cover story featuresfiles.pharmtech.com/alfresco_images/pharma/2019/02/11/... · 2019-07-22 · 2...

The Road to RAFA 2015Exploring the latest advances

in food analysis

2 7th International Symposium on Recent Advances in Food Analysis (RAFA 2015) The 7th International Symposium on Recent Advances in Food Analysis (RAFA 2015) will take place at the Clarion Congress Hotel, Prague, Czech Republic, on 3–6 November 2015. This preview reveals what you can expect at the symposium.

Cover Story

Features

15 Authentication and Routine Screening of Ginsenoside Isomers in Functional Food Products: UHPLC Coupled with Ion Mobility Mass Spectrometry

M. McCullagh, R. Lewis, and D. Douce, Waters Corporation This article describes how ultrahigh-performance liquid chromatography

(UHPLC) can be coupled with ion mobility mass spectrometry (IMS-MS) to prof le phytochemicals contained within ginseng and conf rm quality.

20 The Essentials: Optimizing Sample Introduction for Headspace GC An excerpt from LCGC’s e-learning tutorial on headspace gas chromatography (GC) at CHROMacademy.com

11 Analyzing Persistent and Emerging Contaminants in Food Preventing environmental contaminants from getting in to the food chain

is of paramount importance to us all. Yelena Sapozhnikova, a Research Chemist at the Agricultural Research Service, United States Department of Agriculture (USDA) in Wyndmoor, PA, USA, spoke to The Column about her research into the development and evaluation of analytical methods for persistent and emerging organic chemical contaminants in food samples.

Regulars5 News

The benef ts of blueberries to dental health, characterizing breast cancer cells using GC–MS, and the latest company news and news in brief are featured.

8 Tips & Tricks How to Care for Your GPC/SEC Instrument Daniela Held, PSS Polymer Standards Service GmbH Following some simple rules can give better results in less time. This article explains more.

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

23 Training Courses and Events

25 Staff

5 October 2015 Volume 11 Issue 18

ES675995_LCTC100515_001.pgs 09.24.2015 19:32 ADV blackyellowmagentacyan

7th International Symposium on Recent Advances in Food Analysis (RAFA 2015)The 7th International Symposium on Recent Advances in Food Analysis (RAFA 2015) will take place at the Clarion Congress Hotel, Prague, Czech Republic, on 3–6 November 2015.

The 7th International Symposium on

Recent Advances in Food Analysis (RAFA

2015) will provide an overview of the

current state-of-the-art on analytical and

bioanalytical food quality, safety control

strategies, and introduce the challenges

and novel approaches in this field. The

programme will be tailored to provide

networking opportunities as well as

exploring the latest results from the food

analysis community. Presentations will be

given by leading scientists through keynote

lectures and contributed oral and poster

presentations. The following areas will be

covered:

• Food quality and safety: Allergens;

industrial contaminants; metals and

metalloids; mycotoxins; marine and

plant toxins; packaging and processing

contaminants; pesticide residues; and

veterinary drug residues.

• General food analysis issues:

Authentication and fraud; bioactivity

measurements; flavour and sensory

analysis; foodomics; food forensics;

nanoparticles; novel food and

supplements; organic crops and

foodstuffs; QA/QC; micro- and

nano-food sensors; chemometrics; and

data interpretation.

The conference programme will also be

accompanied by several satellite events

including:

• Workshops on novel analytical

strategies: The 3rd European workshop

on “Ambient mass spectrometry in

food and natural products”; workshop Ph

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RAFA Event Preview2 Douce et al.15Q&A: Sapozhnikova11Tips and Tricks8News5The Essentials20 Staff25CHROMacademy222222 Training & Events232323


The Column www.chromatographyonline.com

on “Infrared and Raman spectroscopy,

and chemometrics for monitoring

of food and feed products, bringing

the lab-to-the-sample”; 1st European

workshop on the “Analysis of

nanoparticles in food, cosmetics, and

consumer products”; workshop on “The

application of micro/nano systems in

food safety control”; and a workshop

on “Smart data sets processing in food

analysis”.

• Interactive seminar: This interactive

seminar will be on the topic of

“Sample-prep, separation techniques,

and mass spectrometric detection in

food quality and safety: step-by-step

strategies for fast development of smart

analytical methods”.

• Food Authorities’ summit, EU and

beyond: An FAO/IAEA workshop: Food

safety — challenges for developing

countries; an United States Department

of Agriculture (USDA) seminar on “Food

safety issues beyond the EU”.

• Reference laboratories colloquium:

A workshop on “Experiences,

achievements, and challenges of EU

Reference Laboratories”.

• EU Framework programme seminar:

Tutorial for newcomers in HORIZON

2020, the EU framework Programme for

Research and Innovation: a discussion

platform mediating networking and

joint planning of projects within the

Societal challenge “Food security,

sustainable agriculture, and forestry,

marine, and maritime and inland water

research and the bioeconomy” (chaired

by an EC representative and supported

by the Czech National Contact Point).

The keynote speakers have been announced

and will include: Paul Brereton (Fera Science

Ltd., York, United Kingdom) on “Food Fraud

— Old Problems New Solution”; Christopher

Elliott (Queen’s University Belfast, Belfast,

UK) on “Elliott Review into the Integrity and

Assurance of Food Supply Networks — Final

Report, A National Food Crime Prevention

Framework”; Carsten Fauhl-Hassek (Federal

Institute for Risk Assessment, Berlin, Germany)

on “Food Authentication: Challenges in Off cial

Control”; Jana Hajslova (University of Chemistry

and Technology, Prague, Czech Republic) on

“Pleasures Offered by Ion-Mobility MS to Food

Chemists”; Thomas Hofmann (Technische

Universität München, München, Germany)

on “Taste from Mother Nature and Culinary

Art — Analytical Decoding by Means of the

SENSOMICS Approach”; Christian Klampf

(Johannes Kepler University Linz, Linz, Austria)

on “Ambient Ionization Mass Spectrometry:

Ten Years after Introducing DART and DESI”;

Jacob van Klaveren (National Institute for

RAFA Event Preview

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Public Health and the Environment (RIVM),

Bilthoven, The Netherlands) on “Exposure

Assessment to Multiple Chemicals and Future

Mixture Testing”; Rudolf Krska (University

of Natural Resources and Life Sciences,

Vienna, IFA-Tulln, Austria) on “How Does

Climate Change Impact on the Occurrence

and the Determination of Natural Toxins”;

Erich Leitner (Graz University of Technology,

Graz, Austria) on “Food Packaging Material

and the Interaction with the Packed Good

and the Analytical Challenges”; Luigi

Mondello (University of Messina, Messina,

Italy) on “Comprehensive Chromatography

(GC×GC, LC×LC) Techniques Coupled

to Mass Spectrometry for the Analysis

of Food Samples”; Michel Nielen (RIKILT

Wageningen UR, Wageningen, Netherlands)

on “Ambient Mass Spectrometry Imaging of

Food Contaminants”; John O’Brien (Nestlé

Research Centre, Lausanne, Switzerland)

on “Challenges and Opportunities in Food

Analysis: Industry Perspective”; Petter Olsen

(Nof ma, Tromsoe, Norway) on “Fighting Food

Fraud — When All You Have is a Hammer,

Everything Looks Like a Nail”; Bert Popping

(Mérieux NutriSciences Corporation, Tassin la

Demi-Lune, France) on “Out with the Old, In

with the New: Novel Approaches in Allergen

Detection Using MALDI-ToF-ToF and Mass

Spectrometry”; Michael Rychlik (Technische

Universität München, München, Germany) on

“Complementary Approaches in Food omics

Towards New Horizons in Food Analysis”;

Michele Suman (Barilla Food Research Labs,

Parma, Italy) on “Summary & Discussion

Platform: Industry Perspectives”.

An exhibition of recently introduced

instrumentation in food analysis and other

valuable equipment will be available during

the symposium. Vendor seminars will also be

organized to introduce recent developments

and scientif c strategies for advanced food

quality and safety control.

Young scientists are encouraged to

present their scientif c work. The prestigious

RAFA Poster Award will be given for the best

poster presentation by a young scientist,

along with other sponsored poster awards.

E-mail: [email protected]: www.rafa2015.eu

RAFA Event Preview

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Ohio State University Joins Waters’ Centers of

Innovation

Waters Corporation (Massachusetts, USA) has announced that

the Campus Chemical Instrument Center headed by Professor

Vicki Wysocki at Ohio State University (Ohio, USA) has joined the

Waters Center of Innovation Program. Caroline Whitacre, Vice

President for Research at Ohio State, said: “We are honoured

to join the Waters Centers of Innovation Program as a partner

and applaud the state-of-the-art instrumentation and technical

support that Waters has provided.”

www.waters.com

Trajan Collaborates with Australian Academia

and Government

Trajan Scientif c and Medical (Trajan) (Melbourne, Australia) has

announced a new strategic collaboration with the University

of Adelaide, supported by the South Australian Government,

to develop a research and development and manufacturing

hub based on a new generation of specialty glass products for

the global science and medical equipment market. Professor

Mike Brooks, Deputy Vice-Chancellor (Research), University of

Adelaide, said: “Trajan’s skills in advanced manufacturing —

including processes and systems, quality control, and logistics —

combined with our research expertise and facilities, will enable

transition of research outputs from the University and its partners

into commercial manufacturing.”

trajanscimed.com

Gas chromatography–mass spectrometry (GC–MS) could be used to aid the characterization of breast cancer cells

according to a new study published in the journal Scientif c Reports.1 The study authors report that the levels of

13 volatile organic compounds (VOCs) in the headspace above breast cancer cell lines varied in vitro and were

indicative of different disease markers including stage of development, receptor expression, and doubling time.1

Breast cancer is a leading cause of death in women worldwide and early detection is essential to effective

treatment. Targeted personalized treatment depends on identifying a number of factors such as the upregulation

of receptors, and requires a number of techniques such as f uorescence in situ hybridization (FISH). An alternative

approach is to use breath analysis using GC–MS — a noninvasive screening method for detecting a range of

diseases including cancer — but marker compounds are usually present at very low levels and can be masked

by other compounds in the breath. Eugenio Martinelli, University of Rome Tor Vergata, told The Column: “This

study had two main purposes. The f rst one is the characterization of VOCs as diagnostic tumour markers. The

second one is the set-up of new technology based on a chemical sensor optimized for detection and analysis of

VOCs associated with tumour cancer cells.”

The study, in addition to a temperature modulated metal oxide gas sensor measurement, performed GC–MS on

samples taken from the headspace of six breast cancer cell lines in vitro to identify 13 VOCs that could be used to

discriminate between cell lines by cell doubling time, transformed condition, estrogen and progesterone receptor

expression, and HER2 overexpression. Martinelli said: “Our results demonstrated that VOCs could give information

regarding the expression of breast tumour markers that have [a] high impact in the clinical management of breast

cancer patients; currently the analysis of these markers is expensive and time consuming.”

The team are going to be working on improving the method and testing it with more breast cancer cell lines and

biological samples from patients. He said: “Moreover we will characterize the metabolic pathways involved in the

production of the breast cancer cell-associated VOCs identif ed by GC–MS. This could give relevant information

either concerning the use of VOCs as tumour markers or regarding new molecular modif cations implicated in

breast cancer progression.” — B.D.

Reference

1. L. Lavra et al., Scientific Reports 5(13246), DOI: 10.1038/srep13246 (2015).

GC–MS of Breast Cancer Cell Lines

5




Extracts of wild blueberries (Vaccinium angustifolium Ait.) contain bioactive molecules

that could be used in the development of new therapeutic treatments for the dental disease

periodontitis, according to research published in the Journal of Agricultural and Food Chemistry.1

Researchers from the Univeristé Laval in Quebec, Canada, found that extracts of wild blueberries inhibited the activity

of the bacterium Fusobacterium nucleatum and also reduced the inflammatory response of the immune system that is

associated with symptoms of the disease.

Periodontitis is a disease caused by inflammation of the gums, in response to bacterial infection that damages the soft

tissue and bone surrounding teeth. It causes shrinking of the gums and loosening of the teeth; if left untreated can result

in tooth loss. The bacterium Fusobacterium nucleatum is associated with the disease and so is a potential target for new

therapeutics. Corresponding author Daniel Grenier from the Univerité Laval told The Column that up to 35% of adults

in North America are affected by periodontitis. He said: “Given emerging data indicating that there is a relationship

between periodontal diseases and systemic health problems such as diabetes, cardiovascular diseases, and preterm birth,

studies on preventive and therapeutic strategies targeting periodontal diseases are highly relevant.”

An extract of wild blueberries (with the sugar removed) was characterized by high performance liquid chromatography–

mass spectrometry (HPLC–MS) to determine phenolic and flavonoid composition. The paper reported that the extract was

composed of 16.6% phenolic acids, 12.9% flavonoids, and 2.7% procyanidins. The same extract was then used in assays

to assess the effect on the growth of the bacterium and the authors found that the extract reduced the ability of the

bacterium to form a biofilm, thus reducing its defence. Commenting on the study findings, Grenier said: “Moreover, the

blueberry extract attenuated the inflammatory response of human macrophages challenged with F. nucleatum, resulting

in a decreased secretion of inflammatory cytokines (IL-1β, IL-6, TNF-α) and tissue destructive enzymes (MMP-8, MMP-9).

Evidence was brought that this property is likely related to the ability of the blueberry polyphenols to block the activation

of the NF-κB signalling pathway that play a key role in inflammatory reactions.”

Work is now ongoing to isolate and characterize bioactive molecules in the extract. Grenier said: “These molecules

could then be used for localized application into diseased periodontal sites, through irrigation or insertion of a

slow-release drug device.” — B.D.

Reference

1. A.B. Lagha, S. Dudonńe, Y. Desjardins, and D. Grenier, Journal of Agricultural and Food Chemistry 63, 6999−7008 (2015).

Blueberry Extracts Dental Health

Markes International Opens New USA Off ce

Markes International (Llantrisant, UK) has announced

that it has opened a second off ce in the USA, near

Sacramento, California. This off ce will provide more

local support to West Coast customers, and following

the opening of Markes’ Cincinnati off ce in 2012, ref ects

the extent to which the company’s business is growing

within the USA, according to the company.

Ken Umbarger, Markes’ VP of Sales and Service for

the Americas, said: “A large part of our business and

potential business is in California, so having a West

Coast presence for sales and service means we can assist

local customers much more eff ciently”. He added: “The

California off ce also provides a closer connection with

our distribution partners, all of whom have major sites in

the San Francisco Bay area”.

www.markes.com

Agilent Collaborates with Weill Cornell Medical

College on ALS Research

Agilent Technologies Inc. (Santa Clara, California, USA)

has agreed to support research by Steven Gross, a faculty

member in the Department of Pharmacology at Weill

Cornell Medical College (New York, New York, USA),

into amyotrophic lateral sclerosis (ALS), also known as

Lou Gehrig’s disease. Agilent will provide the latest mass

spectrometry (MS) technology to support this research,

which aims to achieve an understanding of how the most

common form of this disease develops in the body.

Gross is an internationally recognized expert in the use of

MS-based metabolomics. His expertise is in pharmacology

and cell biology, particularly in relation to the role of nitric

oxide as a signalling molecule. Through the partnership,

Agilent will provide two mass spectrometers for Gross’s

laboratory. www.agilent.com Ph

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News In BriefLCGC TV Highlights

Peaks of the Week


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Determination of Bisphenols in Ready-Made MealsResearchers from the European Commission

in Belgium have developed and validated a

stable-isotope dilution liquid chromatography–

tandem mass spectrometry (LC–MS–MS) method

for the determination of bisphenols in ready

made meals. According to the study published in

the Journal of Chromatography A, the method

detected bisphenol A (BPA) in a number of ready

meals purchased from supermarkets in Belgium.

DOI: 10.1016/j.chroma.2015.08.037

HILIC–MS Analysis of Shellf sh ToxinsScientists from the Swedish Defence Research

Agency in Sweden have published a study in

the Journal of Chromatography A outlining the

determination of shellf sh toxins in a range of

food samples using hydrophilic interaction liquid

chromatography–tandem mass spectrometry

(HILIC–MS–MS). According to the study, the

recoveries in tested foods were 36–111%.

DOI: 10.1016/j.chroma.2015.09.029

Unique Peanut and Tree Nut Peptide DiscoveryA new study published ahead of print in the

journal Food Chemistry describes the discovery

of highly conserved unique peanut and tree nut

peptides using liquid chromatography–tandem

mass spectrometry (LC–MS–MS). According to

the paper, the approach can detect all tree nut

and peanut allergens in one analysis.

DOI: 10.1016/j.foodchem.2015.07.043

The LCGC Blog: The Acid Test — More Useful Calculations for HPLC Eluent Preparation —

Mobile-phase preparation for LC–MS requires careful consideration to ensure the correct pH values and

concentrations are obtained. This blog installment details the difference between using volume percent and

weight percent to make up 0.1% solutions of trif uoroacetic acid and the effect it has on the pH. Read Here>>

Developments in Gas Chromatography Using Ionic Liquid Stationary Phases — Ionic liquids (ILs) have

become recognized in gas chromatography (GC) as stable and highly polar stationary phases with a wide

application range. Having customizable molecular structures, ILs also offer a particular tunability that provides

additional selectivity, and therefore may improve separation for neighbouring analytes. This article presents specif c

properties of IL phase capillary GC columns, including polarity scale and inner surface morphologies of IL columns.

Application of IL phases in achiral and chiral GC, and multidimensional GC, are highlighted. Read Here>>

Slideshow: Seven Common Faux Pas in Modern HPLC — Seven outdated traditional practices that

should not be performed without considering alternative approaches that can improve results, provide

lower operation costs, or give faster run times. Instead of working harder, analytical scientists should work

smarter. Learn more by clicking through the slideshow. Read Here>>

LCGC TV: Advancing Chromatographic MethodsKate Rimmer of the National Institute of Standards and

Technology (NIST), discusses separations science research carried out at NIST — on how the molecular properties of the stationary phase correlate with chromatographic behaviour, the use of 2D LC for the quantitation of polycyclic aromatic hydrocarbons (PAHs), and more. Watch Here>>

LCGC TV: Mary J. Wirth on Slip Flow,

Part 1 — How It WorksIn this video from LCGC TV, Mary J. Wirth of Purdue University explains the phenomenon of slip flow: what it is, how it can improve separations — particularly of proteins and monoclonal antibodies —

and where it may take us in the future. Watch Here>>

August 2015

Volume 28 Number 8

www.chromatographyonline.com

New Directions in GCExtending the role of gas chromatography using

ionic liquid stationary phases

PERSPECTIVES IN

MODERN HPLC

High-throughput characterization in drug discovery

LC TROUBLESHOOTING

LC column overload and

detector overload

COLUMN WATCH

Modern SFC

7



Tips & Tricks GPC/SEC: How to Care for Your GPC/SEC Instrument

Columns are at the heart of a gel permeation/size-exclusion chromatography (GPC/SEC) system. Software is used to evaluate the data, to understand and present the results, and to act as an interface between the analyst and the system. But what about GPC/SEC pumps, injection systems, and detectors? They should run uninterrupted 24/7, without the analyst having to think about them or to deal with them, but these components also need care. Following some simple rules can give better results in less time.

Daniela Held, PSS Polymer Standards Service GmbH, Mainz, Germany.

Modern gel permeation/size-exclusion

chromatography GPC/SEC pumps, injection

systems, and detectors are (in general) very

robust and stable against lots of different

solvents/mobile phases. Some precautions are

required, however, in daily operation, even

when using the best components. Nearly all

of these precautions are associated with the

mobile phase that is applied.

Mobile Phase Selection

All solvents should be of the highest quality

(HPLC-grade). Even if these reagents are more

costly, the difference in purity is marked. It

is very important that the mobile phase is

free of particles and dust because this might

otherwise cause blockages in the system or

the columns.

It is good practice before using a new

solvent to verify that all parts of the GPC/

SEC system are compatible with the solvent

and, when using aqueous systems, can be

used at the pH value applied. This is especially

important if components are used that have

originally been designed for high performance

liquid chromatography (HPLC). Unfortunately,

GPC/SEC analysis often requires “exotic”

organic GPC/SEC mobile phases that might

cause problems with seals or other parts of

the components.

If the application allows, the preferred

choice of solvent should be a non-corrosive

solvent. For example, if an application can be

run in tetrahydrofuran (THF) or in chloroform,

from an instrument point of view the choice

should be THF.

Many GPC/SEC applications require the

addition of salt, acidic, or basic additives.

The concentration of these additives should

always be kept as low as possible. In the

case of dimethylacetamide (DMAc) or

dimethylformamide (DMF), lithium (Li) salts

are often used as additives. Because of the

increased solubility of LiBr compared to LiCl,

bromide is recommended in many application

notes. However, it is more corrosive than LiCl Ph

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and therefore potentially more dangerous for

stainless steel instrumentation.

In aqueous systems, the addition of

an additive preventing algae growth is

recommended. Adding 0.05% NaN3 or

acetonitrile will suppress the growth of

microorganisms that could otherwise block

tubing or columns.

Seal wash options should be installed if

mobile phases with salts are used. The best

seal wash liquid is in most cases the pure

solvent. Seal wash liquids should also be

exchanged regularly and if the seal wash seals

and holders are installed it is important that

they are not running dry.

If very high salt concentrations or extreme

pH values are required it might be a good

investment to use stainless-steel free

instrumentation, which is now commercially

available in many f avours and for nearly all

detection options.

Preventive Maintenance

Preventive maintenance should be performed

regularly to avoid unexpected instrument

downtime, to ensure highest data quality, and

to avoid costly repairs as a result of secondary

damages as a consequence of not replacing

worn parts.

In every GPC/SEC system there are at least

some in-line f lters, pump seals, and injection

rotor seals that require frequent exchange.

In addition, check valves, pistons, tubing,

and lamps should be checked regularly and

replaced if required.

The cycle for replacing of worn parts

depends again on the application. As a rule

of thumb we can say that one preventive

routine maintenance a year is suff cient for

a system with salt-free applications that is

running regularly. If the application is more

demanding and corrosive solutions are used,

a reduced cycle of 6 months (or even less) is

recommended.

Idle Mode, Weekend, and Vacation

Users sometimes have to decide if it is worth

powering down the GPC/SEC system or not.

Reducing the f ow-rate is a good option

that helps to save mobile phase, but still

allows the instrument to be started up again

very quickly.

For a short shutdown period of a few days

or less, it is also possible to run the system in

recycle mode. In this case, the eff uent from

the detector is redirected into the solvent

reservoir. However, running a GPC/SEC

system should only be done if no injections

are performed and the mobile phase is free

from salt and additives (with the exception of

algae prevention additives). Even in the case

of running the instrument in recycle mode,

the mobile phase should be exchanged

regularly.

Tips and Tricks

9




If an instrument is not needed for several

weeks or months it can be powered off

completely.

Special care is required for instrumentation

and columns when mobile-phases with salts

or other additives are used. As long as there

are salts or additives in the system, a low

f ow-rate should always be applied (at least

0.05–0.1 mL/min) to prevent corrosion of the

instrument or the columns.

If the pump is going to be completely turned

off, the salt solution should f rst be replaced

by pure eluent. For this, at least 3–5 column

volumes of pure mobile phase, if not more,

should be used.

If the instrument is not used for a long

time the separation columns can be detached

and stored tightly plugged with their original

plugs in the refrigerator (without freezing

them). In the case of corrosive mobile phases

(chloroform), the instrument can be switched

to a different mobile phase (isopropanol) prior

to shutdown. Restarting the GPC/SEC system

then requires a switch back to the original

mobile phase. For new sample runs the GPC/

SEC mobile phases and buffers should be

prepared freshly on the day required. Starting

an analysis with solutions that are several

days (or even weeks) old and have been

run in recycle mode will most probably end

with low quality data with drifting and wavy

baselines.

If the columns were reinstalled it is good

practice to f rst apply a low f ow-rate (to

ensure that there is no air from storage

trapped) and to f ush the columns with at least

3–5 column volumes before attaching them

to the detectors. It is also worth checking the

calibration with a checkout sample. If in doubt

a new calibration should be performed.

Summary

• Preventive maintenance helps to increase

instrument uptime.

• GPC/SEC instruments always need a low

f ow-rate if mobile phases with additives or

salt are used. They should only be turned off

once removal of all salts and additives using

the pure mobile phase has been performed.

• Veriå cation of solvent and pH compatibility

is required particularly for “exotic” organic

phases. Dedicated instrumentation for

extreme pH values or applications with high

salt content is available.

Daniela Held studied polymer chemistry

in Mainz, Germany, and works in the PSS

software and instrument department. She is

also responsible for education and customer

training.

E-mail: [email protected]

Website: www.pss-polymer.com

Tips and Tricks

10


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Analyzing Persistent and Emerging Contaminants in FoodPreventing environmental contaminants from getting in to the food chain is of paramount importance to us all. Yelena Sapozhnikova, a Research Chemist at the Agricultural Research Service, United States Department of Agriculture (USDA) in Wyndmoor, PA, USA, spoke to The Column about her research into the development and evaluation of analytical methods for persistent and emerging organic chemical contaminants in food samples.

Q. You recently developed a

rapid sample preparation and gas

chromatography tandem mass

spectrometry (GC–MS–MS) method

for the analysis of pesticides and

environmental contaminants in fish.

Can you tell us how you developed

this method?

A: We try to be proactive in identifying

potential hazardous contaminants that are

not under surveillance yet, but that may

potentially cause adverse or chronic health

problems. For example, the European Food

Safety Authority (EFSA) scientific opinion

on emerging and novel brominated flame

retardants (FRs) indicated that because of

the lack of available analytical techniques

for brominated FRs, and, therefore, lack of

information on their occurrence in foods,

a risk characterization was not possible.1

At the same time, some of the brominated

FRs have been shown to be genotoxic and

carcinogenic, while others were identified

as bioaccumulative, requiring monitoring

in the environment and foods. We tried

to fill this gap by developing the method

for the analysis of a wide range of diverse

FRs along with other classes of persistent

organic pollutants (POPs) and pesticides.

Our goal was to develop a new

advantageous method for more than

200 contaminants in fish and seafood,

including a diverse range of pesticides, and

persistent and emerging environmental

contaminants. Environmental contaminants

and pesticides were previously analyzed

11




by separate methods, requiring either a

different sample preparation technique

or an additional chromatographic run.

Integrating these contaminants into

a multi-class, multi-residue method

allows for a faster, less expensive, and

higher-throughput analysis.

We selected pesticides from different

classes: stable organochlorines,

organophosphate insecticides,

nitrogen-containing herbicides, and

pyrethroids. Polychlorinated biphenyl

(PCB) congeners were chosen based on

the World Health Organization (WHO)

list,2 including dioxin-like PCB congeners;

polybrominated diphenyl ether (PBDE)

congeners were selected to represent

the most common congeners used in

consumer products including banned

penta-, and octa- congeners. Polycyclic

aromatic hydrocarbons (PAHs) were

selected based on the US EPA list3 and

included PAHs identified as carcinogenic.

Novel FRs were selected based on the

proposed lists of prioritized FRs for

environmental risk assessment, and

included chlorinated, brominated, and

organophosphate chemicals.1,4–6

The extraction method was based on

“quick, easy, cheap, effective, rugged,

and safe” (QuEChERS) with acetonitrile,

which allows nonpolar and relatively

polar contaminants to be extracted,

while also decreasing the amounts of

co-extractive fat compared to commonly

used non-polar solvents like hexane or

ethyl acetate. A dispersive solid-phase

extraction clean-up (d-SPE) approach

with a zirconium-dioxide-based sorbent

provided ~70% of co-extractive material

removal, and resulted in cleaner

extracts and greater robustness for

the gas chromatography tandem mass

spectrometry (GC–MS–MS) analysis, and

also lower instrument maintenance and

idle time. Low pressure vacuum outlet

GC (LPGC) provided fast separation of

more than 200 analytes and 12 internal

standards in 10 min.7–9 The majority of

contaminants had excellent recoveries,

even at low spiking levels, making

the method applicable for analysis at

environmentally relevant concentrations.

Q. What were the challenges you

faced and how did you overcome

them? What are the advantages of this

approach compared to other methods?

A: The task of identifying potentially

hazardous but not yet monitored

contaminants is a challenge in itself. This

requires intensive research into the newest

publications, different countries’ proposed

regulations, scientific guidance panels,

the contaminant’s chemical properties,

and so on. Obtaining analytical standards

for method development when they are

not commercially available is another

challenge. Creating an efficient and

rugged method covering a large amount

of contaminants from different classes

with satisfactory method performance

was possible by selecting acetonitrile as an

extraction solvent, and zirconium-dioxide

based sorbent for clean-up.

While both the sample preparation

and the analytical run in our method

was rapid, data analysis for more than

200 analytes generated an enormous

amount of data points for each sample,

and data processing and review was a

bottleneck. This is a challenge we have

yet to overcome.

The advantages of our method lies

in its simplicity, speed, low cost, and

high throughput. By using this method,

one analyst can prepare a batch of 12

pre-homogenized samples in 1 h in

a few simple steps. Using disposable

polypropylene tubes for extraction and

d-SPE clean-up, there is no glassware to

clean afterwards — who likes washing and

solvent rinsing glassware?

The instrumental analysis using LPGC

takes only 10 min to run one sample

for more than 200 analytes, plus 2 min

for cooling and re-equilibrating, which

translates to 40 samples for each 8 h shift,

or 120 samples for a 24 h cycle — this is

a very rather high throughput! Remember

that typical laboratories use conventional

GC with 30–40 min runs.

In comparison with traditional methods

for pesticides and POP analysis based

on pressurized fluid extraction (PLE),

gel permeation chromatography (GPC),

solid-phase extraction (SPE) clean-up,

and conventional GC, our method is less

expensive, faster in terms of both sample

preparation and GC analysis time, and

produces less hazardous organic solvent

waste, which reduces the environmental

impact.

Q. In another study, you evaluated

different variables affecting

extractability of incurred contaminants

in fish samples. Could you talk a little

about this? What were your results?

A: When new analytical methods are

developed and validated for contaminants

in food or environmental matrices, samples

are spiked (fortified) in the laboratory,

and method performance is accessed

based on the spiked samples. However,

analytes are more easily extracted from

laboratory-spiked samples than from

incurred samples, in which analytes are

Q&A: Sapozhnikova

12




incorporated into the matrix and have

stronger analyte-matrix interactions

to overcome. Therefore extraction

efficiency of spiked samples can be

different from extraction efficiency of

incurred samples. Accurate results for

real samples depends on all aspects of

the analytical process, including sample

processing, but unfortunately, this part is

often ignored during analytical method

development and validation. Even after

method implementation, quality control

samples used to check ongoing method

performance are typically spiked samples,

not incurred. Standard reference materials

(SRMs) with certified contaminant

concentrations are an excellent means to

determine true extractability, but most of

the time SRMs are not available for many

contaminants and sample types.

The goal of our study was to investigate

variables impacting QuEChERS-based

extraction yields of incurred pesticides and

environmental contaminants in fish with

different lipid content. The variables we

assessed included sample size, sample/

solvent ratio, extraction times, and

extraction devices.10

Our results showed that 2 g test

portions (rather than 10–15 g used

in typical QuEChERS extraction) were

adequate for the analysis of the incurred

contaminants. Smaller subsample size

often translates into faster, easier, and

less wasteful methods, as long as the

test portion meaningfully represents the

original sample. Reduced sample size

and smaller amounts of organic solvents

needed for extraction produce less organic

solvent waste, leading to greener, more

environmentally friendly methods. In terms

of other variables, our results showed that

1 min extraction with the pulsed-vortexing

shaker was sufficient for extraction of

the 35 incurred contaminants detected

in the fish.

Q. In your view, what are the main

challenges associated with the

analysis of contaminants in food

samples?

A: In my opinion, we should pay

more attention to emerging, often

yet unrecognized contaminants. Many

potential contaminants may be missed

during regular targeted monitoring

regardless of their levels of contamination

and toxicity.

For decades, pesticides and persistent

organic pollutants (POPs) have been

monitored in the domestic and

imported food supply to ensure safety

of consumed food. However, other

previously unrecognized contaminants

have been emerging and have become

a greater regulatory concern in food

safety programmes as a result of their

persistence in the environment, and

ability to bioaccumulate in tissues and

biomagnify in the food chain, as well as

potential adverse effects on human health

and the ecosystem. It is important that

these previously unrecognized emerging

contaminants are included among

traditionally monitored chemicals in foods

to provide risk assessment data for the

better protection of human health and the

environment.

I mentioned some challenges before,

such as identifying potentially hazardous

contaminants, and obtaining analytical

standards for method development

when the standards are not commercially

available.

Trying to cover a wide range of

contaminants with different properties

in one uncomplicated high throughput

method is not an easy task; finding the

golden mean with all contaminants

achieving satisfactory method performance

can also be a daunting one.

Food composition complexity is

another factor that complicates analysis.

Co-extractive materials from food samples

(for example, lipids, fats, sugars, pigments,

etc.) complicate the analysis, resulting

Q&A: Sapozhnikova

13


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Traditional mass spectrometers are often perceived

as being too costly and complicated for routine use in

QC labs, but this is no longer the case. Compact mass

detectors that are easy to use and which integrate into

existing LC workfows are now available and provide

unique benefts for the detection of weak UV absorbers

and for resolving analytes from matrix interferences in

herbal and dietary supplements.

Key Learning Objectives

n Understand how mass detection is complementary to UV detection for the analysis of herbal and dietary supplements

n Understand that mass detection provides enhanced selectivity for poor UV absorbing compounds and how this is benefcial in complex product matrices

n Understand how chromatographers can make use of mass data to better understand their samples

Who Should Attend

n Quality Control Lab Managers

n Method Development Chemists

n Quality Control Chemists

Sponsored by Presented by

Mass Detection for the Masses How Compact Mass Detectors Are Improving the Analysis ofHerbal and Dietary Supplements

LIVE WEBCAST: Wed., Oct. 21, 2015 1pm EDT/ 12pm CDT/ 10am PDT

For questions, contact Kristen Moore at [email protected]

Presenters:

James Traub

Senior Business Development ManagerNatural Products Moderator:

Alasdair Matheson

Editor in ChiefLCGC Europe



Hydrocarbons (PAHs). http://www.epa.gov/osw/

hazard/wastemin/priority.htm

4. California Environmental Contaminant

Biomonitoring Program (CECBP) Scientif c

Guidance Panel (SGP). Materials for the

December 4-5, 2008 Meeting. Brominated

and chlorinated organic chemical compounds

used as f ame retardants. http://oehha.ca.gov/

multimedia/biomon/pdf/120408f amedoc.pdf

5. P.R. Fisk, A.E. Girling, R.J. Wildey. Prioritisation

of f ame retardants for environmental

risk assessment. 2003 https://www.gov.

uk/government/uploads/system/uploads/

attachment_data/f l... (accessed 1 July 2012)

6. EFSA Panel on Contaminants in the Food

Chain (CONTAM). Scientif c Opinion on

Polybrominated Biphenyls (PBBs) in Food. 8 (10)

(2010) 1789. http://www.efsa.europa.eu/en/

scdocs/doc/1789.pdf

7. Y. Sapozhnikova, Journal of Agricultural and

Food Chemistry 62, 3684–3689 (2014).

8. Y. Sapozhnikova, LCGC North America 32,

878–886 (2014).

9. Y. Sapozhnikova and S.J. Lehotay, Analytica

Chimica Acta 758, 80–92 (2013).

10. Y. Sapozhnikova and S.J. Lehotay, Journal of

Agricultural and Food Chemistry 63, 5163–5168

(2015).

Another project we are undertaking

focuses on food packaging (FP)

contaminants — chemicals migrating from

FP materials into packaged foods. While

these chemicals are often uncharacterized,

they can potentially be hazardous,

leading to unintentional exposure of the

consumer.

All of these projects have one common

denominator — to develop effective

technologies to enhance food safety

control and protect public human health.

Disclaimer: The views and opinions

expressed in this interview are those

of the author and do not necessarily

reflect the views of USDA or U.S.

government.

References

1. European Food Safety Authority “Scientif c

Opinion on Emerging and Novel Brominated

Flame Retardants (BFRs) in Food” (2012).

2. M. Van den Berg, L.S. Birnbaum, M. Denison,

M. De Vito, W. Farland, M. Feeley, H. Fiedler,

H. Hakansson, A. Hanberg, L. Haws, M. Rose,

S. Safe, D. Schrenk, C. Tohyama, A. Tritscher,

J. Tuomisto, M. Tysklind, N. Walker, and R.E.

Peterson, Toxicological Sciences 93, 223–241

(2006).

3. United States Environmental Protection Agency.

Off ce of Solid Waste, Polycyclic Aromatic

E-mail: [email protected]: http://www.ars.usda.gov/pandp/people/people.htm?personid=47132

in retention time shifts, matrix effects,

and inaccurate results. We need to find

better ways to produce cleaner extracts

and account for matrix effects by using

isotopically labelled internal standards,

matrix-matched calibration curves, and

analyte protectants in GC, all while

keeping the analytical methods relatively

simple, fast, and cost-efficient. There are

plenty of challenges in food analysis to

overcome, there is never a dull moment,

and that is what makes it interesting

and fun.

Q. What are you currently working on?

A: We are currently modifying and

validating the method we developed for

fish and seafood for cattle, swine, and

poultry. Emerging contaminants like FRs

are not routinely monitored in meats,

but they are large volume production

chemicals with lipophilic properties,

which have been detected in wastewater

and sludge, marine mammals, and have

also been reported to bioaccumulate

in tissues.

We are also evaluating a fast

high-throughput flow injection analysis

technique for rapid screening of

contaminants in foods. This approach

provides ultra-fast (1–2 min) screening for

multiple contaminants.

Yelena Sapozhnikova

is a Research Chemist

at the Agricultural

Research Service, United

States Department of

Agriculture (USDA) in

Wyndmoor, PA, USA.

Dr. Sapozhnikova’s research focuses

on the development and evaluation of

new, advantageous analytical methods

for persistent and emerging organic

chemical contaminants in food and

environmental samples. Her research

involves improving all aspects of sample

processing, preparation, clean-up,

and chromatographic separation with

mass spectrometry detection to make

the analysis more efficient, fast, and

cost-effective. Dr. Sapozhnikova has

developed novel methods for analysis

of pesticides, diverse environmental

contaminants, environmental estrogens,

flame retardants, synthetic musk

fragrances, pharmaceutical and personal

care products, and other emerging

contaminants to improve food and

environmental safety and reduce health

risk factors.

Q&A: Sapozhnikova

14



Authentication and Routine Screening of Ginsenoside Isomers in Functional Food Products: UHPLC Coupled with Ion Mobility Mass Spectrometry

Ginseng has been used worldwide for thousands of years. Thought to

possess therapeutic effects, it has been marketed as a natural product for

the treatment of disease. This article describes how ultrahigh-performance

liquid chromatography (UHPLC) can be coupled with ion mobility mass

spectrometry (IMS-MS) to prof le phytochemicals contained within ginseng

and conf rm quality.

M. McCullagh, R. Lewis, and D. Douce, Waters Corporation, Wilmslow, UK.

The popularity of nutraceutical and

functional food products — found in foods,

roots, and herbs — continues to increase

globally bringing about the introduction

of new legislation for analyzing active

compounds in natural products. Such

legislation was brought into effect on

30 April 2011 and resulted in the ban of

hundreds of traditional herbal remedies in

Europe under the Directive 2004/24/EC.1

Regulations have recently been restricted

further, allowing only well-established and

quality-controlled medicines to be sold, as

well as products assessed by the Medicine

and Healthcare Products Regulatory Agency

(MHRA). Manufacturers now have to prove

that their products have been made to strict

standards and contain a consistent and

clearly marked dose.

The roots of ginseng plants have been

used for medicinal purposes for thousands

of years and there are a number of different

species that are thought to possess different

therapeutic properties — including CNS

stimulant activity, hypoglycemic properties,

and sedative effects.2 Korean ginseng is one

of the most widely used herbs in the world.

Its scientif c name is Panax ginseng, which

is the species from which Chinese, Korean,

red, and white ginseng are produced. Ph

oto

Cre

dit

: To

ng

Ro

Im

ag

es/

Ge

tty I

ma

ge

s

15




Chinese and Korean ginseng are the same

plant cultivated in different regions, and

have slightly different properties according

to Chinese medicine. White ginseng is

simply the dried or powdered root of Korean

ginseng, while red ginseng is the same root

that is steamed and dried in heat or sunlight.

Red ginseng is said to be slightly stronger

and more stimulating in the body than

white, according to Chinese herbalism.

Ginsenosides are part of a diverse

group of steroidal saponins with a four

ring structure similar to steroids that can

be classif ed into two main groups: the

panaxadiol (Rb1 group) that includes Rb1,

Rb2, Rc, Rd, Rg3, Rh2, and Rh3; and the

panaxatriol (Rg1 group) that includes Rg1,

Re, Rf, Rg2, and Rh1. American ginseng

is richer in the Rb1 group of ginsenosides

whereas Korean ginseng is richer in the

Rg1 group.3 The phytochemical prof le of

the two species can also be affected by the

time of harvest, storage conditions, and

production processes. Manufacturers are

therefore required to determine the quality

and potency of ginseng products in line with

regulations, to ensure the phytochemical

content is as labelled.

This article demonstrates how

ultrahigh-performance liquid

chromatography (UHPLC) coupled with

ion-mobility mass spectrometry (IMS-MS)

can be an ideal method for prof ling

complex mixtures from natural products.

IMS-MS is a rapid orthogonal gas phase

separation technique that allows another

dimension of separation to be obtained

within an LC timeframe and differentiates

compounds based on size, shape, and

charge. A screening assay to explore the use

of UHPLC separations with ion mobility mass

spectrometry for the characterization of the

distribution and content of mono-, di-, and

tetra-glycosides in raw material or processed

products is presented and illustrated. In this

case, the aim is to illustrate how the quality

and potency of ginseng products can be

determined.

Screening and Conf rming Isomer

Markers with Collision Cross-Section

Values

A collision cross-section (CCS) value is a

robust, measurable physicochemical property

Douce et al.

16


80000

3.47

5.43

6.457.13

7.30

7.73

7.88

8.77

8.99

9.30

8.32

5.95

3.21

3.66

2.12

2.091.25

0.51

1.09

1.71

2.283.07 4.30

4.43

4.82

3.70 4.99

9.83

10.39

10.51

11.24 11.93

13.06

13.24

13.80

12.58

161514131211109876543210

12.22

14.56

14.6715.27

15.75

16.47

16.17

–15.79

0.39

60000

Retention time (min)

Inte

nsi

ty (

Co

un

ts)

40000

20000

Figure 1: UHPLC–IMS-MS electrospray negative mode conventional base peak ion chromatogram obtained for analysis of undiluted Korean ginseng tea extract.

Retention time

Mobility

reso

lutio

n

11

10

9

8

7

6

5

4

3

22

4

6

8

10

12

1416

Figure 2: UHPLC–IMS-MS electrospray negative mode plot of drift time (ion mobility resolution) versus retention time for Korean ginseng tea extract.



of an ion that is an important distinguishing

characteristic related to its chemical structure

and three-dimensional conformation. This

article presents TWCCSN2 values (derived

from ion mobility drift times) as a new

identif cation parameter that can distinguish

ginsenoside isomers as well as unknowns.4

Method: Non-targeted UHPLC–IMS-MS

was used to generate travelling wave

collision cross-sections using a nitrogen

buffer gas (TWCCSN2), accurate mass

precursor/fragment ions, and retention

times to profile ginsenoside standards

Rb1, (Rb2, Rc), (Rd, Re), (Rf, Rg1), and

Rg2 (100 pg/μL). The CCS measurements

generated were then entered into a

scientific library within the software

UNIFI (Waters), allowing the expected

and determined TWCCSN2 values to be

used to screen and confirm the presence

of ginsenoside isomer markers. Korean

ginseng tea (extract in 20 mL of H2O),

gingko biloba, and red panax (undiluted)

extracts were analyzed. These were

screened against the created ginsenoside TWCCSN2 library in UNIFI to determine

the presence/unequivocal identification

of ginsenoside isomers. A Waters Acquity

UPLC I-Class System and Synapt G2-Si Mass

Spectrometer was used in the analysis.

Results: Figure 1 shows the UHPLC–IMS-MS

electrospray negative mode conventional

base peak ion chromatogram obtained for

the analysis of undiluted Korean ginseng

extract to show the complexity of the

sample prof led. Figure 2 presents the

UHPLC–IMS-MS electrospray negative mode

plot of drift time (ion mobility resolution)

versus retention time for the Korean

ginseng tea extract and illustrates how an

ion mobility separation, orthogonal to a

chromatographic separation, can increase

peak capacity.

The retention time region between

6 min and 10 min shows that there are a

large number of compounds that are now

resolved compared to the same region on

the conventional base peak ion extracted

mass chromatogram of Figure 1. The

true complexity of the sample prof led is

illustrated when ion mobility resolution

and UHPLC chromatographic resolution are

combined.

Resolving Co-Eluting Compounds with Ion

Mobility: Figure 3 shows that the combined

peak capacity of UHPLC and ion mobility

can have other advantages in addition to

serving as an additional identif cation point.

The retention time (7.73 min) and drift time

(9.93 ms), aligned precursor, and product

ion spectra for Rc ginsenoside marker

isomer is shown. The spectra presented

only result from the Rc ginsenoside because

chromatographically co-eluting compounds

are resolved using ion mobility. The ion

mobility spectral cleanup makes it clear

that the unknown isomer at 7.88 min has

the same characteristic fragment ions as

ginsenoside Rc, but it can be differentiated

using ion mobility. It is also worth noting

that these acquisitions are non-targeted;

hence the TWCCSN2 values are generated for

all knowns and unknowns. The characteristic

information acquired and processed for an

unknown isomer is shown in Figure 4. The

candidate component summary, mobility

trace, and precursor/mobility product ion

spectra for the unknown isomer with TWCCSN2 = 358.80Å2 at retention time

7.88 min are illustrated. The TWCCSN2

value for the unknown isomer can be

entered into the scientif c library, along

with the TWCCSN2 values generated for

all of the compounds detected during the

chromatographic run.

Identif cation with Collision Cross-Section

Measurements: CCS measurements can

increase conf dence in identif cation as

demonstrated by the results obtained

from prof ling Korean ginseng over

two consecutive weeks. For the marker

ginsenoside isomer pairs (Rb2, Rc), TWCCSN2

Douce et al.

17


1.5e6

1e6

5e5

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500

1617.38667

1.89e6

1.53e6

1279.43799

1077.58627

89.02348149.04524 621.43550

945.54201

999.46182

1078.59030

1130.49650

1132.50403

1184.417751296.66596

1628.87768

Mass error: -0.3 mDa783.48888

Mass error: -1.1 mDa

1131.50016

1126.59930

1124.59578

1123.59365

1077.58236

1076.54660

1600 1700

0

Inte

nsi

ty (

Co

un

ts)

1.5e6

1e6

5e5

0

Inte

nsi

ty (

Co

un

ts)

Observed mass (m/z)

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700

Observed mass (m/z)

Figure 3: Retention time (7.73 min)/drift time aligned (9.93 ms) precursor and ion mobilityproduct ion spectra for Rc ginsenoside marker isomers, where their characteristic fragmentation pattern and proposed fragmentation pathways are shown.



resolved from coeluting components. In

this case the reason for such a screening

approach is to generate a new analysis to

enable the characterization and content of

mono-, di-, and tetra-glycosides in the raw

material or processed functional food or

nutraceutical products. This assay approach

could add conf dence when assessing

quality, potency, and consistency of a f nal

product, incorporating ingredients such as

gingko biloba, Korean ginseng, and red

panax.

While also gaining conf dence in the

identif cations made from the use of

accurate mass measurement and CCS

measurements, there is also the clear

potential to reduce the amount of

high purity standards required, where

conf rmation relies on retention time and

accurate mass measurement. The use of a

CCS screening approach has the potential

to provide signif cant cost savings across

many application areas. In further studies to

prof le ginsenosides, Rb1, Rb2, Rc, Rd, Re, Rf,

Rg1, and Rg2, the consumption and costs of

high purity standards has been signif cantly

reduced within the laboratory where the

study was performed.5

References

1. DIRECTIVE 2004/24/EC OF THE EUROPEAN

PARLIAMENT AND OF THE COUNCIL of 31

measurements of 361.77 Å2/350.58Å2

have been determined. For Rd, Re, 328.89

Å2/333.11 Å2 were determined. For Rf, Rg1,

304.7 Å2/295.83 Å2 were obtained in week

one. In week two, comparative results were

obtained. When comparing the expected

against the measured TWCCSN2 results

determined (for the eight ginsenosides

prof led in the extracts), the measurement

errors were typically <0.5%. It is therefore

possible to distinguish the marker isomer

pairs of ginsenosides in the extracts of the

specif ed products analyzed with conf dence

using TWCCSN2 measurements.

Summary

In conclusion, the results clearly show the

benef ts of using CCS measurements and

the combined peak capacity of UHPLC with

IMS-MS. Coeluting analytes and isomers

were resolved and identif ed in the three

extracts prof led. In addition it was possible

to acquire the cleaned up mobility specif c

product ion spectra, which are ion mobility

March 2004 http://ec.europa.eu/health/files/

eudralex/vol-1/dir_2004_24/dir_2004_24_

en.pdf

2. T. Ligor, A. Ludwiczuk, T. Wolski, and B.

Buszewski, Anal. Bioanal. Chem. 383(7–8),

1098–105 (2005).

3. A.S. Attele, J.A. Wu, and C.S. Yuan, Biochem.

Pharmacol. 58, 1685–1693 (1999).

4. S.D. Pringle, K. Giles, J.L. Wildgoose, J.P.

Williams, S.E. Slade, K. Thalassinos, R.H.

Bateman, M.T. Bowers, and J.H. Scrivens,

International Journal of Mass Spectrometry

261, 1–12 (2007).

5. M. McCullagh, R. Lewis, and D. Douce

Investigating UPLC Ion Mobility Mass

Spectrometry: A new Approach To

Authentication and Routine Screening

of Ginsenoside Isomers In Functional

Food Products. Waters application note

720005422en

6. The use of Collision Cross Section (CCS)

Measurements in Food and Environmental

Analysis. Waters Technical Note No.

720005374en, April 2015.

Michael McCullagh has worked

for Waters Corporation since 2001,

performing sample analysis in the food and

environmental, pharmaceutical/life science

and validation departments. Using time

of flight technology, he has gained

experience in a number of application

Douce et al.

18


Figure 4: UNIFI Component Summary illustrates the candidate component summary, mobility trace, and precursor/mobility product ion spectra for an unknown isomer TWCCSN2 = 358.80Å2 at retention time 7.88 min.



areas, such as metabolite ID, impurity

profiling, natural product profiling,

authentication profiling, and pesticide/

veterinary residue screening. His

experiences in these application areas have

been used to explore the utility of ion

mobility mass spectrometry.

Rob Lewis has 21 years of experience in

the MS industry, working on quadrupole

and time-of-flight LC–MS systems. For the

last 12 years he has been working in the

MS systems evaluation department, which

is responsible for ensuring the development

of MS instruments meet user performance

requirements. During this time he has

worked on many application solutions

including a natural product application

solution where he gained experience

working with compounds such as the

ginsenosides.

David Douce obtained his Ph.D in 1998

from Sheff eld Hallam University (UK) in

the area of environmental monitoring using

HPLC and GC–MS. After working for an

environmental testing laboratory and a large

CRO he moved to Micromass/Waters in

2000. He initially worked in the applications

laboratory performing sample analysis on

a wide range of application areas before

moving into the evaluation group where

all new mass spectrometric products (both

quadrupole and time of f ight-based) are

tested before they are released. His current

areas of interest include source technologies

and the practical use of these in a diverse

number of application areas.


Website: www.waters.com

Douce et al.

19



The Essentials: Optimizing Sample Introduction for Headspace GCAn excerpt from LCGC’s e-learning tutorial on headspace gas chromatography (GC) at CHROMacademy.com

Static headspace sampling is typically

used for the determination of volatile and

semivolatile analytes in liquids and, more

rarely, solid matrices. Application examples

include the analysis of alcohols in blood,

residual solvents in pharmaceuticals,

flavours and taints in food and beverages,

and fragrances in perfumes and detergents.

Samples are heated and agitated at a set

temperature for a set time, after which an

aliquot of the headspace gas is analyzed by

gas chromatography (GC) to determine the

concentration of the analyte of interest in

the headspace, which can then be related

to the concentration in the original sample.

The vapour pressure of a compound

above a solution is directly proportional

to its mole fraction in that solution

multiplied by an activity coefficient. The

activity coefficient relates to the degree

of intermolecular attraction between

the analyte and the other species within

the sample.

The following equations are most often

used to describe the basis of headspace

determination:

CG = CO/(K + VG/VL) [1]

where CG is the analyte concentration

in the gas phase, CO is the analyte

concentration in the original sample, K

is the partition coefficient (2), VG is the

volume of headspace gas, and VL is the

sample volume.

K = CS/CG [2]

where CS is the analyte concentration in

the sample liquid and CG is the analyte

concentration in the headspace gas.

To determine K it is necessary to calibrate

instrument response by analyzing standards

containing a known amount of analyte.

It is very important that the standard is

matrix matched to the analyte because the Ph

oto

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: Jo

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Pe

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20




matrix components can significantly affect

the activity coefficient of the analyte as

described above.

When determining ethanol in water a K

value of around 500 is not unusual, indicating

that there is around 500 times more ethanol

in the water at equilibrium than in the

headspace, again not unusual given the high

solubility of ethanol in water because of the

comprehensive hydrogen bonding between

the analyte and matrix. When determining

hexane in water, K values of 0.01 are not

unusual, meaning there is 100 times (1/0.01)

more hexane in the headspace. How would

these f gures be affected by changing the

various experimental variables?

Sample Volume

Increasing sample volume will not

significantly affect the headspace

concentration for analytes with high values

of K. For intermediate values of K (~10), the

increase in sample volume is approximately

linear and for analytes with low values of

K an increase in sample volume will give a

large proportional increase in headspace

concentration.

Low analyte headspace concentrations

caused by good analyte solubility in the

matrix cannot be significantly improved

by increasing sample volume. Use around

10 mL of sample (if available) in a 20-mL

headspace vial. This also makes the phase

ratio (β = VG/VL) equal to 1 and simplifies

calculations.

Temperature

Samples with a high value of K will be

significantly affected by temperature,

and increasing temperature is a good

way to improve headspace concentration.

However, to obtain good precision,

one needs to carefully and accurately

control the equilibration temperature

and for analytes with K values of 500, a

temperature accuracy of ±0.1 °C is required

to obtain a precision of 5%! With analytes

where K is low, increasing the temperature

has a lesser effect and can even cause

a reduction in analyte headspace

concentration.

One special note here is that as

temperature is increased when using

aqueous samples, the overall headspace

pressure can increase markedly and the

sudden release of pressure on inserting the

sampling needle may case loss of analyte

or a significant dilution effect.

Equilibration Time

Headspace equilibration time will

depend on analyte vapour pressure,

concentration in the sample, phase ratio,

and temperature or agitation. Do not be

tempted to draw a correlation between

equilibration time and partition coefficient

value. Each analyte or sample combination

and sample to headspace ratio will need

to be investigated to determine the time

required to reach equilibrium for each

analyte.

Salting Out

The partition coefficient of polar analytes

in polar matrices can be significantly

reduced by adding a very high

concentration of salt (potassium chloride is

typical) to the sample matrix.

Instrument Variables

When using autosampler devices, use

the smallest volume sample loop that

gives the required signal-to-noise ratio.

The sample, loop, transfer line, and inlet

temperatures should be offset by at least

+20 °C to avoid sample condensation. If

the signal-to-noise ratio allows, applying

a small split flow of 10:1 often improves

analyte peak shape and makes peak area

measurement more reproducible.

Get the full tutorial at www.CHROMacademy.com/Essentials (free until 20 October).

Variable Headspace Analyte Concentration

Increase sample volume — low K

Increase sample volume — mid K

Increase sample volume — high K

Increase temperature — low K

Increase temperature — mid K

Increase temperature — high K

Figure 1: Headspace analyte concentration in a sample vial as a function of sample volume and temperature.

The Essentials

21



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