Vitamin B12 Immunoaffinity Column (CDM040412)

Regulatory status: For research use only, not for use in diagnostic procedures.

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10 Columns
Principles of Testing
Many methods of Vitamin B12 determination based on HPLC-UV detection show low selectivity if problematic matrices are applied.
This method of content determination of Vitamin B12 combines the high selectivity of immunoaffinity columns with its potential to concentrate elute and of purification by HPLC column.
Detection Limit
0.1 to 5μg/g vitamin B12
Assay Procedure
Sample Preparation:
Vitamin B12 samples are to be extracted and analysed with the method of Li et al. [H.-B. Li, F. Cheng, Y. Jiang J. Chromatogr. A 2000; 891:243- 247], e.g. vitamin tablets, liquid vitamin preparations, cell culture extracts. Example: 25g vitamin containing tablets are dissolved in 100ml PBS. The resulting extract may be filtered through a 0.45μm membrane filter.

Enrichment Step IAC:
4ml extract (containing the quantity of Biotin from a 1g sample if above-mentioned sample preparation is followed) is diluted with a total volume of 20ml PBS and then applied in a reservoir on top of the BioTeZ-Immunoaffinity Column. The optimal flow rate through the gel is between 1 to 3ml/min.
According to application and contents expected the applied extract volumes could vary. E.g. extracts may be diluted 1+1 with PBS or 1+4 as mentioned above. In case of very low contents even extract volumes of 200ml may be applied without significant loss of analyte as long as resulting pH is fairly neutral and alcohol or acetonitrile content lies under 15%.

After the whole sample has passed through the gel, the latter is washed with 5ml of PBS. Remaining liquids in the gel are removed by applying either pressure from top of the column or under-inflation from the bottom.

The sample reservoir on top of the BioTeZ- Immunoaffinity Column is removed, and an appropriate vial is placed below the affinity column. The bounded vitamin B12 is eluted by using a total volume of 3ml of HPLC grade methanol. The elution process is performed in two steps. First, an amount of 1ml methanol is applied. Once this amount has passed through the column, there should be a waiting time of 30 seconds. After that, the second portion of 2ml of methanol is eluted through the column. The remaining methanolic solutions should be eluted by application of slight under- or overpressure. All methanolic fractions are unified to give the column elute.
The column elute may be injected into the HPLC directly or, if concentrations are very low, concentrated by evaporation (e.g. using VLM evaporator), re-dissolved in HPLC solvent and finally injected into the system. For the latter case, please see the sample calculation in which the sample concentrate is re-dissolved in 0.4ml HPLC solvent.
Recovery rates are >85% when vitamin B12 in buffer mixtures is analysed in the range of 0.1 to 5μg per IAC.
Analytical Method
Machine: Shimadzu; Column: Trentec Reprosil- Pur RP C18 120 ODS3 5μm; 125x3,0mm with guard column; Mobile Phase A: acetonitrile /water (70:30 v/v) (use only for cleaning purposes at the beginning and at the end of analytical series); Mobile Phase B: 0.03M potassium phosphate, pH 7.0-methanol (80/20 v/v); Gradient: 0.01min B 100%; 30min B 100% (isocratic); Flow Rate: 0.5ml/min; Time of Analysis: 30min; Injector Volume: 100μl; Detection: λ ABS [nm]: 361nm.


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Effect of drug physicochemical properties on swelling/deswelling kinetics and pulsatile drug release from thermoresponsive poly(N-isopropylacrylamide) hydrogels


Authors: Coughlan, DC; Quilty, FP; Corrigan, OI

The effect of drug physicochemical properties on swelling/deswelling kinetics and pulsatile drug release from a thermoresponsive hydrogel was examined. Hydrogels were loaded with drug and thermally triggered swelling/deswelling and release experiments were performed. Two series of drugs of contrasting hydrophilicity and varying physicochemical properties were examined. Benzoic acid (BA), its methyl and propyl esters, and diltiazem base were used as model hydrophobic drugs. Sodium benzoate (NaB), diltiazem HCl (DHCl), vitamin B12 (VB12) and various dextrans (MW 4300, 10,200, 42,000, 68,800) were used as model hydrophilic agents of increasing size. The hydrogel swelling rate was slowed by the presence of the hydrophobic drugs and this decreased rate was solubility dependant for the benzoates. The hydrophilic series increased the rate of swelling compared to the unloaded system. In all cases, the magnitude and rate of hydrogel contraction were proportional to the extent of swelling prior to temperature switch. Drug release was by diffusion below the lower critical solution temperature (LCST), while a solubility-dependent drug pulse release on temperature switch was observed for the hydrophobic series. Effectiveness of thermal control of hydrophobic drug release increased with increasing solubility. The hydrophilic series produced a molecular size-dependent drug pulse on temperature switch above the LCST. Pulsatile on-off drug release was shown with DHCl, VB12 and the various dextrans. Drug solubility, size and chemical nature were shown to be of particular importance in the control of hydrogel swelling and drug release from thermosensitive hydrogels. (C) 2004 Elsevier B.V. All rights reserved.

Hyperhomocysteinemia is key for increased susceptibility to PND in aged mice


Authors: Zhao, Guangchao; Deng, Jiao; Shen, Yuan; Zhang, Peng; Dong, Hailong; Xie, Zhongcong; Xiong, Lize

Background Postoperative neurocognitive disorder (PND) is a severe postoperative complication with no effective therapy that affects up to 19-52% of senior patients. Age and surgery type have been identified as risk factors. However, what caused the increased risk in the elderly is poorly understood. Methods We utilized a PND model in aged mice undergoing experimental laparotomy with general anesthesia to evaluate the causal relationship between hyperhomocysteinemia and increased PND susceptibility. PND was assessed by Novel Object Tasks, Fear Conditioning Tests, and Barnes Maze Tests. Serum homocysteine (Hcy) as well as vitamin B12 and folate acid levels were tested before, immediately after surgery and from day 1 to day 29 after surgery by ELISA. The effectiveness of preventative strategy including diet supplementation of vitamin B12 + folic acid (Vit B12 + FA) and S-adenosylmethionine (SAM) injection targeting hyperhomocysteinemia were also tested. Results PND in aged mice lasted for at least 2 weeks after experimental laparotomy, which was not observed in young adult mice. Serum Hcy results indicated a significant correlation between postoperative cognitive performance and perioperative Hcy level. Preoperative supplementation with VB12 and folic acid (FA) in the diet or S-adenosylmethionine (SAM) injection reduced perioperative serum Hcy level and inhibited the development of PND in aged mice. Conclusions Serum homocysteine accumulation is a fundamental cause for increased susceptibility of PND in aged mice. Preoperative diet supplementation of VitB12 + FA can effectively reduce PND in aged mice, which may be a promising prophylaxis treatment in clinical settings.

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