Butanol Methanol Research Articles

Isolation of Mitochondrial Lipids and Mass Spectrometric Analysis.

Mitochondria participate in many important metabolic processes in the body. The lipid profile of mitochondria is especially important in membrane regulation and pathway signaling. The isolation and study of these lipids can provide unparalleled information about the mechanisms behind these cellular processes. In this chapter, we describe a protocol to isolate mitochondrial lipids from homogenized murine optic nerves. The lipid extraction was performed using butanol-methanol (BUME) and subsequently analyzed using liquid chromatography-mass spectrometry. Further analysis of the raw data was conducted using LipidSearch™ and MetaboAnalyst 4.0.

Improved Butanol-Methanol (BUME) Method by Replacing Acetic Acid for Lipid Extraction of Biological Samples.

Extraction of lipids from biological samples is a critical step in lipidomics, especially for shotgun lipidomics where lipid extracts are directly infused into a mass spectrometer. The butanol-methanol (BUME) extraction method was originally developed to extract lipids from plasma samples with 1% acetic acid. Considering some lipids are sensitive to acidic environments, we modified this protocol by replacing acetic acid with lithium chloride solution and extended the modified extraction to tissue samples. Although no significant reduction of plasmalogen levels in the acidic BUME extracts of rat heart samples was found, the modified method was established to extract various tissue samples, including rat liver, heart, and plasma. Essentially identical profiles of the majority of lipid classes were obtained from the extracts of the modified BUME and traditional Bligh-Dyer methods. However, it was found that neither the original, nor the modified BUME method was suitable for 4-hydroxyalkenal species measurement in biological samples.

Separation of Steroidal Constituents of Oviductus Ranae by One-Step Method High-Speed Counter-Current Chromatography

The dichloromethane extracts of Oviductus Ranae were treated simply and two steroidal compounds were prepared by means of high-speed counter-current chromatography (HSCCC) directly. The two-phase solvent system was composed of petroleum ether–ethyl acetate–butanol–methanol–water (3.5:0.3:0.5:2.5:0.4, v/v/v/v/v). The upper phase was used as stationary phase and the lower phase was used as mobile phase. The dichloromethane extracts of Oviductus Ranae were separated and purified by utilizing the solvent system above. High-performance liquid chromatography analysis showed that the purity of each of the two compounds had reached 95%. Their chemical structures were confirmed as 7-hydroxycholesterol and 7-ketocholesterol according to the 1H-NMR and 13C-NMR data in literature. The high-speed counter-current chromatography proposed in this paper can be used to prepare the main steroidal components in Oviductus Ranae. The method also has advantages in simplicity, speed, and efficiency, which can be applied to the preparation of the high purity 7-hydroxycholesterol and 7-ketocholesterol. In this study, HSCCC was used to separate the substances in Oviductus Ranae for the first time, which provides a foundation for the separation of Oviductus Ranae by means of HSCCC.

Synthesis of trinuclear CuII-complex with tris(4-triphenylmethylphenoxy)-substituted hemihexaphyrazine and radioprotective properties of its water-soluble form

A condensation of 2,5-diamino-1,3,4-thiadiazole with the products of the in situ reaction of 1,2-dicyano-4-(4-triphenylmethylphenoxy)benzene with sodium alkoxides in a mixture of butanol—methanol furnished a trisubstituted ABABAB-type macroheterocycle (hemihexaphyrazine). A 3 : 1 trinuclear complex was synthesized by the reaction of the macrocycle with copper(II) acetate. The compounds obtained were characterized by IR spectroscopy, UV-vis spectroscopy, mass spectrometry, as well as by elemental analysis. Radioprotective properties of the water-soluble form of the complex were studied

Automation Highlights from the Literature

Automation Highlights from the Literature

Automation Highlights from the Literature

Automation Highlights from the Literature

The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma

Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profiles of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroform-based reference method. Lipid recoveries were linear from 10-100 µl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 µl butanol:methanol (BUME) mixture (3:1) followed by two-phase extraction into 300 µl heptane:ethyl acetate (3:1) using 300 µl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, high-throughput BUME method can replace chloroform-based methods, saving both human and environmental resources.

Synthesis of expanded alkylphenoxythiadiazole macroheterocycles

New substituted macroheterocyclic compounds of the ABABAB type were synthesised by condensation of 2,5-diamino-1,3,4-thiadiazole with the products of interaction of 4-(4-tert-butylphenoxy)- or 4-(3,5-dimethylphenoxy)phthalonitriles with sodium alkoxides in a butanol–methanol mixture.

Isolation and Identification of Phenolic Compounds in the Fruit of Benincasa hispida by HSCCC

Separation of phenolic components from the fruit Benincasa hispida was conducted using high‐speed countercurrent chromatography (HSCCC) with two solvent systems, n‐hexane–n–butanol–methanol–water (10:16:5:20, v/v) and n‐hexane–ethyl acetate–methanol–water (1:1:1:1, v/v). Three compounds, astilbin (120 mg), catechin (103 mg), and naringenin (79 mg) were obtained from the separation of 2 g of crude phenolic component extract of the fruit. Chemical structures of these three compounds were confirmed by electrospray ionization ion trap multiple mass spectrometry (ESI‐MS) and NMR.

Improved Thin-Layer Chromatographic Method for the Separation of Cholesterol, Egg Phosphatidylcholine, and Their Degradation Products

Degradation products of egg phosphatidylcholine (EPC) and cholesterol were analyzed with different normal- and reversed-phase thin-layer chromatography (TLC) systems. The best separation, in terms of the highest number of degradation products from both analytes, was obtained with a reversed-phase system, using butanol-methanol-water-96-98% (v/v) acetic acid (40 + 40 + 20 + 4, v/v/v/v) as the mobile phase after overnight saturation at 25 degrees C. A special development technique was used. After a first development, the plate was dried and a second development was performed in the same direction. This method enabled us to separate lysophosphatidylcholine, several free fatty acids and hydroperoxides, and several undefined degradation products of EPC and cholesterol. All products were visualized after the plate was dipped in a 1% (v/v) solution of 4-methoxybenzaldehyde in 98% sulfuric acid-96-98% (v/v) acetic acid-ethanol-water (2 + 10 + 60 + 30), presenting a blue color or a white spot against a colored background. After activation at 110 degrees C, a stable color for both analytes was reached after 12 min. Precision of <5% was obtained at 2 levels of analysis. Good linearity was obtained in the range of 5-30 microg for EPC (r = 0.991) and 5-40 microg for cholesterol (r = 0.991). These results show that TLC can be an inexpensive and easy alternative for the analysis of EPC and cholesterol.

Development of a screening method for five sulfonamides in salmon muscle tissue using thin-layer chromatography

A thin-layer chromatographic (TLC) method was developed for the analysis of five sulfonamides [sulfadiazine (SDZ), sulfamerazine (SMRZ), sulfamethazine (SMTZ), sulfadimethoxine (SDMX) and sulfapyridine (SP)] in salmon muscle tissue. “Matrix solid-phase dispersion” was employed whereby the tissue sample was ground with C 18-derivatized silica gel. This material was packed into a column and washed with 10% toluene in hexane (discarded) followed by dichloromethane which was evaporated. The residue was chromatographed on a high-performance TLC plate using ethyl acetate— n—butanol—methanol—aqueous ammonia (35:45:15:2, v/v). Sulfonamides were detected after spraying the plate with a solution of fluorescamine. Method parameters were determined by analyzing spiked salmon muscle tissue samples. The method detection limits at the 99% confidence level were 0.11, 0.44, 0.07, 0.13 and 0.13 ppm for SDZ, SMRZ, SMTZ, SDMX and SP, respectively. The lowest-detectable levels were approximately 0.04 ppm for SDZ, SMTZ, SDMX and SP, and 0.10 ppm for SMRZ. The average recoveries of analytes were 61, 63, 60, 63 and 57% for SDZ, SMRZ, SMTZ, SDMX and SP, respectively, and were found to be analyst-dependent. The method was found to give linear detector responses for all analytes over spiking levels ranging from 0 to 2 ppm.

Evaluation of solvents used in summarized chromatograms

Based on some concepts from the Information Theory, the solvents used for antibiotic substances by Ishida etal and Betina were evaluated relative to each other by means of summarized chromatography. The information that a summarized chromatogram system supplies should depend mainly on three factors: (1) distribution of the R F of the reference substances in each solvent; (2) interrelationship between the R F values that a given substance shows in the different solvents; and (3) experimental error. Thus, admitting a constant error, it was found that among the 11 solvents tested, those that gave the most information were water; butanol; 3% aq. ammonium chloride; benzene—methanol (4:1) and butanol—methanol—water (4:1:2) with and without helianthin. The phenolic solvents were less informative; ethyl acetate is mediocre in all cases. Although the information is very high in some of the 11 solvents tested (up to 85% of H max), only water supplies information independent of the others, which are redundant among each other.

Chromatographie des produits de radiolyse de la thymine

Chromatography of thymine radiolysis products [ 14C]Thymine in aqueous solution (10 −3 to 10 −4 M) was irradiated with γ rays of 60Co at doses between 10 2 and 10 6 rad. Different chromatographic techniques for separation of the radiolysis products are described. For the paper system solvents such as butanol—water (86:14) and acetonitrile—phosphate buffer (0.2 M NaH 2PO 4 and 0.2 M Na 2HPO 4) (85:15) were employed. Bidimensional thin-layer chromatography with cellulose powder was also used. For this experiment the best solvents were either butanol—methanol—water—NH 4OH (60:20:20:1) or propanol—water (10:3) in the first dimension, and isopropanol—water-saturated (NH 4) 2SO 4—1 M K 2HPO 4 (3:77:20) in the second dimension. The separation is rapid and can be obtained at 4°. We have identified a new photoproduct of thymine: 5-methyl-5-hydroxy hydantoin. R F values of this compound are given.