Developing Good Pipetting Techniques

hOW IMPORTANT IS IT TO DEVELOP GOOD Pipetting Techniques?

In order to achieve optimal and reliable results for our diagnostic services we ensure all staff are
trained to a high degree in basic laboratory skills, such as liquid handling.

Pipettes are used daily in our lab for liquid handling. Two factors contribute to good laboratory
pipetting: tools and techniques. At the Bio-analysis centre we use mainly single channel manual
‘transferpette’ pipettes (Figure 1) which are known to be highly accurate at various volumes; however, the
most important factor is the expertise of the operator.

developing good pipetting techniques

Fig. 1 Single Manual Channel Transferpette

There are two main pipetting techniques: forward pipetting and reverse pipetting (/back pipetting).
Forward pipetting aspirates from the first stop and ejects until the second, whereas reverse
pipetting aspirates from the second stop and ejects until the first.

Other factors include:

– Immersion angle and depth
– Pre-rinsing
– Speed
– Temperature of environment and samples
– Performing pipette leak testing daily

Prolonged poor ergonomics while using pipettes can result in repetitive strain injuries (RSI) and
carpel tunnel syndrome (CTS) (El-Helaly et al., 2017). Studies have shown how higher work duration
increases the risk of developing RSI and CTS (El-Helaly et al., 2017, Bjorksten et al., 1994) . During our
training courses we recognise this issue and work with you to improve your pipetting technique
giving you the confidence to work comfortably in a laboratory environment.

Good laboratory pipetting is fundamental in our work producing accurate and precise data from the
HPLC and MS-LC. We are experienced in pipetting difficult solutions such as plasma and serum,
which are notoriously bubbly and therefore difficult to pipette!

Click here to find out more about our 1 day lab skills course.

References:
BJORKSTEN, M. G., ALMBY, B. & JANSSON, E. S. 1994. Hand and shoulder ailments among laboratory
technicians using modern plunger-operated pipettes. Appl Ergon, 25, 88-94.

EL-HELALY, M., BALKHY, H. H. & VALLENIUS, L. 2017. Carpal tunnel syndrome among laboratory
technicians in relation to personal and ergonomic factors at work. J Occup Health, 59, 513-
520.

Liquid Chromatography Method Development

Why is Method Development important? 

In the pharmaceutical sector, liquid chromatography procedures are used to assay compounds and quantify impurities present within medicinal products. Method development allows the creation of the best method to test for impurities and compounds which help ensure the quality, safety and efficacy of new products and drugs.

Liquid Chromatography Method Development 

Method development is the process by which the optimal conditions are found for a particular compound to separate it from any contaminants. To design the best method for a particular sample, the following parameters and conditions must be optimized:

  • Selection of chromatographic mode (Reverse Phase, Normal Phase,
    Size exclusion, HILIC, etc.)
  • Selection of stationary phase (C18, C8, Phenyl-hexyl, Biphenyl,etc.)
  • Selection of detector (UV, RID, MS, ELSD)
  • Selection of mobile phase (which buffers; pH of buffers)
  • Flow rate
  • Preparation of samples (protein precipitation, Solid Phase Extraction,
    etc)
  • Injection volume
  • Concentration
  • Calibration Range

HPLC Method Development

Our Nexera XR HPLC system (Fig.1) is ideal for developing procedures for a variety of material, ranging from complicated combinations of tiny molecules or proteins to single chemical purification.

HPLC method development

Fig. 2 Nexera XR HPLC System

Mass Spectrometry Method Development

We have the expertise to design methodologies to quantify most drug-like substances using our Shimadzu LCMS-8040 (Fig.2).

Mass spectrometry method development

Fig.2 Shimadzu LCMS-8040

Our manager Dr. Carolyn Hyde has over 20 years of experience developing methods on HPLC and LC-MS. Click here to contact us for more information on our services. 

References

Avoomeen. Why Pharma Companies Must Invest in Method Development & Validation (Internet). (Cited on 16th July) Available from: https://www.avomeen.com/pharma-companies-invest-method-development/

 

 

 

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

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

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.

 

 

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