services

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.

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.

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.

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).

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/

After learning the correct way to pipette Bio Analysis invited me back to continue training. Watch the video to find out how I did and don’t hesitate to contact us about any enquires.

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