Hi Nitesh,
We measure vitamin B1 and B6 using HPLC with particular extraction and detection methods. I'm not sure how exactly vitamin B12 is measured. But this article here might be helpful. Good luck!
Article Isolation and Analysis of Vitamin B12 from Plant Samples
You might find this article useful
Article Can supplementing vitamin B12 improve mental health outcomes...
Estimating vitamin B12 in plant samples can be challenging due to the low levels of this vitamin naturally present in plant-based foods. However, several methods can be employed for this purpose, each with its advantages and limitations. Here are some common methods used for estimating vitamin B12 in plant samples
Microbiological Assay:
This method involves using specific microorganisms that require vitamin B12 for growth.
The plant sample is first extracted to release vitamin B12.
The extract is then added to a growth medium containing the microorganism.
The growth of the microorganism is measured, and the amount of vitamin B12 in the sample is determined based on the growth response.
This method is highly sensitive and specific for vitamin B12 but requires specialized equipment and expertise.
Chromatographic Techniques:
a. High-Performance Liquid Chromatography (HPLC):
HPLC can be used to separate and quantify various compounds, including vitamin B12.
The plant sample is first extracted to release vitamin B12.
The extract is then injected into an HPLC system equipped with a suitable detector (e.g., UV-Vis or fluorescence).
Vitamin B12 is separated from other compounds based on its retention time and detected quantitatively.
HPLC is highly sensitive and can provide accurate quantification of vitamin B12 but requires expensive equipment and skilled operators.
Chromatographic Techniques:
Liquid Chromatography-Mass Spectrometry (LC-MS):
LC-MS combines chromatography with mass spectrometry for the separation and identification of compounds.
The plant sample is extracted, and the extract is injected into an LC-MS system.
Vitamin B12 is separated based on its retention time and detected using mass spectrometry.
LC-MS offers high sensitivity, specificity, and the ability to identify and quantify multiple forms of vitamin B12 simultaneously.
However, LC-MS equipment is expensive, and the method requires technical expertise.
Immunoassay:
Immunoassays involve the use of antibodies to specifically detect and quantify vitamin B12.
The plant sample is extracted, and the extract is added to a microplate coated with antibodies against vitamin B12.
A secondary antibody linked to a detection system (e.g., enzyme or fluorescence) is added to the microplate.
The amount of bound vitamin B12 is measured based on the signal generated by the detection system.
Immunoassays are relatively simple, rapid, and suitable for high-throughput analysis but may lack specificity compared to chromatographic methods.
Spectrophotometric Assay:
Spectrophotometric methods involve measuring the absorbance of vitamin B12 at specific wavelengths.
The plant sample is extracted, and the extract is analyzed using a spectrophotometer.
Absorbance measurements are compared to standard curves prepared using known concentrations of vitamin B12 to quantify its amount in the sample.
Spectrophotometric assays are simple, cost-effective, and suitable for routine analysis but may lack sensitivity compared to chromatographic methods.
Considering the complexity of plant matrices and the low levels of vitamin B12 in plant samples, a combination of methods, such as microbiological assay and chromatographic techniques (e.g., HPLC or LC-MS), may be most suitable for accurate estimation of vitamin B12. Additionally, sample preparation techniques, such as solid-phase extraction or immunoaffinity chromatography, may be employed to enhance the sensitivity and selectivity of the analysis. It is essential to validate the selected method for accuracy, precision, and recovery using appropriate reference materials and quality control samples.
Nitesh Kumar Yadav I would also recommend this article
Article Can supplementing vitamin B12 improve mental health outcomes...