meeting report - Separation Science Group

The Advances in Clinical Analysis meeting 2014, a popular meeting in the centre of London, was held
on the 29th floor of the Guy’s Hospital Tower, London. Having overcome the queue for the half a
dozen lifts, speakers from hospitals, industry and academic researchers presented updates to this
vital and well-established field of analytical science. The technique of liquid chromatography- mass
spectrometry (LC-MS) was a major focus, which is combines the power to analyse complex mixtures
one-at-a-time with detection that can quantify the chemical compound of interest, and perform
chemical reactions to understand its structure. Lewis Couchman of the Royal Society of Chemistry
Separation Science Group and Kings College Hospital, London, opened the day, handing over to
Richard Kay from LGC, Fordham. Richard compared traditional immunoassay (IA) with LC-MS, using
Insulin Growth Factor 1 (IGF 1) as an example, with good agreement between results from either
method. It was therefore suggested that LC-MS/MS can validate traditional methods (rather than
replace), since immunoassays can be subject to interference from enzymes. Richard suggested
future IA could be validated against such methods.
Neil Dalton of the Evelina Children’s Hospital, London, presented on Dried Blood Spot and Dried
Urine spot analyses. Neil described simple systems of sample collection on dedicated filter paper,
which can be sent to the lab via conventional post, and contrasted this with classical methods such
as colorimetry of whole blood where haemoglobin interferes, and large quantities of blood might be
required from the patient. He concluded that DBS analyses can have a large societal impact, offering
population-based health surveys for a variety of conditions.
Vendors supporting this event included ThermoFisher Scientific, Agilent Technologies, Sigma Aldrich
and Phenomenex; also HiChrom, Crawford Scientific and Gilson. Mike Oliver of Thermo pitched their
SOLA solid phase extraction (SPE) for sample preparation, Peter Christensen of Agilent their
RapidFire ultrafast autosampler with in-built SPE, and Jason Wrigley of Sigma described a Vitamin D
analysis method using Hybrid SPE and their Ascentis express fused-core UPLC chromatography
column. A major focus of sample preparation was on phospholipids. Although these underpin our
life as we know it by making up cell membranes, to the analytical chemist they can be troublesome.
They ion-suppress in MS, are amphiphilic thus tricky to precipitate with organic solvent, and can coelute with the compound of interest. James Rudge of Phenomenex focused on these species,
presented their T Phree phospholipid removal plates. Norman Ramsey of Thermo took the
discussion in a different direction, outlining multiple case studies of electrochemical detection with
sensitivity and specificity comparable to MS.
Lunch was a generous platter based on British home cooking: Yorkshire puddings loaded with roast
beef and horseradish sauce, and balls of chicken Kiev loaded with garlic butter, followed by skewers
of fruit with compote. The sun drove off the grey clouds, revealing the stunning view from that
height. A senior academic quietly suggested it was like looking down on a model train set, that he
might pick one up and face it the other way. Nicola Gray of Imperial College, London presented work
on Amino Acid analysis by LC-MS for metabolite phenotyping. Their strategy involved a non-targeted
approach for exploratory profiling, followed by a targeted approach for analyte-specific, absolute
quantitation. Most amino acids do not absorb UV light, so their non-targeted methods used
derivativisation using an aminoquinoline moiety. To reduce solvent consumption and environmental
impact, they moved from UPLC separations with narrow (2.1mm internal diameter compared to
HPLC 4.6mm internal diameter) columns to 1mm i.d. columns, improving sensitivity 3 fold. Nicola
reported validated separation and quantitation of 29 amino acids within a 7.5 minute run.
Advances in Clinical Analysis meeting report
05 November 2014
Andrew Davison of the Royal Liverpool & Broadgreen University Hospitals discussed the preparation
of biological samples for clinical analysis. He suggested that budget size is a real problem for
hospitals in the North of England, and that the workload for their lab is high. For Vitamin D analysis,
their workload is over 35,000 analyses a year, with a turnaround time under 5 days required.
Andrew emphasised good sample preparation enhances column life and reduces instrument
downtime, and presented collaborative work on ‘loading columns’. A cation-exchanger with high
affinity for phospholipids was used to load, and compounds of interest could be backflushed for
separation on a porous graphitic carbon column with a 12 minute runtime.
Sarah Belsey from Viapath in London presented the application of internal calibration LC-MS to the
analysis of the anti-psychotic drug clozapine and its pharmacologically active metabolite
norclozapine. Sarah highlighted the current practice to comply with FDA guidelines requires timeconsuming use of internal quality controls at regular intervals, in addition to blanks, zero samples
and many non-zero samples. They found calibrators labelled with four or eight deuterium atoms
were required due to the presence of chlorine on the compounds of interest, due to interference
from 35Cl/37Cl. Sarah discussed a novel approach to use the collision energy profile to downtune the
MS for equal mass response for each analyte, which proved controversial to some members of the
Mike Morris of Waters Corporation and the British Mass Spectrometry Society discussed the need to
standardise and harmonise LC-MS practice, ideally to link a clinical result in a direct chain to the
respective SI unit. Mike deftly enlivened this somewhat dry aspect of analysis with a slide asking if
LC-MS is perceived as Gandalf or Gollum. Mike suggested LC-MS can be all people think it to be (the
reference method, giving ‘the right answer’, with accuracy and precision) but only if methods are
done properly. Comparing results from eight ‘reputable’ labs, he presented calibration curves, each
of which was perfectly straight but with different slopes. Mike discussed how individual labs making
in-house matrix calibrators by weighing powers on their own balances can be improved upon, and
suggested solution calibrators bought in as a solution.
Zoltan Takats of Imperial College London presented his group’s work on direct mass spectrometric
imaging for direct profiling of tissues, biological fluid and bacteria. Zoltan’s desorption electrospray
imaging (DESI), where a nitrogen gas stream acquires charge and is sprayed onto a sample. The
aerosol droplets are collected by the MS inlet and analysed. Zoltan mentioned they had issues with
morbidity of lab animals but solved this for early human trials by coupling to the pre-existing
technique of electrosurgery. His video of the robot prototype was reminiscent of a scene from
‘Goldfinger’, but Zoltan described success when volunteering himself to remove a growth and test
the technique. Though not a quantitative technique, he described DESI performing histopathology
within 0.5 seconds, assigning the characteristic compounds of a sample to tissue type.
I would like to thank Paul Russell from the Royal Society of Chemistry Separation Science group for
arranging a generous student bursary to attend this meeting. This gave me opportunities to meet
peers and established researchers in the field and learn how I might apply my own project on
analysis of polar molecules by HPLC, including alternative detectors such as charged aerosol
detection, to future research.
Advances in Clinical Analysis meeting report
05 November 2014