Structure elucidation of platelet activating factor derived from human neutrophils

Platelet activating factor (PAF) synthesized by human neutrophils challenged by opsonized zymosan or calcium ionophore was isolated from cells and buffer using Bligh and Dyer extraction following the addition of tracer amounts of tritiated-PAF. The extract was subjected to TLC separation of phospholipid classes, followed by reverse phase HPLC for molecular species separation. All fractions were measured for radioactivity, biological activity and fast atom bombardment mass spectrometry. While the radioactive tracer PAF could be separated into three molecular species, PAF biological activity eluted as a single component which was characterized as 1-O-hexadecyl-2-acetyl-glycero-3-phosphocholine. The lack of molecular species heterogeneity of PAF produced in response to stimuli implies a higher degree of control of biosynthesis than previously suspected.     

肺机械扩张增进表面活性物质分泌机制的研究

采用大鼠离体肺灌流模型,观察肺泡灌洗液中肺表面活性物质(PS)含量的变化。结果表明:在增大潮气量机械扩张肺时PS分泌显著增加(p<0.05),该分泌增加现象能被细胞松弛素B及硝苯吡啶所抑制(p<0.05),但不为细胞外缺钙所抑制(p>0.05)。从而提示扩张所引起的PS分泌增加可能与细胞内的收缩结构有关,而与细胞外钙离子无关。     

Comparison of three detergent-free protein extraction protocols for white adipose tissue

A comparative study of three detergent-free protein extraction protocols—a differential centrifugal fractionation, a delipidation protocol based on the Bligh and Dyer method, and the trifluoroethanol addition as cosolvent to an aqueous buffer—was performed on white adipose tissue. The performance of the protocols directly compatible with liquid chromatography–electrospray ionization–mass spectrometry (LC–ESI–MS) was evaluated based on the total protein extraction yield and the protein recovery from different functional and cellular compartments. The most suitable method for the extraction of white adipose tissue proteins from a wide range of cellular and structural compartments was the delipidation protocol based on the Bligh and Dyer method.     

Evaluation of green solvents: Oil extraction from oleaginous yeast Lipomyces starkeyi using cyclopentyl methyl ether (CPME)

Cyclopentyl methyl ether (CPME) was evaluated for extracting oil or triacylglycerol (TAG) from wet cells of the oleaginous yeast Lipomyces starkeyi. CPME is a greener alternative to chloroform as a potential solvent for oil recovery. A monophasic system of CPME and biphasic system of CPME:water (1:0.7) performed poorly having the lowest TAG extraction efficiency and TAG selectivity compared to other monophasic systems of hexane and chloroform and the biphasic Bligh and Dyer method (chloroform:methanol:water). Biphasic systems of CPME:water:alcohol (methanol/ethanol/1‐propanol) were tested and methanol achieved the best oil extraction efficiency compared to ethanol and 1‐propanol. Different biphasic systems of CPME:methanol:water were tested, the best TAG extraction efficiency and TAG selectivity achieved was 9.9 mg/mL and 64.6%, respectively, using a starting ratio of 1:1.7:0.6 and a final ratio of 1:1:0.8 (CPME:methanol:water). Similar results were achieved for the Bligh and Dyer method (TAG extraction efficiency of 10.2 mg/mL and TAG selectivity of 66.0%) indicating that the biphasic CPME system was comparable. The fatty acid profile remained constant across all the solvent systems tested indicating that choice of solvent was not specific for any certain fatty acid. This study was able to demonstrate that CPME could be used as an alternative solvent for the extraction of oil from the wet biomass of oleaginous yeast. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1096–1103, 2017     

Incorporation of tandem mass spectrometric detection to the analysis of peptide mixtures by continuous flow fast atom bombardment mass spectrometry

The ability to acquire structurally informative daughter ion spectra for individual peptides undergoing separation and analysis by continuous flow fast atom bombardment (CF FAB) is demonstrated. To illustrate the potential of this methodology, tryptic and chymotryptic digests of the 29-residue peptide glucagon were analyzed by CF FAB using mass spectrometric and tandem mass spectrometric detection in consecutive analyses. Daughter ion spectra were recorded using B/E linked scans for the major hydrolysis products observed by liquid chromatography/mass spectrometry. The peptide mixtures were separated by gradient capillary high-performance liquid chromatography with the FAB matrix being added post-column using a coaxial flow interface between the column and flow probe. The entire effluent (3 microl min(-1)) was sampled by the mass spectrometer. Results obtained using less than 300 pmol of digested glucagon indicated several advantages to tandem mass spectrometric detection including the ability to confirm identities for products of enzymatic digestion and the potential use of this method for tandem sequence analysis of peptide mixtures.     

Quantification of ceramide species in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry

We present an optimized and validated liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the simultaneous measurement of concentrations of different ceramide species in biological samples. The method of analysis of tissue samples is based on Bligh and Dyer extraction, reverse-phase high-performance liquid chromatography separation, and multiple reaction monitoring of ceramides. Preparation of plasma samples also requires isolation of sphingolipids by silica gel column chromatography prior to LC-ESI-MS/MS analysis. The limits of quantification were in a range of 0.01-0.50 ng/ml for distinct ceramides. The method was reliable for inter- and intraassay precision, accuracy, and linearity. Recoveries of ceramide subspecies from human plasma, rat liver, and muscle tissue were 78 to 91%, 70 to 99%, and 71 to 95%, respectively. The separation and quantification of several endogenous long-chain and very-long-chain ceramides using two nonphysiological odd chain ceramide (C17 and C25) internal standards was achieved within a single 21-min chromatographic run. The technique was applied to quantify distinct ceramide species in different rat tissues (muscle, liver, and heart) and in human plasma. Using this analytical technique, we demonstrated that a clinical exercise training intervention reduces the levels of ceramides in plasma of obese adults. This technique could be extended for quantification of other ceramides and sphingolipids with no significant modification.     

Rapid diagnosis of phenylketonuria and other aminoacidemias by quantitative analysis of amino acids in neonatal blood spots by gas chromatography-mass spectrometry

A reversed-phase HPLC method compatible with evaporative light scattering (ELS) and electrospray mass spectrometric (ES-MS) detection was developed for separation of phosphatidylserine (PS) molecular species. The method was optimised for separation of three disaturated synthetic species: dipalmitoyl glycerophosphoserine, palmitoyl-stearoyl glycerophosphoserine and distearoyl glycerophosphoserine using isocratic elution with a mixture of 2-propanol, tetrahydrofuran and ammonium formate. Baseline separation was obtained on three different columns: one polystyrene/divinylbenzene (PS/DVB) column and two silica based C(18) and C(30) columns. The best chromatographic resolution was achieved with the C(30) column. The limit of detection for DPPS was 5 microg/ml (S/N=3) with ELS detection and 0.1 microg/ml (S/N=3) with negative ion ES-MS in the single ion monitoring mode. Baseline separation of the five main species in a biological PS sample, bovine brain PS, was obtained with the PS/DVB column. Species identification was done by using the retention times of the intact PS species and their corresponding carboxylate anion fragments obtained by in-source fragmentation. Data have shown that individual PS species can be identified by their retention times using direct ELS detection in a mixture of disaturated PS species. However, for the bovine brain PS electrospray-MS detection was necessary for species identification due to the many possible fatty acid combinations in biological PS.     

A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL

Lipidome profile of fluids and tissues is a growing field as the role of lipids as signaling molecules is increasingly understood, relying on an effective and representative extraction of the lipids present. A number of solvent systems suitable for lipid extraction are commonly in use, though no comprehensive investigation of their effectiveness across multiple lipid classes has been carried out. To address this, human LDL from normolipidemic volunteers was used to evaluate five different solvent extraction protocols [Folch, Bligh and Dyer, acidified Bligh and Dyer, methanol (MeOH)-tert-butyl methyl ether (TBME), and hexane-isopropanol] and the extracted lipids were analyzed by LC-MS in a high-resolution instrument equipped with polarity switching. Overall, more than 350 different lipid species from 19 lipid subclasses were identified. Solvent composition had a small effect on the extraction of predominant lipid classes (triacylglycerides, cholesterol esters, and phosphatidylcholines). In contrast, extraction of less abundant lipids (phosphatidylinositols, lyso-lipids, ceramides, and cholesterol sulfates) was greatly influenced by the solvent system used. Overall, the Folch method was most effective for the extraction of a broad range of lipid classes in LDL, although the hexane-isopropanol method was best for apolar lipids and the MeOH-TBME method was suitable for lactosyl ceramides.     

Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics

Accurate profiling of lipidomes relies upon the quantitative and unbiased recovery of lipid species from analyzed cells, fluids, or tissues and is usually achieved by two-phase extraction with chloroform. We demonstrated that methyl-tert-butyl ether (MTBE) extraction allows faster and cleaner lipid recovery and is well suited for automated shotgun profiling. Because of MTBE's low density, lipid-containing organic phase forms the upper layer during phase separation, which simplifies its collection and minimizes dripping losses. Nonextractable matrix forms a dense pellet at the bottom of the extraction tube and is easily removed by centrifugation. Rigorous testing demonstrated that the MTBE protocol delivers similar or better recoveries of species of most all major lipid classes compared with the "gold-standard" Folch or Bligh and Dyer recipes.     

Mass spectrometric quantification of endogenous beta-endorphin

Fast atom bombardment (FAB) mass spectrometry and multiple reaction monitoring (MRM) in the B/E linked-field scan mode were used to quantify endogenous beta-endorphin (BE) in individual human pituitary extracts. The experimental protocol includes the addition of a stable isotope-labeled internal standard ((2H4-Ile22)BE1-31, human) to the tissue homogenate before extraction, purification of the native BE by a combination of Sep-Pak chromatography and gradient high-performance liquid chromatography (HPLC), trypsin digestion to cleave BE into smaller peptides, and separation of the tryptic fragment BE20-24 (NAIIK) by isocratic reversed-phase HPLC. Mass spectrometric quantification is based upon recording either (a) the [M + H]+ ions of NAIIK and its deuterated analog ((2H4)NAIIK), or (b) the transitions ([NAIIK + H](+)----[NAI]+) and [((2H4)NAIIK + H](+)----[(2H4)NAI]+) using the B/E linked-field scan. Linear calibration curves were obtained using these two mass spectrometric techniques from standard solutions containing 1.25-20 micrograms of BE; each standard solution also contained 10 micrograms of (2H4)BE. The amounts (means +/- s.d.) of endogenous BE in five separate human pituitaries were found to be 156 +/- 84 [( M + H]+ method) and 169 +/- 99 pmol mg-1 protein (MRM method).     

Development of a purification procedure for the isolation of nucleosides from urine prior to mass spectrometric analysis

A chromatographic separation of nucleosides from urine has been developed in order to facilitate their mass spectrometric analysis for clinical diagnosis. A number of chromatographic resins were studied in order to develop an effective and efficient purification procedure. The optimized sequential protocol comprises a centrifugation, acidification and neutralization step, followed by application of an affinity chromatographic column and finally further separation on an acidic cation exchange column and a basic anion exchanger. This scheme shows effective clean-up of a standard radiolabelled nucleoside with a recovery of 92.5%, and recovery of nucleosides added to urine samples before extraction showed recoveries of 72-82%.     

Rapid measurement of plasma free fatty acid concentration and isotopic enrichment using LC/MS

Measurements of plasma free fatty acids (FFA) concentration and isotopic enrichment are commonly used to evaluate FFA metabolism. Until now, gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) was the best method to measure isotopic enrichment in the methyl derivatives of ¹³C-labeled fatty acids. Although IRMS is excellent for analyzing enrichment, it requires time-consuming derivatization steps and is not optimal for measuring FFA concentrations. We developed a new, rapid, and reliable method for simultaneous quantification of ¹³C-labeled fatty acids in plasma using high-performance liquid chromatography-mass spectrometry (HPLC/MS). This method involves a very quick Dole extraction procedure and direct injection of the samples on the HPLC system. After chromatographic separation, the samples are directed to the mass spectrometer for electrospray ionization (ESI) and analysis in the negative mode using single ion monitoring. By employing equipment with two columns connected parallel to a mass spectrometer, we can double the throughput to the mass spectrometer, reducing the analysis time per sample to 5 min. Palmitate flux measured using this approach agreed well with the GC/C/IRMS method. This HPLC/MS method provides accurate and precise measures of FFA concentration and enrichment.     

Liquid chromatography/mass spectrometry of fatty acids as their anthrylmethyl esters

The mass spectra of a series of saturated and unsaturated fatty acids have been recorded as their anthrylmethyl esters using a liquid chromatographic mass spectrometric interface. The spectra show an intense peak for the aromatic nucleus, and a molecular ion. The liquid chromatographic/mass spectrometric separation was performed on a reverse phase column using a solvent system of acetone + acetonitrile. While a complete separation of the fatty acids known to occur in man was not achieved, the recognition of all of these acids is possible using a scanning mode or by ion monitoring.     

Liquid chromatography/mass spectrometry of fatty acids as their anilides

The mass spectra of a series of saturated (C16:0-C30:0) and unsaturated (C16:1, C18:1, C18:2, C18:3) fatty acids have been recorded as their anilides using liquid chromatography/mass spectrometry with the atmospheric-pressure-ionization interface system. The spectra show an intense peak for (molecule + H)+ ion in each case. The liquid chromatographic/mass spectrometric separation was performed on a reverse phase column using a solvent system of methanol alone or methanol + 2-propanol. This method seemed promising for application to both qualitative and quantitative micro-analysis of fatty acids including very long chain fatty acids.     

Lipid sovent systems are not equivalent for analysis of lipid classes in the microeukaryotic green alga,Chlorella

A comparison of three lipid solvent system indicated that they are not equivalent for the analysis of lipid classes in the green alga, Chlorella. Soxhlet extraction (methylene chloride/methanol, 3 h refulx) recovers more neutral lipid than the other methods but is equivalent to the room-temperature Bligh and Dyer (chloroform/methanol/water) extraction modified with phosphate buffer in glycolipid and polar lipid recovery. The Soxhlet method, however, gave a significantly lower recovery of many polyunsaturated fatty acids. The hexane/isopropanol method is selective for algal neutral lipids with poor recovery of membrane lipids (glyco- and polar lipids). Although this selectivity may have some useful applications, for biochemical studies of lipid synthesis in Chlorella, the modified Bligh and Dyer provides the most quantitative and reproducible recovery of all Chlorella lipid classes while minimizing artifacts due to the extraction procedure.     

Development of a Purification Procedure for the Isolation of Nucleosides from Urine Prior to Mass Spectrometric Analysis

Abstract

A chromatographic separation of nucleosides from urine has been developed in order to facilitate their mass spectrometric analysis for clinical diagnosis. A number of chromatographic resins were studied in order to develop an effective and efficient purification procedure. The optimized sequential protocol comprises a centrifugation, acidification and neutralization step, followed by application of an affinity chromatographic column and finally further separation on an acidic cation exchange column and a basic anion exchanger. This scheme shows effective clean-up of a standard radiolabelled nucleoside with a recovery of 92.5%, and recovery of nucleosides added to urine samples before extraction showed recoveries of 72 – 82%.     

Effects of heat-treatment on the stability and composition of metabolomic extracts from the earthworm <Emphasis Type="Italic">Eisenia fetida</Emphasis>

Environmental metabolomics studies employing earthworms as sentinels for soil contamination are numerous, but the instability of the metabolite extracts from these organisms has been minimally addressed. This study evaluated the efficacy of adding a heat-treatment step in two commonly used extraction protocols (Bligh and Dyer and D₂O phosphate buffer) as a pre-analytical stabilization method. The resulting metabolic profiles of Eisenia fetida were assessed using principal component analysis and NMR spectral evaluations. The heated Bligh and Dyer extractions produced stabilized profiles with minimal variation of the extracted metabolomic profiles over time, providing a more suitable method for metabolomic analysis of earthworm extracts.     

Isolation and enzymic assay of choline and phosphocholine present in cell extracts with picomole sensitivity.

Increasing interest in receptor-regulated phospholipase C and phospholipase D hydrolysis of cellular phosphatidylcholine motivates the development of a sensitive and simple assay for the water-soluble hydrolytic products of these reactions, phosphocholine and choline respectively. Choline was partially purified from the methanol/water upper phase of a Bligh & Dyer extract by ion-pair extraction using sodium tetraphenylboron, and the mass of choline was determined by a radioenzymic assay using choline kinase and [32P]ATP. After removal of choline from the upper phase, the mass of residual phosphocholine was determined by converting it into choline by using alkaline phosphatase, followed by radioactive phosphorylation. In addition to excellent sensitivity (5 pmol for choline and 10 pmol for phosphocholine), these assays demonstrated little mutual interference (phosphocholine----choline = 0%; choline----phosphocholine = 5%), were extremely reproducible (average S.E.M. of 3.5% for choline and 2.9% for phosphocholine), and were simple to perform with instrumentation typically available in most laboratories. In addition, the ability to apply the extraction technique to the upper phase of Bligh & Dyer extracts permitted simple analysis not only of choline and phosphocholine, but also of phosphatidylcholine and lipid products of phospholipase C and phospholipase D activity (1,2-diacylglycerol and phosphatidic acid respectively) from the same cell or tissue sample.     

A large-scale purification of paclitaxel from cell cultures of Taxus chinensis

A large-scale purification method was developed for producing paclitaxel, to guarantee high purity and yield from plant cell cultures. The complete method for mass production was a simple and efficient procedure, for the isolation and purification of paclitaxel from the biomass of Taxus chinensis, consisting of solvent extraction, synthetic adsorbent treatment, and two steps of precipitation, followed by two steps of high performance liquid chromatography (HPLC). The organic solvent extraction of biomass obtained crude extract containing paclitaxel. The use of synthetic adsorbent treatment and precipitation in the prepurification process allows for rapid and efficient separation of paclitaxel from interfering compounds and dramatically increases the yield and purity of crude paclitaxel for HPLC purification steps compared to alternative processes. This prepurification process serves to minimise solvent usage, size, and complexity of the HPLC operations for paclitaxel purification. The paclitaxel of over 99.5% purity can be simply obtained with high yield from crude paclitaxel by HPLC using reverse-phase separation on C18 as the first step and normal-phase separation on silica as the second step.     

Drug detection in hair by chromatographic procedures

This article reviews the analysis of 31 drugs and drug metabolites in human hair by thin-layer chromatography, high-performance liquid chromatography, gas chromatography, gas chromatography—mass spectrometry and mass spectrometry. The most important detection method after chromatographic separation of the components is the mass spectrometry because of its sensitivity and specifity. Washing steps to exclude external contamination, extraction, derivatization, stationary phases, detection modes and detection limits of the mass spectrometric and gas chromatographic-mass spectrometric procedures are presented in five tables. Additionally, a method for a gas chromatographic-mass spectrometric screening procedure is presented.