Folate Analysis Service

Why Folate Analysis is Important?

Folates, also known as B9 vitamins, are essential elements in the metabolism of all living organisms. Like many other vitamins, mammals are unable to synthesize it and acquire it from diet. In contrast, plants and prokaryotes have in vivo biosynthesis of folates and do not require to uptake it from the food. Basically, the in vivo biosynthesis of folates begins with the synthesis of the pterin ring, which is catalysed by GTP cyclohydrolase I and this reaction is followed by other five reactions catalysed by five distinct enzymes that convert GTP into 7,8-dihydrofolate, which is reduced by dihydrofolate reductase to produce 5,6,7,8-tetrahydrofolate. p-aminobenzoic acid, which is attached to the pterin moiety by dihydropteroate synthase, is produced by two enzymatic steps from chorismate and makes a connection between the folate and the shikimate pathways. But all of the biological functions of folates in vivo are performed by tetrahydrofolate and its methylated derivatives. Hence folate must first be reduced to tetrahydrofolate via four electron reduction. Folate is important during periods of frequent cell division and growth and its deficiency links to various human diseases including cancer and diabetes.

Liquid chromatography-mass spectrometry (LC-MS), an analytical technique, is considered to be a highly precise and sensitive tool utilized in folate analysis. The method primarily involves the separation of folate molecules present in a sample by employing liquid chromatography, and subsequently, the detection and quantification of these molecules are carried out via mass spectrometry.

LC-MS, owing to its high sensitivity, is particularly suitable for the analysis of folate in complex matrices like food, biological fluids, and tissues, wherein it can detect even trace amounts of folate. The technique's capability to differentiate between different forms of folate, such as methylated and non-methylated forms, is crucial for a deeper comprehension of their biological activity and nutritional significance.

LC-MS offers numerous benefits over other analytical methods, such as high accuracy and precision, low detection limits, and the ability to analyze multiple forms of folate simultaneously. In addition, coupling LC-MS with other separation techniques, including solid-phase extraction and immunoaffinity chromatography, can augment the sensitivity and selectivity of the analysis.

At Creative Proteomics, our team of experienced scientists utilizes state-of-the-art LC-MS equipment and techniques for folate analysis, ensuring the highest quality standards for accurate and reliable results. Whether you require folate analysis for nutritional labeling, clinical diagnostics, or drug development, our LC-MS method offers unparalleled sensitivity and specificity for the detection and quantification of folate.

Applications of Folate Analysis Service

• In the food industry, it is critical that folate analysis be used to develop and test fortified foods and supplements. It is also used to monitor the nutrient content of crops and to assess the impact of agricultural practices on folate levels.
• In the pharmaceutical industry, to facilitate the development and testing of folate-based drugs for the treatment of cancer, inflammation and other health conditions.
• In the biotechnology industry, to help develop and produce folic acid-based diagnostic tests and therapies.

Folate Assay Platform in Creative Proteomics

Creative Proteomics has developed a state-of-the-art folate content assay platform that combines advanced techniques and meticulous protocols to ensure accurate and precise measurement of folate levels in various sample types.

Sample Preparation

Accurate quantification of folic acid content begins with proper sample preparation, and Creative Proteomics offers tailored sample preparation protocols for different types of samples, including blood, tissue, food and supplements. These protocols have been optimized to efficiently extract and purify folic acid from complex matrices while minimizing potential interferences.

During sample preparation, Creative Proteomics utilizes robust extraction methods to ensure the recovery of folate compounds from the sample. Purification techniques are used to remove unwanted impurities and contaminants that may interfere with accurate folate content measurements.

High Performance Liquid Chromatography (HPLC)

Creative Proteomics employs an HPLC method that effectively separates the various forms of folate present in a sample.

The separation of folic acid is achieved based on its unique physicochemical properties, such as polarity and molecular size. By employing appropriate mobile phases and chromatographic conditions, we ensure optimal resolution and minimal co-elution of folic acid compounds.

Mass Spectrometry (MS) Analysis

To improve the sensitivity, selectivity and accuracy of folate analysis, Creative Proteomics combines HPLC with mass spectrometry (MS) detection. Mass spectrometry allows for the precise identification and quantification of folate compounds based on their unique mass-to-charge ratio. We use a high-resolution mass spectrometer capable of accurate mass measurements and high sensitivity. This allows the detection of even trace amounts of folate compounds in complex samples and also allows the characterization of folate isomers and derivatives that may have different biological activities.

Figure 1. The overall workflow of folate biosynthesis

Sample Requirements of Folate Assay

The sample requirements are tailored to different sample types and are designed to optimize the extraction, purification, and analysis of folate compounds. Here are the general sample requirements for folate analysis:

Blood Samples:

• Volume: Depending on the assay sensitivity and desired analysis, typically 1-2 mL of whole blood or serum/plasma is required.
• Storage: Blood samples should be collected in appropriate anticoagulant tubes, such as EDTA or heparin tubes, and stored at 4°C to preserve folate stability. It is essential to avoid repeated freeze-thaw cycles.

Tissue Samples:

• Weight: The recommended tissue sample weight varies depending on the specific requirements and sensitivity of the assay. Typically, 50-100 mg of tissue is sufficient for folate analysis.
• Processing: Tissue samples should be immediately frozen after collection and stored at -80°C until analysis to preserve folate stability. Prior to analysis, the tissue should be homogenized and processed using appropriate extraction protocols.

Food Products and Supplements:

• Quantity: The amount of food product or supplement required for analysis depends on the specific requirements and desired sensitivity. Sufficient quantity should be provided to ensure representative sampling.
• Preparation: Food products and supplements need to be homogenized or finely ground to ensure sample uniformity. If required, specific extraction procedures may be necessary to release folate compounds from the sample matrix.

Other Samples:

Depending on the specific sample type (e.g., urine, cerebrospinal fluid, cell culture media), specific requirements for volume, storage, and processing may apply. It is essential to consult with Creative Proteomics to determine the optimal sample requirements for accurate folate analysis.

Report Delivery

• Experimental protocols
• Instrumental factors of LC-MS
• The raw data files of LC-MS and the summary of LC-MS data quality
• Bioinformatics analysis report