Putting The Pieces Together - PharmTechfiles.pharmtech.com/.../TheColumn_January122016EUsm.pdf2019/02/08 · than the number of peptides in our digest. If we consider a digest with

Putting The Pieces TogetherThe power of 2D LC for

peptide mapping

2 The Power of 2D LC Protein biopharmaceuticals have seen an enormous growth in the last decade,

and as a result, separation scientists are giving increased attention to methods for characterizing biopharmaceuticals. One powerful technique for analyzing proteins is two-dimensional liquid chromatography (2D LC). Gerd Vanhoenacker of the Research Institute for Chromatography (RIC) in Kortrijk, Belgium, has been conducting research into peptide mapping of therapeutic monoclonal antibodies (mAbs) using 2D LC. He recently spoke to The Column about this work.

Cover Story

Features

20 The LCGC Blog: Column Overload in Gas Chromatography with Vacuum Ultraviolet Detection

Kevin A. Schug, University of Texas Arlington Column overload is a very commonly encountered issue in gas chromatography (GC) for

beginners. Changes in peak symmetry, generally observed as peak fronting, can be subtle in the sharp peaks generated by GC, but the result can be significant shifts in retention times, loss of resolution, and error in peak integration. LCGC Blogger Kevin Schug explains more.

15 Extending the Detection Limits for the Analysis of Organotin Contaminants Using Soft Ionization

Laura McGregor, Steve Smith, and David Barden, Markes International This article presents a gas chromatography coupled with time of fl ight mass spectrometry

(GC–TOF-MS) method with soft ionization for trace-level detection and quantitation of organotins in complex matrices.

23 The 10th Balaton Symposium on High-Performance Separation Methods: A Review

Ira Krull, Northeastern University

A review of the 10th Balaton Symposium on High-Performance Separation Methods,

which was held 2–4 September 2015 at the Hotel Azúr, Siófok, Hungary.

Regulars9 News The benefi ts of pattern modulation in GC×GC, a detection method for early-stage ovarian cancer using UHPLC–HRMS, and the latest news in brief are featured in this issue.

12 Incognito Reproducibility of Research — Do We Have a Problem Houston? Incognito talks about reproducibilty in research.

26 CHROMacademy Find out what’s new on the professional learning site for chromatographers.

27 Training Courses and Events

28 Staff

19 January 2016 Volume 12 Issue 1

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The Power of 2D LCProtein biopharmaceuticals have seen an enormous growth in the last decade, and as a result, separation scientists are giving increased attention to methods for characterizing biopharmaceuticals. One powerful technique for analyzing proteins is two-dimensional liquid chromatography (2D LC). Gerd Vanhoenacker of the Research Institute for Chromatography (RIC) in Kortrijk, Belgium, has been conducting research into peptide mapping of therapeutic monoclonal antibodies (mAbs) using 2D LC. He recently spoke to The Column about this work.

Q. In a recent article you mention

that within the current decade you

expect a much larger share of drug

approvals to be of a biological nature,

specifically monoclonal antibodies.1

Why do you think this is the way the

therapeutics market is going?

A: Protein biopharmaceuticals have

emerged as important therapeutics for

the treatment of cancer, cardiovascular

diseases, diabetes, infection, and

inflammatory and autoimmune disorders.

Given their obvious benefits in terms of

safety and efficacy, they are reshaping

the pharmaceutical market. The majority

of these proteins are, and will be,

monoclonal antibodies (mAbs) and many

of these have already received approval in

Europe and the United States.

In a recent supplement from LCGC

Europe, edited by Pat and Koen Sandra,

a brief overview of current and future

trends in drug development and

sales was given.2 Trends for different

classes of pharmaceuticals speak for

themselves: Small-molecule drug sales

are stagnating while recombinant protein

pharmaceuticals sales have increased

by over 25% between 2008 and 2013.

Sales for mAbs have nearly doubled over

the same period of time. Big pharma

organizations are now more and more

focused on biopharmaceutical products.

Of course, they will continue to invest in

the development of key small molecule

formulations, but a major part of

their research is now focused on large

biomolecule drugs in general, and mAbs

in particular.

Q. This shift would obviously

represent an analytical challenge. Do

you feel the pharmaceutical industry

is sufficiently experienced and

prepared for such a shift?

A: The analysis of these big,

2

Q&A: Vanhoenacker 2 The LCGC Blog20Barden et al.15Incognito12News9Balaton Symposium Review23 Staff28CHROMacademy262626 Training & Events27272

3


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heterogeneous biomolecules will often require a shift in analytical approach and technique compared to the analysis of small-molecule pharmaceuticals. This shift in approach is needed at nearly all levels going from sample storage and preparation to analysis and data treatment. Most regulatory guidelines were originally developed for small-molecule drug products and formulations. To enable researchers in academia and industry to set up and validate their methodologies, separate guidelines for biopharmaceutical drug substances and products have been issued such as the International Conference on Harmonization (ICH) Q6B guideline on test procedures and acceptance criteria for biotechnological and biological products, and ICH Topic Q5C, D, and E.

I believe there are opportunities for products and techniques that can automate or accelerate all steps involved in the bioanalytical process. A lot of progress has been made over the last decades, but the present achievements are definitely not the endpoint. Advances towards faster analyses and even more accurate and detailed data generation will be necessary and will be made. Innovations in mass spectrometry (MS), chromatography (particularly

ultrahigh-pressure liquid chromatography [UHPLC] and two-dimensional liquid chromatography [2D LC]), and software are most important. The first two might seem obvious, but the last is equally important. A critical part of biopharmaceutical analysis lies in the data handling and interpretation. Data analysis software is playing a very prominent role in research on various biopharmaceuticals and this is a major difference compared to the analysis of small-molecule pharmaceuticals. It is a field in which various instrument and software developers are investing considerably.

Q. Peptide mapping is obviously a very useful technique, can current one-dimensional (1D) LC methods deal with the large amount of peptides that comprise a large protein drug?A: The power of state-of-the-art 1D LC techniques should not be underestimated. A well-developed (U)HPLC method can be very valuable and robust for peptide mapping. However, when sample complexity increases and the chromatogram becomes populated with a larger number of peptides, as is the case for mAbs, problems with co-elutions will be evident. Method development for one biopharmaceutical product will

Q&A: Vanhoenacker

44

Q&A: Vanhoenacker 2 Q&A: Vanhoenacker 2 The LCGC Blog20 The LCGC Blog2020Barden et al.15 Barden et al.1515Incognito12 Incognito12News9 News99Balaton Symposium Review23 Balaton Symposium Review2323 Staff28 Staff2828CHROMacademy262626 CHROMacademy2626 Training & Events272727 Training & Events27

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not necessarily, and will probably not, be

applicable to another product. These are

the limitations to 1D LC that researchers

encounter today.

We need a much larger peak capacity

than the number of peptides in our

digest. If we consider a digest with

100 peptides, a peak capacity of about

10,000 is required to have a good

chance of separating all of them with

chromatography. The introduction of

high-end MS systems has alleviated the

need for this extreme chromatographic

performance but once methods are

transferred from research to routine

use, MS is out of the picture and the

chromatography needs to do the job.

However, for relatively simple digests

1D LC can be suitable. More complex

samples such as digests of very large

biomolecules or mixtures thereof will

seldom not require more than one

analysis to completely profile the peptide

map, even with MS installed. Combining

different separations (that is, selectivities)

is what 2D LC is essentially doing. This

makes it a very valuable means to analyze

such complex samples.

Q. What does two-dimensional

chromatography offer that

one-dimensional chromatography

cannot in the analytical study of large

proteins?

A: The considerably increased separation

power is what it is all about. Achieving

resolution in the second dimension of

compounds that are not separated in

the first dimension is the most obvious

advantage. However, 2D LC can be used

in different modes.

First, there is comprehensive 2D LC

(LC×LC) where the complete effluent from

the first dimension is sampled in discrete

fractions and each of these fractions is

then analyzed in the second dimension.

The main goal here is to increase peak

capacity. In theory, the 2D LC peak

capacity will be the product of each of the

individual peak capacities.

This approach should increase separation

power dramatically. In reality, however,

the total peak capacity needs to be

corrected for what is called undersampling

and incomplete orthogonality. This has

a serious impact and the practical peak

capacity is significantly lower than the

theoretical peak capacity. Nonetheless,

the peak capacity in comprehensive

2D LC will be considerably higher than

what can be achieved with 1D LC. This

high peak capacity gives a comprehensive

view of the sample constituents and

resolves compounds that are not resolved

with only the first- or second-dimension

selectivity. The goal here could be to

create a generic method to screen

mAb digests for differences in amino

acid sequence or post-translational

modifications.

Other 2D LC techniques are based on

heart-cutting approaches where only

one or a number of fractions (peaks) are

transferred to the second dimension.

The advantage of such a method is that

the second dimension analysis time, and

therefore chromatographic performance,

is more or less detached from the first

dimension. In comprehensive LC×LC,

the available time to perform the

second dimension separation is very

limited and, consequently, the full

potential of this second dimension is

unable to be exploited. This is not the

case in heartcutting 2D LC approaches,

so the choice of analytical conditions

(column dimension, flow-rate, analysis

time) can be tailored according to the

analytical needs at hand. Applications

for large-biomolecule separations

include the combination of a first

dimension ion-exchange mechanism

with a second-dimension MS-compatible

reversed-phase separation. The inorganic

salt present in the first dimension is

removed after passing through the

second dimension. In this way, a specific

peak containing one or more peptides or

proteins can be transferred on-line to the

second dimension where an additional

separation can take place before MS

detection. Various ways to do this are

available and the user-friendliness is

superior compared to off-line methods

where the peaks of interest have to be

collected and then re-analyzed on another

system following some manipulation.

Q. Your research tested three

different 2D LC combinations,

namely strong cation exchange ×

reversed-phase LC, reversed-phase LC

× reversed phase LC, and hydrophilic

interaction liquid chromatography

(HILIC) × reversed-phase LC. Did any

of these combinations stand out as

particularly effective for peptide

mapping of large proteins?

A: Both reversed-phase LC × reversed-phase

LC and strong cation exchange ×

reversed-phase LC are very suitable. In

reversed-phase LC × reversed-phase

LC, compatibility of both dimensions is

excellent and effi ciency on both columns

is relatively high. It is the easiest of these

three combinations and results in a robust

method with high peak capacity. One of

the disadvantages is that full orthogonality

Q&A: Vanhoenacker

5



is impossible to achieve because of the

similarity in separation mechanisms.

However, there are plenty of tools to

incorporate some orthogonality into this

approach, by modifying factors such as

the stationary phase, mobile phase pH and

organic modifi er type, and temperature.

A simple option is to modify the second

dimension gradient during the analysis. In a

reversed-phase LC × reversed-phase LC set

up this makes sense because compounds

that are more strongly retained on the

fi rst dimension will also require stronger

elution conditions in the second dimension.

Combining all of these features results in

powerful methods for detailed peptide

mapping.

The strong cation exchange ×

reversed-phase LC combination will provide

better orthogonality because the separation

mechanisms are not correlated. Peptide and

protein fi rst dimension retention will be

driven mainly by ionic interactions, while

in the second dimension hydrophobicity

of the compound will be the major factor

for retention and separation. Strong cation

exchange for peptides and proteins is

generally performed using aqueous mobile

phases, which makes the transfer to and

injection onto the second dimension

relatively straightforward. Possible

drawbacks of this approach include the low

chromatographic effi ciency some strong

cation exchange columns can provide.

Also, the high buffer and salt load that is

generally used in strong cation exchange

separation of peptides and proteins will be

introduced onto the second dimension and

potentially the MS detector. The choice of a

good fi rst dimension column can obviously

overcome the fi rst drawback. The potential

interference of inorganic mobile phase

additives can somewhat be minimized by

selecting the relevant window in which

2D LC needs to be performed and by using

an additional diverter valve on the mass

spectrometer.

HILIC × reversed-phase LC is the most

difficult combination of the three. It will

provide good orthogonality but there can

be some compatibility issues. Since HILIC

operates with high acetonitrile amounts,

the transfer of these fractions onto the

reversed-phase second dimension can

cause polar peptides to breakthrough.

However, various modifications can be

made to reduce or even avoid this. It is

definitely an interesting approach but I

would advise this combination only in

the case of specific analytical challenges

such as differentiation of protein and

peptide glycosylation. For general work,

the reversed-phase LC × reversed-phase

LC and strong cation exchange ×

Q&A: Vanhoenacker

6


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addition to the required packing chemistry

and particle size. This is where column

manufacturers could step in. For the first

dimension column we like to work with a

column internal diameter between 1 mm

and 2.1 mm. This is done to keep the first

dimension flow rate as low as possible

to decrease volume loading onto the

second dimension. The range of available

stationary phases packed in narrow- and

especially in micro-bore format is limited

and frequently the ideal column will not

be found in a catalogue. Custom ordering

can be the solution but this takes time

and can be costly.

Second-dimension columns to be used

in heartcutting 2D LC approaches can

be of any dimension and the choice here

is plentiful. For comprehensive 2D LC

applications this is not the case. The

second dimension column should be able

to provide good quality chromatography

in as short an analysis time as possible

(below 1 min), preferably with reasonably

low fl ow rates and back pressure: Short

columns packed with small particles or with

superfi cially porous particles seem to be the

obvious choice here. However, one has to

be aware that one comprehensive 2D LC

analysis will result in numerous injections

(typically 50–150 modulations/run) of

relatively large volumes (20–80 μL) onto this

reversed-phase LC combinations will be

much easier to work with.

Q. You considered two options

(formic acid and trifluoroacetic acid

[TFA]) when replacing the mobile

phase for MS detection. However, as

noted, both options have potential

drawbacks. Are there any alternatives

that could have been used and, if

so, in what circumstances would you

recommend their use?

A: I would like to go back one step before

answering this. For peptide mapping and

protein analysis with reversed-phase LC,

the use of a water–acetonitrile mobile

phase with trifluoroacetic acid (TFA) is still

frequently used. The use of TFA results in

good retention for polar peptides because

of its ion-pairing properties. However,

it can produce interfering noise on the

baseline when ultraviolet (UV) detection

is used. This is a result of absorbance of

UV light at 214 nm. Formic acid has a

similar though less hindering effect. When

running very fast gradients (generally

around 30 s) as we do in comprehensive

2D LC, the excessive baseline noise and

drift will lead to poor quality 2D LC

plots. It will be difficult to detect small

compounds in such data. This can be

partly overcome by subtracting blank runs

but this is limited and not very convenient.

This is the reason we prefer to use

phosphoric acid in the second dimension

reversed-phase LC. With this additive a

stable baseline is obtained and retention

and selectivity are very close to those

acquired with formic acid. In my opinion

it is an excellent choice for 2D LC when a

UV detector or diode-array detector (DAD)

is used.

When MS is required, the use of

phosphate or other inorganic mobile

phase additives should be avoided. Here

we need to replace phosphoric acid with

a volatile and organic alternative. It is

known that the ion-pairing effect of TFA

can lead to ionization suppression. Formic

acid is an excellent alternative because

it will give nearly identical selectivity

compared to phosphoric acid. So by using

phosphate in UV work and formate in MS

work we can easily compare results and

identify peptides and proteins detected

by UV based on their MS and MS–MS

spectra.

For the best MS sensitivity, when

analyzing peptides and proteins, positive

ionization is preferred in combination

with an acidic mobile phase. This

is also the reason why we use high

pH reversed-phase LC (ammonium

bicarbonate pH 8.2) as the first

dimension in our reversed-phase LC ×

reversed-phase LC setup. Fractions of this

separation are then transferred to acidic

conditions, which is ideal for positive

ionization with electrospray.

Q. The method you developed showed

a particular aptitude for identity,

purity, and comparability assessments

of biopharmaceuticals and

biosimilars.1 Are there any limiting

factors in the use of this method?

A: The main practical limitations of 2D LC

lie in the compatibility of the dimensions

and column availability. For the analysis

of peptides and proteins a combination of

reversed-phase LC × reversed-phase LC or

strong cation exchange × reversed-phase

LC will hardly ever disappoint. While

compatibility in this case is not an issue,

in other combinations the efforts required

to overcome incompatibility between two

dimensions (for example, flow splitting,

mobile phase addition, combinations,

trapping) could make the technique less

accessible for routine use. That is why

we generally start with reversed-phase

LC × reversed-phase LC or strong

cation exchange × reversed-phase LC

combinations.

In some cases it can be problematic to

find a column with suitable dimensions, in

Q&A: Vanhoenacker

7



second dimension column. For this reason,

the choice of a stable stationary phase with

a slightly larger particle size, and thus end

frits, could be justifi ed. The small loss in

effi ciency will be largely compensated for

by the improved robustness and column

lifetime. In my opinion the ideal column for

second dimension for biopharmaceuticals

and protein digests is about 2 mm to 3 mm

wide and 30 mm to 50 mm long packed

with 3.5-μm particles. The use of a larger

internal diameter increases loading capacity

from the fi rst dimension but requires

high fl ow rates to keep the analysis time

low enough (typically 3–5 mL/min). Such

high fl ow rates lead to signifi cantly high

solvent consumption (several hundred mL/

analysis), which is a disadvantage from an

environmental as well as an economical

point of view.

Of course, the price of 2D LC

instrumentation and consumables and

dedicated software is also something

to keep in mind. The dedicated data

analysis software for multidimensional

chromatography is powerful and very

useful, but it is not as developed as

software developed for protein and

peptide characterization and identification.

Both software platforms often have to be

used in combination, which can make data

analysis more complicated.

Q. Are you planning to develop this

methodology further and perhaps

attempt to address some of those issues?

A: We have recently published an

application note on strong cation

exchange × reversed-phase LC analyses of

E.coli tryptic digest and intact proteins.3

Strong cation exchange conditions were

optimized compared to the published data

on the mAb digests. With the optimized

conditions we were able to generate a

practical peak capacity of about 2250 in

less than 4 h. This is outstanding and very

useful for complex samples such as large

biomolecule digests.

We have also recently used 2D LC in

host cell protein (HCP) characterization

and in determining the pharmacokinetic

properties of antibody fragments. An

interesting overview of the potential

of 2D LC for the analysis of mAbs has

recently been provided by our group.4

The use of heartcutting techniques

has already proven very useful for

hyphenating methods with inorganic

mobile phases with MS after desalting

on the second dimension separation. A

typical application is identification of

impurities and unknowns detected in the

first-dimension separation. We are now

also investigating the use of heartcutting

for large biomolecules. Several separation

E-mail: [email protected]: www.richrom.com

principles commonly used for protein

characterization (reversed-phase LC,

strong cation exchange, size-exclusion

chromatography [SEC], hydrophobic

interaction chromatography [HIC]) can

be used in the first dimension and

well-defined fractions or peaks can be

transferred on-line and analyzed using

reversed-phase LC–DAD–MS.

Multi-dimensional LC is very powerful

and flexible and new ideas will always

surface as applications are developed. I

am convinced that many applications will

follow in the near future.

References

1. G. Vanhoenacker, I. Vandenheede, F. David,

P. Sandra, and K. Sandra, Analytical and

Bioanalytical Chemistry 407(1), 355–366

(2015).

2. K. Sandra and P. Sandra, Advances in

Biopharmaceutical Analysis 28(s10)

(2015).

3. G. Vanhoenacker, K. Sandra, I. Vandenheede,

F. David, and P. Sandra, Agilent Application

Note 5991-5179EN (2014).

4. K. Sandra and P. Sandra, Bioanalysis 7(22),

2843–2847 (2015).

Gerd Vanhoenacker is

the LC Product Manager

at the Research Institute

for Chromatography

(RIC) in Kortrijk,

Belgium. He studied

Pharmaceutical Sciences

at the Katholieke Universtiteit Leuven and

obtained a Ph.D. degree in Pharmaceutical

Sciences from the Ghent University in

2004.

He is (co-)author of over 40 scientific

papers covering different areas of

separation science. His expertise

includes liquid chromatography (HPLC,

UHPLC), liquid chromatography–mass

spectrometry (LC–MS), supercritical

fluid chromatography (SFC), capillary

electrophoresis (CE), and sample

preparation.

In recent years a significant part of

his activities have included method

development for 2D LC. He has practical

experience with 2D LC for the analysis of

a variety of samples.

Q&A: Vanhoenacker

8


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Eastern Analytical Symposium Summary

The 54th Eastern Analytical Symposium and Exhibition was

once again held at Garden State Exhibition Centre in Somerset,

New Jersey, USA, and proved to be another exceptional

meeting of analytical chemists. There was a wide and varied

programme of presentations, short courses, and posters

on offer with a notable plenary lecture by Nobel Laureate

Professor Kurt Wüthrich. The 2015 EAS President Oscar Liu

described the Nobel Laureates talk as “inspiring” and was

pleased with the “enthusiasm and energy of participants

[who] kept the level of engagement high”. However, the EAS

does not just exist to provide a venue for cross-disciplinary

knowledge sharing, career development, and networking,

but also to recognize those individuals who have advanced

their respective fields in an exceptional manner. The following

individuals were all deemed to have enhanced their fields of

study with remarkable work:

2015 American Microchemical Society Benedetti Pichler Award — Apryll Stalcup, Dublin City University, Ireland.

2015 EAS Award for Outstanding Achievements in Mass Spectrometry — Emile A. Schweikert, Texas A&M University, USA.

Outstanding Achievements in the Fields of Analytical Chemistry Award — Professor Chris Enke, Michigan State University, USA.

Outstanding Achievements in Separation Science Award — Professor David S. Hage, University of Nebraska, USA.

Pattern Modulation Offers Alternative to Pulse Modulation in GC×GC New research into fl ow modulation methods in valve-based two-dimensional gas chromatography (GC×GC) has produced

an effective alternative to traditional pulse modulation.1 Described as “pattern modulation”, this new method increases

effl uent to the secondary column with fl ow rates compatible with most chromatographs and spectrometers.

Researcher John Seeley from Oakland University in Rochester, Michigan, USA, said the new approach is easy to

implement with existing instrumentation. “Pattern modulation can be produced with the exact same valve-based hardware

used to conduct conventional pulse modulation separations”, he said, “and requires only simple software commands”.

Standard gas chromatographs require a modulator to produce comprehensive separations. These modulators convert

effl uent peaks emerging from the primary column into a series of sharp pulses injected into the secondary column.

However, pulse generation with valve-based modulation requires a large increase in secondary column fl ow rate or only a

small amount of primary effl uent being transferred to the secondary column.

Today, most GC×GC separations are performed with thermal modulation, but that is an expensive approach. As Seeley

commented, “When cost is not a factor thermal modulation will provide the best performance. But when resources

are tight, valve-based modulation in all of its forms can be an extremely effective tool for generating high-resolution

separations.”

Unlike pulse modulation, where narrow pulses are injected, pattern modulation uses an intricate injection pattern.

This approach allows the majority of the primary effl uent to reach the secondary column. However, the

detector signal generated from this process must be transformed to extract a conventional

pulsed signal. This is key to the analysis.

Initial results using pattern modulation were incredibly positive, but the research

recognizes that there is a point where the complexity of the sample can overwhelm

the signal transformation process. At this point, traditional pulse modulation is

preferred.

Currently, Seeley and his team are trying to establish quantitatively when

pattern modulation is superior to pulse modulation. He said, “We want

to be able to recognize when pattern modulation is the best alternative

for producing valve-based GC×GC separations”. — L.B.

Reference

1. J.V. Seeley and S.K. Seeley, J. Chrom. A 1421, 114–122 (2015).

9


Metabolomic Detection of Early-StageOvarian CancerA detection method for early-stage

ovarian cancer has been developed using

ultrahigh-pressure liquid chromatography–

high-resolution mass spectrometry (UHPLC–

HRMS) in combination with tandem

MS–MS.1 Ovarian cancer is the biggest

killer of all gynaecological cancers and the

fi fth-leading cause of death among women

in the United States. Current screening

methods are inadequate, requiring lengthy

procedures that are inaccurate.

A considerable challenge to researchers,

ovarian cancer is “almost always

asymptomatic in the early stages,” noted

researcher John McDonald of the Georgia

Institute of Technology in Atlanta, USA.

Survival rates for late-stage ovarian cancer

is low but if the disease is detected early,

overall survival rates increase dramatically

(>92%). “Being able to accurately detect

the disease at early stages will make a big

difference,” said McDonald.

In developing the new method,

researchers used UHPLC–MS and MS–MS

in combination with a customized support

vector machine to identify 16 diagnostic

markers that produced an accuracy level

of 100% within the test group. “We are

currently initiating studies to determine if our

test can prospectively detect ovarian cancer

at early stages in high risk women, for

example women who are BRCA positive,”

said McDonald, referring to mutations of

the BRCA 1 and BRCA 2 genes, which are

associated with higher risks for breast and

ovarian cancer. “The frequency of ovarian

cancer onset in these women is quite high,

making this a good cohort in which to test

the clinical utility of our test,” he continued.

The study results also provide evidence

for the importance of lipid and fatty acid

metabolism in ovarian cancer. “It’s still not

completely clear what the signifi cance is

of the observed changes in lipid and fatty

acid metabolism in ovarian cancer patients,”

McDonald told The Column. “However, there

is a growing body of evidence indicating

that these changes may play a regulatory

role in fostering a cascade of molecular

changes that promote the development of

ovarian cancer.” It is hoped that targeting

the enzymes involved in lipid and fatty acid

metabolism will be a promising new area for

ovarian cancer therapy in the near future.

The research team are currently planning

to expand this study to include a wider

range of ethnic and racial groups because

of a recognized lack of diversity within

the test group. The learning algorithm

approach used by the team to identify

the biomarkers is designed to identify

which metabolites are optimally predictive

of disease among the group of women

analyzed in the study. “While we strove

to include women from broad geographic

areas in our study, not all ethnic and racial

groups were represented,” he said. “Thus,

at this point, we cannot be assured that

the 16 biomarkers identified in our study

will be 100% accurate in predicting early

ovarian cancer across all women.” This

may prove difficult because ovarian cancer

is rarely identified at the early stages and

therefore serum samples are extremely

rare. — L.B.

Reference1. D.A. Gaul et al., Sci. Rep. 5, 16351; doi: 10.1038/

srep16351 (2015).

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News The Column www.chromatographyonline.com

10


News In BriefLCGC TV Highlights

Peaks of the Week

Like us Join us Follow Us

A new detection method has been developed for gas chromatography (GC) by researchers from The University of Texas. Vacuum ultraviolet (VUV) detection features full spectral acquisition in a wavelength range of 115–240 nm, where virtually all chemical species absorb. The technique has real world applications in the analysis of fatty acid methyl esters (FAME). The research details the advantages of the detection method and profiles several food oil samples to demonstrate its applicability. doi:10.1016/j.foodchem.2015.08.004

Researchers have investigated the opportunistic foodborne pathogen Cronobacter sp. using gas chromatography–mass spectrometry (GC–MS) to understand its biofilm formation mechanism. Cronobacter sp. are linked to high mortality in neonatal and premature infants as a result of an association with powdered infant formula. Forming a biofilm enables survival in harsh conditions and the resistance of cleaning agents. doi:10.1016/j.foodcont.2015.11.029

Researchers from the University of Seville, Spain, have developed an efficient extraction, clean-up, and analysis method for the determination of environmental contaminants in human placental tissue. Perfluorinated compounds (PFCs) were quantified using UHPLC–MS–MS from 25 randomly selected women. PFCs are a public health concern because of their persistence, bioaccumulation, and toxicity.doi:10.1016/j.talanta.2015.12.020

The LCGC Blog: In Defense of Nitrogen as a Carrier for Capillary GC — There has been much

written about the use of nitrogen as a carrier gas for capillary GC. Formerly, to say it wasn’t any good.

Latterly to say that it’s pretty good and a better alternative to helium than hydrogen from a practicality

standpoint. Read Here>>

Are You Getting the Most Out of Your HPLC Column? — This article provided guidance for

working with the low-dispersion, small-volume columns that were gaining popularity in 2003. These

considerations are still appropriate today with the short, narrow HPLC and UHPLC columns now in

vogue. Read Here>>

What Is “Dead” Volume and Why Should Chromatographers Worry About It? — Dead-volume

effects can cause serious trouble for chromatographers. But if you understand what dead volume is and

how it affects chromatographic results, you can take control of it. Read Here>>

LCGC TV: Bob Kennedy, Part 4: Analytical Chemists in the “Omics” WorldYes, the LC in LC–MS matters in metabolomics and proteomics — it’s not all about the MS. Bob Kennedy discusses how to be an analytical

chemist in a world of biological data.Watch Here>>

LCGC TV: Joe Glajch on Biosimilars, Part 3: Strategies for Demonstrating Similarity of BiologicsHow fully can we characterize a biopharmaceutical? Joe Glajch offers strategies for demonstrating similarity, including statistical approaches, and what

the fi rst US approval of a biosimilar tells us about the FDA’s thinking.Watch Here>>

News The Column www.chromatographyonline.com

11


12


Reproducibility of Research — Do We Have a Problem Houston?Incognito talks about reproducibility in research.

I’ve been trying this week to reproduce some

experiments from a paper by a well-known

research group, and whilst I have results

(fi nally), they would appear to be pointing to

a very different conclusion than that drawn by

the paper’s authors. My aim was to start by

reproducing the results from the paper and

then trying to adapt the methodology to use

a different sampling technique to improve the

sensitivity of the method — a situation, the

like of which, I’m sure, happens globally on a

daily basis. However, as I have found several

times in my career, I was unable to reproduce

the original experiments and therefore unable

to validate my starting position for the new

experiments.

So, one of two things is true: the original

research and data is fl awed, or I am not

capable of replicating that data because of

fl aws in my own experimentation.

I’m unable to tell which is true here — but I

do know that I have wasted a couple of days’

work. The original paper was diffi cult to follow,

with what I thought to be several key variables

and pieces of methodological information

missing. I’m not blaming this — the issues

could well be with my own work — but I’m still

cross, whoever is to blame.

So cross in fact, that I went back to re-read

an excellent recent edition of Nature, regarding

the issues of reproducibility in scientifi c

publications.1 Not the statistical measure of

repeatability, rather the ability of another group

to repeat and substantiate the work of the

originators.

In modern research and development,

it’s all too easy to jump to conclusions and

fi nd patterns in what may otherwise be

considered to be random data, as so often we

have a vested interest in the data — a PhD

thesis, tenure, further funding, advancing a

commercial project, maintaining the reputation

of the department (academic or industrial),

kudos, etc. This makes us sound like a

thoroughly unscrupulous lot, but that’s not

what I’m alleging.

Psychologist Brian Nosek of the non-profi t

Center for Open Science in Charlottesville,

Virginia, USA, which works to increase the

reproducibility of scientifi c research, puts it

much better than I can: “As a researcher,

I’m not trying to produce misleading results, Ph

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but I do have a stake in the outcome.”2 I’m suggesting then that we might be pre-disposed to certain outcomes in our work which leads to actions and decisions that do not give a true re� ection of what the data is telling us, or that we can choose those experiments which lead us to substantiate our theories at the exclusion of other, more rigorous, experiments.

So, what can be done, and what does Nature tell us about what analytical science can do to avoid fooling itself and wasting time?

Well � rst of all, science has always operated on the postulation of a theory or conclusion from experimentation, which has then been repeated and validated or refuted by other groups who will go on to expand upon the original work or postulate an alternative theory. Fine — that’s how things work, but there has been some shocking failures at reproducing academic research on a large scale, which brings into question how much time and resource is wasted producing meaningless data that does not advance science and in fact may even be holding up the good research.

In 2012, researchers at biotechnology � rm Amgen in Thousand Oaks, California, USA, reported that they could replicate only 6 out of 53 landmark studies in oncology and haematology.3 In 2009, workers at the Meta-Research Innovation Center at Stanford University in Palo Alto, California,

described how they had been able to fully reproduce only 2 out of 18 microarray-based gene-expression studies.4

So, what can be done?

Be More Rigorous with Academic PublicationsHere’s the advice to authors on Materials and Methods in a leading academic journal:5 Provide suf� cient detail to allow the work to be reproduced. That’s it. Enough? I really don’t think so. We need checklists to ensure even the most esoteric details are addressed so that reproduction is possible. Checklists would include minor experimental details, experimental design, method performance via statistical analysis, etc. Is there a restriction on space or content for method details? If so — abolish it. What about electronic submission of raw data? In review — blind the author’s names and institutions to avoid bias or deference.

How about replicating papers prior to publication? I can hear the gasps from readers already, but why not? Set aside funding, allow publication of replication studies (more publications for sound science), give replicated publications more research credits, establish replication groups. Where advanced equipment is used, allow replicators to take charge of original equipment to repeat experimentation. Non-replication needn’t

Incognito

1313

Q&A: Vanhoenacker 2 Q&A: Vanhoenacker 2 The LCGC Blog20 The LCGC Blog2020Barden et al.15 Barden et al.1515Incognito12 Incognito12News9 News99Balaton Symposium Review23 Balaton Symposium Review2323 Staff28 Staff2828CHROMacademy262626 CHROMacademy2626 Training & Events272727 Training & Events27

www.gerstel.com

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methods and design prior to any practical

work, perhaps with an “in-principle”

promise of publication. It’s not really any

different to having a methods scrutiny panel

or a good laboratory manager in industry!

t� Adversarial Collaboration — Invite your

rivals to work with you, or on a competing

hypothesis, which will ultimately result in

a joint publication. For all of you thinking,

“They will never agree on enough results

or conclusions to write a joint paper”,

ask yourself if its healthy to have beliefs

so entrenched that pure experimentation

and data analysis cannot be used to prove

or disprove either side’s argument. These

entrenched beliefs are prevalent and deeply

unhealthy.

t� Blinding — There are many ways to “blind”

our data analysis. Mixing up columns in

databases, introducing dummy or biased

data, and mixing up data into different

sets are all possible. Once the data have

been analyzed, the blinds are lifted to

see if the conclusions remain valid. The

obvious example is the use of “placebo”

medications in clinical trials or bioanalytical

work, where the placebo subjects are not

revealed until after the data analysis.

I feel that writing a clever conclusion

to this piece will do nothing other than

detract from the thoughts and theories

contained within. I’d just ask you to

consider if you recognize, in your own

work, any of the problems or biases

presented and if you agree or disagree

with the proposed solutions. Just having

you think about the issue of reproducibility

is a good start; if this piece then allows

you to adopt measures to avoid any of the

problems, then even better.

Oh, and by the way, these issues don’t

just apply to research in an academic

environment!

References

1. Nature 526(7572), 164–286 (2015).

2. Regina Nuzzo, Nature: http://www.nature.com/

news/how-scientists-fool-themselves-and-how-

they-can-stop-1.18517

3. C.G. Begley and L.M. Ellis, Nature 483,

531–533 (2012).

4. J.P.A. Ioannidis et al., Nature Genet. 41,

149–155 (2009).

5. https://www.elsevier.com/journals/journal-

of-chromatography-a/0021-9673/guide-for-

authors

Contact author: IncognitoE-mail: [email protected]

necessarily disbar work from publication, but

some notifi cation of the failure to reproduce

should be made. This approach would

foster and encourage collaboration as well

as introducing rigour and help to underpin

good science. The most I ever learn about

our science is when troubleshooting issues

that arise from method transfers to client

laboratories — would this be any different?

Recognize Cognitive Bias and Build in

Safety Measures

There are many ways in which we can fool

ourselves or have underlying bias in our work.

Even the most ethical of researchers are

susceptible to self-deception; outlined below

are a few of the reasons why:

t� Hypothesis Myopia — A natural inclination

to favour only one hypothesis and look for

evidence to support it, whilst playing down

evidence against it and being reluctant

to adjust or propose more than one

hypothesis.

t� Sharpshooter — Fire off a random series

of shots, then draw a target around the

bullet holes to ensure the highest number

of bullseyes. Getting some encouragement

from your on-going experimental data and

deciding that this must be the correct path

to go down, without realizing that the

data could actually support many different

conclusions from the one you are drawing.

t� Asymmetric Attention (Disconfi rmation

Bias) — Giving the expected results smiling

approval, whilst unexpected results are

blamed on experimental procedure or

error rather than being accepted as a true

challenge to your hypothesis.

t� Just-So Storytelling — Finding rational

explanations to fi t the data after the fact.

The problem is, we can fi nd a story to fi t

just about every type of data — it doesn’t

mean to say the story is true! Also known

as JARKing — “justifying after the results

are known” — because it’s really diffi cult to

go back and start again once we are at the

end of the process.

t� The Ikea Effect — Everyone has a vested

interest in loving the furniture they built

themselves. Is it the same with our

analytical data?

So what strategies might we employ to

overcome these innate biases?

t� Strong Inference Techniques — Develop

opposing or competing hypotheses and

develop experiments to distinguish which is

correct. Not having a favourite child avoids

Hypotheses Myopia and cuts down on the

need for Just-So Storytelling.

t� Open Science — Publication of methods

and raw data for various groups to

scrutinize. Even more radical — publish

and seek approval or revision of research

Incognito

14


Extending the Detection Limits for the Analysis of Organotin Contaminants Using Soft Ionization

The inherent sensitivity and selectivity of time-of-fl ight mass spectrometry (TOF-MS) can be augmented by soft electron ionization (EI) to provide ultratrace-level quantitation of organotins in complex environmental extracts. These organotin species are a focus of current concern as environmental contaminants, but analysis using conventional 70 eV ionization energies is made diffi cult by their propensity to undergo extensive fragmentation. The use of soft EI helps to solve this problem by producing simplifi ed spectra with enhanced diagnostic ions.

Laura McGregor, Steve Smith, and David Barden, Markes International, Llantrisant, Wales, UK.

Organotins (stannanes) are anthropogenic

chemicals that are attracting attention from

environmental analysts because of their

high toxicity and ability to interfere with

the endocrine system, along with their

persistence in the environment. Use of

organotins in anti-fouling paints fi rst caused

concern in the 1970s as a result of a decline

in populations of marine molluscs. The use

of such paints has now been restricted or

banned by many countries, but other sources

remain, such as PVC products, disinfectants,

and agricultural pesticides.1

The current annual average of organotins in

water, as stated by the EU Water Framework

Directive, is just 0.2 ng/L, with a maximum

allowable concentration of 1.5 ng/L.2

This means that highly sensitive detection

methods are required, with environmental

analysts constantly seeking improvements to

instrumentation and technological advances

that could lower reporting limits.

Time-of-fl ight mass spectrometry (TOF-MS)

offers signifi cant advantages in such

scenarios, with instruments using direct

extraction (rather than the inherently less Ph

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effi cient orthogonal extraction) providing

a distinct sensitivity advantage. However,

certain analytes remain challenging even with

such technologies because of their propensity

to fragment extensively at conventional

(70 eV) ionization energy.

This problem can now be addressed by the

use of a soft electron ionization (EI) technique

that offers the advantages of soft ionization

for gas chromatography coupled to MS

(GC–MS), without any of its historic

disadvantages — such as hardware

changes and additional reagent gases. The

technique uses an ion source design in the

TOF instrument that allows the ionization

energy to be varied on a sliding scale from

conventional 70 eV to lower energies.3 The

physical properties of most small molecules

means that relatively small differences in

ionization energies between 10 eV and

20 eV can have signifi cant differences in the

fragmentation pattern. Typically, however, an

ionization energy of about 12–14 eV retains

an useful degree of fragmentation while

avoiding a loss in sensitivity relating to the

unavoidable drop-off in ionization effi ciency

as the ionization potential is approached.

This technique has already been applied

to analytes ranging from petrochemical

hydrocarbons4 to emerging environmental

contaminants.5 This article explains how this

soft ionization method can be applied to

organotin compounds. These are particularly

challenging because of the requirement for

high sensitivity to meet the demands of the

low detection limits, in conjunction with their

extensive fragmentation at 70 eV making

speciation diffi cult. Using soft ionization

provides enhanced “diagnostic” ions and

reduced chemical noise, which leads to

lower detection limits, as well as simplifi ed

spectra for more confi dent qualifi cation. A

further boost in sensitivity is demonstrated

by reduced ionization of common

background/carrier gases at low ionization

energies, leading to minimal chemical noise

and improved signal-to-noise ratios for

compounds of interest.

Experimental

Samples: A selection of some of the most

common organotins were analyzed in this

work: dibutyl tin (Bu2SnH2), tributyl tin

(Bu3SnH), monooctyl tin ([C8H17]SnH3),

and tetrabutyl tin (Bu4Sn). Tri-substituted

organotins such as tributyltin are used as

pesticides,6 while mono- and di-substituted

organotins are used as PVC stabilizers,

as catalysts, and in glass coatings.7 The

lower-substituted organotins can also be

formed in the environment by metabolism

and degradation of higher-substituted

analogues.6 Environmental water samples

were spiked with organotins (50 ng/L) and

ethylated with sodium tetraethylborate

prior to extraction to make the tin hydrides

suffi ciently volatile for analysis by GC–MS. A

dilution series (ranging from 0.1–20 ng/L) was

prepared from the stock solution.

GC: Carrier gas: Helium, constant fl ow at

0.9 mL/min; mode: splitless for 2.0 min (then

150 mL/min purge); temperature: 200 °C;

septum purge: on, 3 mL/min; column:

20 m × 0.18 mm, 0.18-μm Rxi-5Silms

(Restek); oven: 50 °C (2.5 min), 20 °C/min to

300 °C (1 min); total run time: 16 min.

Barden et al.

16


(a)

Retention time (min)


Inte

nsi

ty (

x 1

04 c

ou

nts

)In

ten

sity

(x 1

03 c

ou

nts

)

15

10

5

04 6 8 12

(C8H17)SnEt

Bu4Sn

Bu3SnEt

Bu2SnEt2

1200

900

8.75 8.80 8.85 8.90

600

300

6

2

1

08 10

0

1410

(b)

Figure 1: GC–TOF-MS analysis of the 5 ng/L spiked sample: (a) TIC and (b) EIC (m/z 263+291) highlighting the organotin targets. The inset shows the excellent peak shape for (C8H17)SnEt.


TOF-MS: Instrument: BenchTOF-Select

(Markes International); fi lament voltage: 1.8 V;

ion source: 250 °C; transfer line: 280 °C; mass

range: m/z 40–500; data rate: 4 Hz.

Software: TOF-DS software for BenchTOF

(Markes International) was used for full

instrument control and data analysis.

Results and Discussion

Figure 1 shows the GC–TOF-MS analysis of

the 5 ng/L spiked sample, with an extracted

ion chromatogram (EIC) indicating the elution

order and excellent peak shape and intensity

for the organotin targets, even at this trace

level (5 pg on-column).

Spectral Quality: The spectra obtained for

each of the organotin target compounds

at 70 eV and 14 eV are compared in

Figure 2. The results show that softer

(14 eV) ionization simplifi es the spectra

and increases the intensity of the higher

m/z ions. This is important because these

larger fragments are useful for determining

compound structure.

Furthermore, the preservation of a degree

of fragmentation provides more information

than other soft ionization techniques (such

as chemical ionization) that may produce

spectra containing solely the molecular ion.

Library-searching can therefore be performed

Barden et al.

17


100(a) (b)

70 eV 70 eV

14 eV

70 eV 70 eV

14 eV14 eV

14 eV

0

100

0

100

0

100

0

100

0

100

0

100

0

100

0

41

41

41

41

56

56

121

121

151

121

121

Bu2SnEt2151

151

151

Forward match 904Reverse match 911

Bu3SnEtForward match 907Reverse match 917

(c) (d)C8H17SnEt3

Forward match 911Reverse match 916

Bu4SnForward match 902Reverse match 923

179

179

179

179

179

149

179179

177

207

207

207

207

235

235

235

235

207

207

263

263

263

235

263

291

291

291

291

291

291

41

56

56

83

83

Figure 2: Spectral comparisons for the organotin targets from the 5 ng/L standard, at 70 eV (top) and 14 eV (bottom), illustrating the simplifi ed spectra and enhanced selectivity achieved by soft ionization. Forward and reverse match factors against the NIST 14 database are indicated. Note that for Bu2SnEt2 and Bu3SnEt m/z 207 is a prominent ion at 70 eV, but because it is a common interference ion from column bleed, it would not be ideal for quantitation.

14 eV


Inte

nsi

ty (

x 1

03 c

ou

nts

)

5

4

3

2

1

0

70 eVBu2SnEt2

Bu3SnEt2

C8H17SnEt3

Bu4Sn

7.5 8.0 8.5 9.0

Figure 3: GC–TOF-MS EIC (m/z 263+291) overlays for the organotin targets at 70 eV (blue) and 14 eV (red) for the 5 ng/L spiked sample, showing the improvement in peak intensity achieved at low ionization energy.


at low eV to add another level of confi dence

in compound identifi cation.

Figure 3 shows the EIC (m/z 263+291)

overlays for 14 eV and 70 eV, illustrating

the improved detection limits at low eV.

Note the reduced baseline at 14 eV, as a

result of reduced ionization of common

background/carrier gases, further improving

signal-to-noise ratios for the peaks of interest.

Linearity: The set of derivatized organotins

was analyzed at six dilution levels by GC–

TOF-MS at ionization energies of 70 eV

and 14 eV. The resulting calibration curves

(Figure 4) display excellent linearity at both

ionization energies, with all R2 values

over 0.997. Note in particular the steeper

calibration curves at 14 eV, which result

in increased analyte response factors and

therefore lower quantitation limits.

None of the organotins at the lowest

dilution level (0.1 ng/L) could be reliably

detected at 70 eV (and so this data point is

Barden et al.

18


Inte

nsi

ty (

cou

nts

)In

ten

sity

(co

un

ts)

(a)

1 ng/L

0.1 ng/L(b)

400

60

40

20

0

8.30 8.35 8.40

14 eV

14 eV

70 eV

70 eV

300

200

100

0


8.30 8.35 8.40


Figure 5: GC–TOF-MS EIC (m/z 291) chromatograms showing overlays of the 70 eV and 14 eV analyses of Bu3SnEt at (a) the 1 ng/L level and (b) the 0.1 ng/L level.

5

4

3

2

1

Bu2SnEt2 Bu3SnEt(a) (b)

(c) (d)

Pe

ak

are

a (

x 1

04 c

ou

nts

)P

ea

k a

rea

(x

10

4 c

ou

nts

)

Pe

ak

are

a (

x 1

04 c

ou

nts

)P

ea

k a

rea

(x

10

4 c

ou

nts

)

14 eV

70 eV

14 eV

70 eV

14 eVR2=0.9991 R2=0.9984

R2=0.9974R2=0.9990

R2=0.9987

R2=0.9995

R2=0.9981

R2=0.9996

Concentration (ng/L)

70 eV

14 eV

70 eV

0

0

1

2

3

4

5

6

7

0 5 10 15 20


0 5 10 15 20


0 5 10 15 20


0 5 10 15 20

3

2

1

0

4

2

0

6

8

10

C8H17SnEt3 Bu4Sn

Figure 4: Calibration curves for the organotin targets at both 70 eV (n = 5) and 14 eV (n = 6).


not shown in Figure 4), but the enhanced

sensitivity at 14 eV easily enabled detection

at this level (Figure 5).

Conclusion

This article has demonstrated the

performance of this GC–TOF-MS method

for trace-level detection and quantitation of

organotins in complex matrices.

The soft ionization technology used

provided a level of sensitivity and selectivity

not achievable with other methods, and

spectra exhibiting enhanced diagnostic

ions for all organotins at 14 eV. Excellent

linearity was achieved (R2 > 0.997) for

both conventional (70 eV) and soft (14 eV)

ionization. Moreover, the enhancement in

quantitation ions and reduced background

at 14 eV enabled detection limits to be

extended down to just 0.1 pg on-column for

all organotins in this study.

References

1. H.K. Okoro, O.S. Fatoki, F.A. Adekola, B.J.

Ximba, R.G. Snyman, and B. Opeolu, in

Reviews of Environmental Contamination and

Toxicology, (vol. 213, ch. 2, 2011) pp. 27–54.

2. Directive 2000/60/EC of the European

Parliament and of the Council of 23 October

2000 establishing a framework for Community

action in the fi eld of water. See http://

ec.europa.eu/environment/water/water-

framework/index_en.html

3. L. McGregor, N. Bukowski, and D. Barden,

Current Trends in Mass Spectrometry,

supplement to LCGC North America, LCGC

Europe, and Spectroscopy, 12(1), 16–19 (2014).

4. L. McGregor and D. Barden, Analyzing

crude oil: Improving compound speciation,

Hydrocarbon Engineering, July 2014, http://

www.energyglobal.com/downstream/gas-

processing/31072014/Crude-oil-markes-

analysis/

5. L. McGregor, A. Gravell, I. Allan, G. Mills,

D. Barden, N. Bukowski, and S. Smith, The

Analytical Scientist April 2015.

6. S. Dobson and R. Cabridenc, Tributyltin

compounds (Environmental Health Criteria 116),

World Health Organization (1990).

7. S. Dobson and P.D. Howe, Mono- and

disubstituted methyltin, butyltin, and octyltin

compounds (Concise International Chemical

Assessment Document 73), World Health

Organization (2006).

Laura McGregor received an M.Chem.

in chemistry from the University of St

Andrews, UK, followed by an M.Sc.

in forensic science at the University

of Strathclyde, UK. Her Ph.D. in

environmental forensics, also at the

University of Strathclyde, focused on the

chemical fingerprinting of environmental

contamination using advanced techniques

E-mail: [email protected]: www.markes.com

such as GC×GC−TOF-MS. Laura joined

Markes International in 2013 as a sales

support specialist, and is now product

marketing manager for Markes’ TOF-MS

product portfolio.

Steve Smith studied in Bristol, UK,

for both his B.Sc. and Ph.D., which he

obtained in 2008 on innovative work

profiling volatile organic compounds for

disease diagnosis. Following post-doctoral

positions at the University of the West

of England and Bristol University, Steve

joined Markes International as a senior

applications specialist for thermal

desorption and TOF-MS in 2011, where he

now specializes in GC×GC−TOF-MS.

David Barden is a technical copywriter

at Markes International, having joined

the company in 2011. David studied

natural sciences at the University of

Cambridge, UK, and remained there for

his Ph.D. in organic chemistry, which

he received in 2003. A placement at

the European Journals Department of

Wiley-VCH, Weinheim, Germany, was

then followed by seven years in journal

publishing at the Royal Society of

Chemistry, UK.

Barden et al.

19


The LCGC Blog: Column Overload in Gas Chromatography with Vacuum Ultraviolet Detection

Column overload is a very commonly encountered issue in gas chromatography (GC) for beginners. Changes in peak symmetry, generally observed as peak fronting, can be subtle in the sharp peaks generated by GC, but the result can be signifi cant shifts in retention times, loss of resolution, and error in peak integration. LCGC Blogger Kevin Schug explains more.

Kevin A. Schug, University of Texas Arlington, Texas, USA.

Split injectors were invented to ensure that

wall-coated open-tubular capillary gas

chromatography (GC) columns are not

overloaded. Because it is not practical to

reduce actual injection volumes much lower

than tenths of microlitres and the capacity

of thin-fi lm stationary phases coated on the

capillary wall surface should not be exceeded,

the ratio of gas fl ows in the injection port

directed through the column versus to waste

(through the split vent) is adjusted to set an

appropriate split ratio. Split ratios are generally

reliable between 10:1 and 400:1 (where the

majority of analyte is split to waste) to ensure

that the target analyte of interest exhibits good

peak symmetry. In fact, column overload is

a very commonly encountered issue in GC

for beginners. Changes in peak symmetry,

generally observed as peak fronting, can

be subtle in the sharp peaks generated by

GC, but the result can be signifi cant shifts in

retention times, loss of resolution, and error in

peak integration. Traditionally, it is preached

that column overloading conditions should be

avoided for those reasons; however, in cases

where the greatest sensitivity is desired to

perform ultratrace analysis, a splitless injection,

where all of the analyte is transferred onto the

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column (the split vent is closed for a minute or

two during the injection phase of the analysis),

can be performed. One can imagine that the

choice between the use of split or splitless

injection would be driven by monitoring peak

area and peak shape, and then choosing the

conditions that give a nice symmetric peak and

signifi cant signal for the target analyte over the

desired concentration ranges for analysis. In our

experience with the new vacuum ultraviolet

(VUV) absorption detector for GC, these

considerations may not be so critical.

A VUV detector was introduced into the

market in 2014.1 I have written previously

about the concept and the general operational

advantages it provides.2,3 Since then, we

have applied GC–VUV for the investigation of

permanent gases,4 pesticides,5 and fatty acids.6

We, as well as other groups around the world,

are currently looking at several other application

areas. The majority of VUV detectors to date

primarily have been sold in the petroleum and

petrochemical industries.

In short, the principle of VUV spectroscopic

detection is full-range absorption measurements

between 120 nm and 240 nm, where all

chemical entities absorb and have unique

gas-phase absorption signatures. VUV detection

is quite complementary to mass spectrometry

(MS), because it can well discern isomeric

(isobaric) species that are often indistinguishable

based on electron-ionization MS spectra. Further,

standard concepts from Beer’s law apply — the

magnitude of absorption is directly proportional

to the amount of analyte present and its

absorptivity (cross-section), and absorption for

overlapping signals is additive. This latter point

means that unresolved components can be easily

deconvolved if the reference spectra for each

are present in the VUV spectral library. A ton of

effort need not be placed in fully resolving all

peaks of interest. The strength of this capability

is impressive. For example, we are currently

drafting a manuscript on our effort to fully

speciate Arachlor (previously manufactured by

Monsanto) samples, which are complex mixtures

of polychlorinated biphenyl compounds (PCBs).

Each of the 209 PCBs has a unique spectrum —

they can be well differentiated from one another,

even if they chromatographically overlap.

Returning to the question at hand, the

deleterious effects of GC column overload

should be quite well handled for GC–VUV

analyses. Signifi cant peak fronting can

compromise resolution; it can cause overlap of

neighbouring peaks that would be very diffi cult

to deconvolve using any other GC detector. Yet,

we have shown in most of our applications that

two or three (and probably more are possible)

distinct analytes can be completely resolved from

coeluted peaks into their respective contributions

to the overall signal. Assigning the retention time

to the peak apex will cause the retention time to

shift more and more as overloading is increased.

This is not really a major problem, except for

the context of analyzing a number of samples

with a wide range of analyte concentrations.

In such cases, where some peaks might front

(high analyte concentration) and some might be

symmetrical (low analyte concentration), it would

be necessary to understand that there is potential

for the retention time to shift.

At some point, when larger and larger

amounts of analyte are eluted through the

column into the VUV fl ow cell, the absorption

signal might saturate the detector, especially

in regions of the absorption spectrum where

absorptivity of the molecule is very high.

However, because quantitative analysis is typically

performed through averaging the signal across

a wide wavelength range (in contrast to typical

UV quantitation, where quantitation is often

performed at a predefi ned peak maximum),

when one spectral feature goes off scale (that

is, it becomes saturated), the quantitation

performed by the VUV detector can be defi ned

based on absorption across less-absorbing

wavelengths in the spectrum. Because we know

the shape of the absorption spectrum for an

analyte across the full range of 120–240 nm,

and because the ratios of intensities for

electronic transitions across that range remain

constant, an off-scale response in one region

of the absorption spectrum does not matter.

It is a simple matter to model the shape of

the response of that off-scale region based on

the magnitude of response in other regions of

the spectrum. This treatment could effectively

increase the dynamic range of the detector.

These concepts have not been shown

empirically using GC–VUV; these are simply

my thoughts on the subject. However, there

is nothing to prevent methods from being

conceptualized where column overload is used

to achieve lower detection limits in GC–VUV

analysis. Currently, VUV detection is about as

sensitive as MS in a scan mode (50–200 pg

on-column LODs, depending on the analyte

chromophore). In truth, the main limitation might

actually be the volume of the injection port

liner and the thermal expansion of the various

sample solvents used. In other words, you can

only increase injection volume so high (unless

you become quite creative and experienced in

performing methodically slow splitless injections)

before the capacity of the liner will be reached

and vaporized sample solutions will overfl ow

into other parts of the injection port — an

undesirable situation. Of course, there are

large-volume injectors commercially available.

Some more obscure standard methods (I can

think of one for disinfection by-products by GC

with negative chemical ionization MS) require this

type of hardware for ultratrace detection limits.

Further, the dynamic range of the response for

the method can be extremely large, considering

that the strategy above can be used when

detector saturation is reached.

The LCGC Blog

21



So, I need to get my students to try this

approach (or perhaps one of you readers with

access to a VUV system could try it): Prepare

a series of solutions ranging from absurdly

dilute analyte concentration up to some

concentrations a few orders of magnitude

higher. Use large volume splitless injection

to see how low you can go and how wide

the dynamic range of response could be. I

purposely use “dynamic range” here, because

nothing says that the response has to be linear

all the way up to high concentrations. As long

as the response changes in a predictable, even

if nonlinear, fashion across the concentration

range, then a calibration curve could be

constructed. The progression of peak shape

and size should go from small and symmetrical

up to very large and fronting. At some point,

the detector will saturate in part of the

wavelength range, but that problem can be

mitigated with some modelling of the response

using intensities in less-responsive regions of

the spectrum. There is some precedent for

this technique in the literature using lower

abundance isotope species in MS,7,8 but it is far

from commonplace. Dare I say, it would be fun

to try it and see what performance would be

like.

References

1. K.A. Schug, I. Sawicki, D.D. Carlton Jr., H. Fan,

H.M. McNair, J.P. Nimmo, P. Kroll, J. Smuts, P.

Walsh, and D. Harrison, Anal. Chem. 86,

8329–8335 (2014).

2. K.A. Schug, The LCGC Blog 11 September 2014.

http://www.chromatographyonline.com/lcgc/

Blog/The-LCGC-Blog-My-New-Obsession-Gas-

Chromatography-/ArticleStandard/Article/detail/853

093?contextCategoryId=50130

3. K.A. Schug and H.M. McNair, LCGC North

Am. 33(1), 24–33 (2015). http://www.

chromatographyonline.com/gc-detectors-thermal-

conductivity-vacuum-ultraviolet-absorption

4. L. Bai, J. Smuts, P. Walsh, H. Fan, Z.L. Hildenbrand,

D. Wong, D. Wetz, and K.A. Schug, J. Chromatogr.

A 1388, 244–250 (2015).

5. H. Fan, J. Smuts, P. Walsh, and K.A. Schug, J.

Chromatogr. A 1389, 120–127 (2015).

6. H. Fan, J. Smuts, L. Bai, P. Walsh, D.W. Armstrong,

and K.A. Schug, Food Chem. 194, 265–271 (2016).

7. H. Liu, L. Lam, L. Yan, B. Chi, and P.K. Dasgupta,

Anal. Chim. Acta 850, 65–70 (2014).

8. H. Liu, L. Lam, and P.K. Dasgupta, Talanta 87,

307–310 (2011).

Kevin A. Schug is an Associate Professor and

Shimadzu Distinguished Professor of Analytical

Chemistry in the Department of Chemistry &

Biochemistry at The University of Texas (UT) at

Arlington, USA.

E-mail: [email protected]: www.chromatographyonline.com

The LCGC Blog

22


The 10th Balaton Symposium on High-Performance Separation Methods: A Review

A review of the 10th Balaton Symposium on High-Performance Separation Methods, which was held 2–4

September 2015 at the Hotel Azúr, Siófok, Hungary.

Ira Krull, Northeastern University, Boston, Massachusetts, USA.

The Hungarian Society for Separation

Sciences, headed by Professor Attila

Felinger (Department of Analytical and

Environmental Chemistry, University of

Pécs, Hungary) organized the 10th biennial

Balaton Symposium meeting, which was

held 2–4 September 2015 at the Hotel

Azúr, on the shores of Lake Balaton, Siófok,

Hungary. The Diamond Congress of Hungary

coordinated the symposium, which included

presentations from notable scientists,

plenary lectures, and keynote lectures. In

addition, awards were presented to student

posters that were deemed exceptional.

Technical sessions and poster presentations

were combined with a full social agenda

including meals with unique entertainment

from Zoltán Orosz, a Hungarian accordionist,

and a four-piece band and singer. The

symposium concluded with an evening

buffet-style barbeque by the outdoor hotel

pool, complete with drinks.

The biennial conference was attended

by 285 scientists, mainly from Europe,

but with a few attendees from the USA,

Canada, Asia, and Israel. In addition, there

was a very successful exhibition comprising

14 vendors exhibiting instrumentation,

ultrahigh-pressure liquid chromatography

(UHPLC) columns and accessories, and

supplies related to separation sciences of all

types.

The opening ceremony dedicated

the event to the late Professor Georges

Guiochon, who sadly passed away in

2014. Professor Guiochon was a signifi cant

sponsor and attendee at many past Balaton

Symposia. His wife, Lois Beaver, attended

and participated in the conference.

Following these initial proceedings, the

Halász Medal Award and the Csaba Horváth

Memorial Award were individually awarded

to Professors Janusz Pawliszyn and Peter

Schoenmakers, respectively.

The 2013 Halász Medal Award was

awarded to Professor Nobuo Tanaka, who

could not attend the Balaton Symposium

two years ago. The three awardees Ph

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presented their 30-minute award lectures

before the Thursday sessions began in

earnest. There was further respect and

remembrance for Professor Guiochon

throughout the subsequent sessions, with

many presentations made by his former

students and colleagues.

The emphasis throughout the conference

was on the latest innovative advances in

different areas of separations science. These

included gas chromatography (GC), UHPLC,

high performance liquid chromatography

(HPLC), supercritical fl uid chromatography

(SFC), high performance capillary

electrophoresis (HPCE), and variations

thereof, with numerous applications to

small molecules, biopharmaceuticals,

low-molecular-weight (MW)

pharmaceuticals, and other interesting and

important topics. The talks were generally

followed by meaningful questions for the

speaker, as well as discussions amongst

the attendees, often quite interactive and

thought-provoking. There was also a large

number of poster papers from separation

scientists at many universities and colleges

around the world. This was mainly an

academically oriented conference, but

there were also several presentations from

scientists from industry, instrument and

column vendors, and some government

laboratories.

Two parallel sessions were run

simultaneously over the course of the

conference. The fi rst day included the

aforementioned welcome and awards

ceremony for both the Halász and Horváth

Medals, followed by plenary talks by the

recipients and others to complete each

plenary session. Oral talks were arranged

so that they interrelated with a common,

general topic or theme, which was also

attempted with the poster papers (available

to view on all three days).

Several talks worthy of mention include

one by Gerard Hopfgartner of the University

of Geneva in Geneva, Switzerland, who

spoke on target and non-targeted LC–MS–

MS approaches including differential ion

mobility spectrometry to support -omics

studies. Another combined separations

presentation of interest was that of Dora

Palya of the Wessling International Research

and Education Center in Budapest, Hungary,

entitled “Miniaturized Silica Gel Column

Chromatography Combined with Large

Volume Gas Injection-Gas Chromatography–

Mass Spectrometry as a Chemical

Fingerprinting Method”, a truly hyphenated,

combined chromatography (2D) analytical

approach. A collaborator with Professor

Lindner’s group, Dr. Zs. Gecse of the Institute

of Pharmaceutical Chemistry at the University

of Szeged in Szeged, Hungary, presented

a joint talk on high-performance liquid

chromatographic enantioseparation of cyclic

beta-3-amino acids applying chiral stationary

phases based on cinchona-alkaloids.

This new and novel approach to chiral

recognition described new and improved

chiral stationary phases in HPLC for a wide

variety of natural product analytes. Several

talks included a separation step combined

with on-line MS, such as that by T. Baygildiev

and co-workers of the Department of

Chemistry at the Lomonosov Moscow State

University in Moscow, Russia, which dealt

with simultaneous hydrophilic interaction

liquid chromatography (HILIC) tandem mass

spectrometry methylphosphonic and alkyl

methylphosphonic acids determination

after derivatization with p-bromophenacyl

bromide. This talk dealt with the advantages

of interfacing HILIC-type separations on-line

with MS in terms of improved sensitivity,

lowered detection limits, and improved

identifi cations of trace level impurities,

if present. Finally, several talks centred

around the recent revival of supercritical

fl uid chromatography (SFC) — termed, at

times, as ultraperformance convergence

chromatography (UPC2). This talk was

titled: Chiral Recognition of Dapoxetine

Enantiomers Using Ultraperformance

Convergence Chromatography (UPC2),

and was presented by András Darcsi of

the Department of Pharmacognosy at

Semmelweis University in Budapest,

Hungary, and co-workers. The above is

but a sampling of the total programme

but indicates the breadth and thoroughness

of the meeting, which covered many areas

in analytical chemistry, with specifi c

applications of widespread interest and

importance.

The emphasis throughout was on

UHPLC, HPLC, and high effi ciency and

high throughput separations involving

the same. By and large, the majority of

oral presentations dealt with some form

of LC, involving newer column types

(core–shell, monolithic, charged surface

hybrid, and others), multidimensional

techniques (2D LC, 3D LC), and a few

featuring miniaturized separations (capillary,

nano, micro, and so forth). Many of these

talks dealt with possible applications

for low MW pharmaceuticals and/or

biopharmaceuticals, especially those

from the University of Geneva. One such

example was given by a current graduate

student of Jean-Luc Veuthey and Davy

Guillarme from the University of Geneva in

Switzerland; Aurélie Periat spoke about the

performance of hydrophilic interaction liquid

chromatography–mass spectrometry

(HILIC–MS) versus reversed-phase LC–MS for

the analysis of pharmaceutical compounds.

Balaton Symposium Review

24



There was a great deal of emphasis on

HILIC approaches, and how this might be

interfaced with a second dimension, usually

reversed-phase LC. There were a few papers

that discussed preparative or process-scale

chromatography, and several that dealt with

SFC. Very few papers dealt with regulatory

issues or quality-by-design (QbD). There

were also very few orals dealing with

‘omics topics of any type, though, as above,

many dealt with both low and high MW

pharmaceutical analysis.

One disappointing aspect of this

conference was the lack of oral or poster

presentations dealing with any form of

MS. Given that MS has become a premier

separations approach, whether used alone

or hyphenated with an initial separation

step, and is now widely employed in most

industrial and academic laboratories, one

would have expected more MS papers at

this Balaton Symposium. Perhaps next time?

An interesting question came up at this

conference related to the defi nition of

supercritical fl uid chromatography (SFC).

A number of papers discussed the use of

CO2 as a mobile-phase additive, sometimes

in the form of a supercritical fl uid but

not always. In particular, there seems to

be some confusion about whether the

analytical instrumentation actually performs

SFC. The use of UHPLC instrumentation

to perform true SFC is possible only if the

amount of the organic modifi er is about

2–3% or less, with the pressure needed to

maintain a supercritical fl uid as the mobile

phase. However, more and more papers are

appearing in the literature, and at meetings

such as this, that do not really differentiate

between SFC and UHPLC using CO2 as

a modifi er. It is possible that with higher

and higher percentages of an organic

solvent, one is no longer dealing with a

true supercritical fl uid at all but rather, a

CO2-modifi ed UHPLC mobile phase of high

organic content that is performing HPLC.

In summary, this was a very up-to-date

scientifi c conference with quite high

standards, mostly aimed at serving the

European separations community, which

it has done well for 20 years (every other

year). It was a thoroughly enjoyable social,

academic, intellectual, and practical

experience, with a signifi cant amount of

new and important topics presented and

discussed. There were lively discussions

between the speaker and audience after

each oral talk, as well as at the posters,

which were usually presented by graduate

students or postdocs. There was a

successful mix of academics, industrial-, and

government-separations oriented scientists

present, which led to interesting talks and

discussions throughout the meeting.

Having attended innumerable

national, local, and international

separations-oriented meetings over

too many decades (four or five in 2015

alone), I will attest to this event being

one of the best, most stimulating, open,

and technically sophisticated meetings

in my memory. There was an intellectual

atmosphere obvious at all times, and

everyone came away with more knowledge

than they had when they arrived. I am

indebted to the organizers for giving me

the opportunity to attend my first ever

Balaton symposium in 2015. I look forward

to attending the next Balaton Symposium

in 2017. More information about the

specific programme, vendors, and other

registration information can be found at

the website: www.balaton.mett.hu.

Further information is available, via the July

2015 issue of LCGC Europe (volume 28,

issue 7).

Ira S. Krull is a Professor Emeritus with the

Department of Chemistry and Chemical

Biology at Northeastern Univeristy in

Boston, Massachusetts, USA, and is a

member of LCGC’s editorial board.

E-mail: [email protected]

Balaton Symposium Review

25


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Putting The Pieces Together - PharmTechfiles.pharmtech.com/.../TheColumn_January122016EUsm.pdf2019/02/08 · than the number of peptides in our digest. If we consider a digest with