The 1-MNA was detected by LC-MS/MS with AB SCIEX Triple QuadTM 4500MD mass spectrometry system. For intracellular 1-MNA, cells first were treated with methyl alcohol and the supernatant was collected by centrifugation at 10,000 rpm for 15min. Then, 250μL of 1% zinc sulfate solution with stable isotope labelled internal standard (N-MNA-d4) was added into 50μL cell supernatant or culture medium. After shaken at 400 rpm for 30 min and centrifuged at 14,000 rpm for 20 min, the supernatant sample was transferred to glass vials for LC-MS/MS. Liquid chromatography separation was performed by injecting 5μL sample into the Eclipse XDB-C18 column (4.6x150 mm, 5μm; Agilent) connected to JasperTM (SCIEX) LC system. The MS/MS detection of 1-MNA and N-MNA-d4 (internal standard) was performed in multiple reaction monitoring (MRM) mode. A series of concentration standards (1-MNA) was used for the quantification of 1-MNA in samples. The final intracellular 1-MNA concentration is quantified based on the protein of the cell.
Triple quad 4500 md
Manufactured by AB Sciex
Sourced in United States
The Triple Quad 4500 MD is a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) system designed for clinical diagnostic applications. It provides high-sensitivity detection and quantification of analytes in complex biological matrices.
Automatically generated - may contain errors
Lab products found in correlation
Formic acid, by Thermo Fisher Scientific (3 mentions)
Eclipse xdb c18 column, by Agilent Technologies (1 mentions)
Salicylic acid, by Merck Group (1 mentions)
Bcaa detection kit, by Abcam (1 mentions)
L valine, by Merck Group (1 mentions)
L isoleucine, by Merck Group (1 mentions)
Mini beadbeater, by Biospec (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Synergy uv water purification system, by Merck Group (1 mentions)
Jasper lc system, by AB Sciex (1 mentions)
Hplc grade methanol, by Merck Group (1 mentions)
Ammonium formate, by Merck Group (1 mentions)
Centrifuge 5430 r, by Eppendorf (1 mentions)
Lc 20ad, by AB Sciex (1 mentions)
Beh c18 column, by Waters Corporation (1 mentions)
9 protocols using triple quad 4500 md
1
Quantification of Intracellular 1-MNA by LC-MS/MS
2
Cord Blood Metabolic and Immune Profiling
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Five milliliters of cord blood was collected and stored at −80°C for later use when the newborn's umbilical cord was broken. In this study, SCIEX Triple QuadTM 4500MD liquid chromatography tandem mass spectrometry (AB SCIEX, Framingham, MA, USA) was used to detect levels of 25(OH)D3, and the cobas e 601 electrochemiluminescence automated analyzer as well as an IgE (IgE II) detection kit (Roche, Basel, Switzerland) were used to detect total IgE levels in the cord blood samples.
3
BCAA and BCKA Quantification in Human and Animal Plasma
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The human plasma BCAA and BCKA were measured by Hangzhou Calibra Diagnostics (China) with isotope dilution LC-MS as previously described [20 (link)]. Standard l -leucine, l -isoleucine, l -valine, α-ketoisovaleric acid, α-keto-β-methylvaleric acid, α-ketoisocaproic acid, L-13C1-leucine and salicylic acid were purchased from Sigma-Aldrich. In brief, the plasma samples were pretreated with protein precipitation. LC-MS was performed with an Agilent Zorbax SB-C18 column and Sciex TripleQuad 4500MD. The animal plasma BCAA and cellular BCAA were measured with a BCAA detection kit (Biovision, USA) according to the manufacturer's instructions. The animal plasma BCKA and cellular BCKA were determined by HPLC as previously described [21 (link)].
4
Vitamin D Measurement in Sarcopenia
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Study nurses collected the sarcopenia older participants anthropometric details and blood samples. The fasting blood samples were routinely drawn from participants using venipuncture. Blood samples were protected from light and isolated the serum immediately. Stabilized samples were stored at −80°C. Vitamin D in the serum is in the form of 25(OH)D2 and 25(OH)D3, which were measured using a liquid chromatography–triple quadrupole mass spectrometry (LC-MS/MS, SCIEX Triple Quad 4500MD). The concentration of 25(OH)D was the sum of 25(OH)D2 and 25(OH)D3. According to the manufacturer's notes, the coefficient of variance of intra- and inter assay were both <15%. The linear ranges were 2.0–100 ng/mL for 25(OH)D2 and 4.0–200 ng/mL for 25(OH)D3.
5
Quantification of Staphylococcus aureus Signaling Molecules
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Overnight cultures of S. aureus 1:200 were grown in TSBG at 37°C with or without 50 μM clemastine, and samples were prepared for analysis as described for c-di-GMP with slight modifications (10 (link)). Briefly, the equal weights cell pellets were washed twice with cold molecular grade water (Corning) and lysed by homogenized for 5 rounds using Mini-Bead beater (Biospec, Bartlesville, OK, USA) at 4,800 rpm. The samples were centrifuged at 21,000 × g for 5 min. Supernatants were removed and stored at −80 °C until HPLC analysis. Samples were run similar to c-di-GMP (46 (link)) on an AB SCIEX TRIPLE QUAD 4500MD and peaks were quantified at 260 nm.
6
Comparative Urine and Oral Fluid Toxicology
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Urine toxicology testing was performed by an independent laboratory using liquid chromatography tandem mass spectrometry (Triple Quad 4500 MD, AB Sciex). If a patient was not able to provide a urine sample, oral fluid was collected for analysis. Both urine and oral fluid samples were examined for the presence of prescribed opioids, benzodiazepines, illicit drugs, and their respective metabolites. Chromatographic tests are specific and are not susceptible to cross-reactions; thus, false positive results are rare [22 (link)]. The detection window is substantially shorter for oral fluid testing vs urine testing (eg, morphine is detectable 2-5 days after use in urine vs 1-36 hours in oral fluid [23 ]). When a drug is within the detection windows for both UDT and oral fluid testing, the detection rates are believed to be similar [24 (link)].
7
Quantifying DDX-DNP and DDX-OH Compounds
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An HPLC–MS/MS-system Triple Quad 4500 MD (AB SCIEX, USA) was utilized to identify DDX-DNP and DDX-OH in the reacted solution. The MS parameters were optimized as follows: for DDX-DNP , Q1 Mass 487.1Da, Q3 Mass 440.0 Da, Declustering Potential (DP) 110.0 volts, collision energy (CE) 50.0 volts; and for DDX-OH , Q1 Mass 321.2 Da, Q3 Mass 277.0 Da, DP 110.0 volts, CE 53.0 volts. The mobile phase was composed of A (0.1% v/v formic acid aqueous solution) and B (acetonitrile). Next, the HPLC elution conditions were optimized as follows: 0–1 min: 90% A; 1–2.5 min: 90–0% A; 2.5–4 min: 0% A; 4.0–5.5 min: 0–90% A; and 5.5–6.5 min: 90% A. The flow rate and the column temperature were set to 0.3 mL/min and 40 °C, respectively. Subsequently, the solutions of DDX-DNP and DDX-OH and the reacted solution of DDX-DNP and NaHS were injected, respectively. Finally, all the data were collected and processed using Analyst 1.6.3 software.
8
LC-MS/MS Quantification of Catecholamine Metabolites
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The LC–MS/MS analysis utilized a Jasper LC system (Paolo Alto, USA) and an AB Sciex Triple Quad 4500 MD with electrospray ionization. The column used was the Venusil CAs column, 100 mm × 2.1 mm, 5 μm, from Bonna-Agela Technologies, Tianjin, China. Eppendorf Centrifuge 5430R with low-temperature and high-speed was used. MNs (1 mg/mL in methanol, Lot: C-110), 3-MT (1 mg/mL in methanol, Lot: M − 171), (±)-Metanephrine-d3 hydrochloride (100 μg/mL in methanol, Lot: M − 148), (±)-Normetanephrine-d3 hydrochloride (100 μg/mL in methanol, Lot: N-068), and 3-Methoxytyramine-d4 (100 μg/mL in methanol, Lot: M − 172) were purchased from Cerilliant (Texas, USA). HPLC grade methanol and acetonitrile were from Merk, USA. Formic acid was from Thermo Fisher Scientific (Waltham, MA, USA), and ammonium formate was from Sigma (St. Louis, MO, USA). Deionized water was purified using a Millipore Synergy UV water purification system (Billerica, MA, USA). 24-h Urine samples from apparently healthy volunteers were collected in clean containers containing 5 g of boric acid, and 1 mL samples were stored at −80 °C until analysis.
9
TMAO Quantification by LC-MS/MS
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TMAO plasma and brain levels were measured using liquid chromatography mass spectrometry. Briefly, 60 μL of samples were aliquoted into 1.5 mL Axygen tubes and mixed with 10 μL of a 1ug/mL internal standard consisting of d9-TMAO in methanol. Proteins in the samples were precipitated and supernatants (5 μL) were analyzed by injection into a Waters BEH C18 column (2.1 × 50 mm, 1.7 μm; Cat. No. 03433919615148, Massachusetts, USA) at a flow rate of 0.4 mL/min using an LC‐20AD Shimadzu pump system and SIL‐20AXR autosampler interfaced with a Triple Quad 4500MD mass spectrometry (AB SCIEX, Framingham, MA). By mixing solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in methanol) in different ratios, starting at 10% B, a discontinuous gradient was generated to separate the analytes and then linearly increased to 80% B over 1.0 min, then hold for 1.8 min, then return to 10% B. Quantification of TMAO was performed using multiple reaction monitoring (MRM) transitions at m/z 76.1→59, d9‐TMAO at m/z 85.1→68.1.
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