The Novichok A-series degradation products, O-methyl (1-(diethylamino)-ethylidene)phosphoramidic acid (MOPAA), O-ethyl (1-(diethylamino)ethylidene)phosphoramidic acid (EOPAA), N-(1-(diethylamino)ethylidene)-methylphosphonamidic acid (MPAA), O-methyl (bis(diethylamino)methylidene)phosphoramidic acid (MOPGA), O-ethyl (bis(diethylamino)methylidene)phosphoramidic acid (EOPGA), and MPGA, were synthesized by newly developed methods in our laboratory [18 (link)]. All aqueous solutions were produced using LC–MS-grade water (FUJIFILM Wako Pure Chemical Co., Osaka, Japan). Drug-free urine from five individual donors (three males and two females) was purchased from Lee Biosolutions, Inc. (Maryland Heights, MO) and stored at − 20 °C until use. All other chemicals were used as received. The urine samples were spiked with Novichok agent degradation products and left at room temperature for 1 h or longer before the analyses.
Lc ms grade water
Manufactured by Fujifilm
Sourced in Japan
LC-MS grade water is a high-purity water specifically designed for use in liquid chromatography-mass spectrometry (LC-MS) applications. It is purified to meet the stringent requirements of LC-MS analysis, ensuring minimal interference and optimal performance.
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6 protocols using lc ms grade water
1
Synthesis and Analysis of Novichok Degradation Products
2
Quantification of Stable Isotope-Labeled Oxytocin
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Oxytocin (OT) and arginine vasopressin (AVP) were purchased from the Peptide Institute (Osaka, Japan). Stably labelled isoleucine [13C, 15N] OT (designated as [Ile13C, 15N]OT) was synthesized by Sigma-Aldrich Chemical Japan (Ishikari, Hokkaido, Japan). Another stably labelled OT, in which the seventh proline and eighth leucine were replaced with [13C, 15N] proline and [13C, 15N] leucine, was obtained from Scrum Co. Ltd. (Tokyo, Japan). We designated this OT isotope as [Pro&Leu13C, 15N]OT and used as an internal standard (I-STD) for the quantitation of [Ile13C, 15N]OT in mouse serum samples. LC-MS grade water, acetonitrile (ACN), formic acid, trifluroacetic acid (TFA), trichloroacetic acid (TCA), 4-kDa FITC-dextran were purchased from Wako Chemicals (Tokyo, Japan).
3
Metabolite Extraction and Derivatization
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Whole tissues were ground using Multibeads shocker (Yasui Kikai) with LC/MS‐grade methanol and water in equal proportion by volume and then centrifuges at 13,000 g for 5 min at 4°C. Supernatant was mixed with an equal volume of chloroform. The upper aqueous phase was dried using Speedvac SPD 1010 (Thermo). For LC/MS experiments, the dried pellet of sample was dissolved in 60 μl LC/MS‐grade water (Wako) and then passed through a filter with 0.45‐μm Millex filter unit (Millipore). For GC/MS experiments, the derivatization of samples was carried out in two steps. In the first step, carbonyl functional groups were protected by methoximation using 20 μl of 20 mg/ml solution of methoxyamine hydrochloride in pyridine at 30°C for 90 min. In the second step, after adding 80 μl N‐methyl‐N‐trimethylsilyltrifluoroacetamide with 1% trimethylchlorosilane (MSTFA + 1% TMCS; Pierce), samples were incubated at 37°C for 30 min for derivatization.
4
Comprehensive metabolite profiling by LC-MS and GC-MS
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Whole tissues were grinded by Multi Beads Shocker (Yasui Kikai) with LC-MS grade methanol and water in the proportion of 1:1 (by volume). After the centrifugation, the supernatant was mixed with the same volume of chloroform, and the aqueous phase was dried by SpeedVac SPD1010 (Thermo). For the LC/MS analysis, the dried sample was reconstituted by 50 μl LC/MS grade water (Wako) and filtered with 0.45 μm Millex filter unit (Millipore). For the GC/MS analysis, two-step derivatization was carried out. First, carbonyl functional groups were protected by methoximation using
20 μL of 20 mg/mL solution of methoxyamine hydrochloride in pyridine at 30°C for 90 min. Next, the samples were derivatized using 80 μL of N-methyl-N-trimethylsilyltrifluoroacetamide with 1% trimethylchlorosilane (MSTFA + 1% TMCS, Pierce) at 37°C for 30 min.
5
Metabolite Extraction and Identification
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Frozen samples were powdered using dry ice powder via a mill cutter (Tube Mill control and MT 40; IKA, Staufen, Germany) and were then lyophilized by a freeze-dryer (dry chamber, DRC-1000; freeze-drying instrument, FDU-2100; EYELA, Tokyo, Japan). Four milligrams of powdered samples were weighed (AP324W, Shimadzu Corporation, Kyoto, Japan) and placed into a 2-ml tube with 5-mm zirconia beads. One milliliter of extraction solvent with 0.1% (v/v) formic acid in 80% (v/v) methanol and internal standards (8.4 nM of lidocaine and 210 nM of 10-camphorsulfonic acid) was added into the tube, and the metabolites were extracted using a bead-shocker (Shake Master NEO, Biomedical Science, Tokyo, Japan) for 2 min at 1,000 rpm, followed by centrifugation at 9,100 � g for 1 min. The extracted solutions were evaporated using a liquid handling system (MicrolabSTARplus, Hamilton Company, Reno, NV, USA), and the residual extract was redissolved with LC–MS grade water (FUJIFILM Wako Pure Chemical Corporation) so that the final diluted solution would be 40-fold. The redissolved solution was filtered (MZHVN0W50; Merck Millipore, Darmstadt, Germany). One microliter of the solution including 100 ng of the sample was subjected to widely targeted metabolomics.
6
Metabolite Extraction and Derivatization for LC-MS and GC-MS
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Metabolites were extracted from cells with water/methanol/chloroform (25:25:50 by volume). After centrifugation, the aqueous phase was isolated and dried using a SpeedVac SPD1010 (Thermo). For LC-MS analysis, the dried sample was reconstituted with 50 μl LC-MS grade water (Wako) and filtered through a 0.45 μm Millex filter unit (Millipore). For GC-MS analysis, two-step derivatization was carried out. First, carbonyl functional groups were protected by methoximation using 20 μL of 20 mg/mL methoxyamine hydrochloride in pyridine at 30°C for 90 min. Next, samples were derivatized using 90 μL of N-methyl-N-trimethylsilyltrifluoroacetamide with 1% trimethylchlorosilane (MSTFA + 1% TMCS, Pierce) at 37°C for 30 min.
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