Purification of fatty acid ethyl esters by solid-phase extraction and high-performance liquid chromatography

We have developed a two-step method to purify fatty acid ethyl esters (FAEE) using solid-phase extraction (SPE), with a recovery of 70±3% (mean±S.E.M.) as assessed using ethyl oleate as a recovery marker from a standard lipid mixture in hexane. The first step of the SPE procedure involves application of a lipid mixture to an aminopropyl-silica column with simultaneous elution of FAEE and cholesteryl esters from the column with hexane. Gas chromatographic analysis of FAEE without interference from cholesteryl esters may be performed using the eluate from the aminopropyl-silica column, thus eliminating the need for an octadecylsily (ODS) column in this case. The FAEE can then be separated from the cholesteryl esters, if necessary, by chromatography on an ODS column and elution with isopropanol-water (5:1, v/v). Both the aminopropyl-silica and ODS columns were found to be effective for up to four uses. To permit isolation of specific FAEE species following isolation of total FAEE by the two-step SPE method, we have also developed a purification scheme for individaal FAEE by high-performance liquid chromatography (HPLC). Thus, this simple method allows for reproducible isolation of total FAEE by SPE and isolation of individual FAEE species by HPLC.     

Cold-induced phase separation for the simple and reliable extraction of sex hormones for subsequent LC-MS/MS analysis

Sex hormones, including androgens, estrogens, and progestogens, are important biomarkers for various diseases. Quantification of sex hormones is typically conducted by LC-MS/MS. At present, most methods require liquid-liquid extraction or solid phase extraction for sample preparation. However, these pretreatments are prone to compromise LC-MS/MS throughput. To improve on the current standard practices, we investigated cold-induced phase separation for sex hormone extraction. After protein precipitation with acetonitrile and adjusting the solution constitution with water, samples were stored at ?30°C for 10 min to generate two distinct phases: an acetonitrile-rich layer on top of a water-rich layer. During this process, the hydrophobic sex hormones spontaneously separate into the upper layer. This simple and reliable cold-induced phase separation-based LC-MS/MS methodology was used here to simultaneously detect estrone, estradiol, estriol, testosterone, androstenedione, dehydroepiandrosterone, progesterone, and 17-hydroxyprogesterone in serum. Validation of this method indicated satisfactory performance, including acceptable linearity, accuracy, precision, and tractability. Compared with the mainstream liquid-liquid extraction-based method, this new method exhibits significant progress in throughput, which shortens the time cost of sample preparation from 90 to 40 min. We propose that this method can be an excellent alternative for sex hormone analysis in routine clinical laboratories.     

Floral meristems as a source of enhanced yield and quality of DNA in grasses

Floral meristems of Lolium and Festuca grasses give a 5- to 19-fold enhancement in yield of extracted DNA in comparison with leaves. Meristems also provide highly pure DNA samples. The method could be useful for applications in molecular genetics in many species of the Gramineae. Received: 14 April 1998 / Revision received: 8 June 1998 / Accepted: 10 July 1998     

Analytical tools for the analysis of β-carotene and its degradation products

Abstract

β-Carotene, the precursor of vitamin A, possesses pronounced radical scavenging properties. This has centered the attention on β-carotene dietary supplementation in healthcare as well as in the therapy of degenerative disorders and several cancer types. However, two intervention trials with β-carotene have revealed adverse effects on two proband groups, that is, cigarette smokers and asbestos-exposed workers. Beside other causative reasons, the detrimental effects observed have been related to the oxidation products of β-carotene. Their generation originates in the polyene structure of β-carotene that is beneficial for radical scavenging, but is also prone to oxidation. Depending on the dominant degradation mechanism, bond cleavage might occur either randomly or at defined positions of the conjugated electron system, resulting in a diversity of cleavage products (CPs).

Due to their instability and hydrophobicity, the handling of standards and real samples containing β-carotene and related CPs requires preventive measures during specimen preparation, analyte extraction, and final analysis, to avoid artificial degradation and to preserve the initial analyte portfolio. This review critically discusses different preparation strategies of standards and treatment solutions, and also addresses their protection from oxidation. Additionally, in vitro oxidation strategies for the generation of oxidative model compounds are surveyed. Extraction methods are discussed for volatile and non-volatile CPs individually. Gas chromatography (GC), (ultra)high performance liquid chromatography (U)HPLC, and capillary electrochromatography (CEC) are reviewed as analytical tools for final analyte analysis. For identity confirmation of analytes, mass spectrometry (MS) is indispensable, and the appropriate ionization principles are comprehensively discussed. The final sections cover analysis of real samples and aspects of quality assurance, namely matrix effects and method validation.     

The Use of Fungal Biomass Agaricus bisporus Immobilized on Amberlite XAD-4 Resin for the Solid-Phase Preconcentration of Thorium

Solid-phase extraction method was developed for the preconcentration of thorium (Th). Fungal biomass Agaricus bisporus was immobilized to Amberlite XAD-4 as solid-phase sorbent. The critical parameters such as pH of the sample solution, flow rate of the sample, volume of the sample, and the effect of major ions that affect the preconcentration of thorium in this system were evaluated. The optimum pH for the sorption of Th is 6.0, and quantitative elution occurs with 1.0 mol L^?1HCl. The loading capacity was determined as 0.079 mmol g^?1. The optimized method was validated through analysis of the certified reference material of tea leaves (NCS ZC73014) and successfully applied to the determination of Th in a real ore sample with satisfactory results.     

Evaluation of methods for extraction of bacteria from soil

Abstract Several methods for dispersion of soil were tested for possible use in procedures for extraction of bacteria. Physical cell damage on cells and efficiency in extraction of indigenous cells from soil, were investigated. Cell damage by the dispersion methods was investigated by measuring the physical cell integrity and viability of pure cultures of Escherichia coli and Bacillus subtilis , as well as soil bacteria extracted from soil, when dispersed in slurries of γ-sterilized soil. Separation of bacteria and soil particles on the basis of buoyant density was conducted with the nonionic density gradient medium Nycodenz. When slurries of γ-sterilized soil with added pure cultured cells were centrifuged (10000 × g ) over cushions of Nycodenz (1.3 g ml⁻¹), practically all the added cells were recovered in a layer on top of the cushion. This proves that a reversible attachment and cosedimentation is not an important phenomenon in this procedure. The efficiency of the different dispersion methods for the extraction of indigenous soil bacteria, was assessed after separation of dislodged and attached soil bacteria. This separation was done either on the basis of sedimentation rate by low speed centrifugation, or buoyant density by Nycodenz density gradient centrifugation. The physical dispersion by ultrasonic treatment and chemical dispersion by the use of a chelating agent together with a detergent, were inferior to physical dispersion either by Waring blender (for large volumes) or a rotating rubber pestle treatment (for smaller volumes). The physical dispersion did not appear to be destructive to the cells tested.     

Reactivation of Horseradish peroxidase in organic solvent using extraction

A novel extraction method was developed aiming at increasing the stability of enzymes in organic solvent media. Horseradish peroxidase (HRP), inactivated in a tetrahydrofuran (THF)/water (1:1, v/v), regained and maintained its activity when HRP was extracted by adding a THF/benzene mixture to the original solution. However, the HRP activity was drastically lowered in the enzyme-free blank solution that had been formed by employing the same extraction procedure. As a result, the reactivation after the extraction is believed to depend on enzyme history, and might be arisen from an irreversible structural change of the enzyme.     

Extracting the time-dependent transmission rate from infection data via solution of an inverse ODE problem

The transmission rate of many acute infectious diseases varies significantly in time, but the underlying mechanisms are usually uncertain. They may include seasonal changes in the environment, contact rate, immune system response, etc. The transmission rate has been thought difficult to measure directly. We present a new algorithm to compute the time-dependent transmission rate directly from prevalence data, which makes no assumptions about the number of susceptible or vital rates. The algorithm follows our complete and explicit solution of a mathematical inverse problem for SIR-type transmission models. We prove that almost any infection profile can be perfectly fitted by an SIR model with variable transmission rate. This clearly shows a serious danger of overfitting such transmission models. We illustrate the algorithm with historic UK measles data and our observations support the common belief that measles transmission was predominantly driven by school contacts.