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Trends in analysis of vitamin B-12
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.
... 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 ...
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.
... Quantification of vitamin B12 can be done using microbiological, radioisotopic dilution, electrochemical, chromatographic and spectroscopic methods. 3,4 Few drawbacks of these methods are that these methods require advanced instruments and trained technicians, expensive and cannot be performed on site. Most electrochemical methods are based on the redox activity of the cobalt metal ion attached to the corrin ring. ...
... Change in the oxidation state of the Co(III) ion to Co(II) or Co(I) releases the group attached to sixth site in vitamin B12. 1,3 As reported previously, reduction of Co(III) to Co(II) or Co(II) to Co(I) can be used for the electrochemical quantification of vitamin B12. 1,3 In addition to Co(III) reduction, the oxidation of the corrin ring to form cationic radical can also be used for the quantification of this vitamin. ...
... 1,3 As reported previously, reduction of Co(III) to Co(II) or Co(II) to Co(I) can be used for the electrochemical quantification of vitamin B12. 1,3 In addition to Co(III) reduction, the oxidation of the corrin ring to form cationic radical can also be used for the quantification of this vitamin. 1 cost electrochemical paper-based microfluidic device (µPAD) to quantify vitamin B12 in food samples. ...
This paper reports the development of a simple, sensitive and low-cost microfluidic paper-based electrochemical sensing device developed for the detection of vitamin B12 in foods and pharmaceutical products. This hydrophobic barrier device was constructed on paper using commercially available varnish paint. The working electrode was constructed using a mixture of varnish and graphite powder. Silver conductive ink and a 6B pencil were used to fabricate the reference and counter electrodes, respectively. Cyclic voltammograms of standard vitamin B12 and real samples were collected from -2 V to +2 V at a scan rate of 200 mV/s. Currents of cyclic voltammograms at +2 V for the concentrations of vitamin B12 from 5 – 25 mM produced a linear relationship (r^2 of 0.98). The calculated limit of detection of this paper-based device is 3.7 mM.
... The measurement of B12 in food products is problematic for two main reasons: one, its low concentration in non-fortified foods and two, the possible inclusion of B12 analogues when measured with non-specific methods (Stabler and Allen 2004;Ball 2006). In addition, the noncyano forms of B12 are sensitive to light, thus complicating the accurate quantification of the individual forms (Kumar et al. 2010). As CNCbl is relatively stable, the native B12 compounds in samples are converted into CNCbl by heat extraction with cyanide (Ball 2006). ...
... Alongside MBA, colorimetric and spectrophotometric methods were also used for the analysis of B12 in food and microbial materials (Fisher 1953;Shaw and Bessell 1960). In the last three decades, several chromatographic methods utilising different detection techniques in combination with improved sample preparations have been developed and are being increasingly applied for B12 analysis (Ball 2006;Kumar et al. 2010;Green and Miller 2014). Although modern liquid chromatographic methods are more selective, MBA is still one of the most sensitive methods (detection limit: 1 20 pg/mL) for B12 quantification (Kumar et al. 2010;Sych et al. 2016) and is an official method recommended by the AOAC (2006). ...
... In the last three decades, several chromatographic methods utilising different detection techniques in combination with improved sample preparations have been developed and are being increasingly applied for B12 analysis (Ball 2006;Kumar et al. 2010;Green and Miller 2014). Although modern liquid chromatographic methods are more selective, MBA is still one of the most sensitive methods (detection limit: 1 20 pg/mL) for B12 quantification (Kumar et al. 2010;Sych et al. 2016) and is an official method recommended by the AOAC (2006). Recent advances in liquid chromatographic separation, e.g. ...
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http://urn.fi/URN:ISBN:978-951-51-2753-2
... 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). ...
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
... 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. ...
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. ...
... The official methods for its determination are microbiological assay and LC-UV [14,15]. Other methods include radioimmunoassay (RIA) [16], LC-ICPMS [17,18], AAS [16], LC-MS/ MS [19,20], capillary electrophoresis with UV detection [21] and biosensors [22,23]. With the exception of LC-MS/MS, these methods lack specificity and sensitivity and, in case of RIA and biosensor, they may be too expensive for routine analysis [16,22]. ...
... Other methods include radioimmunoassay (RIA) [16], LC-ICPMS [17,18], AAS [16], LC-MS/ MS [19,20], capillary electrophoresis with UV detection [21] and biosensors [22,23]. With the exception of LC-MS/MS, these methods lack specificity and sensitivity and, in case of RIA and biosensor, they may be too expensive for routine analysis [16,22]. Furthermore, LC-ICPMS is an indirect method with the inconvenience of lower ionization efficiency of organic Co in comparison to the inorganic one [17]. ...
Cyanocobalamin (CNCbl) is an active form of vitamin B12, commonly employed for the preparation of multivitamin supplements and fortified food. In this study, we present a novel analytical method for its determination based on stable isotope dilution liquid chromatography electrospray tandem mass spectrometry (ID LC-MS/MS). Isotopically enriched ¹³C¹⁵NCbl was synthesized in-house and used as internal standard. The method was validated using NIST SRM 3280 multivitamin reference material and by comparison with an independent methodology based on LC-ICPMS. The proposed method provided a detection limit of 57 pg/g and could be applied for the determination of trace level of CNCbl in multivitamin supplements with a relative standard uncertainty better than 3%. The novel ID LC-MS/MS is a primary ratio method that could become a reference for CNCbl determination in multivitamins and food supplements. The method was applied for the characterization of two NRC multivitamin tablet Certified Reference Material (CRM) candidates, VITA-1 and VITB-1 whose CNCbl levels were quantified as 2.64 0.09 and 1.75 0.12 μg/g, respectively.
... The name vitamin B12 refers to the form with the cyano ligand, which does not occur in nature but is formed from other cobalamins during extraction with sodium cyanide (Battersby and Leeper, 1999). The other cobalamins are converted to the cyanocobalmin to increase stability, which protects the vitamin from degradation during heat extraction (Kumar et al., 2010). Cyanocobalamin is readily converted into coenzyme forms in the human body (Obeid et al., 2015). ...
... The buffer contained 100 μL of 1% NaCN to convert all upper ligands of the cobamides to their most stable cyano forms. This was performed to prevent losses of the compounds due to heating (Kumar et al., 2010). ...
... 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. ...
... 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. ...
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. ...
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). ...
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]. ...
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). ...
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]. ...
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). ...
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:
... At specific time intervals after either I.M injection or buccal application, 1.5-ml blood samples were withdrawn from the ear vein (using 26-gauge needles) into prelabeled heparin-beaded tubes. Serum levels of CBL were investigated using ELISA Accu Diag TM Vitamin B12 ELISA Kit [25]. The procedures were accomplished with the aid of METER TECH, model 6 DC 12 v at biochemistry laboratory, Deraya University, Minya, Egypt. ...
Attempting to prepare a convenient bioavailable formulation of vitamin B12 (cyanocobalamin), seventeen tablet formulations were prepared by direct compression. Different concentrations of hydroxypropyl methyl cellulose (HPMC), carbopol 971p (CP971p) and chitosan (Cs) were used. The tablets were characterized for thickness, weight, drug content, hardness, friability, surface pH, in-vitro drug release and mucoadhesion. Kinetic analysis of the release data was conducted. Vitamin B12 bioavailability from the optimized formulations was studied on rabbits by the aid of enzyme-linked immunosorbent assay. Neurotone® I.M. injection was used for comparison. HPMC (F1-F4), CP971p (F5-F8) and HPMC/CP971p (F12-F15) based formulations showed acceptable mechanical properties. The formulated tablets showed maximum swelling indices of 232 ± 0.13. The surface pH values ranged from 5.3 ± 0.03 to 6.6 ± 0.02. Bio-adhesive force ranged from 66 ± 0.6 mN to 150 ± 0.5 mN. Results showed that CP971p based tablets had superior in-vitro drug release, mechanical and mucoadhesive properties. In-vitro release date of selected formulations were fitted well to Peppas model. HPMC/CP971p based formulations showed bioavailability up to 2.7-folds that of Neurotone® I.M. injection.
... Liquid chromatographic methods are better suited for the measurement of active B12 in food materials, particularly for samples containing the B12 analogues in which the MBA analysis is unreliable (Kumar, Chouhan, & Thakur, 2010). In our earlier work (Chamlagain, Edelmann, Kariluoto, Ollilainen, & Piironen, 2015), we reported a sensitive and selective method suitable for the determination of active B12 in fermented products, using ultra-high performance liquid chromatography (UHPLC) after purifying sample extracts on B12specific immunoaffinity columns. ...
The in situ production of active vitamin B12 was investigated in aqueous cereal-based matrices with three strains of food-grade Propionibacterium freudenreichii. Matrices prepared from malted barley flour (33% w/v; BM), barley flour (6%; BF), and wheat aleurone (15%; AM) were fermented. The effect of cobalt and the lower ligand 5,6-dimethylbenzimidazole (DMBI) or its natural precursors (riboflavin and nicotinamide) on active B12 production was evaluated. Active B12 production was confirmed by UHPLC–UV–MS analysis. A B12 content of 12–37 μg·kg−1 was produced in BM; this content increased 10-fold with cobalt and reached 940–1,480 μg·kg−1 with both cobalt and DMBI. With riboflavin and nicotinamide, B12 production in cobalt-supplemented BM increased to 712 μg·kg−1. Approximately, 10 μg·kg−1 was achieved in BF and AM and was increased to 80 μg·kg−1 in BF and 260 μg·kg−1 in AM with cobalt and DMBI. The UHPLC and microbiological assay (MBA) results agreed when both cobalt and DMBI or riboflavin and nicotinamide were supplemented. However, MBA gave ca. 20%–40% higher results in BM and AM supplemented with cobalt, indicating the presence of human inactive analogues, such as pseudovitamin B12. This study demonstrates that cereal products can be naturally fortified with active B12 to a nutritionally relevant level by fermenting with P. freudenreichii.
... When the group is cyanide, it is called cyanocobalamin—represents the most stable active form of vitamin B12 and the most popular synthetic form. Other active forms in the human body are 5-deoxyadenosylcobalamin—with 5ʹ-deoxyadenosine; methylcobalamin—with methyl group; and hydroxocobalamin—with hydroxyl group[6,7]. ...
Vitamin B12 is a water-soluble vitamin and an important micronutrient with critical role in DNA, protein, and lipid synthesis. It is responsible for one-carbon metabolism and cell division of nervous and hematopoietic cells. Among its various functions, the role as immunomodulator in cellular immunity, especially in elevating the number of CD8+ cells and NK cells, attracts scientific interest. Many alternative anticancer and anti-inflammatory treatments involve the use of B12 together with other vitamins and nutrients, but still the scientific information is too obscure and insufficient. Controversial data link tumorigenesis with either increased or decreased B12 blood levels in different types of cancer. Dietary intake and additional supplement with the vitamin do not protect against cancer risk, but the dominant opinion is to integrate B12 as part of rational and healthy nutrition to ensure proper function of the immune system. This chapter will review in brief the most important facts for vitamin B12 functions and properties. We will try also to present in concise way the human immune system and the exact role of B12 in immune activity with emphasis on the questionable participation of vitamin B12 in the process of carcinogenesis and its significance as anticancer immunotherapy.
... Cobalamin, or vitamin B 12 , is an essential nutrient for all higher animals and some microorganisms [27]. Vitamin B 12 is a critical nutrient in many biological processes including; the maintenance of the myelin sheath surrounding nerve cells, cell development, fat and carbohydrate metabolism, and is essential for the rapid synthesis of DNA during cell division [28]. The doping of horses with cobalt has recently been widely debated in the Australian media Fig. 4. Signal intensity and stability of a 100 ng mL −1 Rb solution delivered through the chipLC (᭹ plotted against the left axis). ...
The Agilent Chip Cube Interface is a microfluidic chip-based technology originally designed for nanospray molecular mass spectrometry in which the sample enrichment, nano-column, tubing, connectors and spray tip were integrated into a single biocompatible chip. Here we describe the hyphenation of the Chip Cube Interface to ICP-MS via modification of the standard HPLC chip design and a new total consumption nebuliser suitable for flow rates as low as 300 nL min⁻¹. The potential of the instrument to eliminate common nanoLC − ICP-MS shortcomings such as leaks, blockages and band-broadening was demonstrated via analysis of cyanocobalamin in equine plasma. The method was linear over three orders of magnitude with an r² of 0.9999, the peak area repeatability was 1.9% (n = 7), and the detection limit was 14 ng mL⁻¹. This novel configuration of the Chip Cube Interface coupled to ICP-MS is a suitable platform for the analysis of biomolecules associated with trace metals and speciation applications.
... Briefly, the wet-cell mass from equal volumes of cultures was weighed and suspended with 10 mL of extraction buffer (pH 4.5, 8.3 mM sodium hydroxide, and 20.7 mM acetic acid). Subsequently, 100 µL of 1% sodium cyanide was added in order to convert all the upper ligands of vitamin B12 and its analogs to the most stable cyano forms, preventing loss of the compounds during heat extraction (Kumar et al., 2010). The suspension was vortexed and then extracted in a boiling water bath. ...
Propionibacterium freudenreichii is a traditional dairy bacterium and a producer of short chain fatty acids (propionic and acetic acids) as well as vitamin B12. In food applications, it is a promising organism for in situ fortification with B12 vitamin since it is generally recognized as safe (GRAS) and it is able to synthesize biologically active form of the vitamin. In the present study, vitamin B12 and pseudovitamin biosynthesis by P. freudenreichii was monitored by UHPLC as a function of growth in food-like conditions using a medium mimicking cheese environment, without cobalt or 5,6-dimethylbenzimidazole (DMBI) supplementation. Parallel growth experiments were performed in industrial-type medium known to support the biosynthesis of vitamin B12. The production of other key metabolites in the two media were determined by HPLC, while the global protein production was compared by gel-based proteomics to assess the effect of growth conditions on the physiological status of the strain and on the synthesis of different forms of vitamin. The results revealed distinct protein and metabolite production, which reflected the growth conditions and the potential of P. freudenreichii for synthesizing nutritionally relevant amounts of active vitamin B12 regardless of the metabolic state of the cells.
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.
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.
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.
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.
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.
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.
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.
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.
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...
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.
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.
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.
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.
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.
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.
The reaction of cyanocobalamin (CNCbl) with sodium hydroxymethanesulfinate (HMS) was studied over a wide range of pH (4-11) under aerobic conditions. CNCbl is destroyed in the presence of HMS in aqueous solution to form uncolored substances. The accumulation of stable yellow corrinoids (SYCs) preceded these changes at pH ≥ 8. The major stable yellow corrinoid is (15R)-Coα, Coß - dicyano-13-dehydro-15-hydro-l5-hydroxycob(III)alamin. The yield of this SYC is 25%, and the stability of this compound decreases significantly with increasing concentrations of HMS, pH and temperature.
A new electrochemical sensor for sensitive and selective detection of vitamin B12 (VB12) was constructed by the electropolymerization of pyrrole (Py) in the presence of ferromagnetic nanoparticle-incorporated triazine dendrimer (FMNPs@TD) on a gold electrode (Au). The gold/polypyrrole/ferromagnetic nanoparticles/ triazine dendrimer (Au/PPy/FMNPs@TD) electrode showed electrocatalytic activity for the reduction of VB12 in Britton-Robinson buffers. The performance and interaction of the Au/PPy/FMNPs@TD with VB12 were investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), differential pulse voltammetric (DPV), UV-visible spectroelectrochemistry, and density functional theory calculations. The resulting sensor exhibited excellent performance for determination of VB12 with a wide linear range (2.50 nM to 0.5 µM), highly reproducible response (RSD of 2.3%), low percentage of interferences, and long-term stability. The limit of detection (LOD) and limit of quantitation (LOQ) values for the determination of VB12 were 0.91 and 3.00 nM, respectively. The electron transfer rate constants (ks), charge transfer coefficient (α), surface concentrations of electroactive species (Г), and disproportionation equilibrium constant (KD) for Au/PPy/FMNPs@TD-modified electrodes were calculated. Finally, the constructed sensor was applied to the determination of VB12 in food samples.
Vitamin B12 is a very important micronutrient essential for health (due to its functions) supplied by the diet, mostly, from animal sources. Its deficiency may cause critical health problems, whereby it is important to incorporate this vitamin in food supplements and additives. On the other hand, vitamin B12 is a sensitive compound and can be easily degrade during food process and storage. Through microencapsulation it is possible to overtake some of these limitations and improve its application in food industry. The aim of this work was to produce microparticles with vitamin B12 (1, 2, 3, 4 and 5% (w/w)) and to study its release namely in simulated gastric conditions (SGF). Modified chitosan was chosen as encapsulating agent, considering its biocompatibility and good solubility in water. The microparticles were prepared by a spray-drying technique and then characterized in terms of morphology (Scanning Electron Microscopy - SEM) and particle size (Laser Granulometry Analysis). An UV spectrophotometric method was validated, to evaluate the vitamin release and its stability. The average diameter of the particles varies with vitamin concentration, ranging from 3 to 8 μm, considering the differential volume distribution, and from 0.1 to 0.9 μm considering the differential number distribution. The product yield of the spray-drying process was around 57% for the microparticles with vitamin. The SEM images show microparticles with a regular round shape and a smooth surface. The release time increases with increasing of pH, and decreases with decreasing temperature. The Weibull kinetic model fits very well the experimental results obtained in SGF at 37 °C. Microparticles stability was evaluated after 3 and 6 months and a small decrease in the amount of vitamin released was observed. Vitamin losses are < 10% for 3 months and < 20% for six months storage. So, microparticles of vitamin B12 with a good stability can be produced by spray-drying using modified chitosan.
This study aimed to formulate and evaluate vitamin B12- loaded buccal mucoadhesive hydrogel films. Various film formulations were prepared using chitosan and polyvinyl alcohol. The prepared films were characterized for thickness, weight variation, drug content, percentage moisture uptake and moisture content, surface pH, mechanical properties, in vitro release and mucoadhesion. Vitamin B12 bioavailability from the optimized formulation was studied on rabbits by the aid of enzyme-linked immunosorbent assay (ELISA). Neuroton® I.M injection was used for comparison. The films had acceptable mechanical and mucoadhesion properties. The percentages of moisture content of the optimized formulation were 3.2 ± 0.95 while the percentage drug released was 98.59% ± 1.41 at the end of 40 minutes. FTIR revealed the incidence of drug/polymer interaction. DSC revealed the possibility of the dispersion of cyanocobalamin in a molecular state with complete amorphization in the polymers. The estimated AUC0-8h showed 1.5-fold increases in the bioavailability of cyanocobalamin from the optimized formulation compared with the marketed I.M injection . These findings warrant that vitamin B12 buccal film formulation can be considered as an effective alternative portal with non-invasive and more convenient characteristics compared with the I.M injection dosage form.
Maternal diet and lifestyle choices may affect placental transfer of cobalamin (Cbl) to the fetus. Fetal liver concentration of Cbl reflects nutritional status with regards to vitamin B12, but at these low concentration current Cbl measurement methods lack robustness. An analytical method based on enzymatic extraction with subsequent RP-HPLC separation and parallel ICP-MS and ESI-Orbitrap-MS to determine specifically Cbl species in liver samples of only 10-50 mg was developed using 14 pig livers. Subsequently 55 human fetal livers were analyzed. HPLC-ICP-MS analysis for cobalt (Co) and Cbl gave detection limits of 0.18 ng/g and 0.88 ng/g d.m. in liver samples respectively with a recovery of >95%. Total Co (Cot) concentration did not reflect the amount of Cbl or vitamin B12 in the liver. Cbl bound Co contributes only 45 +/- 15 % to Cot. XRF mapping and XANES analysis confirmed the occurrence of non-Cbl cobalt in pig liver hot spots indicating particular Co. No correlations of total cobalt nor Cbl with fetal weight or weeks of gestation were found for the human fetal livers. Although no gender difference could be identified for total Co concentration, female livers were significantly higher in Cbl concentration (24.1 +/- 7.8 ng/g) than those from male fetuses (19.8 +/- 7.1 ng/g) (p=0.04). This HPLC-ICP-MS method was able to quantify total Cot and Cbl in fetus liver and it was sensitive and precise enough to identify this gender difference.
Traceable and precise quantitative measurements of cyanocobalamin (CN-Cbl) have been hampered by the lack of well characterized standards and pure materials of this bio-inorganic analyte that belongs to the water-soluble vitamins of the B-group known as vitamin B12. Measurement of cobalt and/or phosphorus content of vitamin B12 offer an approach for its quantitation that is traceable to the International System of Units (SI) with low measurement uncertainty. Cobalt and phosphorus measurements of CN-Cbl were carried out by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) and Ion Chromatography (IC). Use of a mixed bed ion exchange column coupled with post column reaction, IC provides a means to differentiate free cobalt from the cobalt complexed inside the corrin ring of the CN-Cbl molecule. In the case of ICP-AES and IC, a prerequisite for quality measurement is the purity of the starting vitamin B12 material. The relative expanded uncertainties (% U) expressed at 95% confidence for these analyses range from 0.3 to 1%.
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.
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.
The identification of structural markers for Bââ/protein interactions is crucial to a complete understanding of vitamin Bââ transport and metabolic reaction mechanisms of Bââ coenzymes. Fourier transform infrared spectroscopy can provide direct measurements of changes in the side chains and corrin ring resulting from Bââ/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 cmâ»Â¹ in DâO and ca. 1675 cmâ»Â¹ 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 cmâ»Â¹ (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.
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.
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.
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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 B12 are complete corrinoids, whose nucleotide at the lower face of the corrin ring is not coordinated to the cobalt. These corrinoids were ≥ 103 times less efficiently recognized by intrinsic factor or transcobalamin than vitamin B12, which contains a Co-coordinated nucleotide. Pseudovitamin B12, 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.
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.
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.
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.
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.
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.
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.
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.
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%.
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.
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.
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.
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.
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.
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.
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.
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.