METABOLITES ASSAY

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

 

 

 

MYCOTOXIN ASSAY

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

GUT MALABSORPTION ASSAY

Gut permeability is an issue when there are changes and the absorption of nutrients is altered.

The Theory:

A solution of 3 sugars are given to patients at a set concentration, then either urine or blood samples are taken at set time points for analysis. The ratio of the Lactulose (disaccharide) to either Rhamnose or Mannitol (monosaccharides) indicates if and where there is a problem in the gut. These 3 sugars are not metabolised by the body, they just permeate across the gut wall.

How we are involved:

We have developed two analysis methods, based on published work, to measure these sugars in either urine or plasma. We have a lower limit of quantitation of 15µg/ml in Urine and 7.5µg/ml in Plasma, however we would advise that the actual lower levels should be 25µg/ml for urine and 15µg/ml for plasma to ensure that the lowest levels do not fall outside the calibration range. Figure 1 shows the timing and peaks for the sugars being sent round the HPLC.

Figure 1: Graph from the HPLC showing the 3 sugars separation and their associated retention times.

We are working with Mike Hewetson from the Royal Veterinary College to adapt the plasma method for use with equine serum. At present, there is no easy way of determining where a problem is in the equine gut and it is hoped that this test will offer an alternative to submitting the horse for a surgical procedure.

WHAT CAN WE DO FOR YOU?

Here at the Bio Analysis Centre we have the capability to run samples through our UHPLC (Ultra High Performance Liquid Chromatograph) or LCMS (Liquid Chromatograph Mass Spectrometer). Shimadzu (1), the manufacture of the machinery, ran a Multiple Residue Analysis of 210 Pesticides in Food Samples by Triple Quadrupole UHPLC – MS/MS.

 

Pesticide residue on food can severely affect human health, so governments and food manufacturers established regulations which set the limits in a range of 0.01 – 10 mg / kg depending on the toxicity of the pesticide. For food used in the production of baby foods, the detectable level of pesticide must be less than 0.01 mg / kg along with prohibiting the use of certain toxic pesticides.

 

Linearity was investigated over a nine point calibration, with samples ranging from 0.5 μg / kg – 0.2mg / kg or 0.5 – 200 parts per billion (ppb) analysed in duplicate; calibration samples were injected once in increasing order and once in decreasing order. From that the Mass Spectrometer calculated from the area of the peak the concentration by correlating to the calibration curve associated with the specific pesticide.

 

This exact assay can be conducted here at the Bio Analysis Centre, if you have a similar project or any enquiries for any other assay, please contact us.

 

References

  1. Multi-Residue Analysis of 210 Pesticides in Food Samples by Triple Quadrupole UHPLC-MS/MS [Internet]. Shimadzu; [cited 29 January 2020]. Available from: https://www.shimadzu.com/an/literature/lcms/jpo113017.html?fbclid=IwAR0vWv-SXKE4BKlRmm_RgKE7JkAFXR9i-JJn3KsZRGb2sYxMwxiyaWKkNWk
Bio-Analysis at the One Nucleus Bio Wednesday 2019

Bio-Wednesday One Nucleus hosted by LBIC at the RVC

We were delighted to be asked to join the panel last night at the One Nucleus event hosted by LBIC at the RVC.

 

The topic, ‘Staffing the 21st Century Life Sciences R&D Industry.’ bio-analysis HPLC and LCMS

As an employer of several graduates, undergraduates and summer placements in our 5 years of business, we were interested to be part of the discussion and hear other people’s point of view.

We have found the quality of students coming through to be excellent and a real asset to us. In a business like ours they receive a very hands on and varied approach to the commercial world of bio-science.  (A hugely different experience from starting out in a Big Pharma Organisation!)

Discussion topics resonated around what the future looks like for graduates and post graduates; what skills employers and recruitment professionals are looking for: risk takers, lateral thinkers, commercial acumen; what could be taught, what should be taught and the changing foot print of career paths.

For a business like ours, people skills are essential, along with self motivation and common sense.

 

We pride ourselves in offering an excellent technical and customer service.  Our HPLC and LC-MS training can be combined with method development and bespoke consultancy to help advance your Research and Development.  Once trained, clients can come back and use the equipment to continue their research projects.

As a business and an employer, we have a lot to offer so please get in touch.

 

Bio-Analysis at the On Nucleus Bio-Weds - HPLC and LCMS

 

 

 

Atypical Myopathy Testing Now Available (In Conjunction With the Royal Veterinary College)

 

The Royal Veterinary College (RVC) and the Bio-Analysis Centre are now conducting testing for atypical myopathy as part of the RVC’s work towards improved treatments and management of this disorder, and to enhance the welfare of affected horses. Atypical myopathy of horses is a severe and life threatening equine muscle disorder that is caused by the ingestion of Sycamore tree seeds, leaves or seedlings by horses that are kept at pasture. Risk factors for horses remain unclear. It is, for example, not currently known whether some trees are more toxic than others or whether the amount of toxin varies at certain times of the year or with certain climatic conditions. The RVC is working to help horse owners to gain a better understanding of the condition.

Following research that was supported by The Horse Trust and the RVC’s Animal Care Trust (ACT), the Comparative Neuromuscular Diseases Laboratory at the RVC is now offering testing of seeds, seedlings and leaves for the hypoglycin A toxin known to cause this disorder. To find out if plant samples from your property contain the toxin known to cause atypical myopathy, you can now send samples directly to the lab where they will be tested at a subsidised cost of £50.   In addition, the Comparative Neuromuscular Diseases Laboratory is also offering testing of horse blood and urine samples, submitted by your vet, if they suspect atypical myopathy or in field companions. This should help to establish a much more rapid and accurate diagnosis, and subsequent treatment, than with previous tests.

Professor Richard Piercy, Professor of Comparative Neuromuscular Disease, said: “We’re really pleased to be able to launch our testing service for owners who may be concerned about their horses. With the support of the Horse Trust and ACT, and through working with owners in this way, we hope to be able to improve the understanding of atypical myopathy and improve the welfare of horses with this severe condition.”

Full details, including prices and packaging instructions are available here.

 

 

2018 Update: The work carried out by Carolyn and Imogen in developing this assay has recently been published in Plos One, along with two others from the Neuromuscular Research Group at the RVC. You can read it here.

 

 

GDPR Update

Bio-Analysis Centre is reviewing and updating our privacy policies to ensure that your stored data is used securely and transparently. Please view our updated Privacy Policy here.

 

deltaDOT’s High Performance Capillary Electrophoresis

The Bio-Analysis Centre offers High Performance (label free) Capillary Electrophoresis (HPCE) system from deltaDOT to its clients.

Whilst electrophoresis is the process during which ions undergo movement in a fluid or gel under the influence of an electric field, capillary electrophoresis is a technique that separates these ions based on their electrophoretic mobility with the use of an applied voltage. This mobility is dependent on the atom’s radius, the charge of the molecule, and the molecule’s viscosity. The rate at which the charged particle moves is directly proportional to the applied electric field – as the field strength increases the mobility increases also.

Read More
method development

Chromatographic analysis – An Intern’s Perspective

Chromatographic analysis is often an indispensable technique for a life scientist.

The gadgetry and the skills required, for the successful application of such techniques, are less so common. Having spent the summer months of 2017, working for the Bio Analysis Centre, I have been a very keen observer of the company’s modus operandi. I have been most impressed by the assembly of systems, developed and maintained by the laboratory manager, Dr. Hyde. This framework for the BAC ensures the smooth running of the services it provides.

Read More