ArticleLiterature Review

Trends in analysis of vitamin B-12

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

Vitamin B(12) is an organic compound containing cobalt and essential nutrient for all cell development and human growth. The daily requirements of vitamin B(12) are very low and deficiencies reported to be at picogram level, thus it necessitates detecting vitamin B(12) at high sensitivity in biological samples. It is also reported that several functional groups in the vitamin B(12) and analogs make more difficult to analyze in biological samples for routine analysis, as analogs are not useful for human metabolism. Many methods have been reported for its analysis like radioisotope, high performance liquid chromatography (HPLC), spectrophotometry, fluorimetric assay, capillary electrophoresis (CE) and atomic absorption spectrometry (AAS). These conventional analytical techniques found to be time consuming, tedious, less safe, low sensitivity and expensive, where as combination of immuno-chemiluminescence and biosensor based analysis found to be ultra sensitive having wide application for the detection of vitamin B(12). This review aims to present a concise survey of articles for all analytical practitioners for better understanding in trends of analysis in vitamin B(12). The format selected for this survey divides coverage into various aspects like introduction of complexity in vitamin B(12) structure with challenges in extraction and analysis by various analytical methods followed by problems in raising antibody against vitamin B(12.) Within the scope of each of these areas, key articles have been selected to describe current practices in analysis of vitamin B(12) with proposed novel approaches.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... Up to now, many analytical techniques have been extensively investigated for VB12 determination [6][7][8][9][10][11][12], e.g., chemiluminescence, ultraviolet-visible spectrophotometry, high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), and flame atomic absorption spectrometry (FAAS). These techniques possess some unique advantages. ...
... These techniques possess some unique advantages. However, some drawbacks exist, such as expensive instruments, a lack of sensitivity and specificity, requiring centralized laboratories, and complicated sample preparation [6][7][8][9][10][11][12]. These methods are unable to meet the requirement for effective monitoring of vitamin B12 in biological samples. ...
... Hence the impacts of oxidation time on oxidation efficiency were investigated in Figure 4a. It was observed in Figure 4a that the reduction current of 1 mg L −1 VB12 with different oxidation times (6,9,12,15,18 ...
Article
Full-text available
Vitamin B12 (VB12) is applied as the cofactors in various important enzymatic reactions and is involved in gene expression regulation mediated by B12-riboswitch and the VB12-dependent photoreceptor. Rapid detection VB12 concertation in a given environment may provide insights in the evaluation of micronutrient levels and the physiological and ecological performances of organisms under the relevant condition. This study demonstrating an amperometric approach to quantify the VB12 in biological samples without complicated sample pretreatment. The electrochemical oxidation step was conducted with a plain graphenic electrode to convert all nitrogen groups within the VB12 molecules to NO3− at 1.3 V vs. Ag/AgCl for 15 min. VB12 was quantified stoichiometrically according to the oxidized nitrate anions, which were reduced with copper oxide nanocrystal decorated graphenic electrode. Cathodic polarization was conducted with a graphite rod electrode before nitrate reduction to eliminate the potential interferences. Under optimized experimental conditions, the presented approach gave a wide detection linear range of 0.15–7378 nmol L−1 and the detection limit was 0.59 nmol L−1. The results for biological samples were comparable to those of the HPLC method. These results indicated that successively combined anodic and cathodic polarization enhanced the detection sensitivity and efficiency of the electrode towards VB12. The proposed electrode shows potential in terms of efficiency, reliability and accuracy for rapid determination of VB12 in biological samples.
... Metabolik işlevlerde metilkobalamin ve deoksiadenozilkobalamin yer alır. Molekül ağırlıkları 1400Da dolaylarında olan kobalaminler çok küçük miktarları ile önemli biyolojik etkiler oluşturan vitaminler olarak bilinir (14,15). ...
... Besin alımında karbonhidrat, protein ve yağlar makrobesin grubunu oluştururken; vitaminler, elektrolitler, eser elementler, esansiyel amino asit ve yağ asitleri ise mikrobesin olarak tanımlanır. Besin alımı, emilimi ve metabolizması insan vücudunda önemli rol oynamakta ve besin metabolizması ile ilgili bozukluklar çeşitli hastalıklar ile ilişkilendirilmektedir (8,15,33,69). ...
Chapter
Full-text available
GIRIŞ Organizmada sinir sistemi ile ilgili hastalıklar nöro-lojik hastalıklar olarak tanımlanır. Nörolojik has-talıklar vücuttaki diğer sistemleri de etkileyerek fonksiyon bozukluklarına neden olabilir. Demans, alzheimer, epilepsi, multiple skleroz, parkinson hastalığı, baş ağrısı bozuklukları, nöro-enfeksi-yonlar, malnutrisyonla ilgili nörolojik bozukluk-lar, inme, travmatik beyin yaralanmaları nörolojik hastalıklar olarak sınıflandırılır (1-3). Demans bi-linçte bozulma olmaksızın, bilişsel fonksiyonların deliryum dışında bir nedenle süregelen, ilerleyici ve genellikle geri dönüşümsüz bozulması olarak tanımlanır. Demans hastalarında hafıza, yargıla-ma, hesaplama, planlama ve organize davranışlar gibi yürütücü işlevlerde bozulma, duygu kontrolü ve çevreye olan ilginin azalması gibi klinik tablolar görülmektedir. Demans hastalarının yaklaşık %50-70'ini oluşturan alzheimer bellek kaybı, konuşma, karar verme işlevlerinde, dikkat, oryantasyon ve kişilik bozukluklarının ortaya çıktığı, ilerleyici ve ölümcül bir hastalık olarak tanımlanır (2, 4). Yaş, kadın cinsiyet ve aile öyküsü gibi faktörler alzhei-mer risk faktörleri arasında belirtilmesine rağmen; hipertansiyon, diyabet gibi kronik hastalıklar, dü-şük eğitim ve sosyoekonomik seviye, metal toksi-sitesine maruz kalma, inflamasyon, oksidatif stres, beslenme yetersizliği, homosistein seviyesinin artması ve B12 vitamini eksikliğinin de alzheimer gelişiminde ve ilerlemesinde etkisi olabileceği ra-por edilmiştir (1, 5, 6). Organizmada çeşitli vitaminlerin eksikliği ya da birikiminde sinir sistemi fonksiyonunun etki-lendiği ve bu durumun alzheimer ve parkinson başta olmak üzere nörolojik hastalıklar ile ilişkili olduğu rapor edilmektedir. Yapılan pek çok ça-lışmada beslenme ve antioksidan bakımından zengin diyetin demans, alzheimer, parkinson, travmatik beyin hasarı, epilepsi, multipl skleroz gibi nörodejeneratif hastalıklarda koruyucu etki-si olduğu bildirilmiştir (7, 8). Diyet ile alınan besin öğeleri arasında protein, yağ ve karbonhidratlar makrobesin; vitamin, elektrolit ve eser elementler mikrobesin olarak nitelendirilmektedir. Mikrobe-sinler arasında yer alan vitamin ve eser element metabolizmasındaki bozukluklar sonucu nöro-dejeneratif hastalıkların gelişiminin arttığı, bu
... However, there are many requirements one must address to apply the previous method such as the pretreatment of VB12 specimens, high cost, interference with other components that are present in the sample, in addition to narrow linear range. Considerable attention is currently being given to electrochemically designed sensors, which offer ease of operation, high sensitivity, and low cost, as well as the portability of the equipment used [16,17,18]. In this regard, different electrode types have been fabricated for the electroanalysis of VB12, such as boron-doped diamond electrodes [19] carbon paste electrodes modified by trans-1,2dibromocyclohexane [20], disposable pencil graphite electrodes with single-walled carbon nanotubechitosan layers [21], fabricated gold electrodes with self-assembled monolayers of mercaptoacetic acid [22], and bismuth film electrodes [23]. ...
... Fig. 4 illustrates the dependence of the electroanalytical response on pH of 29.9 µM VB12 on the PMB/Zn NPs/GC electrode using DPV. When pH was varied from 3.0 to 9.0, the peak potential for VB12 shifted almost linearly toward the negative potential, implying that protons directly participated in the rate determining step of the reduction reaction of B12 [16] which also means no adsorption processes occurred on the electrode surface as observed in a previous study [17]. The linear relationship between the peak potential and pH was found to be Ep/V = 0.0229x + 0.7076 pH (r = 0.9950) (see Fig. 4 B). ...
... Clams, eggs, oysters, fishes, meat, and milk are known sources of Vitamin B12. However, under specific conditions, intestinal microbes produce Vitamin B12, anaerobically [3,4]. ...
... The production of Vitamin B12 from microbial sources is considered as an alternative method because of its simple process. The production of Vitamin B12 has been commercially achieved using bacterial strains such as Pseudomonas, Nocardia and Propionibacterium [8,9] and a higher productivity has been reported from Cobalt-resistant strain of Propionibacteria [4]. Selection of natural Vitamin B12 producers is an endorsed strategy as it does not have any legislative hurdles [10,11]. ...
... As the cobalamins are produced in intracellular form during the pfre fermentation, proper and efficient method for cobalamins extraction has important role. The general protocol mentioned in literature is autoclave extraction and cyanidization of cobalamin molecule (Kumar et al., 2010). Adenosyl, hydroxyl, and methylcobalamin are the natural form of cobalamins which are unstable in extracellular media and all of the mentioned compounds are light sensitive. ...
... The flow rate was 0.75 ml/min at ambient temperature. In the prior studies, UV detection wavelength varies from 254 to 580 nm while the Cyano-Cbl UV scanning shows 3 picks in 278, 361, 550 nm which the 361 nm is the most dominant pick between them (Kumar et al., 2010). Therefore, we applied 361 nm for the detection of Cyano-Cbl. ...
Article
Vitamin B12 contributes many substantial metabolic cycles in the living organisms. Since human beings cannot produce such co-factor by their metabolism, they have to receive this vitamin from foods and supplements. Dimethylbenzimidazole (DMBI) distinguishes the active form of vitamin B12 from pseudo-vitamin B12. De Novo total biosynthesis of vitamin B12 in the bacteria should include DMBI biosynthesis through riboflavin pathway. Propionibacterium freudenreichii can produce vitamin B12 through anaerobic biosynthesis pathway. As vitamin B12 production by P. freudenreichii is the growth-associated phenomena, the effect of different carbon sources (rice bran oil, argan oil), nutrients (DMBI) and amino acids (L-Serin, L-Tryptophan, l-cysteine, l-Methionine) on the growth of Propionibacterium freudenreichii PTCC1674 (pfre) were investigated. Through the statistical analysis of vitamin B12 production, rice bran oil (RBO) was selected as the sole carbon source. By applying Plackett-Burman method, significant parameters of vitamin B12 production were extracted and optimized based on Box-Behnken design of experiments. RBO, DMBI and CaCl2.2H2O concentrations and temperature were the four main effective parameters of vitamin B12 production. Via implemented response to surface methodology (RSM), the response was optimized to 2.94 mg/L, while 14% increase of vitamin B12 (cyanocobalamin) production was obtained at RBO concentration of 8.648% V/V, temperature of 38.3 (°C), DMBI concentration of 55.758 (mg/L) and elemental solution concentration of 2 (mg/L). It was concluded that pfre can grow on rice bran oil as a new carbon source while the changing of culture media composition alters the growth profile. Box-Behnken design effectively optimized parameters achieved from Plackett-Burman screening method.
... It is quite difficult quantifying or demonstrating vitamin production by individual organisms of the human GM using traditional methods (e.g., HPLC, microbiological assays); actually, vitamin B12 determination in real samples is challenging due to limited concentration. Analytical approaches can be addressed to quantitation or assessment of its activity and since quantity of vitamin B12 is not directly proportional to activity, it is important to distinguish between these different aspects [135]. Quantitation is conventionally performed by microbiological assay, and radioisotopic dilution assay (RIDA) which however do not discriminate between different vitamin B12 forms measuring total Cbl with no information about the relative amounts of the different species in the sample. ...
... Quantitation is conventionally performed by microbiological assay, and radioisotopic dilution assay (RIDA) which however do not discriminate between different vitamin B12 forms measuring total Cbl with no information about the relative amounts of the different species in the sample. In addition, application of RIDA can be limited for both analyst safety reasons and the high costs of the equipment dedicated to this methodology [65,135,136]. In Table 2 are reported the main characteristics of some interesting LC methods applied for separation of different forms of cobalamin in feces and correlated samples. ...
... 22 Plants neither synthesize nor require vitamin B 12 ; 23 for this reason, plant-based food is not a source of vitamin B 12 . 24,25 However, due to the symbiosis between plants and certain vitamin B 12 -synthesizing bacteria, it can be found in some plants. 26 One of these bacteria is nitrogen-fixing actinobacterium Frankia alani, which forms endophytic nodules in woody trees and shrubs. ...
Article
Full-text available
BACKGROUND Vitamin B12 (cobalamin) can be produced de novo only by certain bacteria and archaea. It plays a crucial role in the health of animals and humans, which obtain it only through diet, mainly from animal products. This study aimed to identify endophytic bacterial strains capable of synthesizing vitamin B12 and enriching edible plants with it as a potential solution for vitamin B12 deficiency in vegetarians, vegans, and people with poor diets. RESULTS An in silico genome analysis was performed on 66 bacterial genomes, including the reference strain Pseudomonas denitrificans ATCC 13867, a known vitamin B12 producer. The genomes were analyzed using the Rapid Annotations using Subsystems Technology (RAST) server and the MetaCyc database to verify the presence and completeness of the vitamin B12 metabolic pathway. The ability of the strains to produce vitamin B12 was confirmed with a high‐performance liquid chromatography with diode‐array detection (HPLC‐DAD) analysis of pure culture extracts. Eleven strains produced detectable amounts of vitamin B12 under tested conditions. The best performing candidates were further tested for their efficacy in producing vitamin B12 in lettuce grown under sterile conditions on Murashige and Skoog (MS) medium with or without CoCl2 supplementation. Methylobacterium sp. strain P1‐11 produced detectable amounts of vitamin B12 in planta: 1.654 and 2.559 μg per g of dry weight without and with CoCl2 supplementation, respectively. CONCLUSION This is the first time a bacterial endophyte was used to produce vitamin B12 in planta, suggesting that bacterial endophytes could be utilized to enhance the nutraceutical values of plant‐based foods. © 2025 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
... One of the microbiological assays for vitamin B12 analysis is the application of Lactobacillus leishmania [20]. Some disadvantages of the microbial method are time consumption and low specificity [21,22]. A liquid chromatography technique with a UV-visible detector is the recommended and suitable analytical procedure [22]. ...
Article
Reverse-phase high-performance liquid chromatography (RP-HPLC) is among the most widely recommended techniques for analyzing water-soluble vitamins such as vitamins C and B-complexes. The research study was conducted to detect and quantify cyanocobalamin (vitamin B12) from the beef liver and heart muscle extracts using a validated isocratic RP-HPLC procedure. The analytical column was Luna® Phenomenex 5 µm C18 (2) 100 A LC-column (150 × 4.6 mm). The mobile phase consisted of water/ethanol in a ratio of 60:40 (v/v). An enzymatic digestion with 1% potassium cyanide was used for samples (beef liver and heart muscle) extraction. The validated method showed to be linear, R = 0.9977; fast, with a retention time of less than 6.00 min; precise, %RSD of 1.16 and 1.74%; and sensitive, with limits of detection and quantification of 0.00033 and 0.00100 µg/mL. The detected values of cyanocobalamin from the beef liver (BLV) and heart muscle (HRM) extracts were 52.04 ± 0.13 and 42.04 ± 0.29 µg/mL, respectively. BLV extract indicated a higher level of cyanocobalamin. Hence, the validated isocratic RP-HPLC technique can be recommended to analyze cyanocobalamin and related compounds in research laboratories such as diagnostics, foods, and pharmaceuticals.
... In addition, the decreasing concentration of vitamin B 12 is possibly attributed to the changed characteristics of vitamin B 12 during the mixing process over 6 h. Vitamin B 12 is highly sensitive to the exposure of temperature, light exposure, oxygen, and pH levels [44]. Although the condition of the chamber is maintained at the closed chambers without changing in pH, the mixing process allows oxygen exposure and the possibility of change in temperature leading to oxidation and degradation of the vitamin B 12 , thus reducing the concentration of vitamin B 12 . ...
... From the report of [16], a microbiological assay using Lactobacillus leishmania could be another applicable method of this vitamin analysis. The microbial assay method usually has a low specificity in some food matrices and is timeconsuming [15,17]. Also, diverse reverse-phase high-performance liquid chromatography (RP-HPLC) methods for the evaluation of this water-soluble vitamin were reported in milk [18], meat product [6], okra [19], dietary supplement, and ingredients [16,20,21] as well as blood serum [22]. ...
Article
Full-text available
This study developed a rapid reversed-phase high-performance liquid chromatography (RP-HPLC) method equipped with a UV-Vis detector. The aquaponics system›s tilapia and marine snoek fishes were extracted using an autoclaving process and enzymatic treatment. The technique enabled the separation and quantification of cobalamin present in these fishes extracts. Phenomenex Luna® 5 μm C18 (2) 100 A LC-column (150 × 4.6 mm) was used as a stationary phase, while the mobile phase consisted of methanol and phosphoric acid in a ratio of 35:65 (v/v), respectively. The developed method was revealed to be rapid (a retention time of less than 5.0 min), linear (R² = 0.9988), sensitive (limits of detection and quantification showed to be 0.0004 and 0.0011 μg/mL, respectively), precise (percentage relative standard deviation of 0.14% to 0.76%), and accurate (percentage mean recovery of 87.44 ± 0.33% to 97.08 ± 0.74%). The quantified concentrations of this vitamin in extracts of tilapia and snoek fishes were found to be 08.95 ± 0.35 and 14.97 ± 0.04 μg/mL, respectively. Therefore, we suggested that the developed RP-HPLC technique could be applicable for quality control evaluation in the food and pharmaceutical industries. Besides, the method could be vital for diagnostic analysis in clinical laboratories.
... Unlike HHP and PEF, UV-C treatment significantly affected vitamin B12 levels in milk ( Figure 2C) when dose applied is higher than 9 mJ/cm 2 , as expected based on the light sensitivity of the vitamin B12 molecule (Edelmann et al., 2016;Selva Kumar et al., 2010;Toda et al., 2019). The UV treatment at the lowest dose (2 mJ/cm 2 ) for some reason led to a decrease in vitamin B12 in milk, whereas UV-treatment at 4 and 9 mJ/cm 2 presented no significant difference in vitamin B12 concentration compared to the control (CD1, raw milk), However, starting from this dose, there was a clear, statistically significant decrease in vitamin B12 concentration with increasing UV-C light dose, and the milk treated with a light dose of 18 mJ/cm 2 , presented a reduction of 10% in vitamin B12 concentration compared to untreated raw milk. ...
... Vitamins B9 and B12 in grains were determined according to a simple spectrophotometric method described by Ruengsitagoon & Hattanat (2012) and Kumar et al. (2010). Absorption was measured at 460 nm and 275 nm, respectively. ...
... e naturally occurring forms of VB 12 in food are hydroxocobalamin, 5′-deoxyadenosylcobalamin, methylcobalamin, sulphitocobalamin, and a small amount of cyanocobalamin. Among them, cyanocobalamin has the most stable chemical structure, and other chemical variants of VB 12 are photolabile [2,3]. e amount of VB 12 required by the human body is very small. ...
Article
Full-text available
An isotope-dilution liquid chromatography tandem mass spectrometry method was established for the determination of vitamin B12 in milk and dairy products. The samples were spiked with stable isotope-labeled vitamin B12 and digested by pepsin and amylase. The various forms of cobalamin were transformed to cyanocobalamin by potassium cyanide after they were released from the enzymatically digested samples. Cyanocobalamin was extracted and purified by an immunoaffinity SPE cartridge and then measured in multiple reaction monitoring mode (MRM). The linear correlation coefficient (R2) of this method was greater than 0.999 in the range of 2–100 ng/mL. The detection limit and the quantification limit were 0.5 μg/kg and 1.0 μg/kg, respectively. The spiked recoveries ranged from 92.0% to 99.4% at the three spiked levels with the relative standard deviation (RSD) between 1.89% and 4.51%. The measured results of NIST SRM1849a and NIST SRM1869a by the current method are all within the reference value range. The Z value was 0.8 during participating in the FAPAS proficiency test using the developed method in 2021. The method is simple, rapid, accurate, and sensitive, and it is suitable for the determination of vitamin B12 in different types of milk and dairy products such as whey powder, whole milk powder, pure milk, fermented milk, infant formula, and prescription food for special medical purposes.
... From the report of [16], a microbiological assay using Lactobacillus leishmania could be another applicable method of this vitamin analysis. The microbial assay method usually has a low specificity in some food matrices and is timeconsuming [15,17]. Also, diverse reverse-phase high-performance liquid chromatography (RP-HPLC) methods for the evaluation of this water-soluble vitamin were reported in milk [18], meat product [6], okra [19], dietary supplement, and ingredients [16,20,21] as well as blood serum [22]. ...
... Various transition elements have vital importance to the chemistry of living beings, the greatest intimate varieties are copper (Cu), molybdenum (Mo), cobalt (Co) and iron (Fe). Though, cobalt is one of the expected element especially in animal alimentation, it is necessary for biochemical reaction of vitamin B12 and related co-enzymes [1]. The Co is naturally existing in the soils, rocks, water, plants and animals. ...
Article
Highly efficient colorimetric povidone (PVP) mediated Ag nanosensing strategy has been adopted for the sensitive and selective quantification of cobalt ion in aqueous system. PVP functionalized Ag nanoparticles grown by chemo-reductive methodology at ambient conditions. These efficient nanoparticles were confirmed by UV–Vis (UV–Vis) spectroscopic characteristic absorption peak at 390 nm and strong Fourier Transform Infrared (FT-IR) stretching bend at 455 cm⁻¹. The topographical and crystalinity analysis by Field Emission Scanning Electron Microscope (FESEM) and X-ray diffractometer (XRD) analysis reveals that the obtained [email protected] NPs have rough surface and size in range of 30–45 nm respectively. Later [email protected] NPs were employed to develop a highly selective and sensitive colorimetric nanosensor for Co²⁺ detection in the concentration from 0.1 to 5 μM in aqueous environment.
... Vitamin B 12 or cobalamin is an essential vitamin for the human organism, stored in the liver. It can occur in different forms but only methylcobalamin (Me-Cbl), hydroxocobalamin (OH-Cbl), and adenosylcobalamin (Ado-Cbl) are its natural forms (Bosle et al. 2016;Kumar et al. 2010). Cyanocobalamin is a man-made form of vitamin B 12 , supplied by food supplements or fortified food, used to prevent and treat low blood levels of this vitamin (Indyk et al. 2017). ...
Article
Full-text available
In recent years, açai berries ( Euterpe Oleracea M.), goji berries ( Lycium barbarum L.), bilberries ( Vaccinium myrtillus L.), and chia seeds ( Salvia hispanica L.) have increased interest worldwide due to their nutritional value and health benefits. In the present study, SEC-ICP-MS and μ-HPLC-ESI-MS/MS were used for the investigation of cobalt speciation and evaluation of its bioaccessibility in these products. Total cobalt content was determined, and açai berries (0.348 ± 0.042 μg g ⁻¹ ) and chia seeds (0.352 ± 0.036 μg g ⁻¹ ) were found as the best sources of this element. Different elution profiles of the extracts of examined berries and seeds obtained with the use of ammonium acetate, Tris-HCl, and SDS suggested that cobalt is bound by different bioligands in each biomatrix. The bioaccessibility of cobalt species was evaluated by SEC-ICP-MS. On the chromatograms of extracts obtained after simulation of gastrointestinal digestion, peaks corresponding to low molecular mass (17.00–1.35 kDa) cobalt complexes were observed. In the case of goji berries, their intensities were significantly higher on chromatograms of gastrointestinal than gastric extract. In enzymatic extracts, different forms of vitamin B 12 were identified by μ-HPLC-ESI-MS, including its natural forms—methylcobalamin (Me-Cbl) and adenosylcobalamin (Ado-Cbl).
... The most commonly used techniques for the determination of VB 12 include microbiological tests with Lactobacillus leichmannii, radioscopy, high performance liquid chromatography (HPLC), chemiluminescence, fluorimetric tests, capillary electrophoresis (CE), mass spectrometry (MS) and atomic absorption spectrometry (AAS) [7]. The electrochemical techniques have drawn considerable attention due to their easy operation, high sensitivity and low cost as well as the portability of the equipment used [8,9]. ...
Article
Vitamin B12 supplementation is recommended mainly for people who are vegetarian or vegan. The wide extent of the use of this supplementation should act as a warning sign regarding the quality of commercially available products. In this context, a novel electrochemical strategy is proposed for the determination of vitamin B12, where the Co(I/II) redox pair is monitored using a boron-doped diamond electrode for the analysis of supplementation products. The surface of the boron-doped diamond was characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The electrical properties of the electrode are highly influenced by its surface terminations and a cathodic pretreatment of −2.0 V for 30 s in 0.5 mol L⁻¹ H2SO4 before the analysis enhanced the analytical response. The cyclic voltammograms for vitamin B12 obtained at pH 5.0 showed four peaks: two oxidation peaks at −0.74 and +0.18 V, corresponding to Co(I/II) and Co(II/III) oxidations, respectively, and two reduction peaks at −0.12 and −0.75 V, corresponding to Co(III/II) and Co(II/I) reductions, respectively. The experimental parameters (pH, supporting electrolyte, pulse technique) were optimized for the monitoring of the Co(I/II) redox pair. The calibration plot for vitamin B12 was obtained by square wave voltammetry at pH 10.0. It was found to be linear from 0.25 to 5.0 μmol L⁻¹, with a detection limit of 86.0 nmol L⁻¹. A boron-doped diamond electrode with cathodic pretreatment was employed to determine vitamin B12 levels in fortified toothpaste and supplementation tablets. The results were compared with those provided by UV–vis spectrometry. The statistical analysis showed no significant difference between the two methodologies in terms of the precision and accuracy of the data obtained.
... B12 belonging to the group of cobalamin is the only water-soluble vitamin (Jankowska et al. 2017). It is a cobalt-containing (Kumar et al. 2010) organometallic molecule with the similar structure of pigments including chlorophyll and hemoglobin (Samari et al. 2012;Banerjee and Ragsdale 2003;Makarska-Bialokoz 2017). After accumulation in living cells, B12 is transformed into 5′-deoxy adenosylcobalamin (MCM) and methylcobalamin (MS) coenzymes, which take critical roles for methylmalony-CoA mutase and methionine synthase, respectively (Moreno-Garcia et al. 2013). ...
Article
Full-text available
Cu(II) anchored polymeric cryogels are synthesized for the isolation of B12. The poly(2-hydroxyethyl methacrylate) [poly(HEMA)] is used as solid support and N-methacryloyl-l-aspartic acid, is used as a ligand. Synthesis success is proved by the Fourier transform infrared spectroscopy, 1H and 13C nuclear magnetic resonance, scanning electron microscope, N2 adsorption method (Brunauer, Emmet ve Teller), elemental analysis, induction coupled plasma optical emission spectroscopy. In the first part of the work, the polymeric material is synthesized and characterized, and in the second part, adsorptive treatment was carried out to determine the optimum B12 adsorption conditions with this synthesized material in varying conditions. The B12 isolation from cheese is carried out to determine the performance of the polymeric material in the real environment. The antioxidant capacity of the obtained B12 is an indication that the isolation process is successfully carried out.
... Vitamin B 12 (VB 12 ), known as cobalamin, is a complex within a tetrapyrrole ring containing cobalt (Co(II)) as the central atom, which is the only vitamin that contains metal ion in the vitamin family [1]. VB 12 deficiency can cause pernicious anaemia, cardiovascular disease, weakness, fatigue, nerve degeneration and irreversible neurological damage, attributing to the failure in forming red blood cells and repairing the myelin sheath [2]. ...
Article
Full-text available
Phosphorus and nitrogen dually-doped carbon quantum dots (PN-CQDs) were prepared from sucrose, 85% phosphoric acid and 1,2-ethylenediamine as the sources for carbon, phosphorus and nitrogen, respectively. The PN-CQDs possess good water solubility and favorable biocompatibility. The excitation/emission peaks are at 365/451 nm, but bright blue, green, or red emissions are found depending on whether the excitation wavelengths of the laser are set to 408 nm, 488 nm, or 543 nm, respectively. Fluorescence is quenched by both vitamin B12 (VB12) and Co(II) by a combination of inner filter effect and static quenching. The PN-CQDs are shown to be useful nanoprobes for determination of VB12 and Co(II). Response to VB12 is linear in the range of 2.0–31 μM. The response to Co(II) is linear in two ranges, viz. from 1.7–12 μM and from 28 to 141 μM. The limit of detection of VB12 and Co(II) are 3.0 nM and 29.4 nM, respectively. The nanoprobe was successfully applied to the analyses of VB12 in drug samples and of Co(II) in spiked water samples, and it gave satisfactory results. The nanoprobe was also applied to the determination of VB12 and Co(II) in human hepatocarcinoma cells (type SMMC7721), human pulmonary epithelial cells (type BEAS-2B), human adenocarcinoma cells (type A549), and human pheochromocytoma cells (type PC12), respectively. Graphical abstractSchematic presentation of the quenching of the fluorescence of phosphorus and nitrogen dually-doped carbon quantum dots (PN-CQDs) by vitamin B12 (VB12) and Co(II).
... Due to the development of the dietary supplement market and consumers' needs, an increasing number of new dietary supplement products contain "natural form vitamin B 12 " or "high-bioavailable form vitamin B 12 " (such as methylcobalamin) or a mixture of different cobalamins (Obersby, Chappell, Dunnett, & Tsiami, 2013;Kong, Liu, Song, Kuang, & Xu, 2017). Traditionally, vitamin B 12 is analyzed by a non-specific and time-consuming microbiological assay that uses Lactobacillus leichmannii (ATCC 7830) as the test organism (Kumar, Chouhan, & Thakur, 2010). Most of recent published vitamin B 12 analytical methods either focused on determining cyanocobalamin as high-purity dietary supplement/ingredient (Chen et al., 2010) or focused on determining all forms of active vitamin B 12 in serum or food sample for trace-level quantification using high-expense instruments such as mass spectrometry (Schwertner, Valtier, & Bebarta, 2012). ...
... Due to the development of the dietary supplement market and consumers' needs, an increasing number of new dietary supplement products contain "natural form vitamin B 12 " or "high-bioavailable form vitamin B 12 " (such as methylcobalamin) or a mixture of different cobalamins (Obersby, Chappell, Dunnett, & Tsiami, 2013;Kong, Liu, Song, Kuang, & Xu, 2017). Traditionally, vitamin B 12 is analyzed by a non-specific and time-consuming microbiological assay that uses Lactobacillus leichmannii (ATCC 7830) as the test organism (Kumar, Chouhan, & Thakur, 2010). Most of recent published vitamin B 12 analytical methods either focused on determining cyanocobalamin as high-purity dietary supplement/ingredient (Chen et al., 2010) or focused on determining all forms of active vitamin B 12 in serum or food sample for trace-level quantification using high-expense instruments such as mass spectrometry (Schwertner, Valtier, & Bebarta, 2012). ...
Article
Full-text available
Objectives: Vitamin B12 dietary supplement can be critical to the alleviation strategies against micronutrient malnutrition and food insecurity. A high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method has been developed and validated, for the quantitation of four bioactive forms of vitamin B12 (adenosylcobalamin, cyanocobalamin, hydroxocobalamin, methylcobalamin) from dietary ingredients and supplements. Methods: A Plackett-Burman factorial study was used to identify factors that contributed to the extraction of cobalamins. Significant factors were selected to produce the improved HPLC method for cobalamin separation. This method was then subjected to a single-laboratory validation according to the AOAC International guidelines for linearity, suitability, detection limits, accuracy, and precision. Results: The method achieves chromatographic baseline resolution of vitamin B12 forms on a modern column platform without the expensive requirement of an ultra-high pressure liquid chromatography and/or mass spectrometry. The method has a wide analytical range (0.0005% w/w - 85% w/w), high precision (repeatability relative standard deviations ranged from 1.08% to 3.06%), and high accuracy (>96% spike recovery rate). The method detection and quantification limits are less than 0.16 and 0.52 µg/mL, respectively. Conclusions: To our best knowledge, the method is simpler, less time-consuming, and more economical than other published methods for its intended uses. Funding sources: Eurofins Supplement Analysis Center, Eurofins Scientific, Inc.MilliporeSigma. Supporting tables images and/or graphs:
Article
Vitamin B12 plays a significant role in maintaining human health. Deficiency or excess intake of vitamin B12 may cause some diseases. Therefore, it is significant to fabricate sensors for sensitive assay of vitamin B12. In the past few years, a variety of nanomaterials have been developed for the fluorescence detection of vitamin B12 in tablets, injection, human serum and food. In the review, the assay mechanisms of fluorescent nanomaterials for sensing vitamin B12 were first briefly discussed. And the progress of various nanomaterials for fluorescence detection of vitamin B12 were systematically summarized. Furthermore, the sensing performance of fluorescent nanosensors was compared with fluorescent probes. Lastly, the challenges and perspectives about the topic were presented.
Chapter
Vitamin B12 (Vit B12) is a commonly consumed ingredient in food and feed, health care products and is produced by fermentation due to its complexity for synthesis. Therefore, the selection of better microbial strains to produce vit B12 with optimum bioprocessing parameters has been targeted in past decades. This chapter emphasizes the biosynthesis characteristics of vitamin B12 from selected strains for their industrial applications, including Propionibacterium freudenreichii (PF) and Pseudomonas denitrificans.
Chapter
Vitamin B12 is one of the major nutritional sources that is involved in the functions such as cell regeneration and nerve functioning. People with cobalamin deficiency will need its supplement in order to maintain their biological functions. Therefore, many forms of synthetic vitamin supplements are produced, yet they are considered unsafe for human consumption. Thus, biological vitamin supplements are required, especially postbiotic products of microbes that are “Generally Recognized as Safe (GRAS)” are gaining importance. On such approach Propionibacterium freudenreichii is identified to be much efficient in producing vitamin B12 as its postbiotic compound. Moreover, the analysis and quantification of the vitamin produced and its usage are also important to avoid complications such as hypervitaminosis. Thus, this chapter in particular discusses about the method of collecting the postbiotic cobalamin from P. freudenreichii, its analysis and quantification using a recent method, Ultra High-Performance Liquid Chromatography (UHPLC)-Tandem Mass Spectrometer (MS/MS).
Article
As one of derivatives of Vitamin B12, methylcobalamin (MeCbl) is an indispensable "Life Element" and plays an essential role in maintaining human normal physiology function and clinical medicine application. Because of the intricate molecular structure, strong hygroscopicity and optical instability, maintaining its solid stability is a great challenge in pharmaceutical preparation. Based on the structure features of MeCbl hydrates, this study explored the drug solid stability by designing solid-solid phase transformation (SSPT) experiments. Three hydrate powders of MeCbl that had special structure with isolated site and channel water molecules were discovered. It was found that drying condition and surrounding humidity were controlling factors influencing final solid form. The inter-conversion relations relevant to heating-induced and humidity-induced structure changes had been established between the three hydrate powders. Powder X-ray diffraction, thermogravimetric analysis, differential scanning calorimetry, high performance liquid chromatography and dynamic vapor sorption were used to characterize the differences and related properties of stably prepared powders of MeCbl hydrates. The particle size of product could be regulated and controlled by optimizing operating conditions of crystallization process, where ultrasound-assisted and seeding-introduced were applied as promising strategies to enhance solution crystallization process. This study opens up the possibility for the stable preparation and large-scale production of polycyclic macromolecular bulk drugs like methylcobalamin.
Article
Full-text available
Water scarcity is one of the most important abiotic factors limiting rice growth and productivity. The application of nutrients affects various physiological and biochemical mechanisms to curtail water deficiency, but little is known about the ameliorative effects of cobalt (Co) on rice. Our study aimed to investigate the advantageous effects of soaking in Co at the optimum concentration (10 and 44.5 µM) on morpho-physiological traits and oxidative stress, during the vegetative and reproductive stages, also assessing yield attributes, in two rice varieties (Sakha 104, drought-sensitive, and Giza 178, drought-tolerant). Treatments included control (100% field capacity (FC)), moderate drought stress (75 % FC), and severe drought stress (50 % FC) either alone or in combination with Co. A water deficit and oxidative stress affected Giza 178 less than Sakha 104. Co application significantly enhanced the performance of two rice varieties under drought stress by increasing plant height, biomass, water content, tillering, leaf area, pigments (chlorophyll a, carotenoids, total chlorophyll), sugars (soluble sugars, and total carbohydrates), and Co content (shoot and root). Also, Co significantly increased the performance of the antioxidant system by elevating the concentration of total phenols, flavonoids, proline, and antioxidant enzymes (catalase, peroxidase, and polyphenol oxidase), while significantly decreasing chlorophyll b, malondialdehyde, and 2,2-diphenyl-1-picryl-hydrazyl. Yield attributes such as the number of tillers, panicle parameters (number, length, and weight), 100-grain weight, harvest index, and grain nutritive value (sugars, vitamin B, and Co contents) were significantly enhanced by Co in both varieties. However, the maximum performance was observed in Giza 178.
Article
Введение. Физиологические нормы питания и поступление пищи обогащенными разными эссенциальными веществами является предметом изучения многих специалистов, в том числе и технологов. Поэтому важное значение имеет изучение свойств продуктов с применением функциональных групп веществ. Вторичное сырье является источником биологически и физиологически важных веществ, которое может применяться в обогащение продуктов питания, в том числе йогуртных продуктов, с целью уменьшения дефицита эссенциальных веществ, которые могут приводит к нарушению пищевого статуса, а также нести положительный экономический эффект с точки зрения ресурсосберегающей технологии производства. Цель. Изучение технологии производства продукта на основе вторичного молочного сырья - пахты и определение его качественных показателей.Материалы и методы. Исследования проведены на кафедре технологии хранения и переработки продуктов животноводства РГАУ-МСХА им. К.А. Тимирязева совместно с Всероссийским научно-исследовательским институтом молочной промышленности. Сырье для производства йогурта и йогуртного продукта поставлялось из зоостанции РГАУ – МСХА имени К.А. Тимирязева. Исследования сырья и молочных продуктов проводились по общепринятым методам. Результаты. В выработанных продуктах были изучены физико-химические и реологические показатели. На основе полученных данных установлено, что йогурный продукт, с применением вторичного сырья пахты обладает более низкой калорийностью по сравнению с классическим йогуртом из молока. Так же высокое содержание витаминов группы В в пахте, позволяет получать продукт с выраженными биологически активными свойствами. Установлено, что в исследуемых образцах йогуртного продукта присутствовала микрофлора, характерная для традиционного йогурта, это - Str.thermophillus и Lbm.bulgaricus. Жизнеспособность клеток высокая, на конец срока хранения и она составила в среднем 5,04 *107 КОЕ/см3, что подтверждается показателем кислотообразования в продуктах - более 100 °Т Выводы. Разработанная технология производства йогуртного продукта является не только ресурсосберегающей, но и относится к сфере бережного производства. Полученный продукт так же можно рекомендовать в качестве диетического питания, как источник биологически активных веществ.
Article
In this study, vitamers of vitamins B6 (pyridoxine; PN, pyridoxal; PL, pyridoxamine; PM) and B12 (cobalamins) in cooked or processed chicken (n=21) were analyzed and the analytical performance parameters were evaluated. The levels of B6 and B12 vitamers were significantly different in terms of the breeds, cooking method, and the parts of the chicken (p⟨0.05). Ogolgye (boiled) (61.48 μg/100 g) and roasted chicken wings (131.94 μg/100 g) showed the highest levels of total vitamin B6 (PN+PL+PM) among the four breeds of chiken and the cooked or processed chiken, respectively. For cyanocobalamin, Korean native chicken (0.40 μg/100 g) and chicken skewers (0.68 μg/100 g) showed the highest levels among the four breeds of chicken and the cooked or processed chiken, respectively. Analysis of B6 vitamers using high performance liquid chromatography-florescence detector (HPLC-FLD) showed a coefficient of variation (CV) of 0.4-4.6% for repeatability and 4.2-5.9% for reproducibility, showing good precision. Likewise, vitamin B12 analysis using immunoaffinity-HPLC-photodiode array detector (PDA) showed a CV of 5.7% for repeatability and 5.9% for reproducibility. Recoveries of B6 and B12 vitamers were 94.3-100.2%, showing good accuracy. Unlike many previous studies that evaluated PN only, this study provides a more accurate estimation of the total B6 content of cooked or processed chiken, including the contents of PN, PL, and PM, which can be used to revise the Korean food composition table.
Article
Accurate determination of vitamin B12 (VB12) in food matrices remains a big challenge due to the instability of VB12 and extreme complexity of the various matrices. Both the matrix effect and recovery are the main parameters, which are needed to be evaluated meticulously in the measurement. An ultra-high performance liquid chromatography coupled to quadrupole Q-Exactive Plus orbitrap mass spectrometry (UHPLC-QE-Orbitrap-MS) has been developed to detect the VB12 in infant formulas. Besides, four calibration methodologies, including standard addition, standard addition-isotope dilution mass spectrometry, single point isotope dilution mass spectrometry (SP-IDMS), and standard calibration curve isotope dilution mass spectrometry were studied in detail to evaluate the accuracy and precision of the quantification values. The matrix effects of mass spectrometry could be compensated well via a facile SP-IDMS calibration method, which was validated by an F-test and the correction factor. UHPLC-QE-Orbitrap-MS with SP-IDMS calibration method could efficiently determine the VB12 in different infant formula matrices obtained from the supermarket. The precision with reproducibility less than 2.83%, and accuracy with a spiked recovery larger than 97.4% were obtained. The detection and quantification limits of the method were 0.05 and 0.20 μg/kg, respectively.
Article
Relevance. Vitamin B12 (cobalamin) belongs to the group of water-soluble vitamins. This vitamin belongs to the class of tetrapyrroles, biologically active substances consisting of pyrrole rings associated with metal ions. So, in the vitamin B12 molecule, the core is the cobalt ion. A feature of this vitamin is that its synthesis in nature is carried out only by bacteria, and therefore, it is present only in products of animal origin. And one of the sources of its receipt is milk and dairy products, including those enriched with B vitamins. Low concentrations of vitamin B12, even in vitamin-enriched dairy products, at the level of tenths and hundredths of a milligram, make the assessment of its quantitative content a difficult analytical task. The use of reversed-phase liquid chromatography for this purpose, followed by detection by means of a spectrophotometric or diode-array detector, is associated with a time-consuming sample preparation technique. Including repeated concentration of the sample to achieve the required concentrations of the desired substance. In this connection, the development of an analytical method that allows to remove the disadvantages described above, is an urgent task. Methods. This article proposes a method for assessing the content of vitamin B12 by high performance liquid chromatography using a mass-spectrometric detector (HPLCMS). The method developed in this study can be used for laboratory analysis of vitamin B12 in dry formulas for baby food, which will provide the possibility of faster and more efficient results for the control and monitoring of baby food enriched with vitamin complexes of water-soluble vitamins of group B. Results. A method of sample preparation is presented, in which the recovery coefficient of the analyte in the studied samples was 99.12–99.31%. When evaluating the calibration curve, the correlation coefficient was r2 = 99.999. The calculated limit of detection is 0.02 g/kg.
Chapter
Full-text available
For both quantitative and qualitative applications, mass spectrometry (MS) has evolved to become an effective analytical instrument. The first mass spectrometer was designed in 1912 and has since progressed from studying only small inorganic molecules to biological macromolecules, with virtually no mass constraints. Research in drug discovery, in general, depends considerably on MS technologies. The capability of mass spectrometry to analyze proteins and other biological materials is due to the advancements made by establishing soft ionization techniques that can turn biological molecules into ions, such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Consequently, MALDI has the benefit of generating peptide and protein single-charge ions, reducing spectral complexity. Regardless of the cause of ionization, a mass spectrometer's sensitivity is connected to the mass analyzer where ion separation occurs. Both quadrupole and flight time (ToF) mass probes are widely used and can be designed as QToF tandem mass spectrometric instruments combined. As the title suggests, tandem mass spectrometry (MS/MS) is the outcome of conducting two or more concurrent ion separations, typically integrating two or more mass analyzers. The development of high-resolution mass spectrometers resulted in the combination of a quadrupole and flight time Historically, this paper introduces mass spectrometry and describes the benefits and drawbacks of ESI and MALDI along with quadruple and ToF mass analyzers, including scientific mass analyzers. This paper is of an introductory nature and is intended for graduate and senior biochemistry students as well as chemists and biochemists who are not acquainted with mass spectrometry and want to learn the basics; it is not intended for professionals in mass spectrometry. Keywords: Mass spectroscopy, electrospray ionization, matrix-assisted laser desorption ionization, quadrupole, tandem mass spectrometry, proteomics, foodomics.
Chapter
Full-text available
Foodomics has recently emerged as a new discipline that studies food and nutritional products with modern analytical tools, including mass spectrometry (MS), to ensure food quality and safety. Foodomics is currently being used as a powerful tool in food authentication. It has been used to authenticate food items such as cereals, wine, honey, chocolates, and other commercial products. MS is the most suitable technique for authentication of food products because of its precise, accurate, and reproducible analytical power. MS-based techniques are nowadays extensively used for authentication of food and nutritional products. MS-based methods can detect various food components, including nutrients, additives, and toxic contaminants. Due to advancement in separation techniques, ionization and detectors in MS such as liquid chromatography tandem mass spectrometry, gas chromatography mass spectrometry, electrospray ionization tandem mass spectrometry, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, surface-enhanced laser desorption/ionization mass spectrometry, desorption electrospray ionization mass spectrometry, direct analysis in real-time mass spectrometry, extractive electrospray ionization mass spectrometry, and tandem-MS approaches have emerged as the method of choice for authentication of food products. This chapter discusses separation techniques and application of MS to analyze food products.
Article
Vitamins are one of the most essential organic compounds that are necessary for the human body, in order to develop and grow in a healthy way. The aim of this mini-review is to bring together a series of electrochemical sensors (voltametric and amperometric) developed for the determination of vitamins from the families of B, D and K in biological, pharmaceutical or food-related samples. For this mini-review, 16 articles published between 2016 and 2021 were taken into consideration.
Thesis
Full-text available
Background: Coronary artery disease (CAD) is an accumulation of atheromatous plaque in medium and large-sized arteries supplying the myocardium. Iron plays a key role in the maintenance of different cellular functions and mediates various metabolic processes. Iron overload would increase CAD risk by promoting lipid peroxidation. Vitamin B12 acts as a co-factor for the methionine synthase enzyme that catalyzes the conversion of homocysteine to methionine. The lack of vitamin B12 may raise the concentration of homocysteine in plasma which lead to atherosclerosis. Knowledge of the relationship between iron status, vitamin B12 and CAD may contribute to setting priorities and establishing effective national prevention programs to decrease morbidity and mortality from CAD. Objective: To investigate the association of iron and vitamin B12 status with coronary artery disease in Gaza city. Methodology: Case control study was conducted on a sample of 80 persons aged between 30 to 60 years, 40 in each group i.e. patients with CAD and apparently healthy controls. Questionnaire interviews were performed among the study population. Serum ferritin and vitamin B12 were determined using ELISA kits. Serum iron, lipid profile parameters, high sensitivity C reactive protein (hs-CRP) were determined chemically. Low destiny lipoprotein (LDL), and body mass index (BMI) were calculated. Complete blood count (CBC) was also performed. An approval was acquired from local ethical committee to perform this study. All data were analyzed by a computer using SPSS program (version 22). Results: The results showed that the mean level of serum iron in the male cases (71.6 ± 24.7 µg/dl) was lower compared to that of male controls (87.3 ± 28.4 µg/dl) and the difference was statistically significant. Moreover, transferrin saturation percentage in male cases (24.0 ± 8.9%) was lower compared to male controls (29.0 ± 9.9%) and the difference was statistically significant (p = 0.045). Additionally, the mean levels of serum vitamin B12 in male cases (238.8 ± 51.4 pg/dl) was lower compared to male controls (337.3± 108.4 pg/dl) and was statistically significant (p<0.001). The Pearson correlation test showed there was a significant positive correlation between the level of serum iron with the level of vitamin B12 among males but not females (r = 0.28, p = 0.032). Conclusions: The mean difference of transferrin saturation and serum ferritin between the male cases and male controls was statistically significant. In contrast, they were no significant difference among female cases and female controls. The mean levels of serum vitamin B12 in male cases was lower compared to male controls and was statistically significant. While, the difference was not statistically significant between female cases and female controls.
Article
Cobalt as a transition metal ion is a biologically essential trace element, and plays an important role in various biological systems. The silicon nanoparticles (SiNPs) / gold nanoparticles (AuNPs) composite as a simple and efficient fluorescent probe was developed to detect Co²⁺ and vitamin B12 (VB12) based on the selective aggregation and inner filter effect (IFE). The green-emitting SiNPs were synthesized by one-pot hydrothermal method, and the AuNPs were synthesized and modified with thioglycolic acid and cetyltrimethylammonium bromide. The fluorescent probe was fabricated by simple mixing the SiNPs and AuNPs together. In the presence of Co²⁺/VB12, AuNPs are selectively aggregated and result in the enhancement of the local surface plasmon resonance absorption centered at 520 nm, the green fluorescence of SiNPs is significantly quenched via IFE. The fluorescence quenching efficiency of the probe is linearly proportional to the concentration of Co²⁺ in the range of 0.1-80 µM with a low detection limit of 60 nM, which is far lower than the guideline value of Co²⁺ in drinking water (1.7 µM). For vitamin B12 (VB12), its linear relationship is in the range of 0.1-100 µM, and the limit of detection is 69 nM. Furthermore, the proposed method shows good selectivity for the detection of Co²⁺ and VB12, and does not need sophisticated pretreatment, only through simple filter. It has been applied in actual environmental water samples and drug tablets with satisfactory results.
Article
Nitrogen and sulfur co-doped carbon dots (N,S-CDs) with strongly orange fluorescent]ce were fabricated through a facile hydrothermal method using o-phenylenediamine and thiourea as original materials. The N,S-CDs performed excitation-independent photoluminescent...
Article
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.
Article
An X-ray fluorescence spectrometric method for the determination of vitamin B12 in vitamin formulations is described. The method is based on the measurement of Co, the only metallic constituent of vitamin B12. The method is simple, has no spectral interference from any element except Fe and is applicable to both solid and aqueous (dissolved solids) phases. It has a limit of the detection of 2.8 μg/g for Co and 64 μg/g for vitamin B12 in solid pharmaceuticals and 2.0 μg/g for Co and 46 μg/g for vitamin B12 in the liquid (aqueous) counterparts. Several commercial vitamin tablets containing vitamin B12 in 1500-3000 μg/g range are analysed with an accuracy of ∼ 10%. Taking advantage of the water-soluble nature of vitamin B12, some tablets have also been analysed after the ultrasonic extraction of vitamin B12 in aqueous medium. Phosphorous, another distinguishing constituent of vitamin B12, is not found suitable for determining the vitamin due to elevated background and spectral interferences.
Article
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.
Chapter
Vitamin B12 also known as cobalamin is a cobalt-containing corrin ring cofactor required for two enzymes in higher animals, methionine synthase and L-methylmalonyl-Co-A mutase. It is a product of microbial synthesis; therefore, elaborate mechanisms of uptake and transport are necessary to insure availability of this rare nutrient. Vitamin B12 ingested in the diet binds to intrinsic factor (IF) and is internalized in the distal small intestine by the cubam receptor. Human B12 deficiency is caused mainly by lack of animal source food or lack of IF. Human deficiency causes megaloblastic anemia and/or demyelinating neurologic syndromes with deficiency, and methylmalonic acid and homocysteine increase in body fluids. Treatment with parenteral or high dose oral vitamin B12 is effective in malabsorption syndrome and corrects megaloblastic anemia. The development of inexpensive B12-containing foods acceptable for populations unwilling to eat or unable to afford animal source foods would be of benefit.
Article
A sensitive and simple determination method for trace vitamin B12 molecules was developed based on magnetic solid phase extraction (MSPE) by using a newly synthesized magnetic nanoparticles. Determination of target molecules after MSPE was carried out by means of HPLC-DAD systems in food samples. The proposed method has important advantages such as eco-friendly, simplicity, sensitivity, low equipment costs, precise, and accuracy. All experimental variables including extraction time, ionic strength, desorption solvent, and desorption time were investigated in detail. Accuracy of the proposed method was checked by the UV spectra of target molecules, retention characteristics, and by comparing the peak purity with the Vitamin B12 standard. The results showed that utilization of the MSPE step could provide a sensitive (limit of detection of 2.85 ng mL−1), precise (coefficients of variation < 3.60%), and accurate (recoveries >98 %). The reused material displayed a long-term stability after undergoing extraction of 10 times. Recovery values were calculated by using spiked samples and certified reference materials including multivitamin tablets (NIST 3283) and whole milk powders (ERM-BD600) were used for validation of results. Finally, the method was applied to food samples successively.
Article
Vitamin B12 plays a vital role in human metabolism and is an essential vitamin obtained predominantly from food of animal origin. Amongst all animal products, naturally occurring vitamin B12 in milk has the highest bioavailability and dairy products are a broad-access source, especially for vegetarian individuals. The dairy industry requires an accurate and highly sensitive detection method for vitamin B12, however, the extremely low concentration and instability of vitamin B12 creates challenges in analysis. This review discusses the application of modern instrumental techniques for analysis of vitamin B12 in milk as well as a variety of sample preparations, together with their respective advantages and drawbacks.
Chapter
From the chemical point of view, the term vitamin B12 is synonymous with cyanocobalamin, which is by far the most important component of the large family of the cobalt corrinoids. In this review, however, as in most publications, the word vitamin B12 is given a very broad meaning so as to include all the cobalt corrinoids of the cobalamin group. Other cobalt corrinoids which can be considered analogous or precursors of cobalamins are also included.
Article
Microbore reversed-phase HPLC was optimized for the separation of cyanocobalamin, 5′-desoxyadenosylcobalamin (coenzyme B12), methylcobalamin, hydroxocobalamin, aquocobalamin and cobinamide dicyanide isoforms. Various detection modes: UV at 278 nm, ion-spray (IS)-MS and direct-injection nebulization ICP-MS were examined and compared. IS-MS allows one to identify, unambiguously, the eluted compounds, whereas ICP MS was found to be more sensitive than any other on-line detection techniques described for the cobalamin analogues, so far allowing to achieve detection limits down to ca. 10 ng ml−1. The usefulness of the methods developed was demonstrated for the determination of cobalamins present in pharmaceutical preparations.
Article
The identification of structural markers for Bââ/protein interactions is crucial to a complete understanding of vitamin Bââ transport and metabolic reaction mechanisms of Bââ coenzymes. Fourier transform infrared spectroscopy can provide direct measurements of changes in the side chains and corrin ring resulting from Bââ/protein interactions. Using FTIR spectroscopy in various solvent systems, the authors have identified structural markers for corrinoids in the physiological state. They assign the major band (denoted B), which occurs at ca. 1,630 cm⁻¹ in DâO and ca. 1675 cm⁻¹ in ethanol, to the amide I C{double bond} stretching mode of the propionamide side chains of the corrin ring. The lower frequency of band B in DâO versus ethanol is due to the greater hydrogen-bonding properties of DâO that stabilize the charged amide resonance form. Since the propionamides are known to be important in protein binding, band B is a suitable marker for monitoring the interaction of these side chains with proteins. They assign bands at ca. 1,575 and 1,545 cm⁻¹ (denoted C and D) as breathing modes of the corrin ring on the basis of the bands' solvent independence and their sensitivity to changes in axial ligation.
Article
The problem of determining the optimal number, location, and level of treatment for regional domestic sewage treatment plants along an estuary or river is considered. The formulation is one of minimizing the sum of treatment and transport (piping and pumping) costs such that water quality improvement goals for dissolved oxygen are met. Restrictions may also be placed upon the overall level of treatment (required secondary, required uniform, or least cost) if desired. An optimization procedure is developed which utilizes dynamic programing, linear programing, and heuristic location techniques in a series of steps which lead to progressively improved (lower total cost) solutions. The location procedure is intended for use by an engineer-planner during the design stage and requires his participation and skilled judgment during the course of the algorithm. The technique is illustrated for the Delaware estuary for 22 domestic waste sources, nine potential regional sewage treatment plant sites, and 22 industrial waste sources. Results of the case study show considerable savings over previous nonregional treatment schemes.
Article
Three different methods for the measurement of vitamin B12 were compared: two spectrophotometric methods and an HPLC one. When the pure vitamin was used, the results obtained using all three methods were similar, but when samples from microbial material were used, the results were different. The HPLC method could distinguish the true vitamin B12 from the different vitamin B12 analogues whereas the spectrophotometric methods could not.
Article
The pattern of the vitamin B12 requirement of a soil bacterium "Lochhead 38" (provisionally assigned to Arthrobacter) resembled that of the protozoan Ochromonas malhamensis and of higher animals. Of the naturally-occurring B12-vitamins, cyanocobalamin and vitamin B12III are active. Pseudovitamin B12 and Factor A have very little or no intrinsic activity, and when present in relatively high concentrations both compounds depress the rate of the growth response to limiting cyanocobalamin. Factor B, the porphyrin-like nucleus of the vitamin B12 molecule without the nucleotide, is inactive, as are also methionine and deoxyribosides. A disadvantage in the use of Lochhead 38 for assay purposes is that in vitamin-B12-dehcient cultures the organisms flocculate.
Article
A microbial sensor consisting of immobilized Escherichia coli 215 and an oxygen electrode is described for the determination of vitamin B12. When the sample solution is injected into the microbial electrode system, the increased consumption of oxygen by the micro-organisms causes a decrease in the dissolved oxygen around the porous membrane of the oxygen electrode and the current decreases gradually with time until a steady state is reached. The response time for a rate measurement is 2 h. When 0.5 mg of Escherichia coli 215 is immobilized, a linear relationship is obtained between the current decrease and the vitamin B12 concentration between 5 × 10−9 and 25 × 10−9 g ml−1.
Article
Vitamin B12 analogues and aqueous cobalt are separated by liquid chromatography using a reversed-phase column (Spherisorb ODS-2) and a mobile phase consisting of a linear gradient from a 25:75 () mixture of methanol and 0.085 M phosphoric acid solution buffered at pH 5.2 with triethanolamine to a 60:40 mixture over 5 min. The vitamins are selectively detected by measuring cobalt with an atomic absorption spectrometer directly interfaced to the Chromatograph. A simple T-piece is used to compensate with air the difference between the nebulizer uptake rate and the Chromatographic flow-rate (1.5 ml min−1). Recoveries of the components from spiked preparations ranged from 90.8 to 108%. The method is applicable to the analysis of pharmaceuticals for cobalamins.
Article
A fully automated chemiluminescence analyzer for the determination of vitamin B12 in serum has been commercialized and clinically used. To determine the applicability of this apparatus in food analysis, vitamin B12 was assayed in foods by the chemiluminescent method, which was compared with a microbiological method. In shellfishes and spirulina, the values determined by the microbiological method were 6−8-fold greater than the values determined by the chemiluminescence method, although there was good similarity between the values by the two methods in other foods. Except for the shellfishes and spirulina, which contained substantial amounts of vitamin B12-substitutive compounds or inactive vitamin B12 analogues (or both), the observed correlation coefficient between the methods in the foods tested was excellent (r = 0.99, y = 1.2x − 1.1, n = 9). The chemiluminescence method was suitable for the determination of vitamin B12 in foods as well as in serum and was simpler (fully automated) and more rapid (180 samples analyzed per hour), highly selective (use of intrinsic factor, the most specific vitamin B12-binding protein), and reproducible (coefficients of variation of 1.2−6.7%) than the microbiological method. Keywords: Vitamin B12; bioassay; chemiluminescence; intrinsic factor; food
Article
Perhaps no other vitamin has received, in such a short time, the concentrated effort that has been expended on vitamin B12. Yet since vitamin B12 was isolated in 1948 (9), the analytical determination of the vitamin in natural substances has remained a difficult problem. The rapid spectrophotometric method described here was developed for the determination of vitamin B12 in fresh and dried bacteria cells. Benzyl alcohol or n-propyl alcohol is used to extract vitamin B12 selectivety from dry or moist bacteria cells. The extracted vitamin is then determined by differential spectrophotometric measurement. Assay results are presented in comparison with microbiological results for bacteria cells, and for feed supplements. The new method is simple to operate, and is much faster and more accurate than bioassay. It will be useful for control of vitamin B12 production by fermentation, and for the standardization of finished products.
Article
The new chemical method for determining vitamin B12 was devised to replace the more costly and less accurate biological assay, which uses the accelerating effect of the vitamin on the growth of certain types of microorganisms. The chemical method of assay approaches the biological method in sensitivity and excels it with respect to speed and accuracy. It enables one to determine vitamin B12 in fermentation broths and crude concentrates with a reliability not hitherto achieved by other published methods of analysis. Vitamin B12 is an important growth factor widely distributed in minute quantities. A red metal-containing pigment, vitamin B12 stimulates the formation of red corpuscles in the human body and plays an important role in human and animal nutrition. The search for new sources of vitamin B12 as well as studies relating to the best methods for evaluating the vitamin in human and animal nutrition will be greatly accelerated by this new chemical analysis.
Article
The catalytic hydrogen process has been investigated for vitamin B12a, aquocobalamin, In phosphate buffer (pH 6.8), chloroacetlc acid buffer (pH 2.87), sulfate buffer (pH 1.97), and nonbuffered HCI solutions and for methylcobalamin In chloroacetlc acid buffer (pH 2.87), sulfate buffer (pH 1.97), and nonbuffered HCI solutions. The proposed mechanisms of these processes all Involve protonatlon of the cobalamln species, Irreversible reduction of the protonated species, and a following redox reaction which regenerates a precursor cobalamln species and produces molecular hydrogen. Depending on the solution conditions the regeneration reaction can take place in a reaction layer In solution or with an adsorbed catalyst and a diffusing species. Rate constants are estimated for the catalytic rate determining steps and are within a few orders of magnitude of diffusion limited rates leading to very large catalytic currents and the possibility of ultratrace electroanalysis.
Article
Nonlinear regression was applied to the analysis of steady-state voltammograms obtained at carbon fiber microelectrodes. Reversible regression models were used to analyze data for oxidation of ferrocene in acetonitrile with and without added electrolyte. This analysis constitutes a test for reversibility of the redox couple. An estimate of cell resistance in highly resistive media was obtained by including ohmic drop in the model for reversible electron transfer. This allows computation of „resistance-free” voltammograms obtained in that medium. A general model for steady-state voltammograms of quasi-reversible electrode reactions was used to estimate simultaneously apparent standard heterogeneous electron transfer rate constants (k0′), formal potentials, and electrochemical transfer coefficients for ferrocyanide and the Co(II)/Co(I) couple of vitamin B12r. Precision for ferrocyanide with a k0′ of 0.023 cm/sec was about ±30%. Results on ferrocene suggest an upper limit for estimating k0′ of about 1–2 cm/sec when the signal/noise ratio (S/N) is large. Data for the quasi-reversible Co(II)/Co(I) couple of vitamin B12r showed that results of reasonable precision and accuracy can be obtained for systems with poor S/N.
Article
The determination of cobalamins using capillary electrophoresis inductively coupled plasma mass spectrometry (CE-ICP-MS) was investigated. Both capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) modes of operation were studied. The optimal separation of four cobalamin species (cyanocobalamin, hydroxocobalamin, methylcobalamin, and 5′-deoxyadenosylcobalamin) and a potentially harmful corrinoid analogue (cobinamide dicyanide) was obtained using CZE at a pH of 2.5. Both 20 mM phosphate and 20 mM formate buffers were used with success, although the formate buffer provided improved resolution. The CZE-ICP-MS method was used to quantify cyanocobalamin in a vitamin supplement and the analytical results were in good agreement (±5%) with values obtained by ICP-MS for total Co levels. The solution detection limits for cobalamins using CZE-ICP-MS were approximately 50 ng/ml. MEKC was found to be useful for the screening of vitamin preparations because it provided a rapid means of distinguishing cyanocobalamin (the form most commonly used in vitamin preparations) from free cobalt. The separation of free cobalt and cyanocobalamin using MEKC was achieved in less than 10 min.
Article
C1-metabolizing bacteria were analyzed for their corrinoids. The autotrophic phototrophe Chloroflexus aurantiacus contains predominantly the light-sensitive coenzyme B12. The corrinoid could be teh prostethic group of a methylmalonyl-CoA mutase, which is involved in the CO2 fixing reaction sequence from proplonyl-CoA to succinyl CoA. Methanobacterium thermoautotrophicum and Sporomusa ovata contain only traces of light-sensitive corrinoids, indicating that the demethylation reaction is favored, if these corrinoids are involved in methyl transfer reactions. The chemical structure of the unique p-cresolyl cobamide is specific for the acetogenic bacterium S. ovata, rather than the corrinoid ‘factor III’ for methanogenic bacteria.
Article
The binding of several corrinoids to the binding site of human intrinsic factor, transcobalamin or haptocorrin was investigated. p ‐Cresolyl cobamide and 2‐amino‐vitamin B 12 are complete corrinoids, whose nucleotide at the lower face of the corrin ring is not coordinated to the cobalt. These corrinoids were ≥ 10 ³ times less efficiently recognized by intrinsic factor or transcobalamin than vitamin B 12 , which contains a Co‐coordinated nucleotide. Pseudovitamin B 12 , with a weak Co‐N coordination bond, revealed only moderate affinity to intrinsic factor. From these findings it is concluded that the cobamide binding to intrinsic factor and transcobalamin is strongly affected by the Co‐N coordination bonds of their lower cobalt nucleotide ligands. We suggest that the Co‐N coordination bond positions the nucleotide at a critical distance to the corrin ring, which is recognized by the binding proteins. Human haptocorrin, however, disclosed to distinctive selectivity regarding the different corrinoid structures. The protein bound all corrinoids with similar efficiency, independent of the strength of their Co‐N coordinations, or the structures of their lower Coα ligands. Hence, the corrin ring, rather than a structural feature induced by the Co‐N coordination, has to be considered responsible for the corrinoid binding to haptocorrin.
Article
An investigation has been carried out to establish a rapid method for the determination of vitamin B12 as cobalt in solid pharmaceutical samples by electrothermal atomization atomic absorption spectrometry with the solid sampling technique using an inner miniature cup. Calibration graphs of peak area versus mass of the element were constructed by use of a synthetic reference material (SyRM). The SyRM is prepared by coprecipitation of cobalt ions with magnesium(II) 8-quinolinate. In order to determine the accuracy of the proposed method, three pharmaceutical preparations were analyzed according to the proposed method using standardization against the SyRM and the results obtained compared with those when solutions of the same samples were analyzed by other techniques. There is good agreement between the results obtained from the proposed and the other method. The detection limit for cobalt in a solid pharmaceutical preparation is 0.15 ng/mg (i.e. 4 ng/mg of vitamin B12) for a typical sample mass of 1.0 mg.
Article
The coupling of liquid chromatography and electrothermal atomic absorption spectrometry (LC-ETAAS) lowers the detection limit for identification of vitamin B12 analogues. Cobalamins and aqueous cobalt (II) were separated by reversed-phase liquid chromatography using a linear gradient: 2674 (v/v) methanol:0.05 M phosphate buffer (pH 4.2) to 5050 mixture over 8 min. The vitamins were quantitatively determined in the column effluent by measuring total cobalt by ETAAS. The analysis of meat and liver extracts by LC-ETAAS showed that the matrix did not interfere with the determination of cobalt. Hence, recoveries of cobalt in spiked meat and liver samples were satisfactory.
Article
A new solid-phase enzyme-linked competitive binding assay for vitamin B12 (cyanocobalamin) is described. The assay is based on the competition between analyte B12 molecules and a glucose-6-phosphate dehydrogenase-vitamin B12 conjugate for a limited number of R-protein binding sites immobilized on sepharose particles. After appropriate incubation and washing steps, the enzyme activity bound to the solid-phase is inversely related to the concentration of B12 in the sample. Under optimized conditions, the method can detect B12 in the range of 310–10–110–8 M (using 100l sample) with high selectivity over other biological molecules.
Article
A fluorimetric method for the determination of vitamin B12 has been developed. The fluorescence emission was measured at λex/λem275/305 nm in phosphate buffer solution (pH 7.0), and the experimental variables and possible interference were studied. The linear calibration range was 1.000 ng/mL to 100.0 ng/mL with a correlation coefficient of 0.9994 and a detection limit of 0.1 ng/mL. The method is rapid, simple and highly sensitive. It was used to determine vitamin B12 in pharmaceutical preparations. The recovery was 96%–98% and the relative standard deviation was in the range of 1.8%–2.7%. The results agreed with those obtained by spectrophotometry.
Article
A novel chemiluminescence (CL) sensor for vitamin B12 combined with flow-injection analysis is presented in this paper. It is based on the catalytic effect of cobalt(II), liberated from vitamin B12 by acidification, on the CL reaction between luminol, immobilized electrostatically on an anion-exchange column, and hydrogen peroxide electrochemically generated on-line via a negatively-biased electrode from dissolved oxygen in the flow cell. The sensor responds linearly to vitamin B12 concentration in the 1.0 × 10−3–10 mg l−1 range, and the detection limit is 3.5 × 10−4 mg l−1 vitamin B12. A complete analysis, including sampling and washing, could be performed in 1 min with a relative standard deviation of < 3.5%. The system is stable for over 500 determinations and has been applied successfully to the determination of vitamin B12 in pharmaceutical preparations.
Article
Determination of trace amounts of cyanocobalamin (18 ng/g) in fatty foods was performed by solid-phase extraction and high-performance liquid chromatography (HPLC) with visible detection at 550 nm using a membrane filter for the precolumn separation of particulate material. It was found that a membrane filter (HLC-Disk 25, 0.45 μm) was most suitable for the separation of oily particulates. A sample solution was applied to a solid-phase extraction cartridge and then cyanocobalamin was eluted using a 50% aqueous acetonitrile solution followed by HPLC. This method was suitable for the determination of trace amounts of cyanocobalamin in nutrient samples. The proposed method was simple, rapid (extraction time: ca. 12 min, analysis time: ca. 12 min), sensitive (detection limit: ca. 0.15 ng at a signal-to-noise ratio of 3:1), highly selective and reproducible (relative standard deviation: 2.67%) for cyanocobalamin. The calibration graph for cyanocobalamin was linear in the range of 0.1 to 30 ng. Recovery of cyanocobalamin was over 90% by the standard addition method.
Article
Water- and fat-soluble vitamins were separated on a MetaChem Polaris C18-A ( mm, 3 μm particle size) in a single run using combined isocratic and linear gradient elution with a mobile phase consisting of 0.010% trifluoroacetic acid of pH 3.9 (solvent A) and methanol (solvent B) at the flow rate 0.7 ml min−1. A linear gradient profile (A:B) started at 95:5 and was constant in the first 4 min, then linearly decreased up to 2:98 during the next 6 min, then it was constant in the next 20 min and finally linearly increased up to 95:5 ratio of water phase in the last 5 min of the separation. The most suitable detection wavelength for simultaneous vitamin determination was 280 nm. The method was applied for the solid sample of pharmaceutical preparation (B-Komplex), fortified powdered drinks (multi-vitamin) and food samples. The results were in good agreement with the declared values.
Article
A novel method for the determination of vitamin B12 by high-performance liquid chromatography with fluorescence detection is reported. The method was simple and highly sensitive with good precision. Vitamin B12 was analyzed by HPLC on a μBondapak C18 column (300×3.9 mm, 10 μm) with methanol–water (30:70) as mobile phase and fluorescence detection at 305 nm (with excitation at 275 nm). The calibration graph was linear from 1.000 to 100.0 ng ml−1 for vitamin B12 with a correlation coefficient of 0.998 (n=6). The detection limit was 0.1 ng ml−1. The method was successfully applied to the determination of vitamin B12 in vitamin B12 tablets, multivitamin tablets and fermentation medium. The recovery was from 94 to 102% and the relative standard deviation was in the range of 1.8 to 4.1%.
Article
Vitamin analysis is essential for quality control and development of functional foods. In this study, a biosensor-based technology developed by Biacore AB was evaluated for analysis of water-soluble vitamins B2, B12, folic acid, biotin, and pantothenic acid used to supplement infant formula samples. Performance parameters such as accuracy, repeatability and recovery for the five vitamins were studied. The repeatability was measured in terms of relative standard deviation (RSD) and HORRATr value. The RSD for all vitamins was below 2% and the values of HORRATr were 0.16, 0.10, 0.15, 0.11 and 0.22, for B2, B12, folic acid, biotin, and pantothenic acid, respectively. The recovery of vitamins ranged from 94.7% to 109.1%. Linear analyses indicated that the square of the correlation coefficient (R2) for B2, B12, folic acid, biotin, and pantothenic acid were 0.993, 0.997, 0.993, 0.993 and 0.995, respectively. The results showed that the biosensor-based vitamin analysis technology is a sensitive, reliable and realistic alternative to other methods.
Article
Methods for the determination of Vitamin B12 remain limited due to their low sensitivity and poor selectivity. In the present work, a simple and sensitive HPLC-ESI-MS method for determining Vitamin B12 in food products and in multivitamin-multimineral tablets was developed. Vitamin B12 was extracted from food products with 50 mM sodium acetate buffer (pH 4.0) in the presence of sodium cyanide. Total Vitamin B12 content in food product can be obtained by efficient enzymatic hydrolysis to release the bound Vitamin B12. Vitamin B12 was quantified with ginsenoside Re as internal standard (I.S.) after their separations on a C18 column with a gradient of mobile phase made of water and acetonitrile. MS with SIR mode at m/z 930.8 was used for Vitamin B12 quantification. The calibration graphs plotted with five concentrations of Vitamin B12 was linear with a regression coefficient r2 = 0.9994. The intra-assay R.S.D. and the inter-assay R.S.D. were 2.6% (n = 5) and 3.5% (n = 6), respectively. The recoveries evaluated at spiking three different concentrations on fortified products were above 93%. The method has been applied successfully in the determination of Vitamin B12 in fortified food products and multivitamin-multimineral tablets.
Article
A sensitive chemiluminescence (CL) method, based on the enhancive effect of cobalt(II) on the CL reaction between luminol and dissolved oxygen in a flow injection (FI) system, was proposed for determination of Vitamin B12. The increment of the CL intensity was proportional to the concentration of Vitamin B12, giving a calibration graph linear over the concentration from 2.0×10−10 to 1.2×10−6 g l−1 (r2=0.9992) with the detection limit of 5.0×10−11 g l−1 (3σ). At a flow rate of 2.0 ml min−1, a complete determination of Vitamin B12, including sampling and washing, could be accomplished in 0.5 min with the relative standard deviations (R.S.D.) of less than 5.0%. The proposed method was applied successfully to the determination of Vitamin B12 in pharmaceuticals, human serum, egg yolk and fish tissue.
Article
The results obtained by a competitive binding method for the determination of vitamin B12 in food were compared with those obtained by a widely used microbiological assay with Lactobacillus leichmannii A.T.C.C. 7830. Both assays were performed on the same sample extract. The extraction was carried out at pH 4.5, 121°C for 10 min with 0.1 m sodium acetate-acetic acid buffer in the presence of potassium cyanide. A high correlation (r2 = 0.841) between the results from the two methods was found. In the case of pork and yoghurt, the large differences observed could be due to the presence of substances in their extracts which interfere with the assays. The competitive binding assay can therefore be applied to the determination of vitamin B12 in some foods.
Article
High-performance capillary electrophoresis (HPCE) was compared for the identification and determination of corrinoids (hydroxy-, cyano-, 5′-desoxyadenosyl- and methyl-cobalamin and cyano-cobinamide) with high-performance liquid chromatography (HPLC). The within-run reproducibility of the retention times in HPCE and HPLC were similar (2.4 and 2.2%, respectively). The detection limit in HPCE was 20 μg/ml. HPLC can be used, in combination with radioisotope dilution assay, when very low concentrations (100 pg/ml) have to be determined in biological material. HPCE is more efficient than HPLC for the identification of corrinoids after conversion into the CN-cobalamin and CN-cobinamide forms.
Article
A protein-binding assay for the determination of vitamin B12, based on the use of an R-protein-enzyme conjugate and the microtitration plate format, has been developed and applied to fortified foods. The assay limit of detection was 9 pg per well and the assay sensitivity 0.09 μg per 100 g of food. The immobilized phase of the assay, a B12-keyhole limpet haemocyanin passively adsorbed to the plate wells, was synthesized by procedures giving significant advantages over previously reported approaches. The assay was simple to perform.
Article
Aerosol matrix-assisted laser desorption/ionization (MALDI) has been combined with a reflectron time-of-flight mass spectrometer for improved mass resolution. A methanol solution of matrix and analyte was sprayed directly into a reflectron time-of-flight mass spectrometer, and the aerosol particles were irradiated and ionized with a frequency-tripled Nd:YAG laser at 355 nm. Mass resolution of over 300 was observed for the peptides bradykinin, angiotensin II, and gramicidin D and for vitamin B(12). This represents a resolution enhancement of approximately 10-fold over that previously reported for aerosol MALDI with a linear time-of-flight instrument.
Article
Since R protein binds cobalamin (vitamin B12) and cobalamin analogues, whereas intrinsic factor is highly specific for true cobalamin, we compared the serum cobalamin values obtained with these proteins in radioisotope dilution assays. With R protein, eight of 21 patients with cobalamin deficiency had serum cobalamin levels (mean, 204, range, 85 to 355 pg per milliliter) that overlapped with values for 74 normal subjects (mean, 576, range, 220 to 1230). With intrinsic factor, no patient values (mean, 36, range, less than 10 to 78 pg per milliliter) overlapped with the normal values (mean, 322, range, 130 to 785). Paper chromatography showed that these differences were due to the presence of cobalamin analogues. R protein constituted 51 to 85 per cent of the cobalamin-binding protein in 10 commercial serum cobalamin assay kits, which were said to contain "intrinsic factor". Human plasma contains cobalamin analogues that can mask cobalamin deficiency with current radioisotope dilution assays.
Article
A new isomeric form of cobalamins is reported. The conversion of cobalamin to cobalamin (the new form) is achieved by substituting the benzimidazole base by a less bulky group like H2O or CN- and modest thermal treatment. The back conversion of adenosylcobalamin to the corresponding regular form occurs in the "base-off" form at room temperature. It seems that the corrin ring becomes quite flexible in the "base-off" form and the freer axial movement of the cobalt atom flips the corrin ring into a different conformation. The change in conformation is borne out by subtle changes in the proton magnetic resonances on the corrin ring and the base, and very marked variation in the emission Mössbauer spectra. The latter is indicative of appreciable changes in the spatial conformation in the immediate vicinity of the central metal atom. The ultraviolet-visible and infrared spectra of cobalamin are indistinguishable from those of its corresponding regular form. The new conformational isomeric species is present as an impurity in all commercially available cobalamins (including pharmaceutical preparations). It raises the question whether the cobalamins' constitute the real biologically active anti-anemic factor in humans.
Article
Parts-per-trillion detection of several vitamins and amino acids has been achieved through the application of laser-induced molecular fluorescence techniques. Compounds which possess native fluorescence were investigated as well as nonfluorescent compounds which could be labeled with fluorescamine. In all cases the use of a pulsed laser as the irradiation source was shown to improve the minimum detectabilities by at least two orders of magnitude over those achieved with conventional light sources.
Article
1.Low serum B12 levels can be measured with considerable precision by microbiological assay with the Euglena gracilis assay and B12 deficiency can be recognised with a high level of consistency by either the Euglena or L. leichmannii assays. Either method is ideally suited for the assay of large numbers of specimens. The Lactobacillus leichmanii technique requires preliminary extraction of protein and it has been suggested that this may be a source of inaccuracy. 2.The radioisotope dilution assay should be the ideal method of measuring B12 levels in small or moderate numbers of specimens for it is a simple method that can be carried out in any laboratory with suitable counting equipment. After many false starts the conditions required for accurate assay are now understood. Each of 40 to 50 radioisotopic dilution techniques that have been introduced claims to be capable of differentiating B12 deficiency from control subjects but the reported correlations between the actual levels found in the two different assays are variable and the levels may be much higher with some radioisotopic methods. 3.The subnormal serum levels which are found in pernicious anaemia with all these techniques indicate severe reduction of the liver B12 level. A low serum B12 level in other conditions has, in the absence of associated folate or iron deficiency, the same significance. If the fall in the serum B12 level is associated with folate or iron deficiency, the tissue B12 levels are usually reduced but not to the low levels found in B12 deficiency states. 4.In practice, a subnormal B12 level is a valuable pointer not only to unsuspected pernicious anaemia but also to other gastrointestinal or nutritional disorders. The significance of a fall in the B12 level can only be understood if its cause is defined by a full clinical and gastroenterological investigation. 5.Falsely low serum B12 levels are found under certain iatrogenic conditions and B12 levels may be normal in spite of cellular deficiency of B12 under the rare circumstances of pernicious anaemia being associated with chronic myeloid leukaemia or when there is deficiency of TC 2.
Article
A method for the simultaneous determination of cyanocobalamin, cobinamide (Factor B), and hydroxocobalamin in the solid state is described. The method is based on heating at 120 degrees for cobinamide and at 140-145 degrees for cyanocobalamin (15-20 min). The cyano content in the sample is distilled as hydrocyanic acid, trapped in 0.1 M potassium nitrate at pH 12-13, and determined by means of the cyanide ion-selective electrode. The error of this method, statistically established, does not exceed +/-3%.