifuged, and the supernatants were digested overnight with 1 mU of chondroitinase b, 1mU heparitinase, and 1mU keratanase II (Seikagaku). Recovered samples were analyzed by the LC-MS/MS system and normalized by creatinine concentration in urine. Total GAGs refer to total glycosaminoglycans or the additive sum of all the GAGs measured (HS, DS and KS). Disaccharide GAG concentrations were calculated by Analyst 1.5.1 software (AB SCIEX). Each sample was measured in triplicate with three injections for each sample.

Montavon B., Winter L.E., Gan Q., Arasteh A, & Montaño A.M. (2022). Mucopolysaccharidosis Type IVA: Extracellular Matrix Biomarkers in Cardiovascular Disease. Frontiers in Cardiovascular Medicine, 9, 829111.

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MDCK-mdr1 Cell Transwell Assay for Drug Transport

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MDCK-mdr1 cells obtained from the Netherlands Cancer Institute were grown on Transwell type filters (Corning) for 4 days to confluence in DMEM/F12 media containing 10% fetal bovine serum and antibiotics. Compounds were added to the apical side at a concentration of 10 μM in a transport buffer comprising of 1X Hank’s balanced salt solution, 25 mM D-glucose and buffered with HEPES to pH 7.4. Samples were incubated for 1 h at 37 °C and carefully collected from both the apical and basal side of the filters. Compounds selected for MDCK-mdr1 cell assays were infused on an Applied Biosystems API-4000 mass spectrometer to optimize for analysis using multiple reaction monitoring (MRM), as previously described.^{33 (link)} The chromatography was conducted with an Agilent 1100 binary pump with a flow rate of 0.5 mL/min. Mobile phase solvents were 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). The solvent conditions were 10% B for 1 min, followed by a gradient to 95% B over 5 min. Data reported are average values from 2–3 measurements.

George A., Robert W., Amruta M., Vineetha V., Rodney S., Scott R, & Rangan M. (2019). Functionalized 6-(Piperidin-1-yl)-8,9-Diphenyl Purines as Inverse Agonists of the CB1 Receptor – SAR Efforts Towards Selectivity and Peripheralization. Bioorganic & medicinal chemistry, 27(16), 3632-3649.

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MDCK-mdr1 Cell Transport Assay Protocol

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MDCK-mdr1 cells obtained from The Netherlands Cancer Institute were grown on Transwell type filters (Corning) for 4 d to confluence in DMEM/F12 media containing 10% fetal bovine serum and antibiotics as has been described previously.^{36 (link),37 (link)} Compounds were added to the apical side at a concentration of 10 μM in a transport buffer comprising 1× Hank’s balanced salt solution, 25 mM d-glucose, and buffered with HEPES to pH 7.4. Samples were incubated for 1 h at 37 °C and carefully collected from both the apical and basal side of the filters. Compounds selected for MDCK-mdr1 cell assays were infused on an Applied Biosystems API-4000 mass spectrometer to optimize for analysis using multiple reaction monitoring (MRM). Flow injection analysis was also conducted to optimize for mass spectrometer parameters. Samples from the apical and basolateral side of the MDCK cell assay were dried under nitrogen on a Turbovap LV. The chromatography was conducted with an Agilent 1100 binary pump with a flow rate of 0.5 mL/min. Mobile phase solvents were A, 0.1% formic acid in water, and B, 0.1% formic acid in methanol. The initial solvent conditions were 10% B for 1 min, then a gradient was used by increasing to 95% B over 5 min, then returning to initial conditions. Data reported are average values from 2 to 3 measurements.

Jin C., Decker A.M., Makhijani V.H., Besheer J., Darcq E., Kieffer B.L, & Maitra R. (2018). Discovery of a Potent, Selective, and Brain-Penetrant Small Molecule that Activates the Orphan Receptor GPR88 and Reduces Alcohol Intake. Journal of medicinal chemistry, 61(15), 6748-6758.

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Transwell Transport Assay for MDCK-mdr1 Cells

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MDCK-mdr1 cells obtained from The Netherlands Cancer Institute were grown on Transwell type filters (Corning) for 4 d to confluence in DMEM/F12 media containing 10% fetal bovine serum and antibiotics. Compounds were added to the apical side at a concentration of 10 μM in a transport buffer comprised of 1× Hank’s balanced salt solution, 25 mM d-glucose and buffered with HEPES to pH 7.4. Samples were incubated for 1 h at 37 °C and carefully collected from both the apical and basal sides of the filters. Compounds selected for MDCK-mdr1 cell assays were infused on an Applied Biosystems API-4000 mass spectrometer to optimize for analysis using multiple reaction monitoring (MRM), as previously described.^{39 (link)} The chromatography was conducted with an Agilent 1100 binary pump with a flow rate of 0.5 mL/min. The mobile phase solvents were 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). The solvent conditions were 10% B for 1 min, followed by a gradient to 95% B over 5 min. Data reported are average values from two to three measurements.

Amato G., Manke A., Wiethe R., Vasukuttan V., Snyder R., Yueh Y.L., Decker A., Runyon S, & Maitra R. (2019). Functionalized 6-(Piperidin-1-yl)-8,9-Diphenyl Purines as Peripherally Restricted Inverse Agonists of the CB1 Receptor. Journal of medicinal chemistry, 62(13), 6330-6345.

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Montelukast Quantification by LC-MS/MS

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An Applied Biosystems API4000 mass spectrometer was used with turbo ion spray and positive ionization. Mass ratios were 586.4/422.1 amu for montelukast, 592.3/427.1 amu for the motelukast standard, 602.4/438.0 amu for montelukast 1,2‐diol, and for hydroxybupropion, and 592.3/427.1 amu for the montelukast 1,2‐diol standard.

Dennison J., Puri A., Warrington S., Endo T., Adeloye T, & Johnston A. (2018). Amenamevir: Studies of Potential CYP2C8‐ and CYP2B6‐Mediated Pharmacokinetic Interactions With Montelukast and Bupropion in Healthy Volunteers. Clinical Pharmacology in Drug Development, 7(8), 860-870.

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MDCK-mdr1 Transwell Transport Assay

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MDCK-mdr1 cells obtained from The Netherlands Cancer Institute were grown on Transwell-type filters (Corning) for 4 days to confluence in DMEM/F12 media containing 10% fetal bovine serum and antibiotics. Compounds were added to the apical sides at concentrations of 10 μM in a transport buffer comprising 1× Hank’s balanced salt solution with 25 mM d-glucose and buffered with HEPES to pH 7.4. The samples were incubated for 1 h at 37 °C and carefully collected from both the apical and basal sides of the filters. The compounds selected for the MDCK-mdr1 cell assays were infused on an Applied Biosystems API-4000 mass spectrometer in order to optimize them for analysis using multiple-reaction monitoring (MRM), as previously described.^{27 (link)} Chromatography was conducted with an Agilent 1100 binary pump with a flow rate of 0.5 mL/min. The mobile-phase solvents were 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). The solvent conditions were 10% B for 1 min, followed by a gradient to 95% B over 5 min. The data reported are average values from two to three measurements.

Amato G.S., Manke A., Harris D.L., Wiethe R.W., Vasukuttan V., Snyder R.W., Lefever T.W., Cortes R., Zhang Y., Wang S., Runyon S.P, & Maitra R. (2018). Blocking Alcoholic Steatosis in Mice with a Peripherally Restricted Purine Antagonist of the Type 1 Cannabinoid Receptor. Journal of medicinal chemistry, 61(10), 4370-4385.

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Characterization of MβCD Molecules

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To identify the average molecular weight and levels of methylation in side chains of MβCD molecules, we carried out the MS analysis. Initially, 0.4 mg of MβCD powder was dissolved in 3 mL of deionized Millipore (Sigma-Aldrich, St. Louis, MO) water as the stock solution (100 μM). An aliquot of the MβCD stock solution was further diluted with 90% methanol to 10 μg/mL (8 μM) for MS analysis. The diluted MβCD solution was directly infused into the API-4000 Mass Spectrometer (Applied Biosystems, Forster City, CA) at a rate of 10 μL/min. The mass spectrometer was used for Q1 scan with positive ion electrospray mode at 5000V and 100V declustering potential. The MS scan range was adjusted from m/z 1,200 to 1,450 at a scan rate of 2 s. The data analysis was performed by Analyst 1.5.1 software (Applied Biosystems, Foster City, CA).

Li R., Hao J., Fujiwara H., Xu M., Yang S., Dai S., Long Y., Swaroop M., Li C., Vu M., Marugan J.J., Ory D.S, & Zheng W. (2017). Analytical Characterization of Methyl-β-Cyclodextrin for Pharmacological Activity to Reduce Lysosomal Cholesterol Accumulation in Niemann-Pick Disease Type C1 Cells. Assay and Drug Development Technologies, 15(4), 154-166.

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Serum 25(OH)D3 Quantification by LC-MS/MS

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Serum samples were stored at −70°C. Reagents, including methanol, methyl cyanides, n-hexane and anhydrous ethanol (Merck KGaA, Darmstadt, Germany), standard substance (SRM972, Sigma-Aldrich, St. Louis, MO, USA), internal standard product (Advanced Medical Isotopes Corporation, Kennewick, WA, USA) and quality control (RECIPE, Munich, Germany). Serum 25(OH)D₃ levels were measured by liquid chromatography-tandem mass spectrometry on an API 4000 mass spectrometer (Applied Biosystems Life Technologies, Foster City, CA, USA) in the Jinyu Medical Test Center (Guangzhou, China).

ZHAN Y, & JIANG L. (2014). Status of vitamin D, antimicrobial peptide cathelicidin and T helper-associated cytokines in patients with diabetes mellitus and pulmonary tuberculosis. Experimental and Therapeutic Medicine, 9(1), 11-16.

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In Vitro PK Assay for LDT5

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This assay was performed as part of the routine in vitro PK assays,
even knowing that LDT5 (a monohydrochloride salt) was highly water soluble. The study
was performed in a 96-deep well plate by spiking 10 µL of working stock solutions to
990 µL of 50 mM sodium phosphate buffer, pH 7.4. After 2 h, the plate was centrifuged
at 1,000 g for 20 min at room temperature and aliquots were
withdrawn from the supernatant and diluted 1:1 with acetonitrile for analysis by a
validated LC-MS/MS detection method using labetalol as an internal standard, a BDS
Hypersil Phenyl (150*4.6, 5 µm) column and a mobile phase composed of 5 mM ammonium
formate:acetonitrile (40:60, % v/v) with 0.05% formic acid. An API 4000 mass
spectrometer (Applied Biosystems/MDS SCIEX, Canada) was used for detection in a
positive ionization mode and with the following MRM transitions: 357.4→165.2 and
329.2→162 for LDT5 and labetolol, respectively.

Noël F., Nascimento-Viana J.B., Romeiro L.A., Silva R.O., Lemes L.F., Oliveira A.S., Giorno T.B., Fernandes P.D, & Silva C.L. (2016). ADME studies and preliminary safety pharmacology of LDT5, a lead compound for the treatment of benign prostatic hyperplasia. Brazilian Journal of Medical and Biological Research, 49(12), e5542.

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Measurement of PFAS in Serum Samples

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We measured eight PFAS using a modification of a published method (Kuklenyik et al. 2005 (link)) [current acronyms (Buck et al. 2011 (link)) followed by previously used acronyms as applies in parentheses: perfluorooctane sulfonamide (FOSA/PFOSA), 2-(N-ethylperfluorooctane sulfonamido) acetate (Et-FOSAA/Et-PFOSA-AcOH), 2-(N-methyl-perfluorooctane sulfonamido) acetate (Me-FOSAA/Me-PFOSA-AcOH), perfluorohexane sulfonate (PFHxS), perfluorooctane sulfonate (PFOS), PFOA, perfluo-rononanoate (PFNA), and perfluorodecanoate (PFDA/PFDeA)]. Briefly, isotope-labeled internal standards were used for quantification. To compensate for the lack of isotope-labeled internal standard for PFHxS and to account for potential matrix effects, we spiked the calibration standards into calf serum. Analyte concentrations were detected by negative-ion TurboIonSpray–tandem mass spectrometry on an API 4,000 mass spectrometer (Applied Biosystems, Foster City, CA). The limits of detection (LODs) were

0.08 ng / mL

(PFNA),

0.1 ng / mL

(PFOSA, Et-PFOSA-AcOH, PFHxS, PFOA), and

0.2 ng / mL

(PFOS, Me-PFOSA-AcOH, PFDeA). Low-concentration quality control materials (QCs) and high-concentration QCs, prepared from a calf serum pool, were analyzed with the unknown samples and with reagent and serum blanks to ensure the accuracy and reliability of the data (Kuklenyik et al. 2005 (link)).

Lyall K., Yau V.M., Hansen R., Kharrazi M., Yoshida C.K., Calafat A.M., Windham G, & Croen L.A. (2018). Prenatal Maternal Serum Concentrations of Per- and Polyfluoroalkyl Substances in Association with Autism Spectrum Disorder and Intellectual Disability. Environmental Health Perspectives, 126(1), 017001.

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