Development of a reliable analytical method to determine lipid peroxidation biomarkers in newborn plasma samples.

This paper describes a reliable analytical method based on ultra-performance liquid chromatography coupled to tandem mass spectrometry to determine F2-isoprostanes and other total byproducts (isoprostanes, isofurans, neuroprostanes and neurofurans) as lipid peroxidation biomarkers in newborn plasma samples. The proposed procedure is characterized by a simple sample treatment employing a reduced sample volume (100µL). Also, it shows a high throughput and high selectivity to determine simultaneously different isoprostane isomers in a large number of samples. The reliability of the described method was demonstrated by analysis of spiked plasma samples, obtaining recoveries between 70% and 130% for most of the analytes. Taking into account the implementation of further clinical studies, it was demonstrated the proper sensitivity of the method by means of the analysis of few human newborn plasma samples. In addition to this, newborn piglet plasma samples (n=80) were analyzed observing that the developed method was suitable to determine the analyte levels present in this kind of samples. Therefore, this analytical method could be applied in further clinical research about establishment of reliable lipid peroxidation biomarkers employing this experimental model.

Ultra high performance liquid chromatography coupled to tandem mass spectrometry determination of lipid peroxidation biomarkers in newborn serum samples.

Byproducts of arachidonic (AA) and docosahexaenoic acid (DHA) oxidation are highly relevant for the study of free radical associated conditions in the perinatal period. Plasma metabolites can provide the clinician with a snapshot of the oxidant status of patients before and after specific clinical interventions (e.g.: supplementation with oxygen). We describe a new andreliable ultra-performance liquid mass spectrometry method to determine F2-isoprostanes and other byproducts (isoprostanes, isofurans, neuroprostanes, neurofurans) in newborn serum samples. Cord blood samples were obtained from severely depressed newborn infants (Apgar score 1 min < 3; arterial cord pH < 7.00), and aliquoted for serum determination and stored at -80 °C. A UHPLC-MS/MS method was employed. It has a series of technical advantages: simple sample treatment; reduced sample volume (100 µL) which is essential for preterm neonates with low circulating blood volume, high throughput of sample analysis (96 samples in less than 24 h) and high selectivity for different isoprostanes isomers. Excellent sensitivity was achieved within limits of detection between 0.06 and 4.2 nmol L(-1), which renders this method suitable to monitoranalyte concentration in newborn samples. The method's precision was satisfactory; with coefficients of variation around 5-12% (intra-day) and 7-17% (inter-day). The reliability of the described method was assessed by analysis of spiked serum samples obtaining recoveries between 70% and 120%. The proposed method has rendered suitable for serum determination for newborn babies at risk of oxygen free radical associated conditions.

Detection of batch effects in liquid chromatography-mass spectrometry metabolomic data using guided principal component analysis.

Metabolomics based on liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for studying dynamic responses of biological systems to different physiological or pathological conditions. Differences in the instrumental response within and between batches introduce unwanted and uncontrolled data variation that should be removed to extract useful information. This work exploits a recently developed method for the identification of batch effects in high throughput genomic data based on the calculation of a δ statistic through principal component analysis (PCA) and guided PCA. Its applicability to LC-MS metabolomic data was tested on two real examples. The first example involved the repeated analysis of 42 plasma samples and 6 blanks in three independent batches, and the second data set involved the analysis of 101 plasma and 18 blank samples in a single batch with a total runtime of 50h. The first and second data set were used to evaluate between and within-batch effects using the δ statistic, respectively. Results obtained showed the usefulness of using the δ statistic together with other approaches such as summary statistics of peak intensity distributions, PCA scores plots or the monitoring of IS peak intensities, to detect and identify instrumental instabilities in LC-MS.

Science based calibration for the extraction of 'analyte-specific' HPLC-DAD chromatograms in environmental analysis.

Multivariate science based calibration (SBC) has been applied to the resolution of overlapped peaks in liquid chromatography with diode array detection (LC-DAD). Complex river water samples spiked with 11 pharmaceutical substances resulted in poorly resolved chromatograms containing additional peaks from interfering matrix compounds and a change in the background absorbance due to the mobile phase gradient. Applying the present multivariate approach it was possible to resolve all 11 analytes from overlapping peaks, obtaining linear calibration lines (R(2)>0.96). Recovery percentages on spiked samples ranged between 74.6 and 113.5%, which are quite satisfactory taking into account the low concentration ranges considered to 1-7 µg L(-1).

Application of point-to-point matching algorithms for background correction in on-line liquid chromatography-Fourier transform infrared spectrometry (LC-FTIR).

A new background correction method for the on-line coupling of gradient liquid chromatography and Fourier transform infrared spectrometry has been developed. It is based on the use of a point-to-point matching algorithm that compares the absorption spectra of the sample data set with those of a previously recorded reference data set in order to select an appropriate reference spectrum. The spectral range used for the point-to-point comparison is selected with minimal user-interaction, thus facilitating considerably the application of the whole method. The background correction method has been successfully tested on a chromatographic separation of four nitrophenols running acetonitrile (0.08%, v/v TFA):water (0.08%, v/v TFA) gradients with compositions ranging from 35 to 85% (v/v) acetonitrile, giving accurate results for both, baseline resolved and overlapped peaks.

Determination of glycolic acid in cosmetics by online liquid chromatography-Fourier transform infrared spectrometry.

An isocratic online liquid chromatography Fourier transform infrared procedure has been developed for the determination of glycolic acid in cosmetics. The method involves the ultrasound-assisted extraction of glycolic acid from the samples with an acetonitrile:phosphate buffer (25 mM, pH 2.7) (3:97 v/v). The extracts were centrifuged and filtered before their injection into the chromatography system, which was equipped with a C18 column and used a flow rate of 150 microL min(-1). FTIR spectra were acquired using a time-resolved rapid scan mode. To calculate the chromatograms, the spectral area was integrated between 1288 and 1215 cm(-1), with baseline correction established between 1319 and 1150 cm(-1), after correcting for the eluent spectral background. Peak area values of the extracted sample chromatograms were interpolated from an external calibration curve. The method provided a limit of detection of 0.034 mg mL(-1) and a relative standard deviation of 6% for five measurements at the 0.174 mg mL(-1) concentration level. Recovery values obtained by spiking 400 mg of three commercially available samples with amounts of glycolic acid from 3.7 to 9.8 mg ranged between 99.6 and 101%. The results obtained for the commercial samples agree well with their declared concentrations. An attempt to directly determine glycolic acid by attenuated total reflectance measurements using partial least squares calibration showed that results were strongly influenced by compounds coextracted from the matrix.

Determination of lecithin and soybean oil in dietary supplements using partial least squares-Fourier transform infrared spectroscopy.

Lecithin and soybean oil in dietary supplements were determined by Fourier transform infrared spectrometry transmission measurements in dichloromethane in combination with a partial least squares (PLS) regression. Two different PLS models were developed, using 16 synthetic mixtures of analytes in dichloromethane, making measurements in the spectral range from 931.8 to 1252.3 cm(-1) for lecithin and from 911.4 to 1246.9 cm(-1) and 1695.3 to 1774.5 cm(-1) for soybean oil. Seven products from the Spanish market with lecithin concentrations between 21.1% and 99.1% and soybean oil concentrations between 0% and 37.2% were analyzed by the proposed method and the data was compared to a chromatographic reference procedure obtaining accurate results. For samples spiked with amounts between 50 and 250 mg of lecithin and soybean oil recovery percentages between 98.0% and 102.1% and between 93.6% and 102.0% with an average precision of 0.35% and 0.41% were achieved for lecithin and soybean oil, respectively. This method can be applied for the quality control of dietary supplements.

Determination of critical eluent composition for polyethylenglycols using on-line liquid chromatography-Fourier transform infrared spectrometry.

In this work, it has been extended to methanol:water mobile phases, the use of a background correction method for on-line LC-FTIR measurements named Univariate background correction based on the use of a reference spectra matrix (UBC-RSM) and absorbance ratios. It permits to overcome the problem related to spectral changes occurring during the gradient elution, which in the past limited the on-line coupling of LC and FTIR to isocratic elutions. The combined use of the aforementioned background correction technique in on-line isocratic and gradient LC-FTIR, and partial least squares (PLS) has been applied for the search of the critical conditions for polymers. Polyethylenglycol (PEG) has been used as a model example and results found fitted well with previously published ones.

On-line gel permeation chromatography-attenuated total reflectance-Fourier transform infrared determination of lecithin and soybean oil in dietary supplements.

Gel permeation chromatography (GPC) with attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrometry detection has been proposed for the simultaneous determination of lecithin and soybean oil in dietary supplements. The method involves the extraction of analytes with dichloromethane in an ultrasound water bath and the injection of 2 ml of centrifuged and filtered extracts into the system integrated by two Envirogel GPC columns (19 mm x150 mm, 19 mm x 300 mm) coupled on-line. Dichloromethane was used as mobile phase. A method has been developed to select the most appropriated wavenumber to be used for the determination of each considered compound from the calculation of a factor which maximizes the analyte signal minimizing the interferent contributions, being selected the detection wavenumbers of 1034 and 1138 cm(-1) for lecithin and soybean oil, respectively in the first order derivative ATR-FTIR spectra. The method provides limits of detection of 2 and 4 mg ml(-1) for lecithin and soybean oil and repeatability values, measured as relative standard deviation, of 2.5% and 3.4% being extended the linear range till 100 mg ml(-1) for lecithin and up to 50 mg ml(-1) for soybean oil. Accurate results were found for 10 synthetic samples and 7 commercial dietary supplement preparations.

Stainless steel cookware as a significant source of nickel, chromium, and iron.

Stainless steels are widely used materials in food preparation and in home and commercial cookware. Stainless is readily attacked by organic acids, particularly at cooking temperatures; hence iron, chromium, and nickel should be released from the material into the food. Nickel is implicated in numerous health problems, notably allergic contact dermatitis. Conversely, chromium and iron are essential nutrients for which stainless could be a useful source. Home cookware was examined by atomic absorption spectroscopy: seven different stainless utensils as well as cast iron, mild steel, aluminum and enamelled steel. The materials were exposed to mildly acidic conditions at boiling temperature. Nickel was a major corrosion product from stainless steel utensils; chromium and iron were also detected. It is recommended that nickel-sensitive patients switch to a material other than stainless, and that the stainless steel cookware industry seriously consider switching to a non-nickel formulation.

[Ultrastructural changes in nucleoli during embryogenesis of Marsilea vestita]. / Ultrastructural changes in nucleoli during embryogenesis of Marsilea vestita.

The nucleoli of oospheres and young embryos of Marsilea vestita exhibited a typical ultrastructure with segregation into several compact nucleolus-like bodies and small granules of similar appearance. When embryos reached the 16 cell stage, the granules disappeared and the nucleolar-like bodies changed into classical nucleolar components. When the embryos were submitted to hypothermic shock, the nucleoli returned to the characteristic ultrastructure of the very young stages. In ageing oospheres the nucleus was full of very compact clumps. No detectable RNA synthesis occurred in the oospheres and young embryos, and the same phenomenon applied in ageing oospheres and after cold shock treatment. Transcriptional activity started with the 8-16 cell stage in the reference embryos. These results suggest that nucleolar ultrastructure is correlated with rRNA synthesis.

Stored mRNA in early embryos of a fern Marsilea vestita: a paternal and maternal origin.

In early embryos of the fern Marsilea vestita, we have shown that transcription did not begin until the 8-cell stage, even though protein synthesis was taking place. As in animal species, the pre-existence of stored maternal mRNA has been suspected in embryonic cells. To investigate the presence of such long-lived mRNA we have used in situ hybridization method using [3H]polyuridylic acid, [3H]poly(U), as a probe. Temporal and spatial changes in the distribution of poly(A)+ RNA were followed throughout all the different phases of embryogenesis. Poly(A)+ RNA was present in the cytoplasm of both unfertilized and fertilized eggs and early embryos, while the nucleus exhibited no, or a moderate, [3H]-poly(U) binding activity. New poly(A)+ RNA was detected in the nucleus only at the 16-cell stage, when cytological differentiation of the root apical cell became morphologically detectable. After in situ hydridization with the labeled probes, the numerous silver grains detected autoradiographically over the mature sperm, indicate the presence of poly(A)+ RNA molecules associated with the male genome. We discuss the possible role of poly(A)+ RNA of maternal origin in supporting early translation prior to synthesis of new mRNA. Additional studies are needed to elucidate the role of stored paternal mRNA.

Fertilization of a colchicine-treated gamete of the fern Marsilea vestita.

In the fern, Marsilea vestita, the addition of colchicine to the culture medium during the final stages of archegogenesis gives rise to a new distribution of cellular organelles and lipid droplets within the egg cytoplasm. These changes affect primarily the usually free space through which the spermatozoid moves towards the female nucleus. Depending on the length of the treatment this fertilization cone may disappear. Completion of fertilization, i.e. karyogamy, is only observed in the presence of this cone. When it is missing, progress of the male nucleus, which is no longer directly channeled towards the female nucleus, is notably slowed down and karyogamy does not occur. Then, male chromatin decondensation takes place in the egg cytoplasm. This event enables us to describe the structure of the sperm nucleus: it is composed of two cylindrical parallel units which, subsequently, break up into short fragments which may be the "male chromosomes".