Co-culturing Propionibacterium freudenreichii and Bifidobacterium animalis subsp. lactis improves short-chain fatty acids and vitamin B12 contents in soy whey.

The lack of vitamin B12 in unprocessed plant-based foods can lead to health problems in strict vegetarians and vegans. The main aim of this study was to investigate the potential synergy of co-culturing Bifidobacterium animalis subsp. lactis and Propionibacterium freudenreichii in improving production of vitamin B12 and short-chain fatty acids in soy whey. Different strategies including mono-, sequential and simultaneous cultures were adopted. Growth, short-chain fatty acids and vitamin B12 were assessed throughout the fermentation while free amino acids, volatiles, and isoflavones were determined on the final day. P. freudenreichii monoculture grew well in soy whey, whereas B. lactis monoculture entered the death phase by day 4. Principal component analysis demonstrates that metabolic changes in both sequential cultures did not show drastic differences to those of P. freudenreichii monoculture. However, simultaneous culturing significantly improved vitamin B12, acetic acid and propionic acid contents (1.3 times, 5 times, 2.5 times, compared to the next highest treatment [sequential cultures]) in fermented soy whey relative to other culturing modes. Hence, co-culturing of P. freudenreichii and B. lactis would provide an alternative method to improve vitamin B12, acetic acid and propionic acid contents in fermented foods.

Deep-Learning-Assisted Discriminative Detection of Vitamin B12 and Vitamin B9 by Fluorescent MoSe2 Quantum Dots.

A facile and environmentally mindful approach for the synthesis of MoSe2 QDs was developed via the hydrothermal method from bulk MoSe2. In this, the exfoliation of MoSe2 was enhanced with the aid of an intercalation agent (KOH), which could reduce the exfoliation time and increase the exfoliation efficiency to form MoSe2 QDs. We found that MoSe2 QDs display blue emission that is suitable for different applications. This fluorescence property of MoSe2 QDs was harnessed to fabricate a dual-modal sensor for the detection of both vitamin B12 (VB12) and vitamin B9 (VB9), employing fluorescence quenching. We performed a detailed study on the fluorescence quenching mechanism of both analytes. The predominant quenching mechanism for VB12 is via Förster resonance energy transfer. In contrast, the recognition of VB9 primarily relies on the inner filter effect. We applied an emerging and captivating approach to pattern recognition, the deep-learning method, which enables machines to "learn" patterns through training, eliminating the need for explicit programming of recognition methods. This attribute endows deep-learning with immense potential in the realm of sensing data analysis. Here, analyzing the array-based sensing data, the deep-learning technique, "convolution neural networks", has achieved 93% accuracy in determining the contribution of VB12 and VB9.

A robust method for simultaneous determination of eight B vitamins in human serum by liquid chromatography tandem mass spectrometry.

The level of vitamin B group in human serum is an important index of human health. Among B vitamins, cyanocobalamin in serum is unstable and its content is extremely low. Rapid and simultaneous detection of multiple B vitamins including cyanocobalamin is a challenge. Herein, we have developed a rapid and stable method that can realize the determination of thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxic acid, biotin, 5-methyltetrahydrofolate, and cyanocobalamin simultaneously in 6 min. The method was established based on protein precipitation with methanol and then chromatographic separation was achieved using Waters acquity ultra-high-performance liquid chromatography high strength silica T3 column, which was stable and sensitive especially for cyanocobalamin. Limit of quantification, precision, trueness, and matrix effect were validated according to the European Medicines Agency and United States Food and Drug guidelines and Clinical and Laboratory Standards Institute guidelines on bioanalytical method. The limit of quantification for thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxic acid, biotin, 5-methyltetrahydrofolate, and cyanocobalamin was 0.4, 0.4, 0.8, 2.0, 0.4, 0.1, 0.4, and 0.04 ng/mL separately, respectively. Intra- and interday precisions were 1.1%-12.4% and 2.0%-13.5%, respectively. The relative errors were between 0.3% and 13.3%, and the matrix effects were between 2.6% and 10.4%.

Challenges in the determination of total vitamin B12 by cyanidation conversion: insights from stable isotope dilution assays.

Previous methods for vitamin B12 (B12) analysis have extensively used cyanidation conversion with the intention of converting all cobalamins to cyanocobalamin (CNCbl) for total B12 determination. This approach has been favored for its advantages in reducing the number of analytes, increasing analyte concentration, and improving analyte stability. However, the present study revealed underlying limitations associated with this approach. First, a stable isotope dilution assay (SIDA) determining total B12 as CNCbl after cyanidation conversion (conversion SIDA method) was developed. Method validation demonstrated good sensitivity, recovery, accuracy, and reproducibility for the target analyte CNCbl. However, subsequent application of the conversion method to real meat samples showed incomplete conversions of cobalamins. These inconsistencies revealed day-to-day variability and reliability challenges associated with the cyanidation process. It was not possible to identify this issue during method validation as CNCbl was spiked as the sole analyte and it requires no further cyanidation conversion. The application of LC-MS/MS enabled the detection of trace amounts of unconverted cobalamins. Nevertheless, this approach remains restricted by instrument sensitivity and stability as well as the performance of immunoaffinity purification for different vitamers. Further development of a reliable monitoring method is a prerequisite for further optimization of the cyanidation process. However, significant improvements of analytical instrumentation in terms of sensitivity and stability are required to overcome the current limitations.

Determination of Vitamin B12 and Folate Compounds in Commercially Available Edible Seaweed Products.

BACKGROUND: Information on the contents of both vitamin B12 and folate in edible seaweeds is limited, of which deficiencies disrupt methionine biosynthesis to accumulate homocysteine as a risk factor of cardiovascular diseases. METHODS: Both vitamins were determined in commercially available edible seaweed products using high-performance liquid chromatography. RESULTS: Dried purple laver (Neopyropia yezoensis) products contain higher levels of vitamin B12 (approximately 30-60 µg/100 g dry weight) and folate compounds (approximately 880-1300 µg/100 g dry weight) than other seaweed products, such as kombu (Saccharina japonica), hijiki (Sargassum fusiformis), and wakame (Undaria pinnatifida). 5-methyltetrahydrofolate was the major folate compound in purple laver products. 5-formyltetrahydrofolate was found at a moderate level, whereas tetrahydrofolate, 5,10-metenyltetrahydrofolate, 10-formyltetrahydrofolate, and folic acid were found to be minor folate compounds. CONCLUSIONS: These findings suggest that dried purple laver (nori) products are suitable sources of vitamin B12 and folate compounds for humans, especially vegetarians.

Review: State of the knowledge on the importance of folates and cobalamin for dairy cow metabolism.

Synthesis of B vitamins by the rumen microbiota is usually sufficient to avoid the appearance of clinical deficiency symptoms in dairy cows under normal feeding conditions. Nevertheless, it is now generally accepted that vitamin deficiency is much more than the appearance of major functional and morphological symptoms. Subclinical deficiency, which is present as soon as the supply is lower than the need, causes cellular metabolic changes leading to a loss of metabolic efficiency. Folates and cobalamin, two B vitamins, share close metabolic relationships. Folates act as co-substrates in one-carbon metabolism, providing one-carbon unit for DNA synthesis and de novo synthesis of methyl groups for the methylation cycle. Cobalamin acts as a coenzyme for reactions in the metabolism of amino acids, odd-numbered chain fatty acids including propionate and de novo synthesis of methyl groups. Both vitamins are involved in reactions to support lipid and protein metabolism, nucleotide synthesis, methylation reactions and possibly, maintenance of redox status. Over the last decades, several studies have reported the beneficial effects of folic acid and vitamin B12 supplements on lactation performance of dairy cows. These observations indicate that, even when cows are fed diets adequately balanced for energy and major nutrients, B-vitamin subclinical deficiency could be present. This condition reduces casein synthesis in the mammary gland and milk and milk component yields. Folic acid and vitamin B12 supplements, especially when given together, may alter energy partitioning in dairy cows during early and mid-lactation as indicated by increased milk, energy-corrected milk, or milk component yields without affecting DM intake and BW or even with reductions in BW or body condition loss. Folate and cobalamin subclinical deficiency interferes with efficiency of gluconeogenesis and fatty acid oxidation and possibly alters responses to oxidative conditions. The present review aims to describe the metabolic pathways affected by folate and cobalamin supply and the consequences of a suboptimal supply on metabolic efficiency. The state of knowledge on the estimation of folate and cobalamin supply is also briefly mentioned.

Production and characterization of cyanocobalamin-enriched tomato (Solanum lycopersicum) fruits grown using hydroponics.

BACKGROUND: Vitamin B12 is an essential vitamin that is absent in plant-derived foods such as fruits and vegetables. This can result in an increased risk of developing vitamin B12 deficiency in strict vegetarians (vegans). There are several studies that have aimed to enhance nutrients in food crops. The purpose of the present study was to fortify tomato fruits with vitamin B12 (or cyanocobalamin). RESULTS: Tomato plants were grown for 70 days in hydroponic culture pots and treated with 5 µm of cyanocobalamin on days 1-24 after the fruiting, and then harvested for tomato fruits. The ripened tomato fruits contained 4.0 × 10-7  g of cyanocobalamin per 100 g of dry weight and showed a significant increase in glucose and lycopene levels. CONCLUSION: The present study highlights the use of a cyanocobalamin-supplementation system for the production of B12 fortified tomato fruits that can help prevent B12 deficiency in vegetarians. © 2022 Society of Chemical Industry.

Method optimization of the simultaneous detection of B12 congeners leading to the detection of a novel isomer of hydroxycobalamin in seawater.

RATIONALE: More than half of surveyed microalgae and over 90% of harmful algae have an obligate requirement for vitamin B12 , but methods for directly measuring dissolved B12 in seawater are scarce due to low concentrations and rapid light-induced hydrolysis. METHODS: We present a method to detect and measure the four main congeners of vitamin B12 dissolved in seawater. The method includes solid-phase extraction, separation by ultrahigh-performance liquid chromatography and detection by triple-quadrupole tandem mass spectrometry utilizing an electrospray ion source. This method was applied to coastal field samples collected in the German Bay, Baltic Sea and the Danish Limfjord system. RESULTS: The total dissolved B12 pool ranged between 0.5 and 2.1 pM. Under ambient conditions methyl-B12 and adenosyl-B12 were nearly fully hydrolyzed to hydroxy-B12 in less than 1 h. Hydroxy-B12 and a novel, corresponding isomer were the main forms of B12 found at all field sites. This isomer eluted well after the OH-B12 peak and was also detected in commercially available OH-B12 . Both compounds showed very high similarity in their collision-induced dissociation spectra. CONCLUSIONS: The high instability of the biologically active forms of Me-B12 and Ado-B12 towards hydrolysis was shown, highlighting the importance of reducing the duration of the extraction protocol. In addition, the vitamin B12 pool in the study area was mostly comprised of a previously undescribed isomer of OH-B12 . Further studies into the structure of this isomer and its bioavailability are needed.

Combination of high performance liquid chromatography and flame atomic absorption spectrophotometry using a novel nebulizer interface supported T shaped slotted quartz tube for the determination of Vitamin B12.

A novel nebulizer interface (NI) was proposed to combine high performance liquid chromatography (HPLC) and flame atomic absorption spectrophotometer (FAAS). A glass concentric nebulizer was linked to T-shaped slotted quartz tube (T-SQT) using a tubing to transfer the liquid solution eluted from the chromatographic system into the atomization region of FAAS system. T-SQT was also used to intensify the interaction of atoms with the hollow cathode lamp light. Vitamin B12 was selected as an analyte to show the applicability of the new hyphenated system. After optimizing some parameters such as mobile phase flow rate and pH, nebulizer gas flow rate, T-SQT height and injection volume, linear range for the analyte was determined between 4.7 and 92 mg/kg as Co. Limit of detection (LOD) and limit of quantitation (LOQ) for the HPLC-NI-T-SQT-FAAS system were calculated to be 1.6 and 5.3 mg/kg as Co, respectively. Recovery studies were also conducted to verify the accuracy and applicability of the developed method for vitamin tablets and excellent percent recovery results (~ 100%) with low standard deviation values were obtained when matrix-matching calibration strategy was performed for each vitamin tablet. A successful separation and detection of the analyte was achieved within 3.0 min that offers high sample throughput. Two different vitamin tablets were analyzed by the optimized hyphenated system. The developed method also provides low usage of sample solution in contrast to conventional nebulizer in the FAAS system.

Exploring Mechanisms of Biotic Chlorinated Alkane Reduction: Evidence of Nucleophilic Substitution (SN2) with Vitamin B12.

Chlorinated alkanes are notorious groundwater contaminants. Their natural reductive dechlorination by microorganisms involves reductive dehalogenases (RDases) containing cobamide as a cofactor. However, underlying mechanisms of reductive dehalogenation have remained uncertain. Here, observed products, radical trap experiments, UV-vis, and mass spectra demonstrate that (i) reduction by cobalamin (vitamin B12) involved chloroalkyl-cobalamin complexes (ii) whose formation involved a second-order nucleophilic substitution (SN2). Dual element isotope analysis subsequently linked insights from our model system to microbial reductive dehalogenation. Identical observed isotope effects in reduction of trichloromethane by Dehalobacter CF and cobalamin (Dehalobacter CF, εC = -27.9 ± 1.7‰; εCl = -4.2 ± 0.‰; λ = 6.6 ± 0.1; cobalamin, εC = -26.0 ± 0.9‰; εCl = -4.0 ± 0.2‰; λ = 6.5 ± 0.2) indicated the same underlying mechanism, as did identical isotope effects in the reduction of 1,2-dichloroethane by Dehalococcoides and cobalamin (Dehalococcoides, εC = -33.0 ± 0.4‰; εCl = -5.1 ± 0.1‰; λ = 6.5 ± 0.2; cobalamin, εC = -32.8 ± 1.7‰; εCl = -5.1 ± 0.2‰; λ = 6.4 ± 0.2). In contrast, a different, non-SN2 reaction was evidenced by different isotope effects in reaction of 1,2-dichloroethane with Dehalogenimonas (εC = -23.0 ± 2.0‰; εCl = -12.0 ± 0.8‰; λ = 1.9 ± 0.02) illustrating a diversity of biochemical reaction mechanisms manifested even within the same class of enzymes (RDases). This study resolves open questions in our understanding of bacterial reductive dehalogenation and, thereby, provides important information on the biochemistry of bioremediation.

Effects of dietary supplementation of different levels of vitamin B12 on the liver metabolism of laying hens.

BACKGROUND: Vitamin B12 plays an important role in lipid, protein, carbohydrate and nucleic acid metabolism. We investigated the effect of supplementing layers' diets with different vitamin B12 levels on liver metabolism using a liquid chromatography-mass spectrometry-based metabolomic approach to observe and analyse wide-target metabolomics in the liver. RESULTS: We assigned hens to three groups, namely blank control group without vitamin B12 diet (BCG), normal control group with 25 µg kg-1 vitamin B12 (NCG) and vitamin B12 supplement group I with 100 µg kg-1 vitamin (VBSG I). The VBSG I group layers had higher (P < 0.05) vitamin B12 concentration than those from other groups. The egg yolk vitamin B12 concentration increased (P < 0.01) with the increasing vitamin B12 dietary supplemental level. Between the NCG versus BCG, VBSG I versus BCG, and VBSG I versus NCG groups, 11, 20 and 11 metabolites were significantly changed, respectively. The KEGG pathway of vitamin B6 metabolism was significantly impacted in the NCG layers than those from BCG; seven and five pathways were significantly impacted in the VBSG I layers compared with those from BCG and NCG, including pyrimidine metabolism, vitamin B6 metabolism, glycerophospholipid metabolism, etc. CONCLUSION: We concluded that 25 µg kg-1 vitamin B12 supplementation in corn-soybean meal-based layer diet increased the egg yolk vitamin B12 concentration and impacted the vitamin B6 metabolic pathway, and 100 µg kg-1 of it increased the egg yolk and liver vitamin B12 concentrations and impacted vitamin B6 , lipid, nucleic acid and amino acid metabolic pathways. © 2022 Society of Chemical Industry.

One-pot room temperature synthesis of orange-emitting carbon dots for highly-sensitive vitamin B12 sensing.

Fluorescent carbon dots (CDs) with the long-wavelength emission have received increasing attention due to their promising application prospects in the biological field. The synthesis of CDs with long wavelength emission mainly focuses on the high temperature method, but the room temperature synthesis is still rarely studied. Herein, a simple room temperature strategy is developed for the preparation of orange-emitting CDs (O-CDs) by Schiff base crosslinking reaction between methyl-p-benzoquinone and triethylenetetramine. The proposed O-CDs show a strong excitation-dependent emission with a relative quantum yield of about 6.56%. Because of the strong inner filter effect, O-CDs can be well applied for the highly-sensitive detection of vitamin B12 (VB12). The intensity ratio (F/F0) of O-CDs is linear against the concentration of VB12 from 50 nM to 200 µM with a low detection limit of 10 nM. Therefore, the obtained O-CDs nanoprobes provide a promising platform for pharmaceutical analysis applications.

Cross-feeding among microalgae facilitates nitrogen recovery at low C/N.

Although co-culture of microalgae has been found as a feasible strategy to improve biomass production, their interspecies relationships are not fully understood. Here, two algae taxa, Chlorella sp. and Phormidium sp., were mono-cultured and co-cultured in three photobioreactors for 70 days with periodically harvesting to investigate how dual-species interaction influence nitrogen recovery. Results showed that the co-culture system achieved a significantly higher protein production and nitrogen removal rate than those in the individual cultures at a C/N ratio of 3:1 (p < 0.05). Genome-Centered metagenomic analysis revealed their cooperative relationship exemplified by cross-feeding. Phormidium sp. had the ability to synthesize pseudo-cobalamin, and Chlorella sp. harbored the gene for remodeling the pseudo-cobalamin to bioavailable vitamin B12. Meanwhile, Chlorella sp. could contribute the costly amino acid and cofactors for Phormidium sp. Their symbiotic interaction facilitated extracellular polymeric substances (EPS) production and nitrogen recovery. The EPS concentration in co-culture was positively related to the settling efficiency (R2 = 0.774), which plays an essential role in nitrogen recovery. This study provides new insights into microbial interactions among the photoautotrophic community and emphasizes the importance of algal interspecies interaction in algae-based wastewater treatment.

Validation of a quantitative web-based food frequency questionnaire to assess dietary intake in the adult Emirati population.

BACKGROUND AND OBJECTIVE: A culture-specific web-based food frequency questionnaire (FFQ) to assess dietary intake in the United Arab Emirates (UAE) adult population was developed using data from the 2009-2010 national nutrition survey. The objective of this study was to assess the relative validity of the newly developed FFQ for use in the adult Emirati population (AE-FFQ), which contained a list of 139 food lines. METHODS: A convenient sample of 60 (36 females and 24 males) adult Emiratis completed 3 non-consecutive 24HRs over a period of one month, followed by the AE-FFQ, which assessed the intake over the previous month. Relative validity was evaluated by comparing nutrient and food group intakes from the AE-FFQ with the average three 24HRs using Wilcoxon signed-rank tests, Spearman's correlation coefficients (CC), Bland-Altman analysis, and cross-classification. RESULTS: The AE-FFQ overestimated energy and most nutrients and food groups. Bland-Altman analysis showed significant proportional bias between the 2 methods. Deattenuated energy-adjusted Spearman correlation coefficients were poor to good ranging from 0.06 (iron) to 0.62 (fiber) for nutrients, 0.39 median value, and from -0.01 (cruciferous vegetables) to 0.64 (eggs) for food groups, 0.41 median value. A fairly acceptable agreement was obtained, with correct classification into the same or adjacent quartile ranging from 34% (vitamin B12) to 78% (pyridoxine), median 69% for nutrients and from 55% (diet soft drinks) to 87% (soft drinks), median 67% for food groups. CONCLUSIONS: The AE-FFQ is an acceptable tool for ranking UAE adults (aged 18 to 50) according to their dietary intake to investigate the role of Emirati dietary patterns on health and disease. Caution is needed for assessing absolute intake, however, given the bias observed in assessing group-level agreement.

Nicotinamide-Functionalized Carbon Quantum Dot as New Sensing Platform for Portable Quantification of Vitamin B12 in Fluorescence, UV-Vis and Smartphone Triple Mode.

Development of an efficient, portable and simple nanosensor-based systems with reliable analytical performance for on-site monitoring of vitamin B12 (VB12) are still major problems and a challenging work for quality control of manufacturers. Herein, a new fluorescence, UV-Vis and smartphone triple mode nanosensors were designed for the simultaneous detection of VB12 with high sensitivity and accuracy. A novel nanosensor was synthesized through nicotinamide-functionalizing of carbon quantum dot (NA-CQDs) by an one-step microwave-assisted method with green approach. The NA-CQDs sensor showed excellent fluorescence properties and wide linear ranges from 0.1-60 µM with the detection limits of 31.7 nM. Moreover, color changes of NA-CQDs induced by the VB12 could also be detected by UV-Vis spectrophotometer and inhouse-developed application installed on smartphone as a signal reader, simultanusly. The Red, Green and Blue (RGB) intensities of the colorimetric images of NA-CQDs/VB12 system which taken by smartphone's camera converted into quantitative values by the application. A smartphone-integrated with NA-CQDs as colorimetric sensing platform displays good linear ranges (4.16 to 66.6 µM) for on-site determination of VB12 with detection limit of 1.40 µM. The method was successfully applied in the determination of VB12 in complex pharmaceutical supplement formulations without any sample pre-treatment and matrix interfering effects. The recovery results (96.52% to 105.10%) which were in agreement with the reference methods, demonstrating the capability of the smartphone-assisted colorimetric sensing platform in many on-site practical applications of quality controls.

[Determination of cobalt in plasma blood samples by the ICP-MS method after oral intake of dietary supplements containing low doses of cobalt].

Salts of inorganic cobalt (Со) prevent the degradation of the alpha subunit of the hypoxia-inducible factor (HIF), imitating the state of hypoxia in the body and increasing the production of the endogenous hormone erythropoietin (EPO), and are used as doping substances that increase blood oxygen capacity and endurance, which give competitive advantages in sports. Currently, a large number of dietary supplements, including Co-containing ones, are offered on free sale. Their uncontrolled intake can affect not only the professional career of athletes, but also their health, due to the fact that this trace element and its salts are the strongest inorganic poisons and carcinogens. Despite this, their availability on the pharmaceutical market, a noticeable effect of erythropoiesis stimulation and a convenient oral form of administration lead to the need for their detection in modern doping control. The purpose of this research was to develop an approach to differentiate cobalt from vitamin B12, present in the body in its natural state, from the intake of cobalt salts by quantifying and comparing blood levels of vitamin B12 and total cobalt. Methods. The study involved 9 healthy volunteers (women and men) aged 25 to 45 years, leading an active lifestyle. Three of them took 2500 µg/day of cobalamin for 20 days (comparison group), three - dietary supplement containing cobalt asparaginate (100 µg/day in terms of pure cobalt), and the rest - dietary supplements with cobalt sulfate heptahydrate (100 µg/day in terms of pure cobalt) (administration groups) at the same time after meals. Blood samples were taken at baseline and on days 5, 9, 14 and 20. The concentrations of total cobalt in blood plasma samples of volunteers were measured by inductively coupled plasma mass-spectrometry (ICP-MS), the levels of cobalamin were determined on a Cobas 6000 immunochemical analyzer using the Elecsys Vitamin B12 II Assay ELISA kits. Results. It was found that oral intake of of cobalamin at a therapeutic dose significantly exceeding the recommended daily intake (3 µg), there was a regular slight increase in the blood concentration of total cobalt (1.1 times). At the same time intake of dietary supplements containing cobalt in the form of sulfate or asparaginate (about 100 µg per day in terms of pure cobalt) was accompanied by 4-6.7 fold increase in the concentration of total cobalt while unchanged vitamin B12 plasma concentration was observed. The detection of such changes can reliably indicate the use of prohibited salts and, of course, will be in demand for anti-doping control. Conclusion. Long-term monitoring of vitamin B12 and total cobalt levels, similar to hematological module of the Athlete Biological Passport program, will unambiguously detect possible abuse of cobalt salts and can be an additional evidence of the presence of these doping substances to other analytical methods, such as a combination of liquid chromatography and ICP-MS (LC-ICP-MS).

Development of a simple and sensitive HPLC-DAD method for quantification of vitamin B12 fortified in infant food.

To prevent infants from vitamin B12 deficiency, infant food is designed based on cow's milk or cereal with the fortification of vitamin B12. A method for quantitative determination of vitamin B12 in infant food was developed with hydrophilic high performance liquid chromatography (HPLC) coupled with a diode array detector (DAD). The sensitivity of the detector was enhanced by implementing a 60 mm high-sensitivity LightPipe flow cell, and the limit of detection (LOD) and limit of quantification (LOQ) were improved as low as 0.006 µg 100 g-1 and 0.02 µg 100 g-1 respectively. The effect of sample extraction and enrichment, chromatography separation parameters on the analyte, were studied in detail and optimized. Under these conditions, the method performed a good linear analytical range of 0.3-50 µg L-1, and a good repeatability with % RSD below 2.8% and recovery of 90.2-96.5% (n = 6). To the best of our knowledge, for the first time, 60 mm high-sensitivity LightPipe flow cell was included in the HPLC-DAD method for determination of the trace amount of vitamin B12 in infant food. The proposed method was further validated by analysis of FAPAS QC samples (T21120 and T21118), and it was specific and precise for the intended use.

Dual-Emitting Carbonized Polymer Dots Synthesized at Room Temperature for Ratiometric Fluorescence Sensing of Vitamin B12.

Ratiometric fluorescence (FL) probes are highly desirable for highly sensitive and reliable assays. Dual-emitting carbonized polymer dots (CPDs) have great application prospects in building ratiometric FL sensors. However, dual-emitting CPDs are usually synthesized at high temperatures and high pressures, which not only increases the cost but also complicates the structure of CPDs. Here, we developed a facile strategy for the fabrication of dual-emitting CPDs at room temperature using tetrachlorobenzoquinone and ethylenediamine. The formation of CPDs was induced by Schiff base condensation reaction, enabling the following cross-linking polymerization process. The dual-emitting CPDs demonstrate good photostability and antioxidant capacity. Importantly, the typical dual-emission bands of the as-prepared CPDs are found to have a blue emission band at 445 nm with a maximum excitation of 350 nm and a yellow emission band at 575 nm with a maximum excitation of 440 nm. Based on the dual-emitting property of CPDs, a ratiometric FL nanoprobe is obtained for sensitive determination of vitamin B12 (VB12), as the inner filtering and static quenching effects between VB12 and CPDs allow effective quenching of the blue FL of CPDs, while the yellow FL is maintained. The established assay shows linear detection ranges of 0.25-100 µM with a low limit of detection of 0.14 µM. These findings provide new guidance for the facile preparation of CPDs with excellent dual-emitting optical properties, indicating good prospects in biosensing.

Vitamin B12 quantification in human milk - Beyond current limitations using liquid chromatography and inductively coupled plasma - Mass spectrometry.

Vitamin B12 plays a key role in human biological functions and is vital in the neurological development of infants. The assessment of the vitamin B12 intake in exclusively breastfed babies depends on the reliability of its determination in milk. In this report, we present a new accurate and robust method for quantification of vitamin B12 in human milk. A highly specific sample preparation is applied, associated with chromatographic separation and detection by ICP-MS. Excellent sensitivity and accuracy are reported, with recovery values well within acceptability limits (80-120%), within- and between-day variability are lower than 10% and 15% respectively. Strong correlation with a microbiological assay was observed (r2 = 0.9) within the validation range (40-1000 pmol/L, corresponding to 54 to 1355 ng/L). The method can be used to routinely monitor vitamin B12 in clinical or population observational studies, determine infant's intake or assess efficacy of mother's supplementation.

Implementation of Two Chromatographic Methods for Simultaneous Quantitation of Thioctic Acid, Benfotiamine and Cyanocobalamin.

Two accurate and sensitive chromatographic methods have been introduced and validated for the simultaneous determination of thioctic acid, benfotiamine and cyanocobalamin in bulk powders and in their pharmaceutical formulation. Method A is reversed-phase ultra performance liquid chromatographic method with an isocratic elution, where a rapid separation was accomplished on a Zorbax C8 column using a mobile phase of acetonitrile:0.05 M phosphate buffer (pH 6 adjusted by o-phosphoric acid) (23:77, v/v). The retention times (tR) were 0.578, 0.852 and 1.376 for cyanocobalamin, benfotiamine and thioctic acid, respectively. The separated peaks were revealed at 210.0 nm. Method B is a thin-layer densitometric method where the separation of the studied drugs was carried out on silica gel plates using methanol-chloroform-heptane-1-sulphonic acid sodium salt (0.4%) (7:3:0.1, by volume) as a mobile phase, and scanning of the separated bands was done at 240.0 nm. The retardation factor (Rf) values were 0.17, 0.48 and 0.75 for cyanocobalamin, benfotiamine and thioctic acid, respectively. Validation of the methods was achieved following ICH guidelines and the applied methods succeeded to determine the cited drugs in their pure forms and capsules. Results were statistically compared to the manufacturer's method where no significant difference was observed.