For chromatographic analysis, stock solutions (50 µg/mL) of each test sample were prepared in acetonitrile (LiChrosolv®, Merck, Darmstadt, Germany). Chromatographic measurements were performed using the ACQUITY UPLC I-Class system (Waters) with a Xevo G2-XS QTof, ESI + detector, and an auxiliary PDA detector (λ = 254 nm). ACQUITY UPLC BEH C18 columns, 130 Å, 1.7 µm, 2.1 mm × 100 mm (Waters Corp.) were used for separation, operating at 35 °C, flow of 0.35 mL/min, and sample injection of 1 µL. The retention behavior of the analytes as a function of the mobile phase composition range (acetonitrile, LiChrosolv®, Merck + 0.01% HCOOH—water, LiChrosolv®, Supelco + 0.01% HCOOH) was tested. The concentration of acetonitrile, expressed as v/v by volume, varied from 0.25 to 0.50 in constant steps of 0.05. HCOONa solution (20 µg/mL) was used as a reference substance. The components of the analyte were identified on the basis of the monoisotopic masses of molecular ions [M + H]+ determined with an error of <5 ppm. Capacity factors k were calculated according to the formula k = (tR − tD)/tD, where tR is the retention time of the solute, and tD is the dead time obtained using an unretained analyte. Each experiment was repeated three times. Logk, which was calculated from the capacity factor k, was used as the lipophilicity index, converted to the logP scale.
Lichrosolv
Manufactured by Merck Group
Sourced in Germany, United States, Belgium, Italy, Switzerland
LiChrosolv is a line of high-performance liquid chromatography (HPLC) solvents and reagents manufactured by Merck Group. The core function of LiChrosolv products is to serve as components in HPLC systems, providing the necessary solvents and solutions for efficient separation, purification, and analysis of various chemical compounds.
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Lab products found in correlation
Formic acid, by Merck Group (10 mentions)
N hexane, by Merck Group (5 mentions)
Uvasol, by Merck Group (5 mentions)
Milli q system, by Merck Group (5 mentions)
Hipersolv chromanorm, by Avantor (5 mentions)
Hydrochloric acid (hcl), by Merck Group (4 mentions)
Chromasolv, by Honeywell (4 mentions)
Reagentplus, by Merck Group (3 mentions)
Acquity uplc 1 class system, by Waters Corporation (3 mentions)
Formic acid, by Thermo Fisher Scientific (3 mentions)
Edta vacutainer plasma tubes, by BD (3 mentions)
Centrifuge 5804 r, by Eppendorf (3 mentions)
Diisobutyl phthalate, by Merck Group (3 mentions)
Benzophenone, by Merck Group (3 mentions)
2 6 di tert butyl 4 methylphenol, by Merck Group (3 mentions)
Diisobutyl phthalate 3 4 5 6 d 4, by Merck Group (3 mentions)
Potassium chloride (kcl), by Merck Group (3 mentions)
Methanol, by Merck Group (3 mentions)
Dphp d 10, by Merck Group (2 mentions)
β glucuronidase enzyme solution, by Merck Group (2 mentions)
119 protocols using lichrosolv
1
UPLC-QTOF Analysis of Analyte Lipophilicity
2
Analytical Workflow for Antibiotic Determination
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Antibiotic standards, Disodium EDTA (OmniPur®), Oxalic acid (ReagentPlus®), Sodium sulphate (≥99%), n-hexane (Emsure®), Dichloromethane (LiChrosolv®), Methanol (LiChrosolv®) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (≥99.9%) was sourced from Fisher Scientific, Loughborough, Leicestershire, while the SPE columns (200 mg x8 mL tubes) were purchased from Supelco™, Bellefonte, USA. The 0.45 μm GHP ACRODISC 13 mm disposable syringe filter unit was purchased from Agilent, whereas the deionised water (Ultrapure water) used in this study was produced using an Integral 10 Elix Milli-Q system with an LC (Biopak) polisher (Massachusetts, USA). Other apparatuses used include the Waring laboratory blender – Z272205 (Sigma-Aldrich, St. Louis, MO, USA), and the Vortex mixer VM18 (Schiltern Scientific, Beds, UK).
3
Mass Spectrometry Sample Preparation
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Solvents used to prepare the samples were acetonitrile hypergrade for LC–MS LiChrosolv® purity ≥ 99.9% (Sigma-Aldrich, St. Louis, MO), ethyl acetate 99.7% (Sigma-Aldrich, St. Louis, USA), methanol hypergrade for LC–MS LiChrosolv® purity ≥ 99.9% (Sigma-Aldrich, St. Louis, MO). Trifluoroacetic acid was LC–MS grade, and LiChropur was ≥ 99.0% (GC) (Sigma-Aldrich, St. Louis, MO). The ultrapure water used to prepare extracts was purified by the Direct-Q water system (Millipore, Bedford, MA). MALDI matrices were α-cyano-4-hydroxycinnamic acid (CHCA) MALDI-MS grade, purity ≥ 99.0% (HPLC) (Sigma-Aldrich, St. Louis, MO) and 2,5-dihydroxybenzoic acid (DHB) MALDI-MS grade, purity ≥ 99.0% (HPLC) (Sigma-Aldrich, St. Louis, MO). The phospholipid standards used for TOF calibration were 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (ammonium salt) purity ≥ 99% (DOPE), 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, purity ≥ 99% (DMPG),1,2-dipalmitoyl-sn-glycero-3-phosphate monosodium salt, powder, purity ≥ 99% (DPPA), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine sodium salt, powder, purity ≥ 99% (DPPS) obtained from Avanti Polar Lipids, Inc. (Alabaster, Al, USA).
4
Development of Mesoporous Silica Drug Delivery System
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All reagents used, such as orthophosphoric acid 85% EMSURE®, potassium dihydrogenphosphate and disodium hydrogenphosphate dodecahydrate (Merck, Darmstadt, Germany), tetraethylorthosilicate (TEOS), cyclohexyltrimethoxysilane (CHTMS) (99%, pKA = 7.55), (3-aminopropyl)triethoxysilane (APTES, 99%, pKB = 3.63), poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer (Pluronic P-123) and 5-fluorouracil (5-FU, ≥ 99% by HPLC) (Sigma-Aldrich, St. Louis, MO, USA) were at least analytical grade and no further purification was done. Buffer solution with pH = 2 (hydrochloric acid/potassium chloride), according to Veibel was purchased from Honeywell Fluka (Steinheim, Switzerland) and physiological saline with pH = 7 was purchased from Centralchem (Banská Bystrica, Slovak Republic). Toluene (>99%) was purchased from Sigma-Aldrich and dried over zeolite sieves. Methanol LiChrosolv® was of gradient grade for liquid chromatography (Merck). Water for chromatography LiChrosolv® (Merck) was used for the preparation of a mobile phase and all solutions.
5
Quantitative Analysis of Fatty Acids
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FF and FFA standards of 99.8% certified purity were used for the analysis and quantification of the analytes, and chloramphenicol-d5 (CAF-d5) of 97% certified purity was used for the internal standard. All standards were manufactured by Sigma Aldrich, Inc. (now Merck & Co., Darmstadt, Germany)
The spiking solutions of FF, FFA, and CAF-d5 were prepared in a solution of methanol/water (8/2) at a concentration of 5000 ng·mL−1.
Reagents such as water (LiChrosolv®), acetone (Emsure®), dichloromethane (LiChrosolv®), and hexane (LiChrosolv®), were sourced from Merck & Co., Inc., while methanol certified for use in liquid chromatography (HPLC and UHPLC) and spectrophotometry, was sourced from J.T. Baker® (Avantor™ Performance Materials, Inc., Center Valley, PA, USA).
The spiking solutions of FF, FFA, and CAF-d5 were prepared in a solution of methanol/water (8/2) at a concentration of 5000 ng·mL−1.
Reagents such as water (LiChrosolv®), acetone (Emsure®), dichloromethane (LiChrosolv®), and hexane (LiChrosolv®), were sourced from Merck & Co., Inc., while methanol certified for use in liquid chromatography (HPLC and UHPLC) and spectrophotometry, was sourced from J.T. Baker® (Avantor™ Performance Materials, Inc., Center Valley, PA, USA).
6
Quantifying Lipids from Algae Extracts
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Lipids released from algae into the non-polar phase were quantified by measuring liquid component concentrations. Each sample of supernatant was vigorously shaken, and a volume of 50 mL was pipetted into a 250 mL flask, to which 50 mL of a mix (1.6:1 volume ratio) of n-hexane (LiChrosolv, Merck) and isopropanol (LiChrosolv, Merck) was added. Immediately after preparation, all samples of algal pellets were shaken intensely in isopropanol for at least 5 min, and then treated in an ultrasound bath unit for 1 h, where the temperature of samples reached 35°C. Over the night, they were left at room temperature. In the morning, the samples were intensely shaken again and treated in ultrasound bath for another hour. After 5 h of samples rest at room temperature, 3–4 separated phases were visible. In all cases 1 mL of the present most upper phase was filtered through 2 μm syringe filter (Chromafil O-20/25 PTFE, Macherey-Nagel) into the 1.5 mL HPLC vial. The presences of lipid molecules were confirmed by HPLC as described elsewhere (Likozar and Levec, 2014a (link), b (link); Likozar et al., 2016 (link)).
7
Milk Sample Preparation for Elemental Analysis
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Powdered milk samples were initially reconstituted with ultrapure water in a glass flask (1000-mL; particle certified, Thermofisher Scientific) by diluting 25 g test portion in 175 g ultrapure water (Lichrosolv, Merck). Glass flasks covered with a glass lid were shaken for at least 15 min at 40 °C into a shaking water bath (GFL-1083, Milian). Processed liquid and raw milk samples were used as is.
25 mL liquid or reconstituted milk sample and 20 mL ultrapure water (Lichrosolv, Merck) were added into a glass flask (1000-mL; particle certified, ThermoFisher Scientific); 2 mL of multi-enzymatic detergent (Prozyme, Borer Chemie AG) was added and mixed for 2 min at 40 °C in the container; 10 mL of calcium chelating agent sodium ethylene diamine tetra acetate (EDTA-Na 0.5 M, pH 8, Invitrogen, ThermoFisher Scientific) was added and mixed for 3 min at 40 °C in the container; 2 mL of alkaline solution tetramethyl ammonium hydroxide (TMAH 25% v/v Sigma-Aldrich) was added into the glass container that was finally immediately put in a microwave (CD575MWPG, Panasonic NN) at a power of 1000 watts for a maximum of 1 min to ensure a final temperature below 80 °C. The hot digested milk sample was then immediately submitted to filtration process.
25 mL liquid or reconstituted milk sample and 20 mL ultrapure water (Lichrosolv, Merck) were added into a glass flask (1000-mL; particle certified, ThermoFisher Scientific); 2 mL of multi-enzymatic detergent (Prozyme, Borer Chemie AG) was added and mixed for 2 min at 40 °C in the container; 10 mL of calcium chelating agent sodium ethylene diamine tetra acetate (EDTA-Na 0.5 M, pH 8, Invitrogen, ThermoFisher Scientific) was added and mixed for 3 min at 40 °C in the container; 2 mL of alkaline solution tetramethyl ammonium hydroxide (TMAH 25% v/v Sigma-Aldrich) was added into the glass container that was finally immediately put in a microwave (CD575MWPG, Panasonic NN) at a power of 1000 watts for a maximum of 1 min to ensure a final temperature below 80 °C. The hot digested milk sample was then immediately submitted to filtration process.
8
Preparation of UHPLC-Grade Solvents
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UHPLC-grade methanol was purchased from SK Chemicals®® (Pangyo-ro, South Korea) and acetonitrile (Lichrosolv®®) from Merck Millipore (Darmstadt, Germany). Formic acid (Lichrosolv®® 98–100%) was also acquired from Merck Millipore. Absolute ethyl alcohol (99.5% Anidrol®®) was purchased from Soltech (Diadema, Brazil). Ultra-pure water was produced by a Milli-Q system.
9
Extracellular Folate Analysis by HPLC
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Extracellular folate was analyzed using an Agilent 1260 Infinity HPLC System with a diode array detector (DAD; Agilent Technologies Inc., Santa Clara, CA, USA) and a ZORBAX Eclipse XDB-C18 chromatography column (15 cm×4.6 mm, 5 μm; Agilent Technologies, Inc.) at λ=282 nm. The mobile phase was freshly prepared and consisted of water (HPLC grade; LiChrosolv®, Merck KGaA) with glacial acetic acid (0.66 %; EMSURE®, Merck KGaA) and methanol (pure HPLC grade; LiChrosolv®, Merck KGaA), with a ratio of V(water):V(methanol)=70:30 (33 ). The flow rate was 0.8 mL/min. Folic acid standard was obtained from R-Biopharm (provided in the Vitafast folic acid test kit; Pfungstadt, Germany) and used without further purification.
10
Lipid Extraction from Microbial Cells
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All cells were concentrated on GF/C filters with a pore size of 1.2 µm (Whatman, Dassel, Germany) under a reduced pressure of 700 mbar, and filters were immediately transferred into the extraction mix consisting of chilled methanol (LiChrosolv, Merck, Darmstadt, Germany), ethanol (LiChrosolv, Merck, Darmstadt, Germany) and chloroform (HPLC gradient grade, Fisher Chemical, Thermo Scientific, Bremen, Germany) 1:3:1 (v:v:v) [51 (link)]. After ultrasonication for 5 min, extracts were centrifuged at 30,000× g for 15 min. The supernatants were stored at −80 °C.
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