Jul 10

Supercharging reagents – revving up peptide LC-MS analyses

Over the past 5 years, LC-MS has (arguably) become the technique of choice to quantify peptides in biological matrices. The benefits of using LC-MS as a quantitative technique are significant, however, there is one drawback – low sensitivity.

The acquisition of multiple numbers of charge states for peptides in the MS source can result in significant sensitivity loss, as usually only a single charge state is selected for quantitation. Modifying the ionisation characteristics of peptides in the source is difficult, and changing the level of normal mobile phase modifiers has minimal effects.

Supercharging reagents

The addition of supercharging reagents to mobile phases significantly shifts the charge states of peptides and proteins present in the ESI source. This enables the analyst to change the selected precursor for quantitative purposes, with potential sensitivity gains. Sensitivity can be increased if a higher charge state gives a better fragmentation profile. Selectivity can also be improved, where selecting a different precursor removes signal from co-eluting interferents.

Dr Richard Kay, Principal Scientist within Bioanalytical Sciences at LGC, will host a webinar on, Supercharging reagents – revving up peptide LC-MS analyses, on 20th July. Richard will discuss the following during the webinar:

  • A number of examples of the application of supercharging reagents to peptide LC-MS analyses
  • Impact on the quantitative performance
  • Case studies
  • Q&A

LEnPeptideAnalysis

Register for the webinar to learn more about: Supercharging reagents – revving up peptide LC-MS analyses

 

 

Jul 06

Nanotechnology: The big challenge behind the characterisation of the small

Heidi Goenaga-Infante working in the labNanotechnologies are everywhere, from the medicines we take to the food we eat and the sporting equipment we use, but what do we really know about the potential effects when they come into contact with complex matrices and how do we ensure that nanoproducts are safe?

The global market for nanomaterials is estimated by the European Commission to be 11 million tonnes at a market value of €20 billion, and products underpinned by nanotechnology are forecast to grow from a global volume of €200 billion in 2009 to €2 trillion by 2015.

We know that the properties of nanomaterials can change significantly when they are used in complex matrices, such as biological systems, potentially affecting functionality and behaviour. It is these changes that are exploited in nanobiotechnology or nanomedical applications. For example, in some therapeutic applications, protein coated nanoparticles (apolipoprotein E coatings) can target specific locations, such as the brain.

However, there may be other currently unknown biological interactions which could pose a potential risk to human health. These risks are compounded by a lack of robust methods to characterise nanomaterials in complex biological matrices.

In a recent article in Drug Discovery & Development magazine, we explore the legislation that has been introduced in the EU that focuses on nanomaterials and the implications that these have for the nanobiotechnology or nanomedical industries. We outline LGC’s work on the development of methods to characterise nanomaterials for their physical, chemical and optical properties in biological matrices in support of research aiming to understand how nanoparticles interact with biological systems. This three-year multi-national project, ‘Chemical and Optical Characterisation of Nanomaterials in Biological Systems’ (NanoChOp), finished in May.

Visit the Drug Discovery & Development magazine website to read the article.

Jun 26

Food safety and quality – how fresh is your fish?

fishThe moment a fish is caught it begins the natural process of decay, being broken down by enzymes (from the fish itself or bacteria naturally present).

Measuring the resulting chemicals produced gives a measure of the freshness, and thus the quality, of the fish.

Similarly, as LGC’s Michael Walker asks in a recent article for Food Science & Technology, mouldy food served in restaurants or shops is likely to be sent straight back with a complaint, but what happens if the mould isn’t spotted and is unwittingly ingested? There’s more at stake than an unappetising meal and a bad reputation for the restaurant.

Contamination of raw food material by toxic mould metabolites, known as mycotoxins, can cause vomiting and diarrhoea as well as cancer. Careful crop husbandry, timely harvesting and proper storage provide barriers to contamination and statistically valid sampling followed by chemical analysis acts to secure supply chain safety. Yet contamination can still occur.

Accurate analysis of these breakdown products is therefore essential. Measure too low and the produce will appear better quality than it is – potentially affecting shelf life. Measure too high and the fish (in this instance) may have to be discarded. In extremes, certain breakdown products can cause food poisoning, due to biogenic amines like histamine being formed.

The FSA reminds us that histamine, which can cause scombrotoxic fish poisoning, is produced when fish and fish products from the family that includes tuna, mackerel, and herring are not refrigerated correctly. Warmer temperatures allow bacteria to multiply and produce histamine at levels that can make people ill if it is eaten. Unfortunately, cooking the fish will not destroy histamine. Histamine in fish is controlled by Regulation (EU) No 1019/2013 of 23 October 2013 amending Annex I to Regulation (EC) No 2073/2005 as regards histamine in fishery products.

Not all toxins are controlled by Regulation 1881/2006 (though, in his article, Michael handily provides a list of those which are covered).

If you are charged with assessing accuracy of analysis in fish or meat, LGC’s Proficiency Testing team is giving you the opportunity to order test material 748 in our meat and fish scheme (QMAS).

Test material 748:

  • Quality parameters in fish

Target analytes:

  • Histamine
  • Total Volatile Nitrogen (TVN)
  • Trimethylamine (TMA)

Next available:

  • Round MT233
  • Despatch date 21 September 2015

If you would like to register for this test material, please click here, or if you would like further info, email us here.

May 28

Anti-microbial resistance: the importance of measurement accuracy

Imagine a world in which your morning shave or an afternoon pottering in the garden poses a major risk to your life. Or the medical breakthroughs that have led to organ transplantation and cancer treatment are no longer viable and no treatment exists.

This could be a reality if the “ticking time bomb” of anti-microbial resistance (AMR) is left unchallenged.

In less than 40 years, almost half of all worldwide deaths are predicted to be caused by resistant infections. Medicine, as we know it today, will be unrecognisable, as small cuts become potentially life threatening. Routine operations for ailments such as tonsillitis or appendicitis could be abandoned, deemed too risky, and childbirth will become a whole lot more dangerous.

When Sir Alexander Fleming collected his Nobel Prize for the discovery of penicillin, he predicted that a time would come when microbes become resistant to antibiotics. Despite this, there has been no new class of antibiotic discovered since 1987 and some diseases which have been routinely treated by antibiotics, such as gonorrhoea, are becoming resistant and are now on the rise.

It is clear from articles in the press that the warnings and calls for increased funding for research are beginning to be listened to. But while new classes of drugs are desperately required, there is also an urgent need for better management to ensure our existing therapies are used appropriately. The efficacy of these new drugs, and those that are already available, can be maximised if measurement considerations, such as ensuring adequate reproducibility of diagnostic test results that direct their use, are considered from the outset.

In its role as UK designated National Measurement Institute for chemical and bio-measurement, LGC is conducting research into the accuracy of methods for AMR analysis and to determine how best to support reproducible AMR measurement.

This research aims to develop quantitative, validated and highly accurate methodologies to support current and emerging molecular approaches for diagnosis, surveillance and monitoring of infectious diseases.

This will significantly improve the accuracy, reliability and comparability of respiratory pathogen diagnostics, thus contributing to greater confidence in public health monitoring and improved healthcare.

To find out more about this work and the impact it is having, read the latest issue of our Catalyst newsletter (page 4-5) and a recent article in Laboratory News.

May 20

Pasta adulteration: shining a light on new testing methods

Today is World Metrology Day (WMD), a day of celebrations to commemorate the anniversary of the signing of the Metre Convention in 1875. This year, WMD celebrates the central role of light to life and the role that metrology plays in enabling the application and advancement of light-based technologies. In this blog, we look at how LGC scientists have developed multispectral imaging methods to test for pasta adulteration.
penne_pasta_jpg

With more than 300 types of pasta available to consumers, it is no surprise that pasta dishes have been a dinnertime staple and have been regularly voted in the top 10 favourite UK dishes for many years.

There are 13.5 million tons of pasta produced worldwide (Survey carried out by IPO – 2013[1]), and here in the UK we consume 2.5kg per year per capita. It’s clear that pasta is big business but where there is big business there is the potential for fraud.

In the UK, food fraud is committed when food is deliberately placed on the market, for financial gain, with the intention of deceiving the consumer.

Good quality pasta is made from durum wheat – a type of wheat that is very high in protein, low in gluten, and tends to be quite dense. Pasta produced from durum wheat tends to have good cooking quality and stability to overcooking.

In recent years, as farmers faced poor crop yields due to bad weather conditions, the price of durum wheat has soared. This has led to an increase in the substitution of cheaper common wheat for durum wheat. This isn’t a problem if the pasta is labelled correctly and it isn’t sold as premium pasta made from durum wheat, but often it is not. The customer is duped into buying a lower quality product at a premium price.

Authentication of pasta is currently carried out using molecular biology-based techniques focusing on DNA as the target analyte. Whilst proven effective, these approaches can be criticised as being destructive, time consuming, and requiring specialist training for laboratory staff.

LGC scientists have been behind the application of multispectral imaging (MSI) techniques for authenticating pasta. The development of compact imaging platforms with the capability to rapidly differentiate a range of materials, including grains and seeds, based on surface colour, texture and chemical composition, mean that it is possible to detect, for example, minute differences between durum wheat and common wheat grains, destined to be used for the production of pasta.

Our expert scientists recently carried out a study to evaluate the applicability of two MSI instruments for identification and quantitation of durum wheat grain samples. The two instruments tested were capable of rapidly distinguishing between the two wheat types and assigning percentage adulteration levels characterised by low bias and good repeatability. The results demonstrated the potential for MSI based seed or grain adulteration testing to supplement existing standard molecular approaches and improve access to food authentication diagnostic technologies.

Further studies will now take place to investigate the applicability of multispectral imaging as a complementary and alternative diagnostic test for food adulteration in other areas, inclusive of wheat and basmati rice authenticity.

This work was financially supported by Defra, as part of Defra project “Feasibility study for using rapid and automated spectral imaging for food authenticity testing”.

Find out more about World Metrology Day.

[1] http://www.internationalpasta.org/resources/World%20Pasta%20Industry%20Survey/IPOstatreport2013.pdf

 

May 15

ICH Q3D elemental impurities testing: are you prepared for the new ICH/USP regulatory guidelines?

LEnElementalImpuritiesAs a consequence of the new ICH/USP regulatory guidelines concerning testing of elemental impurities the way in which pharmaceutical companies and their suppliers will be required to test their APIs and excipients will change dramatically.

The guidelines, which aim to set out a global policy for limiting metal impurities in drug products and ingredients puts these impurities into various categories. In order to comply with the guidelines sponsors will need to carry out a detailed risk assessment of their materials in terms of these different categories.

USP <231> Heavy Metals test is being replaced with two new chapters, USP<232> Elemental Impurities (Limits) and USP<233> Elemental Impurities (Procedures). As a consequence the use of Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) and Inductively Coupled Plasma-Mass Spectroscopy (ICP-MS) will become the preferred standard approach for the determination of heavy metals in drug products, replacing the much out dated and non-specific / non quantitative wet chemical tests. With the added elemental specificity, accuracy and sensitivity (ppb / ppt range) afforded by ICP-OES / ICP-MS, sponsors will be able to confirm that their materials meet the new compliance criteria.

The new USP directive is scheduled for January 2018 and LGC is already prepared for what will be a very significant industry requirement. Sarah James, Principal Scientist within our CMC Analytical Services team will host a webinar on “Elemental Impurities: Multi-element analysis to ICH Q3D and USP<232><233>” on 27th May 2015. During the webinar Sarah will discuss the following:

 

  • Regulatory context of the guidelines
  • Describe the analytical challenges associated with this type of testing
  • Highlight the key essentials in delivering multi-element quantitative data.

 

Register for the webinar to learn more about the new ICH/USP regulatory guidelines.

 

 

May 11

Hot off the press: Catalyst newsletter is out now

Have you ever wondered about the causes of water pollution or how we can find a solution to drug resistant diseases?

Catalyst-2015-thumbnailTo learn about these and other measurement issues, download the latest issue of Catalyst, a newsletter which brings you highlights of LGC’S activities as a designated National Measurement Institute (NMI) under the UK National Measurement System.

Our work at LGC spans various scientific disciplines with many of our research areas highlighted in this issue of Catalyst. For example, we reveal if DNA sequencing is the solution in the fight against drug resistant diseases, provide an insight into the study of nanomaterials, as well as a two-page feature describing how LGC scientists are using measurement research to help improve the quality of water supplies

All of this and more is included in the latest issue of Catalyst, which is available to download from the NMI section of the LGC website today.

Blog written by Priya Kudhail

May 01

Metabolism of therapeutic peptides

Glucagon peptide2Peptide and protein based drugs are significantly more complicated to work with than small molecules, and as yet do not have specific guidelines for their development with regards to in-vivo metabolites.

The analysis of metabolites from small molecule drugs is defined by the FDA in the metabolites in safety testing guidelines (MIST, 2008). Currently, analytical chemistry laboratories apply the same validation guidelines for regulated bioanalysis of peptides as they do for small molecules, therefore it is prudent to apply similar rules to the metabolite conundrum.

One particular challenge with peptide bioanalysis and metabolite identification is that they are significantly more complicated molecules to analyse than small molecules. However, with the vast improvement in liquid chromatography and mass spectrometric instrumentation over the past 10 years, their analysis has now become easier and even commonplace in standard bioanalytical laboratories.

The combination of two types of mass spectrometer – high resolution systems such as Time of Flight detectors, and quantitative systems such as triple quadrupoles, has enabled the rapid identification of unknown metabolites and their subsequent quantitation relative to the parent molecule.

At the Annual Peptides Congress, Richard Kay, Principal Scientist at LGC presented a poster on Metabolism of therapeutic peptides: from identification to quantitation”. Download the poster to find out more about:

  • Examples of in-vivo and in-vitro derived peptide metabolite ID
  • Quantitation of metabolites
  • Metabolite profiles

 

If you have any questions or would like further information on peptides, please don’t hesitate to contact us.

Apr 15

Environmental testing: Getting the measure of success

Environmental testingPollution is a global issue, with interconnected environmental and health consequences. Outdoor air pollution at current levels in the UK makes a significant contribution to mortality. It has a greater burden, in terms of estimated total population survival time, than the mortality impacts of environmental tobacco smoke or road traffic accidents[i].

Water polluted with heavy metals from industrial processes can result in birth defects or cause cancer. Microbial pollutants can result in infectious diseases through drinking contaminated water and although the quality of our water has improved dramatically over the past few decades, only 27% of our water-bodies in England are currently classified as being of ‘good status’ under new standards set down by the EU Water Framework Directive[ii].

In the latest issue of Laboratory News, we explore how European environmental legislation covers a vast array of pollutants but question how robust the standards are that they are founded upon. The two-page feature outlines the challenge to analytical chemists to fully characterise measurement procedures and explains how metrology is a key component of this. It describes the work that LGC is undertaking in its role as the UK designated National Measurement Institute for chemical and bio-measurement to tackle these measurement challenges.

Visit the Laboratory News website to the read the feature.

 

[i] https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/304641/COMEAP_mortality_effects_of_long_term_exposure.pdf

[ii] https://www.gov.uk/government/policies/improving-water-quality

 

Mar 20

Ultrasensitive immunoassays using single molecule counting technology

Singulex Erenna2Traditional ELISA-based immunoassays have struggled to determine baseline endogenous concentrations for many biomarkers. The Erenna® Immunoassay System is based on the same principles as a plate-based sandwich ELISA but offers 50-1000 fold greater sensitivity through a combination of microparticle bead (MB) capture and single molecule counting of fluorescently labelled detection molecules. MB’s have the dual benefit of having a larger surface area for analyte capture and an elution step which can concentrate the analyte into a smaller volume.

This SMC™ technology platform enables the detection of changes in levels of low abundance biomarkers to support early decision-making and complete PK profiles for protein biotherapeutics.

We have evaluated initially the Erenna instrument at LGC using human IL-6 kits commercially available from Singulex. We have then validated the system and used to develop/validate PK/PD assays in multiple matrixes.

At Biomarkers Congress and the EBF Symposium, our drug development services team presented a poster on:  Ultrasensitive immunoassays using single molecule counting technology

Download the poster to find out more about:

  • Typical assay procedure
  • Single molecule counting
  • Method development
  • Results and conclusions

If you have any questions or would like further information, please visit drug development services or contact us.

 

Article contributed by Salvatore Scarpaci, Senior Scientist, LGC and Michael Naughton, Scientist, LGC.

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