Fluorine-functionalized conjugated microporous polymer as adsorbents for solid-phase extraction of nine perfluorinated alkyl substances.

Perfluorinated alkyl substances (PFASs) are persistent, toxic, ubiquitously distributed, and bioaccumulated substances, which have attracted increasing concern. To investigate the environmental effects of PFASs, there is a need to develop a sensitive, rapid, and efficient method for detecting trace level PFASs. In this study, a conjugated microporous polymer (CMP) with loading of fluorine, fabricated by Sonogashira-Hagihara cross-coupling, was exploited as a solid-phase extraction (SPE) adsorbent. The prepared fluorine-functionalized CMP (FCMP), which showed a large surface area of 1089 m2·g-1, high porosity, and good chemical stability, was used to extract PFASs from water samples. The adsorption mechanism was investigated using a sorption isotherm model, and the main interactions were fluorous and hydrophobic affinity. The FCMP-based SPE combined with high-performance liquid chromatography-tandem mass spectrometry achieved low limits of detection (0.19-0.97 ng·L-1), wide linear range (2-1600 ng·L-1), and good reproducibility (3.4%-12.9%) under the optimal conditions. Furthermore, the approach was utilized for the analysis of three water samples (snow, river water, and irrigation water) to evaluate its reliability, and satisfactory recovery (70.5%-127.5%) was obtained. Thus, FCMP was feasible SPE adsorbents for the selective extraction of PFASs.

Adsorption separation of guanosine 5'-Monophosphate and cytidine 5'-Monophosphate by mixed-mode Resin HD-1: Experimental study and mathematical modeling.

The preparative separation of guanosine 5'-monophosphate (GMP) and cytidine 5'-monophosphate (CMP) on mixed-mode resin HD-1 was experimentally and theoretically investigated. The adsorption mechanisms of the two nucleotides were elucidated by analyzing adsorption equilibria and kinetics at different pH values. The adsorption dynamics of GMP and CMP in a fixed bed packed with resin HD-1 were studied. All nucleotide monovalent cations, zwitterions, and monovalent anions were adsorbed by the resin. Further, a general adsorption isotherm model was developed by considering the adsorption of different nucleotide species and the dissociation equilibrium behaviors of resin ligands. The model fit the adsorption isotherm data of GMP and CMP well. A modified liquid-film linear driving force model with the combined physical adsorption of nucleotides in different dissociation states and ion exchange of Na+ was established. The dissociation equilibria of resin ligands and nucleotides were considered. The model satisfactorily predicted the adsorption kinetic data at different pH values. The values of the efficient diffusion coefficients for GMP and CMP were not significantly influenced by the solution pH. A modified transport-diffusion model with pH gradient elution was proposed. The model accurately predicted the elution chromatographic peaks of GMP and CMP, as well as the pH at the outlet of the fixed bed packed with resin HD-1.

Profile of Nucleotides in Chinese Mature Breast Milk from Six Regions.

This study measured the total potentially available nucleoside (TPAN) content in breast milk from six different regions of China as a part of the Maternal Nutrition and Infant Investigation (MUAI) study. A total of 631 breast milk samples were collected from healthy, lactating women with singleton, full-term pregnancies between 40 and 45 days postpartum in Changchun, Chengdu, Lanzhou, Shanghai, Tianjin, and Guangzhou. TPAN and free 5'-monophosphate nucleotide (5'-MNT) contents were determined by high-performance liquid chromatography. The TPAN content of the Chinese mature milk ranged from 11.61 mg/L to 111.09 mg/L, with a median level of 43.26 mg/L. Four types of nucleotides were identified, and the median levels of cytidine monophosphate (CMP), uridine monophosphate (UMP), guanosine monophosphate (GMP), and adenosine monophosphate (AMP) were 22.84 mg/L, 9.37 mg/L, 4.86 mg/L, and 4.80 mg/L, respectively. CMP was the predominant nucleotide, accounting for 52.9% of the TPAN content, while free 5'-MNT accounted for 18.38% of the TPAN content. The distribution pattern of the TPAN content and level of the individual nucleotides were significantly different among the selected regions (p < 0.05), but the result showed no significant differences in the TPAN level in breast milk (p > 0.05). In addition, no correlation was reported between the geographic distribution and TPAN levels. This result showed that TPAN better reflects the level of total potential nucleosides in Chinese breast milk rather than 5'-MNT in free form. CMP, UMP, GMP, and AMP are the only 4 types of nucleotides reported in all detections. In addition, results revealed a large variation of TPAN levels in Chinese breast milk across six regions, so that the median value may not be the optimal fortification level of TPAN for Chinese infant populations.

Correlating mesophilic counts to the pseudo-CMP content of raw milk / Correlação entre contagem de mesófilos e índice de Pseudo-CMP em leite cru

A presente comunicação objetivou avaliar a quantificação do caseínomacropeptídeo (CMP), bem como diferenciá-lo (devido à adulteração com soro) do pseudo-CMP (devido à proteólise bacteriana) em amostras de leite cru coletadas nos domicílios do sul do Brasil. Os resultados reforçam a necessidade de práticas higiênicas durante a ordenha e estocagem do leite. As amostras de leite estudadas não estavam adulteradas por adição de soro, mostrando que a análise por cromatografia de exclusão por tamanho deve ser complementada a fim de revelar a identidade do peptídeo (CMP ou pseudo-CMP). A contagem bacteriana total (TBC) também se mostrou útil como indicador da contaminação do leite por micro-organismos proteolíticos, uma vez que uma relação diretamente proporcional entre TBC e pseudo-CMP foi estabelecida.(AU)

[Determination of five nucleotides in infant formula milk powder by modified high performance liquid chromatography].

A modified high performance liquid chromatographic (HPLC) method was developed for the determination of the five nucleotides (uridine monophosphate (UMP), adenosine monophosphate (AMP), inosine monophosphate (IMP), guanosine monophosphate (GMP) and cytidine monophosphate (CMP)) in infant formula milk powder. The samples were extracted by water, deproteinized by acetic acid and purified with an HLB SPE cartridge. The analytes were separated by a Waters XBrigde Amide column (150 mm×4.6 mm, 3.5 µ m). Acetonitrile, 10 mmol/L sodium dihydrogen phosphate aqueous solution and 0.12%(v/v) phosphoric acid aqueous solution were used as mobile phases with gradient elution. The detection wavelength of photodiode array detector was set at 254 nm. Five linear calibration curves were obtained with correlation coefficients (r2) of 0.9999. The recoveries were determined at three spiked levels ranging from 86.9% to 105.7%. The limits of quantification (LOQs) were from 5.6 mg/kg to 8.0 mg/kg. The intra-day and inter-day precisions were 0.5%-1.7% (n=5) and 0.6%-1.9% (n=9), respectively. The method is simple, effective, accurate and repeatable. It is suitable for thedetermination of the five nucleotides in infant formula milk powder.

Analysis of Nucleotide 5'-Monophosphates in Infant Formulas by HPLC-UV: Collaborative Study, Final Action 2011.20.

A collaborative study was conducted on AOAC First Action Method 2011.20: 5'-Mononucleotides in Infant Formula and Adult/Pediatric Nutritional Formula. After the successful analysis of National Institute of Standards and Technology (NIST) 1849a Standard Reference Material (SRM) as a practice sample, 12 laboratories participated in the analysis of duplicate samples of six different infant formula products. The samples were dissolved in high-salt solution to inhibit protein and fat interactions, with the nucleotides [uridine 5'-monophosphate (UMP), inosine 5'-monophosphate (IMP), adenosine 5'-monophosphate (AMP), guanosine 5'-monophosphate (GMP), and cytidine 5'-monophosphate (CMP)] separated from the sample matrix by strong-anion exchange SPE, followed by chromatographic analysis using a C18 stationary phase with gradient elution, UV detection, and quantitation by an internal standard technique using thymidine 5'-monophosphate. For nucleotide-supplemented products, precision is within the Standard Method Performance RequirementsSM (SMPR) 2011.008 target reproducibility limit of ≤11%, with the reproducibility RSD (RSDR) estimated at 7.1-8.7% for CMP, 7.9-9.0% for UMP, 2.8-7.7% for GMP, 5.5-10.3% for IMP, and 2.7-6.2% for AMP, and Horwitz ratio (HorRat) values of 0.9-1.0 for CMP, 0.9-1.0 for UMP, 0.3-0.7 for GMP, 0.6-1.0 for IMP, and 0.3-0.7 for AMP.

Analysis of free nucleotide monophosphates in human milk and effect of pasteurisation or high-pressure processing on their contents by capillary electrophoresis coupled to mass spectrometry.

A simple, efficient and green analytical method for the determination of free nucleotide monophosphates in human milk is proposed. It involves centrifugal ultrafiltration (CUF) as sample treatment and capillary electrophoresis-electrospray mass spectrometry (CE-ESI-MS) for separation and simultaneous quantification. The optimised method, applied to the analysis of human milk samples, included their dilution (1:5) with water followed by CUF treatment. No matrix effects were found. The method provided limits of detection between 0.08 and 0.13 µg mL(-1) and limits of quantification between 0.26 and 0.43 µg mL(-1). The intralaboratory repeatability and reproducibility afforded relative standard deviation values lower than 10%. The method was applied to the study of the effects of Holder pasteurisation and high-pressure processing on the nucleotide contents in samples from a human milk bank. The results showed concentration values between 0.5 and 10 µg mL(-1), with higher concentrations for the samples treated by pasteurisation. The effect of freezing time on the content of nucleotides was also assessed.

The role of sodium in the salty taste of permeate.

Many food companies are trying to limit the amount of sodium in their products. Permeate, the liquid remaining after whey or milk is ultrafiltered, has been suggested as a salt substitute. The objective of this study was to determine the sensory and compositional properties of permeates and to determine if elements other than sodium contribute to the salty taste of permeate. Eighteen whey (n=14) and reduced-lactose (n=4) permeates were obtained in duplicate from commercial facilities. Proximate analyses, specific mineral content, and nonprotein nitrogen were determined. Organic acids and nucleotides were extracted followed by HPLC. Aromatic volatiles were evaluated by gas chromatography-mass spectrometry. Descriptive analysis of permeates and model solutions was conducted using a trained sensory panel. Whey permeates were characterized by cooked/milky and brothy flavors, sweet taste, and low salty taste. Permeates with lactose removed were distinctly salty. The organic acids with the highest concentration in permeates were lactic and citric acids. Volatiles included aldehydes, sulfur-containing compounds, and diacetyl. Sensory tests with sodium chloride solutions confirmed that the salty taste of reduced-lactose permeates was not solely due to the sodium present. Permeate models were created with NaCl, KCl, lactic acid, citric acid, hippuric acid, uric acid, orotic acid, and urea; in addition to NaCl, KCl, lactic acid, and orotic acid were contributors to the salty taste.

A validated method for the determination of nucleotides in infant formulas by capillary electrophoresis coupled to mass spectrometry.

In this work CE-ESI-MS is proposed for the identification and simultaneous quantification of several ribonucleotide 5'-monophosphates in infant formula (IF) samples. The target compounds were adenosine 5'-monophosphate, cytidine 5'-monophosphate, guanosine 5'-monophosphate, uridine 5'-monophosphate, and inosine 5'-monophosphate. To our knowledge, the application of CE for the determination of these bioactive compounds in IFs has not yet been described. Optimization of the composition of the electrophoretic separation buffer and -mainly- the injection medium was carried out with a view to obtaining the best sensitivity and separation efficiency for the CE-MS coupling. Different sample treatments were assayed and one based on centrifugal ultrafiltration proved to be the simplest and most compatible with CE separation of the analytes and their ionization by the electrospray source. The whole optimized method (centrifugal ultrafiltration treatment prior to CE-MS) was validated according to the 2002/657/EC decision, obtaining a reliable and robust CE-MS method to determine these compounds in IF samples, with LODs between 0.8 and 1.8 µg/g (S/N = 3) and recoveries in the 90-106% range.

Analysis of genomic methylation level using micellar electrokinetic chromatography with UV detection.

Analytical methods for quantification of 5'-methylcytosine in genomes are important tools to investigate epigenetic changes in gene expression during development, differentiation, aging, or cancer. Here, we report a novel genomic methylation content assay based on enzymatic hydrolysis of DNA and MEKC separation of 5'-deoxyribonucleoside monophosphates (dNMP) using the cationic surfactant CTAB as pseudostationary phase. Calf Thymus DNA was used during method development to determine electrophoretic parameters and electrolyte composition for a complete separation between 2'-deoxycytosine-5'-monophosphate and 2'-deoxy-5'-methylcytosine 5'-monophosphate (d5mCMP). Methylated and not methylated oligonucleotides were used to confirm the identity of each peak and evaluate analytical parameters of the method. The LOD of the method was found to be 12.5 pmol/µL for d5mCMP.

Analysis of 5'-mononucleotides in infant formula and adult/pediatric nutritional formula by liquid chromatography: First Action 2011.20.

A method for the routine determination of 5'-mononucleotides (uridine 5'-monophosphate, inosine 5'-monophosphate, adenosine 5'-monophosphate, guanosine 5'-monophosphate, and cytidine 5'-monophosphate) in infant formula and adult nutritionals is described. After sample dissolution and addition of internal standard, potential interferences were removed by anion-exchange SPE followed by HPLC-UV analysis. Single-laboratory validation performance parameters include recovery (92-101%) and repeatability (1.0-2.3% RSD). The method was approved for Official First Action status by an AOAC expert review panel.

HILIC-MS/MS method for the quantitation of nucleotides in infant formula and adult nutritional formula: First Action 2011.21.

Official Method 2011.21 is for the quantitation of the following nucleotides: adenosine 5'-monophosphate (AMP), guanosine 5'-monophosphate (GMP), uridine 5'-monophosphate (UMP), cytidine 5'-monophosphate (CMP), and inosine 5'-monophosphate (IMP) in infant formula and adult/pediatric nutritional formula. It uses hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS). Preparation of the internal standards was conducted using centrifugal ultrafiltration and the standards are AMP- (13)C10, (15)N5; GMP-(13)C10, (15)N5; UMP-(13)C9, (15)N2; and15 CMP- (13)C9, (15)N3. Data were collected by using multiple reaction monitoring of the product ions of protonated molecules of the five nucleotides generated by positive-electrospray ionization. The HILIC conditions were conducted with ammonium formate (30 mmol/L) in water (pH 2.5, adjusted with formic acid) and methanol. The LOD and LOQ of the standard solution were 0.005-0.01 and 0.01-0.03 microg/mL, respectively. Recovery data were collected for intraday and interday testing and ranged from 98.1 to 108.9% with an RSD of 0.7-5.4%. The analytical range of the method is between 0.04 to 5 microg/mL for standard solution.

[Determination of nucleotides in infant formula milk powder by ion chromatography].

A method was developed for the determination of nucleotides in infant formula milk powder by ion chromatography (IC). The separation was performed on an IonPac AS16 column with KOH solution as the mobile phase at a flow rate of 1.0 mL/min and 25 degrees C. The detection wavelength was set at 260 nm and the sample injection volume was 25 microL. There were good linear relationships between the mass concentrations and the peak areas of cytidine monophosphate (CMP), adenosine monophosphate (AMP), uridine monophosphate (UMP), inosine monophosphate (IMP) and guanosine monophosphate (GMP) in the ranges of 0.09-50, 0.06-50, 0.06-50, 0.09-50, 0.06-50 mg/L, respectively. The limits of detection (S/N = 3) of CMP, AMP, UMP, IMP and GMP were 0.03, 0.02, 0.02, 0.03 and 0.02 mg/L, respectively. The method has been applied for the determination of the five nucleotides in infant formula milk powder with the recoveries of 92.5%-102.4%. This method is rapid, simple and suitable for the determination of real samples.

A liquid chromatographic method for routine analysis of 5'-mononucleotides in pediatric formulas.

An RP-HPLC method for the routine determination of supplemented 5'-mononucleotides (uridine 5'-monophosphate, inosine 5'-monophosphate, adenosine 5'-monophosphate, guanosine 5'-monophosphate, and cytidine 5'-monophosphate) in pediatric formulas and milk products is described. Following sample dissolution, potential interferences were removed by anion-exchange SPE. Chromatographic analyses were performed using a C18 stationary phase with gradient elution, UV detection, and quantitation by an internal standard technique. A single-laboratory validation was performed, with recoveries of 92-101% and repeatability RSDs of 1.0-2.3%. The method was optimized for the rapid, routine analysis of nucleotide-supplemented bovine milk-based infant and follow-on formulas.

Ion-pair high-performance liquid chromatography with diode array detection coupled to dual electrospray atmospheric pressure chemical ionization time-of-flight mass spectrometry for the determination of nucleotides in baby foods.

A liquid chromatography with diode array detection coupled to dual electrospray atmospheric pressure chemical ionization time-of-flight mass spectrometry (HPLC/ESI-APCI-TOF-MS) method is described for the rapid determination of five monophosphate nucleotides (cytidine 5'-monophosphate, uridine 5'-monophosphate, adenosine 5'-monophosphate, inosine 5'-monophosphate and guanosine 5'-monophosphate) in baby foods. The method is based on the deproteinisation of foods and direct analysis of nucleotides by ion-pair HPLC using isocratic elution with a mobile phase of 5% (v/v) methanol and 95% (v/v) 0.1 M formate buffer (pH 5.5) containing 0.01 M N,N-dimethylhexylamine (DMHA) at a flow-rate of 0.7 mL min(-1). The HPLC was hyphenated with two different detection systems, photodiode-array (DAD) and ESI-APCI-TOF-MS in negative mode. The method was validated for linearity, detection and quantitation limits, selectivity, accuracy and precision. The recoveries obtained for spiked samples were satisfactory for all the analytes. The method was successfully applied to the analysis of nucleotides in different baby and/or functional food samples, as cereals, purees and dairy products. A study was also carried out on the stability of nucleotides in acidified dairy infant food with pasteurized yoghourt and follow-on formulae samples stored at room temperature and at 30 degrees C.

Nucleotides and nucleosides in ovine and caprine milk during lactation.

The aim of this study was to determine the nucleoside and nucleotide content in ovine and caprine milks at the colostral, transitional, and mature stages of lactation. Samples from 18 dairy sheep and 18 dairy goats were collected at 1, 2, 3, 4, 5, and 15 d postpartum. Separation and quantitation of the 5'-nucleotides (NT) and the nucleosides (NS) was performed by reverse phase HPLC. For each compound measured, considerable interindividual variation was recorded in both species of milk. The total NS content ranged from 57 to 132 micromol/L and from 54 to 119 micromol/L in ovine and caprine milk, respectively. The major NS identified in both species of milk was uridine, representing more than 60% of the total NS pool. The mean levels of inosine and guanosine were comparable between ewe and goat milk. Instead, the mean level of cytidine across the sampling period was much higher in ewe milk (11.9 micromol/L compared with 4.5 micromol/L in goat milk) and exhibited a peak value on the fourth day of lactation. The adenosine content was at least 3-fold higher in caprine milk compared with its ovine counterpart. The total NS and orotic acid contents did not differ significantly between the 2 species. However, in the case of total NT content, interspecies differences were significant, with NT levels ranging from 294 to 441 micromol/L in ovine milk and from 166 to 366 micromol/L in caprine milk. The NT content in colostrum (1-3 d) of both species was higher than in mature milk (15 d), and uridine monophosphate was the dominant NT in all samples.

The possible role of human milk nucleotides as sleep inducers.

Breast-milk contains a potent mixture of diverse components, such as the non-protein nitrogen fraction which includes nucleotides, whose variation in levels is evident throughout lactation. In addition, these substances play an important role in sleep homeostasis. In the present study, human milk samples were analyzed using a capillary electrophoresis system. The rhythmicity of each nucleotide was studied by cosinor analysis. It was found that the nucleotides 5'AMP, 5'GMP, 5'CMP, and 5'IMP have significant (P < 0.05) circadian rhythms, the acrophases of the first two being during the night, and of the latter two during the day. While 5'UMP did not show a clear circadian rhythm, there was an increase in its levels at night. In conclusion, the rise in nocturnal levels of 5'AMP, 5'GMP, and 5'UMP could be involved in inducing the 'hypnotic' action of breast-milk at night in the infant.

Determination of nucleotides in infant formula by ion-exchange liquid chromatography.

Nucleotide-supplemented infant formula has been shown to positively modify the composition of intestinal microflora, emulating the attribute of human milk. Quantification of nucleotides in infant formula is of interest because of its applicability in quality and safety assessments. There is no standard method for the analysis of nucleotides in infant formula. In the present study, ion-exchange liquid chromatography (IELC)- and centrifugal ultrafiltration (CUF)-based protocols were developed for routine determination of additive nucleotides in infant formula. Five target nucleotides, guanosine 5'-monophosphate (GMP), inosine 5'-monophosphate (IMP), uridine 5'-monophosphate (UMP), cytidine 5'-monophosphate (CMP), and adenosine 5'-monophosphate (AMP) were measured by IELC with a mobile phase of 50 mM diammonium hydrogen phosphate buffer, pH 4.0, with UV detection at 254 nm. The calibration was linear over the range 0.5-50 microg/mL; R(2) = 0.999. The calculated LOD and LOQ were 0.01-0.05 microg/mL and 0.05-0.5 microg/mL, respectively. Recovery values (spiked concentration levels: 0.5, 5, and 10 microg/mL) ranged from 85.0 +/- 1.4% to 92.3 +/- 2.1% using only CUF preparation. This was applied to measure the concentration of five nucleotides in common infant formulas.

Mononuclear phagocytes in the blood of turtles characterized by ultrastructural and cytochemical analyses and by phagocytic activity.

Ultrastructural and cytochemical characteristics of mononuclear phagocyte cells in turtles are not well described in the literature, especially in Phrynops hilarii. Thus, the aim of this study was to evaluate these characteristics in the mononuclear phagocyte cells and their phagocytic activity "in vitro" using the turtle P. hilarii as an experimental animal model. The six turtles used in the study were observed in two seasons, spring and summer. Results showed that mononuclear phagocytes incubated only in diluted solution or with colloidal charcoal have cytoplasm phagolysosomes. The cells incubated with colloidal charcoal and further exposed to the cytochemical reaction for acid beta-glycerophosphatase, showed cytoplasm phagolysosomes filled by charcoal particles being digested and some positively stained lysosomes. Acid beta-glycerophosphatase positive reaction was present in lysosomes and inside the phagolysosomes, while acid cytidine 5-monophosphatase staining occurred in lysosome surroundings. A positive reaction for trimetaphosphatase was also found inside phagolysosomes. In conclusion, the presence of lysosomal enzymes like trimetaphosphatase and cytidine-5'-sodium monophosphate, in the circulating blood of P. hilarii indicate that mononuclear phagocytes participate in the phagocytic process by gathering many phagocytic cells and forming multinucleated giant cells, which probably have a role in the blood "clearance" process.

Separation of cytidine 5'-triphosphate biosynthesized from cytidine 5'-monophosphate on ion-exchange resin and HPLC analysis of cytidine compounds.

Conditions were studied in the biosynthesis of cytidine 5'-triphosphate (CTP) from cytidine 5'-monophosphate (CMP). A 201 x 7 anion ion-exchange resin was applied for the separation of CTP from CMP. Adsorption isotherm and elution conditions (eluant, eluant concentration, flow rate, sample volume loaded) were investigated. At the same time, a new high-performance liquid chromatography on an anion ion-exchange column WAX-1 with UV detector at 260 nm was developed to measure CMP, cytidine 5'-diphosphate (CDP), and CTP. The retention time for CMP, CDP, and CTP are 0.723, 1.448, and 4.432 min, respectively. This new rapid high-performance liquid chromatography (HPLC) method for the analysis of cytidine compounds in biological sample has a wide linear range with high precision and repeatability.