Unlabeled metformin (metformin-d0), unlabeled creatinine (d0-creatinine), trideutero-creatinine, i.e., [methylo-2H3]creatinine (d3-creatinine; declared isotopic purity of >99 atom% 2H), and [guanidino-2H6]dimethylmetformin (declared isotopic purity of >99 atom% 2H) were purchased from Sigma-Aldrich (Steinheim, Germany). Pentafluoropropionic anhydride (PFPA) was from Thermo Scientific (Dreieich, Germany). Daily prepared solutions of PFPA in ethyl acetate (1:4, v/v) were used for the derivatization of metformin. Stock solutions were prepared in deionized water and stored in a refrigerator at 8 °C. Glassware for GC–MS (i.e., 1.5 mL autosampler glass vials and 0.2 mL microvials) and a fused-silica capillary column Optima 17 (15 m × 0.25 mm I.D., 0.25 µm film thickness) were purchased from Macherey-Nagel (Düren, Germany).
Optima 17
Manufactured by Macherey-Nagel
Sourced in Germany
The Optima 17 is a centrifuge designed for general laboratory use. It provides reliable and efficient separation of samples. The core function of the Optima 17 is to facilitate sample preparation and processing through centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Ethyl acetate, by Merck Group (4 mentions)
Trace 1210 series gas chromatograph, by Thermo Fisher Scientific (2 mentions)
As1310 autosampler, by Thermo Fisher Scientific (2 mentions)
Acetone, by Merck Group (2 mentions)
Pentafluoropropionic anhydride, by Merck Group (2 mentions)
Pentafluoropropionic anhydride pfpa, by Thermo Fisher Scientific (1 mentions)
Trace 1310, by Thermo Fisher Scientific (1 mentions)
As 1310, by Thermo Fisher Scientific (1 mentions)
Sodium nitrite, by Merck Group (1 mentions)
6 pentafluorobenzyl bromide pfb br, by Merck Group (1 mentions)
Sodium nitrite 15n, by Merck Group (1 mentions)
Sodium 15n nitrate, by Merck Group (1 mentions)
Glacial acetic acid, by Merck Group (1 mentions)
Bstfa, by Macherey-Nagel (1 mentions)
Methylglyoxal, by Merck Group (1 mentions)
Glyoxal, by Merck Group (1 mentions)
Specord 50, by Analytik Jena (1 mentions)
2 3 4 5 6 pentafluorobenzyl bromide pfbbr, by Merck Group (1 mentions)
Toluene, by Merck Group (1 mentions)
Boric acid, by Merck Group (1 mentions)
13 protocols using optima 17
1
Isotopic Tracer Quantification of Metformin and Creatinine
2
Quantitative Analysis of Derivatized Nitrite and Nitrate
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Derivatized unlabeled and labeled nitrite and nitrate species were measured by GC-MS on an Agilent system model 5980 based on the quadrupole technology. An Optima 17 (15 m × 0.25 mm i.d., 0.25 µm film thickness) from Macherey-Nagel was used. Helium (70 kPa) and methane (200 Pa) were used as carrier and reactand gas, respectively. Aliquots (1 µL) of toluene extracts were injected in the splitless mode. Oven temperature was held at 70 °C for 1 min and then increased to 280 °C at a rate of 30 °C/min. Constant temperatures were kept at the ion source (180 °C), interface (280 °C), and injector (200 °C). Negative-ion chemical ionization (NICI) was used at an electron energy of 230 eV and an emission current of 300 µA (Scheme 2 ). Nitrite and nitrate species were analyzed in the selected-ion monitoring (SIM) mode using a dwell time of 50 ms for each ion (Table 1 ). The sum of peak area values of all ions monitored was set to 100%. Peak area values of selected ions were used to calculate their peak area ratio (PAR).
3
GC-MS Analysis of Toluene Extracts
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One-µL aliquots of the extracts (toluene) were injected splitless into the GC–MS apparatus, which consisted of a single quadrupole mass spectrometer model ISQ, a Trace 1210 series gas chromatograph, and an AS1310 autosampler from ThermoFisher (Dreieich, Germany). A fused-silica capillary column Optima 17 (15 m length, 0.25 mm I.D., 0.25 µm film thickness) from Macherey–Nagel (Düren, Germany) was used. For amino acids, the injector temperature was kept at 280 °C. Helium was used as the carrier gas at a constant flow rate of 1.0 mL/min. Interface and ion-source temperatures were set to 300 and 250 °C, respectively. Interface and ion-source were set to 260 and 250 °C, respectively. Electron energy was 70 eV and electron current 50 µA. Methane was used as the reactand (buffer) gas at a constant flow rate of 2.4 mL/min for negative-ion chemical ionization (NICI). Oven temperature programs were used as described elsewhere (Hanff et al. 2019 (link)). The starting oven temperature for metformin was held at 90 °C for 0.5 min and ramped to 210 °C at a rate of 15 °C/min and then to 320 °C at a rate of 35 °C/min. In quantitative analyses, the dwell time was 100 ms for each ion in the selected-ion monitoring (SIM) mode and the electron multiplier voltage was set to 1900 V.
4
GC-MS Quantification of Acetazolamide
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GC-MS analyses were performed on a single-quadrupole mass spectrometer model ISQ directly interfaced with a Trace 1310 series gas chromatograph equipped with an autosampler AS 1310 from ThermoFisher (Dreieich, Germany). The gas chromatograph was equipped with a 15 m long fused-silica capillary column Optima 17 (0.25 mm I.D., 0.25 μm film thickness) from Macherey-Nagel (Düren, Germany). In quantitative analyses, the following oven temperature program was used with helium (at a constant flow rate of 1 mL/min) as the carrier gas: 1.0 min at 90 °C, then increased to 250 °C at a rate of 35 °C/min and to 320 °C at a rate of 35 °C/min, respectively. The column was held at 320 °C for 6 min. Interface, injector and ion-source were kept constant at 260 °C, 200 °C and 250 °C, respectively. Electron energy was set to 70 eV and electron current to 50 μA. Methane (2.4 mL/min) was used as the reagent gas for NICI. Aliquots (1 μL from the toluene phase) were injected in the splitless mode by means of the autosampler. Quantification of acetazolamide was performed in the NICI mode by selected-ion monitoring (SIM) the ions with m/z 581 and m/z 83 for d0-AZM and m/z 584 and m/z 86 for d3-AZM (IS) with a dwell-time of 50 ms for each ion. The electron multiplier voltage was set to 2025 V. Deviations from the conditions described above are mentioned in the sections Results and Discussion.
5
Quantitative GC-MS Analysis of AGEs
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All synthetic amino acids and derivatization reagents were of analytical grade and were commercially obtained from various manufacturers in Germany (Sigma-Aldrich, Merck, Cayman, Iris Biotech GmbH, Carbosynth, TCI, Chemsolute). Glassware for GC–MS (1.5-mL auto-sampler vials and 0.2-mL microvials) and the fused-silica capillary column Optima 17 (15 m × 0.25 mm I.D., 0.25-µm film thickness) were purchased from Macherey–Nagel (Düren, Germany). Separate stock solutions of amino acids were prepared by dissolving accurately weighed amounts of commercially available unlabeled and stable-isotope labeled AGEs in deionized water. Stock solutions were diluted with deionized water as appropriate.
6
Quantification of Serum Analytes by GC-MS
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The concentrations of all analytes in study serum samples were measured by stable-isotope dilution gas chromatography–mass spectrometry (GC–MS) using previously developed and validated methods [28 (link),29 (link)]. GC–MS was performed on a single quadrupole mass spectrometer model ISQ coupled with a Trace 1210 series gas chromatograph and an AS 1310 autosampler from ThermoFisher (Dreieich, Germany). A fused-silica capillary column Optima 17 (15 m, 0.25 mm I.D., 0.25 μm film thickness) from Macherey-Nagel (Düren, Germany) was used. Quantification was performed by selected-ion monitoring (SIM) the characteristics of mass–to–charge (m/z) ions of unlabeled and stable-isotope labeled analogs that served as internal standards (Table S1 ). To assess the precision of the GC–MS methods, quality control was performed [30 (link),31 (link)]. The quality control (QC) sample was prepared by mixing equal volumes of all serum samples of the study. In total, 12 QC samples for amino acids and 7 QC samples for nitrate, nitrite and MDA were analyzed concomitantly with the study serum samples. The procedures for sample preparation, derivatization and GC–MS analyses and the results of the QC are reported in the Supplementary Materials .
7
Stable Isotope Tracer Analysis
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2,3,4,5,6-Pentafluorobenzyl bromide (PFB-Br), sodium nitrite (purity 99.99+%), sodium [15N]nitrite and sodium [15N]nitrate (declared as 99 atom% at 15N each) were obtained from Sigma-Aldrich (Steinheim, Germany). Toluene was purchased from Baker (Deventer, The Netherlands). Sodium bicarbonate and carbonate, acetone and glacial acetic acid were from Merck (Darmstadt, Germany). 2H-Labelled creatinine ([methylo-2H3]creatinine, >99 atom% 2H) was obtained from Aldrich. PFB-Br is corrosive and an eye irritant. Inhalation and contact with skin and eyes should be avoided. All work should be and was performed in a well-ventilated fume hood. Separate stock solutions of salts were prepared by dissolving accurately weighed amounts of commercially available unlabeled and stable-isotope-labeled salts in deionized water. Stock solutions were diluted with deionized water as appropriate.
Glassware for GC-MS (1.5-mL autosampler vials and 0.2-mL microvials) including the fused-silica capillary column Optima 17 (15 m × 0.25 mm I.D., 0.25-micrometer film thickness) were purchased from Macherey-Nagel (Düren, Germany).
Glassware for GC-MS (1.5-mL autosampler vials and 0.2-mL microvials) including the fused-silica capillary column Optima 17 (15 m × 0.25 mm I.D., 0.25-micrometer film thickness) were purchased from Macherey-Nagel (Düren, Germany).
8
Hypusine Quantification by GC-MS
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The (2S)-Hypusine dihydrochloride (declared chemical purity, ≥ 95% by HPLC), tetradeutero-methanol (CD3OD; declared isotopic purity, ≥ 99.8% at 2H) and penta-fluoro-propionic anhydride (PFPA) were purchased from Sigma-Aldrich (Steinheim, Germany). Methanol (CH3OH) was obtained from Chemsolute (Renningen, Germany). Hydrochloric acid (37 wt%) was purchased from Baker (Deventer, The Netherlands). Ethyl acetate (EA) was obtained from Merck (Darmstadt, Germany). Glassware for GC–MS (1.5-mL auto-sampler vials and 0.2-mL micro-vials) and the fused-silica capillary column Optima 17 (15 m × 0.25 mm I.D., 0.25-µm film thickness) were purchased from Macherey–Nagel (Düren, Germany). Stock solutions of Hyp were prepared by dissolving the commercial preparation in its original glass flask in deionized water. The stock solution of Hyp was diluted with deionized water as appropriate.
9
Quantification of Creatine and Creatinine
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Unlabeled creatine (d0-creatine), unlabeled creatine phosphate 4×H2O, unlabeled creatinine (d0-creatinine) and trideuterocreatinine, i.e., [methylo-2H3]creatinine (d3-creatinine; declared isotopic purity of >99 atom% 2H) were obtained from Aldrich (Steinheim, Germany). Stock solutions of d0-creatine, d0-creatinine and d3-creatinine (each 20 mM) were prepared in deionized water and stored in a refrigerator at 8 °C. BSTFA was obtained from Macherey-Nagel (Düren, Germany). Glassware for GC–MS (i.e., 1.5 mL autosampler glass vials and 0.2 mL microvials) and a fused-silica capillary column Optima 17 (15 m × 0.25 mm I.D., 0.25 µm film thickness) were purchased from Macherey-Nagel.
10
Glyoxal and Methylglyoxal Reaction Protocols
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Glyoxal and methylGlyoxal were obtained from Sigma-Aldrich (Steinheim, Germany) each as 40 wt.% solution in water. L-Lysine, Nε-(1-carboxymethyl)-L-lysine (chemical purity, 95%) and Nε-(1-carboxyethyl)-L-lysine (chemical purity, 95%) were purchased from Cayman (Ann Arbor, Michigan, USA). Tetradeuterated methanol (CD3OD, 99% at 2H) and pentafluoropropionic anhydride (PFPA) were supplied by Sigma-Aldrich (Steinheim, Germany). Methanol was obtained from Chemsolute (Renningen, Germany). Ethyl acetate (EA) was obtained from Merck (Darmstadt, Germany). Hydrochloric acid (37 wt.%) was purchased from Baker (Deventer, The Netherlands) and was used to prepare the esterification reagent (2 M HCl in CH3OH or 2 M HCl in CD3OD). Glassware for GC-MS (1.5-mL autosampler vials and 0.2-mL microvials) and the fused-silica capillary column Optima 17 (15 m × 0.25 mm I.D., 0.25-µm film thickness) were purchased from Macherey-Nagel (Düren, Germany). Spectrophotometric analyses were performed at room temperature on the spectrophotometer model Specord 50 from Analytik Jena (Jena, Germany) using 1-cm cuvettes (UVetten, Eppendorf, Hamburg, Germany). Scans were performed in the range 190–500 nm (1 s per cycle).
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