Simultaneous Analysis of 97 Primary Metabolites on a Pentafluorophenylpropyl Column (PFPP). This assay was performed by Shimadzu (1), the manufacturer of the equipment used, all information shown is from them unless otherwise stated.

What are Metabolites?

‘Metabolites are substances made or used when the body breaks down food, drugs, chemicals, or its own tissue (for example, fat or muscle tissue). This process, called metabolism, makes energy and the materials needed for growth, reproduction, and maintaining health. It also helps get rid of toxic substances’ (2).

Energy at the cellular level is derived by various metabolic processes, including the glycolytic system and the TCA cycle. In order to investigate the metabolites of living systems it is important to quantify the amount of each metabolite.

Table 1: List of Metabolites detected taken from Shimadzu paper (1)

Method and Sampling

Many primary metabolites are hydrophilic and difficult to analyse by reverse phase chromatography.  Usually, Ion pair chromatography is used for hydrophilic compounds, but this is not compatible with LCMS analysis due to the ion pair reagents causing high background signals and sensitivity deterioration.

A method using a PFPP column has been developed to overcome these limitations, it allows not only hydrophobic interaction but also retention of hydrophilic compounds, which is essential for successful analysis of primary metabolites.

The samples provided to Shimadzu were tissue extracts and all measurements were done on a Shimadzu LCMS – 8040 Triple Quadrupole UFMS.

Liver and Heart tissue samples where extracted and immediately frozen in liquid nitrogen, each frozen sample was weighed and homogenised in methanol containing internal standards. After homogenisation, a methanol-chloroform extraction was carried out, the metabolites were collected, and the extracts concentrated.

After preparation, 3 µl of the solution was injected onto the column using the gradient shown in Table 2 at a flow rate of 0.25ml / min.

Table 2: Gradient used for Metabolite Analysis (1)

In each extract over 80 metabolites were detected, the PFPP column was especially effective at separating the amino acids and organic acids (1).

References:

1. Simultaneous Analysis of 97 Primary Metabolites By PFPP: Pentafluorophenylpropyl Column [Internet]. Shimadzu Excellence in Science. 2014 [cited 16 March 2020]. Available from: https://www.shimadzu.com/an/literature/lcms/jpo114088.html
2. NCI Dictionary of Cancer Terms [Internet]. National Cancer Institute. [cited 16 March 2020]. Available from: https://www.cancer.gov/publications/dictionaries/cancer-terms/def/metabolite

This assay was performed by Shimadzu (1), the manufacturer of the equipment used, all information shown is from them unless otherwise stated.

Mycotoxins are severely toxic contaminants which can be found in grains, they’re naturally occurring and are produced by certain moulds found on food (fungi) and present a risk to human health. The most common are Aflatoxins and Ochratoxin A. The World Health Organisation set the maximum level for Aflatoxin in food at 0.5 – 15 µg/kg (2) and for Ochratoxin-A at 1 – 50 µg/mg for food and 100 – 1000 µg/kg for animal feed (3).

Due to the toxicity of Mycotoxins, the rapid determination of their presence is essential. UHPLC-MS/MS offers the best combination of selectivity, sensitivity, and speed for detecting these compounds in complex matrices. In this study (1), a high throughput method for the quantitation of 23 Mycotoxins in various matrices was established.

Method:

5g of dry food (such as grain or animal feed) was mixed with 10ml of water to produce the sample. 10g of wet food (such as fruit) produced the sample.

10ml Acetonitrile was added and the sample macerated, before salts were added to allow phase separation.  The solution was then centrifuged to pellet the solids. The supernatant was transferred to a clean tube and 5 fold diluted with Mobile Phase A (Water + 0.5% acetic acid) and internal standard.

20 µl of the solution was injected onto the column using the gradient shown in Table 1 at a flow rate of 0.4ml / min.

Table 1: Gradient used for Mycotoxin Analysis

This assay could be used for any type of food, and as the method is developed by the manufacturer on the exact machine, we can run this assay. If you have a similar project would like analysed, then please get in contact.

References

1. Rapid simultaneous assay of 23 mycotoxines in a variety of food samples by UHPLC-MS/MS using fast polarity switching [Internet]. 2013 [cited 2 March 2020]. Available from: https://www.shimadzu.com/an/literature/lcms/fro113069.html
2. Mycotoxins [Internet]. World Health Organisation. 2018 [cited 2 March 2020]. Available from: https://www.who.int/news-room/fact-sheets/detail/mycotoxins
3. Egmond V. Worldwide regulations for Ochratoxin A [Internet]. 1991 [cited 2 March 2020]. Available from: https://www.ncbi.nlm.nih.gov/pubmed/1820350