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News from GERSTEL GmbH & Co. KG · Eberhard-Gerstel-Platz 1 · 45473 Mülheim an der Ruhr · Germany · Phone + 49 (0) 2 08 - 7 65 03-0 · gerstel@gerstel.com
No. 14Focus on intelligently
automated sample preparation
Material analysis
Amber, glue
and polymers

Toxicology
Pain management
drugs in urine

Flavor analysis
Rotundone
in wineISSN 1619 - 0076

www.gerstel.com

GDear reader,

Holger Gerstel

Ralf Bremer

Yours sincerely, GERSTEL management.

Content:
Flavor analysis
Searching for the pepper in Shiraz
Rotundone is a key flavor compound of Australia’s Shirazwines. Wine researchers report a simple yet powerful
analysis method and are now looking to determine how
it is formed in order to optimize growth conditions. ............... 3

Focus: Material analysis
Amber authentication, a closer look
Is it real or is it a fake? When it comes to amber this is
not easily determined. Pyrolysis and thermochemolysis
GC/MS with a twist help find the answer..................................... 6

GERSTEL

B

A

No. 14

GmbH &

L

Co. KGA

N

A

L· EberhardGerstel-Platz 1 · 45473

YT

MülheimIC

an der Ruhr

A

L

· GermanyS· PhoneO+ 49 (0)2 08 - 7 65

Foc us on
int ell ige
samplepreparatio nt aut om ati onnMaterial
analysisAmber, glue
and polym
erClinical Chem
Pain manag istryement
drugs in urine

L

U

T

Polymers: Making it stick
I

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- 0076O

erstel.com

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News from

03-0 · gerstel@gerstel.com

ISSN 1619

G

www.g

Eberhard G. Gerstel

In close cooperation with our users in industry, research
institutes and government agencies, GERSTEL has developed numerous innovations in the fields of GC/MS- or
LC/MS related instrumentation and their application to realworld analytical work, in a word: Practical solutions.
Many of these innovations were the result of a request
for a solution to a specific problem, including requests toautomate sample preparation. Solutions for specific tasks
have frequently proven useful in other application areas,
sometimes unexpectedly, leading to entirely new standard
products and solutions.
In recent decades, countless projects and cooperations between GERSTEL experts and laboratory analysts
have been successfully completed. These cooperations
generally proved fruitful for both sides by combining ourtechnical and application know-how with the in-depth
knowledge of the customer as to the products that need to
be analyzed. At GERSTEL we strive to regularly surprise theworld with innovations that offer significant added value
and benefits to the GC/MS and LC/MS analyst.
When we develop new solutions today, we draw on
a variety of resources that have grown and developedside by side under one roof leading to efficient communication and exchange. The R&D Department includes
Technical Construction, Prototyping, as well as Electronics, Mechanical, and Software Development. Analytical
Services, Customer Support, and Customer Training are
equally important departments. The close cooperation
within our organization is evident in the many articles oncustomer applications published in the GERSTEL Solutions
Worldwide magazine and other magazines and journals
over the past several years.
The GERSTEL product portfolio is mainly based on
three pillars. One of them is Thermal Desorption (TD)used for solvent-free extraction and concentration of VOC
and SVOC analytes from a range of matrices. If you can
combine TD with an automated pyrolysis option, a whole
new world of possibilities opens up, such as analysis ofpolymer material structures. You will find more on this key
topic on the pages 6 to 14.
A further pillar and regular main topic in almost all
our publications is automated sample preparation. This
area offers the greatest optimization potential in almost
all laboratories. With the MultiPurpose Sampler (MPS), we
offer our customers a highly productive, efficient and flexible autosampler. The MPS is easily
expanded and adapted to many different tasks using the available modules and options. In combination
with GC-MS and LC-MS/MS systems
we arrive at the third pillar: Customized solutions that match the customer requirements perfectly – wecall them Sample Prep Solutions. If
you are considering automating yoursample preparation or have alreadymade plans to do so, contact theexperts at GERSTEL – the conversation will most probably prove usefulfor you.
With this, we would like to wish
you much pleasure and enjoyment reading the latest
GERSTEL Solutions worldwide Magazine No. 14, which
will again provide glimpses of what GERSTEL technology and GERSTEL solutions offer the modern laboratory.
Enjoy the magazine!

GERSTEL Solutions worldwide 14

To determine the compositionand structure of adhesive tapes,
you need know-how and the right
instrumentation. The authors of thisstory have both...........................................10

Phthalates: Easy extraction ofplasticizers from toys

Flavor analysis
Rotundonin wineAccording to US- and EU regulation,
children’s toys must be free of certain
plasticizers. The analysis method used specifies GC/MS
determination of critical phthalates, using cumbersome andlabor intensive manual sample preparation processes. It canbe done so much easier as reported in our story
on page ...................................................................................................12

Pharmaceutical analysis„Corked“ pills – no thanks!
To track down off-odors in pharmaceutical products andtheir packaging, researchers have developed a highly
sensitive GC-MS/MS method based on
Stir Bar Sorptive Extraction (SBSE) ................................................15

Flavor and Fragrance analysis
Wondrous Vanilla
Vanilla flavor isn’t determined by vanillin alone, but rather
by a large number of compounds, some of which are
newly discovered and presented here. To identify flavor
compounds and to determine product quality and originof vanilla pods and extracts, Headspace and Thermal
Desorption are key techniques.......................................................19

Toxicology
Pain management (PM) drugs in urine –can the analysis be fully automated?
Analyzing urine for pain management drugs and their
metabolites can be hard work – but it doesn’t have to be.
If hydrolysis and clean-up are automated and dispersive
SPE is chosen instead of standard SPE, the process is
accelerated ............................................................................................ 22

News ..........................................................................26
Preview ......................................................................28
Imprint .......................................................................28

Vineyard in One Tree Hill near
Adelaide, Southern Australia.

Searching for the pepper in Shiraz
Rotundone is a key flavor compound with a distinct black pepper note. Rotundone occurs naturally in pepper, butalso in wines such as Australian Shiraz and others from around the world, for example in the Austrian Green Veltliner.
Australian wine scientists are searching for clues as to how the peppery compound is formed in the hope that thisknowledge would enable them to optimize both growth conditions for the Shiraz grape and the ensuing wine-making
process. Finding answers requires an efficient analysis method. Researchers turned to Membrane Assisted Solvent
Extraction (MASE) combined with heart-cut GC/MS for more efficient determination of rotundone in wine and grapes.

W

hen it comes to wine, tasting beats reading reviews. But
a little reading could offer a good starting point when
you set out to differentiate noble drops from decent everyday wines or even inferior ones. When mingling with real
or would-be connoisseurs, the following vocabulary samples
could help you fit in: A wine scientist is called an oenologist;
wine expert in a classy restaurant: Sommelier; and while
merry wine drinkers enjoy wine tasting, highbrow
tasting is referred to as a degustation. But evenexperts can find themselves at wit’s end when sensory impressions do not solve the puzzles
they face and hard facts are required toensure best possible product quality
and product acceptance.
When faced with a wine with
its hundreds of volatile compounds that influence the flavor, the technique of choice forthe analyst is gas chromatogra-

GERSTEL Solutions Worldwide – No. 14

phy with mass selective detection (GC/MS). This is the tool
used by Tracey E. Siebert and Sheridan R. Barter from theAustralian Wine Research Institute in Glen Osmond (Adelaide), Australia. The scientists conduct basic research; they
are not trying to outperform sommeliers.
Siebert‘s and Barter‘s goal is to find out how wine flavor compounds are formed in order to find ways to optimize growth conditions for best taste and bouquet. The
Australian scientists had set their sights
on rotundone, a bicyclic sesquiterpene
containing a conjugated ketone group.
With its characteristic black pepper note, rotundone is considered a
key flavor compound in Australian
Shiraz wine. Shiraz is the number one grape grown in Australia. It is highly valued due to its
high yield and resistance to cold
weather, and of course due to the3

GC/MS-System comparable to the oneused by Siebert and Barter for automated MASE and 2D-GC/MS Rotundone
determination

Structural formula of (-)-Rotundone

excellent tasting wine it produces. Shiraz vines
were originally cultivated in France (Syrah).
Today the wine is not only popular in Australia, but also widely grown in South Africa,
Latin America, the United States, and Canada.

Wine under the influence
Many factors influence quality and characterof the final wine; it is generally accepted that
controlling the grape maturation process iskey. Knowledge of specific factors that influence, for example, how much rotundone isformed in the vine and how growth conditions could be optimized could help improve
quality, taste, flavor, and ultimately productvalue. Such knowledge might enable wine
producers to provide more uniform year-toyear wine quality and taste. But Sieber andBarter are convinced that such insights canonly be gained based on access to efficientand sensitive analysis methods.
The scientists presented their work in
May 2013 in Palm Springs, California, USA
during the 37th International Symposium
for Capillary Chromatography (ISCC) [1].
Previously used analysis methods were labor
intensive and time consuming. Large sample volumes had to be handled and extensive
sample preparation was required for matrix
elimination and analyte concentration since
Rotundone is present only at very low concentration levels.
According to literature: “The flavor
threshold of Rotundone is at 16 ng/L in red
wine and 8 ng/L in water. A limit of quantitation (LoQ) of < 8 ng/L would be necessary
for juices, grapes, mash and wine. These arecomplex aqueous samples that contain sugars,
ethanol, anthocyanins or polyphenols.“ [2].

Critical factor for success: The
extraction
The critical factor that helps ensure analytical success or failure is the extraction step,
according to Siebert and Barter.
Searching for a suitable technique, thescientists came across automated Membrane
Assisted Extraction (MASE). MASE wasdeveloped in cooperation between the Environmental Research Institute Leipzig-Halleand GERSTEL.
This type of liquid – liquid extraction isbased on using a semi-permeable membrane
as a phase boundary, which keeps particulatematter and other matrix components out of
the extraction solvent. Further sample cleanup steps such as filtration and centrifugation are no longer needed. MASE extracts –even of heavily matrix laden samples are clean
and can be injected directly to the GC/MS or
LC/MS system for analysis. Additionally,
MASE enables extraction using sample-solvent mixtures that do not normally result inphase separation. As an example, HPLC compatible polar solvents can be used to extract
aqueous samples in which the solvents arenormally miscible. MASE extends the analytical possibilities considerably [3,4]Schematic diagram of Membrane Assisted Solvent
Extraction (MASE): The semi-permeable membrane insert is immersed into the sample (left).
Extraction solvent is added into the MASE insert
and analytes diffuse through the membrane into
the extraction solvent while particulate matter is
kept in the sample (center).
The extract is aspirated by the syringe for introduction to the GC/MS (right).

4

Experimental
The grape juice/extract or wine sample wasadded to a 20 mL autosampler vial, the MASE
membrane bag placed inside the vial, and the
Sample preparation:
Grapes: A 100 g sample of berries was
gently blended and centrifuged
and the juice supernatant collected. The solid residue was further
extracted by adding 60 mL of an
ethanol/water mixture (50:50)
and internal standard (IS) to thesolids and shaking for 24 hours
before centrifuging and collecting
the supernatant. The supernatantand the juice initially collected
were combined and topped up
with water to a volume of 200
mL. Of the final volume, a 15 mL
aliquot was taken and used foranalysis.
Wine:

A 15 mL sample of wine was taken
and used directly for analysis.

vial capped. Sample preparation and introduction were performed automatically by the
GERSTEL MultiPurpose Sampler (MPS).
MASE was performed by adding 750 µL of
hexane into the MASE membrane bag. The
sample was extracted for 60 min at 35 °C. A
100 µL aliquot of the resulting extract was
injected (Large Volume Injection - LVI) into
a GERSTEL Cooled Injection System (CIS
4) inlet mounted in an Agilent GC 7890.

GERSTEL Solutions Worldwide – No. 14

The separation took place on a multidimensional heart-cut GC
system, based on an Agilent Capillary Flow Technology (CFT) Deans’
Switch configured with two separation columns. A CryoTrap System (CTS, GERSTEL) was connected between the first and second
dimension columns for analyte focusing prior to the analytical separation. An FID monitor detector was used on Column 1 and an Agilent 5975C MSD (Agilent Technologies) was used in Selected Ion

Main FID-Chromatogramm and heart cut MSD chromatogram showing aclearly defined rotundone peak. The method developed by Siebert andBarter was used to determine rotundone in Shiraz grapes and in wine
following MASE analyte extraction.

1st Dimension FID chromatogram

Heart cut

SIM chromatogram

e

on

nd

tu
Ro

2nd dimension
separation of
coelutingcompounds

Rotundone in red wine: calibration and repeatability
3.0

2.5

Instrument method parameters
GERSTEL MPS XL, CIS and CTS
LVI:
100 µL at 0.58 µL/sec
He carrier gas flow: 100 mL/min
CIS:
Glass wool packed liner
CIS:
20 °C, solvent vent, 20 psi (0.12 min);splitless, 47.6 psi, 12 °C/sec to 240 °C(2.5 min); split, 12 °C/sec to 275 °C (2min)
Column 1:
VF-35ms (30 m x 0.25 mm x 0.25 µm),
medium polarity phase; He 47.6 psi
Column 2:
VF-200ms (30 m x 0.25 mm x 0.25 µm),
unique selectivity with dipole-dipole
interactions He 37.7 psi
Column 3:
Deactivated fused silica (0.70 m x 0.1
mm)
GC Oven:
80°C (1 min), 5°C/min to 210°C, cool
15°C/min to 130°C (2 min), 10°C/min to
280°C (10 min)Heart-cut:
26.25 to 26.75 min
CTS:-20°C (34 min), 20°C/sec to 300°C (1 min)MSD SIM:m/z 147, 161, 163, 203, 208, 218, 223

Monitoring (SIM) mode for the analytical separation on Column 2. Stable isotope dilution was performed using d5-Rotundon as the internal standard.
According to Siebert and Barter, the described method combining MASE and LVI minimizes sample preparation workload
significantly. Problems with interferences were compensated using
internal standards. By heart-cutting the key part of the chromatogram for separation onto a second column, a cleanly separated and clearly defined rotundone peak is obtained. The scientists reported the analysis method to be accurate, precise, rugged, and sensitive leading to limits of quantitation in the low ppt
range and high sample throughput. The statistical data speak volumes: “In the Shiraz grapes (0 to 2000 ng/L) as well as in wine
(0 to 500 ng/L), the limits of quantitation (LOQs) were 5 ng/L.
The linear correlation (R2) was >0,999 and the reproducibility
expressed as standard deviation was below 3 % (n=6) both for low
and high concentration values – a very good result”, the wine scientists reported. They went on to say that “the described method
will allow more detailed research on the formation of rotundone
in the grape. This includes the maturation processes involved aswell as whether yeast plays a role by influencing the extraction
process. Such knowledge will make it more likely that we will beable to describe how rotundone is synthesized in the grape andultimately control the rotundone concentration in the wine.” Further investigations are planned and a scientific publication of theresearch results is scheduled to appear in the near future.

Calibration 0 to 500 ng/L

Low reps 20 ng/L

Literature

Response Ratio (U/L)

High reps 200 ng/L
2.0

1.5

y = 2.7898x + 0.0323
R² = 0.9996
1.0

0.5

0.0
0.0

0.1

0.2

0.3

0.4

0.5

0.6

Concentration Ratio (U/L)

GERSTEL Solutions Worldwide – No. 14

0.7

0.8

0.9

1.0

[1] Siebert, T. E. and Barter, S. R.: Determination of the potent flavour
compound rotundone in grapes and wine using MDGC-MS andmembrane assisted solvent extraction, Posterpräsentation, 37th
International Symposium for Capillary Chromatography (2013)
Palm Springs, USA.
[2] Wood, C.; Siebert, T. E.; Parker, M. et al.: From Wine to Pepper:
Rotundone, an Obscure Sesquiterpene, Is a Potent Spicy Aroma
Compound, J. Agric. Food Chem. 56 (2008) 3738-3744[3] Schellin, M. and Popp, P.: Membrane-assisted solvent extraction ofpolychlorinated biphenyls in river water and other matrices combined with large volume injection–gas chromatography–massspectrometric detection, J. Chromatogr. A. 1020 (2003) 153–160[4] www.gerstel.com/en/automated-membrane-extraction-mase.
htm

5

Amber fascinates by its color, refraction oflight, and golden glow, but how can you tell
if it’s the real thing?

Material analysis

Amber authentication, a closer look
Amber is cherished for its beauty and is used in jewelry and works of art. Amber has been thought to have healing
powers and has been widely used for charms to keep bad luck at bay. However, it is not easy to know if the gem inyour hand is real amber or a well-produced fake. Looks can be deceiving and so can the texture of the surface. Ageis what matters and older is better: A million years is the bare minimum - orders of magnitude more the norm. Whileamber doesn’t easily reveal its age or origin, instrumental analysis can shed light on the matter: Pyrolysis coupledwith gas chromatography (GC) and mass spectrometry (MS) is known as a strong complement to spectroscopic
techniques. If you automate the pyrolysis and add a suitable reagent to perform thermochemolysis, detail on theamber’s origin is revealed.

T

he town of Ribnitz-Damgarten lies beautifully situated where the river Recknitz
flows into Lake Ribnitz in the German Stateof Mecklenburg-Vorpommern. The town of
15,000 inhabitants is a very special place as canbe seen by visiting its homepage. Since 2009,
Ribnitz-Damgarten has been officially designated as the “Amber City.” The reason is theAmber Manufacture in the Damgarten section of town and the German Amber Museum

6in the Ribnitz section. The town boasts anamber shopping mile where amber of every
shape and hue can be acquired. In addition
there is the annual Amber Queen Contest;
the International Amber Art Competition;and the world’s longest amber necklace at 120
meters. At the Baltic Seashore not far fromtown, visitors can spend the day hunting foramber, the “Gold of the sea”. Whether or not
amber found on the beach is the real thing canbe determined by a simple density test as recommended by the German Amber Museum.
The following experiments will provide good
indications as to the type of amber found andwhether it is real:• Produce a saturated salt solution by adding
two soup spoons of salt to a glass of water.
• Amber will float in this solution, stone, glass
and polymer materials will sink.
• Compare: Among the different amber

GERSTEL Solutions Worldwide – No. 14

types, clear amber is the heaviest, whereas“amber bone” is the lightest.
• Rub larger amber chunks against wool.
They will build up an electrostatic charge
and attract paper snippets.
• Carefully tap the find against your front
teeth. If it is amber, it is not unpleasant andwill produce a dull sound. Amber produces
the same sensation as if you tap your finger nail on the teeth. A yellow pebble or a
glass shard will sound light and their contact with the teeth will produce a distinctly
unpleasant sensation.

from the Ameki Formation in Nigeria [1],the project is sponsored with a research grantfrom the Alexander von Humboldt Foundation. For the geochemical characterization
and biochemical classification, Sonibare andhis colleagues have previously used Infrared(IR) Spectroscopy and Gas Chromatography(GC) with Mass Spectrometry (MS) detection to determine the molecular compositionof different fossilized plant resins. They are
now looking to automated pyrolysis as wellas thermochemolysis to help move the project forward.

The Johannes Gutenberg University in
Mainz, Southwestern Germany is far awayfrom the Baltic coast, but even here amber ishigh on the agenda. Dr. Oluwadayo O. Sonibare and Prof. Dr. Thorsten Hoffmann fromthe Department of Inorganic and Analytical Chemistry along with Prof. Dr. Stephen
F. Foley from the Dept. of Geosciences andEarth System Research Centre are searching for a more accurate method to determine origin and authenticity of amber. The
recommendations from the German Amber
Museum are useful for tourists and for those
who casually collect amber, but of course notfor university level scientific research on thematter. The stated goal of the scientists is todetermine “The molecular composition andchemotaxonomy aspects of Eocene amber

What is known about amber?
Amber, in mineralogical terms also known as
succinite, was formed when tree resin from a
fir or other coniferous species was hardened
through contact with air millions of years ago.
Large quantities of dried resin wound up in
the oceans and sank into deep sediment layers, which were in turn covered by more layers of sand and rock. Over time and underhuge pressure in an anaerobic environment,
amber was formed. Most amber finds havebeen dated to the tertiary period, around 55
million years ago. However, “younger” species
are known as well. To be classified as amber,at least a one million year maturation period
is required. Baltic amber is among the mostancient of known ambers. Amber is foundAmber, in mineralogic terms called succinite, isfossilized tree resin, which seeped from firs orother conifers millions of years ago

in many countries, for example, in England, Portugal, Spain and Italy, but also in
Canada, Mexico, Japan, the Dominican
Republic, in Madagascar, and in Borneo.
Amber fascinates through the varying coloration, light refraction, and the golden yellow glow after polishing. Amber feels sensuous and warm, and is surprisingly light. It
makes you want to touch it, have it against
your skin, and use it as a hand charm, letting
your fingers run over it. Even today, people on
the Baltic shore actively collect amber, whichis also said to have healing, pain-killing andcalming properties. Sometimes, million-yearold insects or plants are clearly visible, encasedand fossilized in the amber.

Delving into the details

The remains of insects and plants that lived millions of years ago are sometimes found encased andfossilized in amber.

GERSTEL Solutions Worldwide – No. 14

At GERSTEL Headquarters, R&D ProjectManager, Dr. Eike Kleine-Benne and his colleague Yunyun Nie met with Dr. Sonibare
from the Johannes Gutenberg University to
exchange information on the properties ofamber with a view to determining both origin and authenticity. In short: What are thepossibilities for determining the composition and specific properties of amber and thus
finding markers for different types of amber?
Deciding on which technique to use is easier
if you have detailed knowledge of the composition of the material. According to Dr. Sonibare, the fossilization of plant resin is a complex curing and maturation process involving

7

R-COOH

TMAH

-

+

[R-COO] [(CH3)4N]

Δ

R-COOCH3 + (CH3)3N

-

OH

CH3
N

H 3C

CH3
CH3

Methylation of a carboxylic acid using tetramethylammonium hydroxide (TMAH). Shown to the right: TMAH structural formula.

loss of volatile content, polymerization, andcrosslinking of terpenoids over a period of
up to 100 million years. Amber is sometimes
considered a natural polymer, but chemicallyspeaking ambers are heterogeneous mixturesof polyesters classified as terpenoids (mono-,sesqui-, di- and triterpenoids), all of which
are derived from isoprene, a compound that
occurs naturally in plants. In contrast to terpenes, terpenoids contain functional groups.
The analysis techniques mainly used for thecharacterization of amber are infrared (IR)
spectroscopy, Raman spectroscopy, GC/MS
analysis of solvent-based extracts, and pyrolysis-GC/MS. Based on the chemical and physical properties of amber, the scientists decided
to use automated pyrolysis-GC/MS, but also
to take it to the next level: Thermochemolysis, combining pyrolysis and a derivatization
reagent, in this case tetra methyl ammoniumhydroxide (TMAH), a technique previously
described in literature. The hope and theory
was that the derivatized pyrolysis fragments
could be more extensively characterized, providing deeper knowledge of the compositionand enabling a more accurate classificationof amber types and their geographical/geological origin.

IR spectroscopy is used to differentiate Baltic
amber from other ambers, says Eike KleineBenne. The main useful IR differentiator isthe so-called “Baltic shoulder” at 1250 to 1175
cm-1. This absorption band is associated withsuccinic acid, which is found in Baltic amber.
However, with regard to amber classificationand differentiation in general, the IR method
is of limited use, due to the fact that different amber types often yield similar patterns.
Raman spectroscopy is useful for the determination of differences in amber maturation
processes, not for geographical origin.
GC/MS enables a molecular – structural elucidation of the soluble part of amber, which
unfortunately only constitutes 20 % of thetotal.
Pyrolysis-GC/MS gives the analyst the possibility of generating fragments of the polymer parts, i.e. to split insoluble and non-volatile macromolecules into individual fragments, separate these and determine theiridentity based on a detailed mass spectrum.
The fragment pattern and certain markers arethen used to determine the type and origin ofamber samples.
Thermochemolysis combines pyrolysis anda derivatization reagent. This means thatas soon as certain pyrolysis fragments areformed, they are derivatized in order to enable
or improve their determination by GC/MS.
Amber is flammable, it burns generating a
distinct aromatic smell and it was long usedas substitute for incense. Amber shows limited solubility in alcohol, ether, chloroform,
and turpentine oil. Ground amber is negatively charged and has a melting point of
375 °C.

Hunting for data and gathering
information with pyrolysis GC/MS
Eocene amber samples from the Ameki formation in Nigeria were kindly made available
for the project by the University of Glasgow
Hunterian Museum.The sample material was
ground and 200 µg portions were individuallypyrolyzed at 480 °C for 20 seconds. The following pyrolysis-GC/MS system was used:
Agilent GC 6890N with GERSTEL Cooled
Injection System (CIS), Agilent 5795B Inert

Comparison of chromatograms resulting frompyrolysis GC/MS andthermochemolysis GC/MS,
respectively, of Eoceneamber from Nigeria(FAME: Fatty acid methyl
ester / FA: Fatty acid).

8

GERSTEL Solutions Worldwide – No. 14

GERSTEL PYRO provides more in-depth
information on the analyzed amber

The amber team from right to left: Oluwadayo O. Sonibare, Ph.D., Yunyun Nie, and Eike Kleine-Benne, Ph.D..

XL (Triple Axis) MSD, GERSTEL MultiPurpose Sampler (MPS), GERSTEL Thermal Desorption Unit (TDU), and GERSTEL PYRO pyrolysis module mounted
on the TDU. PYRO is a plug-in unit, which
can be mounted on the TDU in just a few
minutes and allows automated sample introduction when used with the MPS. Technicaldetail: The TDU was operated in split modein order to vent excess solvent and volatileorganic compounds (VOCs).The CIS was set
to an initial temperature of 300 °C and used asheated split interface, with a split ratio of 1:20.
The column used was a ZB-5MS fusedsilica capillary column (30 m x 0.25 µm x 0.25mm) from Phenomenex. The GC oven temperature program was set as follows: 60 °C(2 min) – 6 °C/min – 300 °C (10 min) using
Helium carrier gas at 1 mL/min. The MSD
was operated in Electron Impact (EI) ionization mode with an ionization energy of 70 eV,
and source and quadropole temperatures set
to 230 °C and 150°C respectively. Full scan
spectra were acquired from 50 to 650 Da.
Data acquisition and data handling were
performed using ChemStation Software(Agilent Tecnologys) and compound identification performed using both the integrated
mass spectral library and information fromliterature.

• Naphthalene-1-Carboxylic acid1,2,3,4,4a,7,8,8a-octahydro-1,4a,5,6-tetramethyl (FA III)
• Naphthalene-1-carboxylic acid1,2,3,4,4a,5,8,8a-octahydro-1,4a,6-trimethyl-5-methylene (FA V).

Gaining deeper knowledge stepby step
Initially, a ground amber sample was pyrolyzed. In the chromatogram, a long line of the“usual suspects” were identified, which werepreviously described in literature. In this case,a series of fatty acids (FAs), among them:• Norchrysanthemic acid (FA I)• Naphthalene-1-carboxylic acid1,2,3,4,4a,7,8,8a-octahydro-1,4a,6-trimethyl (FA II)GERSTEL Solutions Worldwide – No. 14

To gain better chromatographic performance and more information, amber samples
were pyrolyzed in the presence of tetramethyl
ammoniumhydroxid (TMAH) for in situ
derivatization (methylation) during the 20 s
pyrolysis period. This process is called thermochemolysis; fatty acids are converted into
their corresponding methyl esters (FAMEs):• Norchrysanthemic acid methyl ester(FAME I)
• Naphthalene-1-carboxylic acid1,2,3,4,4a,7,8,8a-octahydro-1,4a,6-trimethyl methyl ester (FAME II)• Naphthalene-1-Carboxylic acid1,2,3,4,4a,7,8,8a-octahydro-1,4a,5,6-tetramethyl methyl ester (FAME III)• Methyl-1,2,3,4-Tetrahydro-1,5,6-Trimethyl-1-Naphthalenecarboxylate(FAME IV)
• Naphthalene-1-carboxylic acid1,2,3,4,4a,5,8,8a-octahydro-1,4a,6-trimethyl-5-methylene methyl ester(FAME V).
• Methyl-16,17-Dinorcallitrisate(FAME VI)
The difference in performance between
pyrolysis and thermochemolysis is explainedby Eike Kleine-Benne as follows:„In the pyrolysis chromatogram, the acids
peaks (FA (I), FA (II), FA (III) and FA (V) are
broad, and partly saw tooth shaped, a typicalphenomenon for highly polar or active compounds such as free fatty acids (FFAs) separated on a non-polar column. FA (IV) and FA(VI) are not found at all. In contrast, the cor-

responding methylated acids elute as well separated sharp peaks in the thermochemolysisbased chromatogram, including FAME (IV)and FAME (VI). It is clear that pyrolysis with
TMAH brings out more detailed information
about the analyzed material.” As Dr. Sonibare reports, an earlier study yielded the firstavailable information on the molecular composition of fossilized resins from the Eocene
Ameki formation in Nigeria with pyrolysis
GC-MS analysis clearly indicating that theamber belongs to class I b, which is derived
from regular labdatriene structures. Anothercharacteristic of class I b ambers is that they do
not contain succinic acid. The pyrolysis products of the amber were dominated by labdanlike diterpenoids and a small number of sesquiterpenoids. The exclusive presence of labdane diterpenoids and the absence of planttriterpenoids in the Eocene Ameki amberpointed to a conifer (Gymnosperm) originof the resin. With the described method, Dr.
Sonibare, Prof. Hoffmann and Prof. Foley havea tool which helps them categorize amber byorigin while delivering further information
for their amber research: New research results
were recently published in the Journal of Analytical and Applied Pyrolysis [2].
Literature[1] O. O. Sonibare T. Hoffmann, S. F. Foley:
Molecular composition and chemotaxonomic aspects of Eocene amber from the
Ameki Formation, Nigeria, Organic Geochemistry 51 (2012) 55–62[2] O. O. Sonibare, R.-J. Huang, D. E. Jacob,
Y. Nie, E. Kleine-Benne, T. Hoffmann, S. F.
Foley: Terpenoid composition and chemotaxonomic aspects of Miocene amber fromthe Koroglu Mountains, Turkey, Journal of
Analytical and Applied Pyrolysis 105 (2014)
100-1079

Material analysis

Making it stick
Thermal desorption combined with gas chromatography is a simple yet powerful tool that can quickly extract low
levels of volatile organic compounds (VOCs) from a wide range of solid matrices, including polymers and adhesives,
providing comprehensive information about VOC content. The addition of a pyrolysis module to the tool kit enables
the analyst to see more, looking deeper into the structure of the polymer or adhesive material while using a standard
GC/MS system.

F

astening, bonding, joining, and mounting of materials is done in a number ofways. For example, materials used in industrial applications and building materials usedin homes and offices are increasingly bonded using adhesive solutions. These replace
more traditional fastening methods, such as
screws, bolts, rivets and even welding.The reasons that adhesives are making such inroads
into these applications are manifold: Adhesive tapes, for example, are flexible, light, andeasy to position or even reposition and arelargely immune to mechanical impact. They
are widely used in the automobile-, electronic-, and paper industries, among professional builders and by do-it-yourself handymen
or -women. Adhesive solutions are as varied
as the applications they are used for. In manyindustries, it is critically important to know
the composition of the adhesive material usedsince it can greatly influence the quality of
the final product. This information can also
be used to identify cheap imitations that cannegatively impact revenues and ruin the reputation of a quality brand.
A typical adhesive tape consists of a carrier film, an anchor coating and the adhe-

10

sive material. If it is a double-sided tape, oneside is typically protected with a separating
film, called a liner. Depending on the application, different types of liners can be used,such as polyethylene (PE), polypropylene(PP) or polyethylene terephthalate (PET).
Carrier films can be made of PE, PP, PET,
or of soft or hard poly vinyl chloride (PVC).
In addition, various additives are used to tailormaterial properties to the specific application.
These include plasticizers added to produce,for example, soft PVC, anti-aging agents, or
anti-blocking agents. The adhesive material
is typically produced from polyacrylate, synthetic caoutchouc, or natural caoutchouc, allof which may contain one or more of the following: Adhesive resin, fillers, crosslinking
agents, and/or plasticizers. As an example, adouble sided tape may be based on a soft PVC
carrier with a resinous polyacrylate adhesive
layer on both sides coated with a PE film asshown in the diagram on the following page.
If the soft PVC carrier and the PE film
are purchased materials, analytical laboratory
resources will be needed for quality control toidentify the raw materials used.
Often, all additives contained in

all materials used must be identified in order
to ascertain their compatibility with the adhesive, which of course must also undergo compositional analysis. The focus of the productquality control efforts would typically be thedetermination of the polymer composition
and the adhesive resin content. For these andsimilar tasks, Pyrolysis-GC/MS has provenparticularly useful. Thermal fragmentation
followed by GC separation and MS determination offers a range of possibilities for qualitative and quantitative analysis as the threefollowing examples show.

Using pyrolysis-GC/MS forpolymer analysis
Initially, the PE film used for the double sided
tape described above was analyzed. Material testing had revealed that the mechanicalproperties did not meet requirements – eventhough the infrared (IR) spectrum for boththe front and back side of the tape showed
only the absorption bands typical for PE. To
clarify the matter, pyrolysis GC/MS was usedwith the following instrument configuration: A GERSTEL MultiPurpose Sampler (MPS) for automated sample preparation and introduction mounted on top of an
Agilent 7890/5975B GC/MSD system. The setup was extended witha GERSTEL Thermal Desorption
Unit (TDU) mounted on a GERSTEL Cooled Injection System (CIS
4), PTV-type inlet. The PE film waspyrolyzed at 700 °C and the formed
fragments separated using a Restek Stabilwax DA column. Both linear and branched aliphatic hydrocarbons were found. A
comparison with a user-generated pyrolysis
library of various polymers revealed: Linear
aliphatic hydrocarbons typically occur in PE
pyrograms; branched aliphatic hydrocarbons,on the other hand, typically occur in polypropylene (PP). Finally, the analysis of a PE/PP
reference mixture revealed that the unknown

GERSTEL Solutions Worldwide – No. 14

“PE” material was in fact a mixture of PE and
PP. Since IR spectra of the front and back of
the tape only produced PE adsorption bands,
it was concluded that the unknown material
was a three layer film with a PP layer between
two PE layers.

Using Pyrolysis GC/MS todetermine additives in polymers
To help assess, whether an adhesive would
be compatible with a PVC carrier film, theplasticizer used in the PVC was determined.

a pyrolysis GC/MS method based on matrix-matched calibration was developed. The
main challenges during method development
were to identify and select suitable pyrolysis fragments as markers for individual adhesive resins and to optimize sample preparation in order to achieve adequate reproducibility. Methyl abietate (m/z = 316) was selected
as marker for rosin methyl ester (see figure).
For hydrated rosin resins, norabietan (m/z =262) was used as quantification marker. Bothmethods were developed as Single Ion Monitoring (SIM) methods in order to achieve bet-

PE liner
Polyacrylate adhesive
Soft PVC carrier
Polyacrylate adhesive

Schematic diagram of adouble-sided adhesive tape
(PE = polyethylene, PVC =Poly vinyl chloride)

A combined analysis procedure of IR spectroscopy and pyrolysis GC/MS was used; in bothcases, methods were selected, which didn’t
rely on solvent-based extraction. Based on
the IR analysis, it was determined that there
was polyurethane in the PVC, but no phthalate. The PVC film was then pyrolyzed at
800 °C and the fragments identified. In addition to the hydrochloric acid fragment, whichis to be expected when pyrolyzing PVC, a highintensity peak for the hexamethylene diisocyanate (HDI or HMDI) fragment was found.
This information made it clear that a polyurethane-based plasticizer, which contained
HMDI as isocyanate component, was used
for the PVC. Based on experience, the plasticizer was deemed compatible for use withthe adhesive.

Quantifying additives with
pyrolysis GC/MS
In order to quantify adhesive resin content, in this case rosin aka colophony resin,

GERSTEL Solutions Worldwide – No. 14

ter signal to noise ratios and higher sensitivity. The sample preparation was optimized
as follows: The adhesive material sample wasweighed on silicone-coated release paper, i.e.
anti-adhesive paper (sample weight: 0.5-1
mg). Then the sample was sprinkled with afine glass powder, which prevents the sample
from adhering to the pyrolysis tube without
interfering with the pyrolysis process andwithout impacting the analysis result. For all
samples and calibration standards, duplicate
analyses were performed and the mean values
used. Calibration standards were generated byadding 5 to 40 % (w/w) rosin resin to resinfree polyacrylate adhesive material made upof n-butyl acrylate, 2-ethylhexyl acrylate andacrylic acid. The calibration standards were
pyrolyzed at 800 °C resulting in a calibration
curve with a correlation coefficient of 0.9916.
The calibrated methods were subsequently
used to determine both the rosin resin and thehydrated rosin resin contents in polyacrylateadhesives. Additional hydrated resin types
were analyzed to determine whether hydra-

Pyrogram of a rosin methyl ester
resin after pyrolysis at 800 °C.
The peak with the highest intensity
was determined to be methyl
abietate (m/z = 316).

ted rosin glycerol ester resin or other esterbased resins could be analyzed quantitatively.
Multiple rosin methyl ester resins from different manufacturers were finally analyzed. For
both methods it was found that the rosin resin
content could be determined with a standard
deviation below 4 %. Given the highly heterogeneous nature of such adhesive resins, thedeveloped pyrolysis GC/MS methods forquantitative determination of resin content
are considered sufficiently accurate. Using thismethod, the rosin resin content of polyacrylate adhesives can be determined for quality control purposes. Further investigations
will determine whether the methods can beapplied to other matrices such as synthetic
caoutchouc materials. It should also be possible to expand the use of the method to determine other resin classes such as polyterpene
resins and hydrocarbon resins.

Conclusions
Pyrolysis GC/MS is a versatile technique,which can be used for qualitative as well as
quantitative analysis of polymers in generaland, as shown in this work, also of adhesivematerials. Of particular benefit to the analyst
is the very limited effort required for sample
preparation as well as the considerable depthof information obtained – even from just asingle analysis.
Authors
Aylin Meß, Vjaceslav Frank and Andreas
Westphal, tesa SE, Quickbornstraße 24,20253 Hamburg, Germany

11Phthalates

Easy extraction of plasticizers from toys
The US Consumer Product Safety Commission’s (CPSC) Test Method CPSC-CH-C1001-09.3 [1] is used by testinglaboratories for the determination of phthalate content in children’s toys and child care articles covered by thestandard set forth in the Consumer Product Safety Improvement Act Section 108. The general manual approach is
to dissolve the sample completely in tetrahydrofuran, precipitate any PVC polymer with hexane, filter and dilute withcyclohexane, and then analyze by Gas Chromatography/Mass Spectrometry (GC/MS). A combined autosampler andsample preparation robot can be used to automate these sample preparation steps.

C

hemical additives are widely used to
impart desirable properties to many products. For some compounds, however, due totoxicity issues, the amounts used need to beclosely monitored.. Phthalic acid esters, also
known as phthalates, are among these. They
are widely used as plasticizers in polymers,
making the material more pliable. Phthalates
influence the physical properties of the polymer material significantly making it softer andmore flexible. PVC, for example, is a highly
durable material. Plasticizers give PVC theflexibility and elasticity that have made it so
widely used and appreciated. Applications
where PVC is used range from flooring, artificial leather, shower curtains, baby care articles, toys, packaging, shoes, sport and leisure12

materials, and medical products such as IV
bags used for medical infusions and blood
storage. Some further uses are: Cable coating,roofing materials, truck tarpaulins and undercoating of vehicles. However, there is a flipside to these benefits: Some phthalates, suchas Di(2-ethylhexyl)phthalate [DEHP] aswell as dibutylphthalate [DBP] and Benzylbutylphthalate [BBP] are considered harmful
to human health and are banned by the European Union (EU) for a wide range of uses [3].
According to EU guidelines, DEHP is harmful to reproduction. Within the EU, restrictions apply to items used by children who
are less than 3 years of age. Toys for this age
group as well as baby care articles that contain the aforementioned phthalates at indi-vidual concentrations higher than 0.1 % w/w
are not allowed into the market place.
The US Congress has permanently
restricted the use of three phthalates (DEHP,
DBP and BBP) as well as (on an interim basis)three additional phthalates (DINP, DIDP
and DnOP) in any amount greater than 0.1
percent (computed for each phthalate, individually) in children’s toys and certain childcare articles. The risk of a negative healthimpact due to the transfer of the additives
from PVC based products is deemed particularly great in small children.
In order to ensure compliance withgovernmental health and safety regulations, products that fall under the regulationmust be monitored for these compounds.

GERSTEL Solutions Worldwide – No. 14

Sample preparation is key
Sample preparation of polymers being monitored for additives is the most time consuming
part of the overall analysis. When phthalates
are determined in polymer matrices following manual extraction procedures describedin CPSC-CHC1001-09.3, these procedures
are often the rate-limiting step in terms ofoverall productivity and throughput, and they
involve the use of hazardous chemicals andsolvents. Typically, liquid dissolution of thepolymer is first performed in order to quantitatively release additives and other compounds of interest from the material. A solvent, in which the polymer is not soluble, isthen added to precipitate the polymer whileleaving the analytes of interest in solution. Filtration of the final extract is performed prior
to analysis by either GC or HPLC. This general procedure can be applied to a wide variety of polymers and plastics. In this work, weshow that the manual procedure established
for the extraction of phthalates from consumer products produced from PVC is easily automated using the GERSTEL MultiPurpose Sampler (MPS). The entire process
is conveniently controlled using MAESTRO
software.
Weigh a 50 mg sample of polyvinylchloride(PVC) in a sealable glass vial.
Add 5 mL of THF to the sample
Mix the sample for at least 30 minutes,
to ensure complete dissolution. Ultrasound and/or mild heating can be used toaccelerate the dissolution process. If thesample hasn’t been completely dissolved,
the mixing period should be extended byanother 2 hours before continuing.
Precipitate the PVC polymer using 10 mL ofhexane for each 5 mL of THF used during
the dissolution step.
Shake and wait at least 5 minutes for thepolymer material to precipitate and settle.
Filter the THF/Hexane solution through a0.45 mm PTFE filter and collect a few mL of
the filtrate in a separate glass vial.
Combine a 0.3 mL aliquot taken from the
THF-hexane mixture with 0.2 mL of theinternal standard, in case this is used, and
top up to 1.5 mL with cyclohexane.
Inject 1 µL of the solution into the GC/MS.

GERSTEL Solutions Worldwide – No. 14

As explained by Ed Pfannkoch, Technical Director, GERSTEL, Inc., Baltimore,
MD: “It was our aim to transfer the manual steps one by one and automate the entire
process while using the PrepAhead functionin MAESTRO to perform sample preparation simultaneously with the GC/MS run
and increase productivity”. To this end, Ed
Pfannkoch and his team used the Dual Headversion of the GERSTEL MPS. One head
was fitted with a 2.5 mL syringe for liquidtransfer used for sample preparation, theother with a 10 µL syringe used for liquid
injection into the GC/MS system (Agilent
7890GC/5975MSD); the PTV inlet used
was a GERSTEL Cooled Injection System(CIS 4). The MPS was further equippedwith a heated agitator, GERSTEL Automated Liner Exchange (ALEX) as well asa Filtration Option. Method and sequencetable setup including all sample preparation
steps were performed using the GERSTEL
MAESTRO Software, completely integrated
with the Agilent ChemStation.
For this method, the PVC sample is dissolved in tetrahydrofuran (THF) and the
PVC precipitated using hexane followedby filtration, dilution with cyclohexane andfinally analysis using GC/MS. The test procedure is listed in the box to the left on thispage.

Greetings from the rubber duck
As research objects, Ed Pfannkoch and his
team chose ordinary rubber ducks as well asother toys made of PVC. Three other samples
were obtained from a local CPSC testing station (CPSC-1, CPSC-2, and CPSC-3). All
PVC samples to be extracted were initially cut
into small pieces of no more than 2 mm particle size. These were weighed in 10 mL glass
vials fitted with screw caps and placed in the
MPS sample tray. Analysis method parameters were established as shown in the box on
the right. The method was calibrated using
standard solutions covering two concentration ranges: Lower range (50-1000 ng/mL)

and upper range (5-100 µg/mL). “Initially,
we tested whether the solvents used for the
extraction were free of phthalates”, Edward
Pfannkoch explained: “We went throughan entire blank extraction procedure, without any sample present, and no phthalates
were detected in the final extract.” Following the initial experiment, the certified reference samples were extracted – first using
the manual sample preparation method andthen using the automated extraction procedure. The results obtained using the two
methods were found to be in good agreement. The automated sample preparation
method combined with GC/MS analysis of
the sample extracts worked reliably and proAnalysis conditions
PTV (CIS 4):

Baffled liner, split (20:1)
or splitless, 50 °C;
12 °C/s; 280 °C (3 min)Column:

30 m HP-5MS (Agilent) di= 0.25 mm df = 0.25 µm

Pneumatics:

He, constant flow = 1.0
mL

Oven:

50 °C (1 min); 20 °C/
min; 310 °C (5 min)MSD:

Full scan, 40-350 amu

SIM Parameters (Mass [m/z], Dwell time [ms])Group 1:5 min (91, 10), (105,10), (149, 10), (167, 10),
(194, 10), (194, 10),(205, 10), (212, 10),
(223, 10)Group 2:

11.7 min (91, 10), (149,10), (167, 10), (206, 10),(279, 10)Group 3:13.7 min (149, 10), (167,10), (261, 10), (279, 10),
(293, 10), (307, 10)13

Benzylbenzoate (IS)Diisononylphthalate
DiethylphthalateA

Di-n-butylphthalate

Di-n-octylphthalate
Diethylexylphthalate
Butylbenzylphthalate

Diisodectylphthalate

BC

duced accurate results. To increase the overall throughput, and to
reduce the use of toxic solvents, Pfannkoch and his team reduced
the amount of sample and solvent used by 50 % without any measurable impact on the analytical results. Finally, the rubber duckand the CPSC samples were taken through the complete automated procedure.
Extracts were analyzed by GC/MS in split and splitless mode.
The MSD was operated in SIM/Scan mode. For the lower concentration range, the instrument was operated in splitless mode.
For the higher concentration range, the split was set to 20:1. Analytes were identified based on their mass spectrum and retention times. Quantification was performed based on the calibration curve.

No need to “duck” hard questions
Chromatogram resulting from automated preparation of a 50 mg sample (A);automated preparation of a 25 mg sample (B); and manual preparation of a
25 mg sample (C). Conclusion: The chromatograms are equivalent.

Acetyl-tri-n-butylcitrate (ATBC)

Diisononyladipate

Diethylphthalate

At the end of every method development, the user has to face up
to the critical question: Was I successful? Edward Pfannkoch is
happy with the results. “Our main aim, to transfer the extraction to
the MPS and to automate it completely was achieved”, he states.
The automated analysis of the certified reference materials gave
accurate results.The automated GC/MS method for the determination of DEHP, DBP, BBP, DINP, DIDP and DnOP delivered
accurate results with a relative standard deviation (RSD) of 1.95.5 percent for all determined phthalates, which is highly precise.
In short, the automated GC/MS analysis following test method
CPSC-CH-C1001-09.3 performed flawlessly. The results weachieved were highly satisfactory – also for real samples. In thesamples CPSC-1 and CPSC-2, we found all six target analytes.
Sample CPSC-3 on the other hand contained DEHP at a highconcentrations level.[4]Literature

Benzylbenzoate (IS)Full Scan chromatogram of a rubber duck sample. The chromatogramillustrates the benefits of using SIM/Scan mode: Even plasticizers, which are
not selected as target analytes appear in the chromatogram. These include:
Diethylphthalate, acetyl-tri-n-butylcitrate and diisononyladipate.

A

Diethylhexylphthalate[1] United States Consumer Product Safety Commission, Test
Method: CPSC-CH-C1001-09.3 Standard Operating Procedure
for Determination of Phthalates, April 1st, 2010.
[2] German Federal Environmental Protection Agency (Umweltbundesamt): Phthalates: Useful plasticizers with undesired
properties, 02/2007; www.umweltbundesamt.de/sites/default/
files/medien/publikation/long/3541.pdf[3] Directive 2005/90/EC of the European Parliament[4] Fredrick D. Foster, John R. Stuff, Jacqueline A. Whitecavage,
Edward A. Pfannkoch: Automated Extraction and GC/MS
Determination of Phthalates in Consumer Products, GERSTELAppNote 4/2013, www.umweltbundesamt.de/sites/default/
files/medien/publikation/long/3541.pdf

Disononylcyclohexan-1,2-dicarboxylate
Di-n-butylphthalate

Butylbenzylphthalate

Benzylbenzoate (IS)

Butylbenzylphthalate

Di-n-butylphthalate

B

Di-n-octylphthalate
Diethylhexylphthalate
Di-n-octylphthalate
Diisodecylphthalate

Full scan chromatogram for sample CPSC-2. Figure B shows extracted
ion chromatograms of SIM data. Target analytes are identified in the
SIM chromatogram. The full scan chromatogram shows the presence ofanother compound: Diisononylcyclohexane-1,2-dicarboxylate.

14

GERSTEL Solutions Worldwide – No. 14

Pharmaceutical analysis“Corked” pills – no thanks!
Pharmaceutical products with an unpleasant smell may be perfectly
safe and effective. However, patients instinctively associate offodors with inferior quality or even the possibility that the productmay harm them. When this happens there is a good chancethat the medicine will not be taken, but simply thrown away.
This hurts the image of the manufacturer and can even prompt
extremely costly product recalls. To track down off-odors andtheir sources in pharmaceutical products and their packaging,scientist have developed and validated a highly sensitive GCMS/MS method based on Stir Bar Sorptive Extraction (SBSE).

W

hen it comes to quality
control, the devices given
to us by Mother Nature are
hard to beat. Anything weingest passes through several sophisticated anatomically based inspection steps.
First a visual inspection, thena sensory evaluation of “mouthfeel” combined with the taste
sensed on the tongue and flavor or odor sensed by our olfactory
bulbs in the nose. The combinationsof all these senses produce a number of responses: Things that provide an overall pleasant experience are favored and eagerly
consumed - sometimes in

GERSTEL Solutions Worldwide – No. 14

15

Electron Impact (EI) spectrum following a 5 ng direct injection of 2,4,6-trichloroanisole.The molecular ion cluster appears as m/z = 210(M), 212 (M+2) and 214(M+4). The main cluster at m/z=195 (M) is the methyl loss peak cluster. Due tosiloxane interferences, the isotope ratio for the three chlorine atoms in the molecule is not properly detected.

Electron impact NIST library spectrum of 2,4,6-trichloroanisole (# 333450). The
molecular ion cluster appears at m/z=210 (M), 212 (M+2) and 214 (M+4)
with the isotope ratio to be expected from an organic molecule containing threechlorine atoms.

larger amounts than advisable. However, it only takes thepresence of a pungent or unpleasant smell to set off our neural alarm network, making us sniff at food and instinctively
turn it away. If you look at the greater picture, olfactory evaluation has played a central role in our survival through theages and continues to be a key guardian of our safety. Evolutionary biologists contend that especially the expansion of
the olfactory center lead to the decisive increase in mammal
brain size [1].

The nose decides
In order for a product to reach the stomach and the heart ofthe consumer, it needs to get by the nose. Products taken orally
should have a pleasant or at least a neutral flavor. This applies
to food and food ingredients as well as to condiments, sweets,
beverages, and of course, pharmaceutical products. Some offodors can set off the olfactory alarm even at ultra-trace levels due to our highly sensitive olfactory early warning system.
Threshold levels can be extremely low and vary widely fromperson to person. In order to accurately and reliably determine
the identity and concentration of off odor compounds at relevant levels, gas chromatography in combination with tandem mass spectrometry (GC-MS/MS) is a widely used technique. This is due to its ability to provide low detection limits
and accurate mass spectral data, especially for known (target)
compounds. For best results, GC-MS/MS is coupled with an
extraction/concentration technique that allows for the unadulterated compounds to be introduced into the system. Ideally,
off-odor compounds should be sniffed out and the productheld back before it is under the nose of the consumer and before
a situation develops that includes negative headlines and product recalls that can cause extensive and expensive damage toa brand. Case in point: A couple of years ago, a global pharmaceutical company was forced to recall tens of thousands ofpackages of different brand name products following customer
complaints concerning a musty, moldy off-odor.The malodorous culprits in question turned out to be halogenated anisoles
and phenols, well-known repeat offenders in the wine industry
leading to corked wines. The line-up included the following
identified suspects: 2,4,6-trichloroanisole (TCA), 2,4,6-tribromoanisole (TBA) and 2,3,4,6-tetrachloroanisole (TeCA),
metabolic offspring of their equivalent phenols, some of whichwere also caught in the dragnet: 2,4,6-trichlorophenol (TCP),
2,4,6-tribromophenol (TBP) and pentachlorophenol (PCP).
The odor thresholds, the lowest concentrations at which compounds are detected by the human olfactory system, in this casein wine or water, are as follows: 1.4-4 ng/L for TCA; 3-8 ng/L
for TBA; and 4-24 ng/L for TeCA [2]. For TCP and PCA
the values are approximately 4000 ng/L [3,4].

On the origins of corky off-odor
Collision induced dissociation (CID) spectrum of 2,4,6-trichloroanisole with precursor
ion m/z=210 (M).

16

The usual suspect as a source of 2,4,6-TCA in wine is the cork
stopper made from the bark of the cork oak tree (Quercus

GERSTEL Solutions Worldwide – No. 14

suber). TCA is a microbial metabolite, formedby methylation of trichlorophenol (TCP) that
may have been applied to the bark as a pesticide. To suspect the cork stopper of introducing TCA to the wine is therefore only logical, but when wine drinkers started experiencing corkiness in wines with modern polymerbased stoppers, experts knew that they had
been barking up the wrong tree.
Over the course of the ensuing research
projects, it was found that various compounds,
mainly halogenated anisoles, would give a
musty, moldy note to the wine. These compounds could be formed from other chlorinated chemicals that are used for cleaning ofwine production equipment or for treatingwooden transport pallets or packaging material.
Until the end of the 1980’s, pentachlorophenol (PCP) was used as a fungicide toprotect wood, including wooden pallets,from microbial decay. Among other byproducts, PCP contained 2,3,4,6-tetrachlorophenol (TCP), a compound that is metabolized microbially to 2,3,4,6-tetrachloroanisole (2,3,4,6-TeCA, TeCA), which causes
corkiness in wine.
In animal tests, PCP was found to be carcinogenic. In Germany, the use of PCP has
been prohibited since 1989. PCP was substituted by 2,4,6-tribromophenol (TBP), a combined fungicide and flame retardant, which
is often used to protect cardboard packaging, polymer materials, paints and coatingseven for building modules used in pre-fabricated homes. As it turns out, microorganisms metabolize TBP to 2,4,6-TBA, a compound given the sensory attributes musty,
earthy, and chemical with a smell of solvent.
TBA is well known to impart a corky odor
and taste to wine.
TBA was indeed the main culprit when
the above mentioned pharmaceutical products fell from grace with discerning consumers. The contamination with 2,4,6-TBA, it
was speculated, could have been introduced
through wood protection agents used in the
production of transport pallets. The off-odor
compound would then have been transferred
to packaging materials transported or stored
on those pallets and subsequently into thepharmaceutical products. History is said torepeat itself, and in this case, the scenario waswell known from previous incidences in thebrewing industry, in which beer bottle caps

GERSTEL Solutions Worldwide – No. 14

had been contaminated leading to the recallof truckloads of product.
Whenever a cause is identified beyond
reasonable doubt, effective remedies can be
put into place to prevent a reoccurrence.
With this in mind, scientists charged
with finding the root cause of the packaging debacle decided to develop and validatea highly sensitive GC-MS/MS method forthe determination of 2,4,6-TCA, 2,4,6-TBA,
2,4,6-TBP and 2,4,6-TCP in tablets as well as
2,4,6-TBA in packaging material [3].

Searching for the best possible
extraction technique
To meet the stringent sensitivity requirementsin the method they developed, Gyorgy Vas andhis team of colleagues from Johnson & Johnson and from McNeil Consumer Healthcare
were looking for a powerful extraction andanalyte concentration technique, which could
span a wide volatility range and result in reliable quantification at ultra-trace concentration levels. During their literature search, thescientists determined that results based onheadspace solid phase micro-extraction (HSSPME) were mainly reported for the relatively volatile halogenated anisoles. In theirpaper in Journal of Chromatography (A) [3],the scientists summarize the possibilities withthis technique as follows: “Compared withliquid-liquid, solvent based extraction methods, HS-SPME has the advantage that it iseasy to automate, simple to perform and canbe used for a wide range of compounds”. The
limitation, however, was determined as lackof extraction efficiency from solid and liquidsamples for many less volatile compounds. To
enable the sensitive and reliable determination of both volatile and less volatile compounds, the scientists turned to Stir Bar Sorptive Extraction (SBSE) using the GERSTELTwister®, which relies on a significantly larger
volume of sorbent phase and had already beenshown to be effective in determining the target compounds in the wine industry [2]. “Wehave found SBSE to be highly efficient for thedetermination of trace level analytes due to thefact that the extraction phase volume (of theTwister) is relatively large compared with thatof SPME (about 5 µL for the 10 mm length
Twister) relative to that of SPME (about 65
nL for the 100 µm fiber),” Dr. Vas and hiscolleagues write in explaining their choice.

Predicting the extraction efficiency
How efficiently analytes can be extractedusing SBSE and the PDMS-based Twister,is best predicted based on their octanol/
water distribution coefficient (Ko/w), a
dimensionless value. The Ko/w value represents the ratio of the concentrations ofthat analyte in the respective phases in
a 1-octanol- water two-phase system at
equilibrium. Ko/w serves to describe thehydrophobic or hydrophilic properties of achemical. The logarithmic value log Kow of
a compound allows predictions of its distribution in a PDMS-Water system, and is areliable indicator of how well a compound
will be extracted into the Twister stir bar.
A high log Ko/w value represents a highlyhydrophobic compound which would be
extracted very efficiently from an aqueous matrix using the GERSTEL Twister.

The straight and narrow path:
Focused development ofvalidated methods
Dr. Vas and his colleagues developed their
methods based on standard addition quantification. To this end, standard solutions containing halogenated anisoles at 20, 40 and
200 pg/µL were generated as well as solutions containing halogenated phenols at 500,
1000 and 2000 pg/µL and finally an internal standard solution containing d5-TBA at
100 pg/µL. These solutions were generated
on a daily basis.
Standards were added to pharmaceutical
tablets of different weight as well as to different packaging materials: Cardboard, polyethylene, polycarbonate and pallet wood. Quantification of the target analytes was performed
with isotope dilution using 2,4,6-d5-tribromoanisole as internal standard. Compounds
were identified and quantified using tandem mass spectrometry detection in Multiple Reaction Monitoring (MRM) mode. The
following mass transitions were monitored:
TBA 346 -> 331 (quantifier) and 346-> 303 (qualifier)
TCA 212 -> 197 (quantifier) and 212 -> 169 (qualifier)TCP 196 -> 132 (quantifier) and 196 -> 160 (qualifier)
TBP 330 -> 222 (quantifier) and 330 -> 250 (qualifier)d5-TBA 349 -> 331 (quantifier)
Validation of the method was performedin accordance with the requirements of the17Agilent 7890A / 7000B Triple Quadrupole GC/MS. The system is mountedwith a GERSTEL MPS Dual Rail sampler
with agitator, MultiFiber Exchange (MFX),
Thermal Desorption Unit (TDU), Twister
Option, SPME fiber bake-out station, andDynamic Headspace System (DHS).

ICH Q2 (R1) guidance (International Conference on Harmonization of TechnicalRequirements for Registration of Pharmaceuticals for Human Use) as well as in accordancewith the with US Food and Drug Administration (FDA). Given the fact that the ICH
Q2 (R1) guidance does not fully apply to analytical validation of methods for the determination of trace concentration and since
TCP is an herbicide, the European Council
Directive 96/23/EG (Commission Decision
2002/657/EC of 12 August 2002) was followed concerning the analysis method anddata analysis. The GC-MS/MS system used
was an Agilent 7890/ 7000B Triple Quadrupole GC/MS system. The GC inlet used
was a GERSTEL Cooled Injection System(CIS) used for cryofocusing and temperature
programmed transfer of analytes to the GC
column (DB-5 MS, UI, 20 m, 0.18 mm, 0.36µm film thickness). A GERSTEL Thermal
Desorption Unit (TDU) mounted on top of
the CIS was used to desorb the GERSTELTwisters (10 mm length, PDMS: 1.0 mm
film thickness). Introduction of the TDU
Liners with the Twisters was performed
automatically using the GERSTEL MultiPurpose Sampler (MPS), which holds up to

196 TDU liners insealed sample tray
positions.
Fo u r t a b l e t s
were placed in separate sample vials each containing formic acid(0.1 %) and 5 µL of standard solution. The
vials were placed in an ultrasound bath for 30
min and subsequently extracted for 90 min
with Twister stir bars at a stirring speed of1000 rpm. The Twisters were removed fromthe sample vials, gently dried with lint-free
paper cloth and placed in individual TDU
liners, which were placed in the MPS sample
tray for subsequent GC-MS/MS analysis.
The packaging material to be analyzed
was cut in pieces of one square centimeter
each, the pieces were then added to a sample vial along with 100 pg/g 2,4,6-TBA.
The vials were sealed and left for 48 hours
to enable TBA to penetrate the packaging
material and to be absorbed by it. An internal standard was added and the sample left
to equilibrate for one hour before the extraction procedure. The sample was then transferred to a 125 mL vial and 100 mL of awater-acetone mixture was added. The vial
was placed in an ultrasound bath for 30 min
and subsequently the water-acetone mixture
was extracted for 90 minutes using a Twisterstir bar with a stirring speed of 1000 rpm.
The Twister was removed from the sample
vial and gently dried using a lint-free paper
cloth and placedin a TDU liner,
which was placedin the MPS sample
tray for subsequent
thermal desorption
and GC-MS/MS
analysis.

Conclusions
Dr. Vas and his colleagues succeeded indeveloping and validating an SBSETD-GC-MS/MS
based method for18

quantification of TCA, TCP, TBA and TBP
in solid pharmaceutical products [3].
The method was validated as a standard
addition method for the analysis of pharmaceutical products for the above mentioned target analytes, relevant to off flavor incidents.
The validated range is 100-1000 pg/tablet for
halogenated anisoles and 2500-10000 pg/tablet for halogenated phenols. Detection limits (absolute amounts) for TCA: 4 pg; TCP:
286 pg; TBA: 9 pg; TBP: 371 pg.
Recovery based on 100 pg of the halogenated anisoles and 2500 pg of the halogenated phenols added; recovery ranges determined in four different solid dosage formulations for TCA: 79-97 %; TCP: 67-89 %;TBA: 68-76 %; TBP: 56-72 %Precision data for repeat measurementson the same sample performed by the sameuser on the same instrument on the same day
using deuterated TBA (d5-TBA) as internal
standard gave the following relative standard
deviations (RSD) in percent for TCA: 6.211.3 %; TCP 3,2-12,9 %; TBA: 3.1-11.0 %;TBP: 6.5–15.6 %.

Sources[1] Timothy B. Rowe, Thomas E. Macrini, and
Zhe-Xi Luo, Fossil Evidence on Origin of theMammalian Brain, Science 20 (2011) 955957[2] H. Rudy, Efficient and sensitive determinationof corkiness and other off-flavours in wine,
LCGC The Column, Vol. 10, Jan. 2014.
[3] Jiun-Tang Huang, Lori Alquier, Joyce P. Kaisa,
Gail Reed, Timothy Gilmore, and Gyorgy
Vas, Method development and validation forthe determination of 2,4,6-tribromoanisole,
2,4,6-tribromophenol, 2,4,6-trichloroanisole,and 2,4,6-trichlorophenol in various drug
products using stir bar sorptive extraction
and gas chromatography-tandem mass
spectrometry detection, Journal of Chromatography A 1262 (2012) 196-204.

GERSTEL Solutions Worldwide – No. 14

Flavor and Fragrance Analysis

Wondrous Vanilla
Vanilla isn’t just vanilla or even plain vanilla. There is a whole plethora of vanillatypes and many different ways to prepare vanilla pods. Their characteristic flavor
patterns aren’t only determined by vanillin, the main known flavor compound,but also by a large number of other flavor compounds, many of which have
not yet been identified. Each type of vanilla has its own characteristics. In
order to determine the origin of vanilla, and for quality control purposes,
laboratories typically rely on Headspace or Thermal Desorption techniques
used in combination with GC/MS. This article describes interesting aspectsof the analysis of vanilla pods and vanilla extracts and reports on newly
discovered vanilla flavor compounds.

No other spice or flavor ingredient is as
widely used and appreciated as vanilla. In
spite of the expression “plain vanilla”, vanillais the king of flavors and it is not only usedin food and sweets, but also in perfumes, cosmetics and pharmaceutical products. Vanillais said to soothe the soul and reinvigorate thebody explaining its widespread use in aroma
therapy. Recent research reports indicate thatvanillin, the key flavor compound in vanilla,
may have cancer inhibiting properties. The
ancient Aztecs considered vanilla the “food of
the gods”, today it is among the most important and most widely loved spices in the world.
One of the reasons why vanilla is so widelyused is because key flavor compounds can besynthesized from cheap ingredients such as
paper pulp. Real vanilla, on the other hand,is very expensive with prices reaching as highas $ 80,000 per ton [1]. However, the amountof naturally grown and fermented vanilladoes not even come close to satisfying world
demand. With prices of the real thing so high,quality and authenticity, i.e. origin and potential adulteration must be carefully monitored.
Traditionally this job has been the responsibility of taste and flavor panels, but increasingly, chemical analysis has come into play.
Producers of food and other products that
contain vanillin as a flavor ingredient can relyon standard chemical analysis techniques for
quality control. The analysis of real vanilla,
however, can present a challenge. Due to thecomplexity of the chemistry and fermentation
processes involved, it typically requires morethan one analytical technique to provide reliable results. Stephen J.Toth, Ph.D. knows thisbetter than most people – and from firsthandexperience. Over the course of his work on his
doctoral dissertation: “Comparison and integration of analytical methods for the characterization of vanilla chemistry“ [1] at theState University of New Jersey, the scientist
strove to find a unified analytical method for

GERSTEL Solutions Worldwide – No. 14

the determination of volatile and semi-volatile organic compounds (VOCs and SVOCs)
in vanilla pods and extracts. The solution hefound was to use two techniques: 1) Liquid
chromatography (LC); and 2) Gas chromatography (GC) in combination with headspace and thermal desorption.

Determination of vanilla flavor
components
For the determination of semi-volatile ornon-volatile flavor compounds in vanilla, suchas vanillin, 4-hydroxy benzaldehyde, vanillicacid and 4-hydroxy benzoic acid, HPLC is
the separation method of choice. In his dissertation, Dr. Toth listed as reference a large
number of journal papers based on HPLC
methods, mainly for the analysis of vanilla
extracts, which served as reference points forhis method development work. The separation was ultimately performed using much
shorter UHPLC columns, which are based on
smaller particle size packing. As a result, theanalysis time for vanillin and related phenolic
compounds was cut from 13.45 to 1.86 minutes, a seven-fold improvement in throughput. In addition, the amount of acetonitrile
used was reduced by about two thirds. Still, for
the wider range of more volatile compounds
that are responsible for the bulk of vanilla’s
flavor, including many unknown compounds,
HPLC is not the technique of choice. For
these, gas chromatography combined withmass spectrometry (GC/MS) must be used
– preferably combined with headspace (HS),HS-SPME or other sample introduction andanalyte concentration techniques.
To determine which technique was best
suited for the analysis, the scientist compared results from the following: Solid Phase
Micro Extraction (SPME), Headspace Sorptive Extraction (HSSE) using the GERSTELTwister, and direct thermal desorption of deep19

A brief introduction to vanillaVanilla belongs to the orchid family.
Vanilla grows in tropical regions andthere are more than one hundred different species of vanilla, only two of which
are commercially relevant. Vanilla Planifolia
and Vanilla Tahitensis are the only orchid
species to produce a commercially usefulfruit. Originally from Mexico, vanilla is mainly
grown in Indonesia and Madagascar today.
Most of us wouldn’t recognize a vanilla plantor its fruit. The green odorless bitter tasting
vanilla fruit is nothing like the vanilla pods
we are exposed to in high-end food stores.
Over the course of a five month complex
and intricate fermentation process, the fruitis transformed into dark brown, extremely
tasty and flavorful vanilla pods. Amongother compounds, vanillin is formed byhydrolysis of glucovanillin. Controlling thefermentation process is key to producing
the right flavor profile, the right taste andan overall quality product. The fermented
vanilla is marketed in two different forms:
As an extract in alcohol solution (extraction
grade) and unprocessed (gourmet grade).
A high quality vanilla pod is characterizedby highly pleasant flavor and taste, moisture
content of 18 – 25 %, and dark chocolatelike color. The surface must be oily and freefrom defects and mold. The vanillin content
should be greater than 2 % as a key indicator to high quality, but this is not always
the decisive factor: Many vanilla types
have excellent overall taste and flavor eventhough their vanillin content is lower than 2%. This leads to the conclusion that otherflavor compounds contribute significantly tothe overall flavor and taste [1].

Headspace Sorptive
Extraction (HSSE)
Headspace Stir Bar Sorptive
Extraction (HSSE) is an addTDU liner
on development of Stir Bar
TDU
Sorptive Extraction (SBSE).
HSSE has been used for flaTwister
vor analysis work involving alarge number of different products [2] and it is based on using
Liner in liner
the GERSTEL Twister as aconnection
passive sampler in the headspace of a sample. In standard
CIS liner
SBSE, the Twister extracts
analytes from a liquid sample while actively stirring it;
CIS
in HSSE, the Twister is posiGC
tioned in the headspace above
columnthe sample from which it indirectly extracts volatile compounds. After equilibrium has
been established between theliquid, headspace and Twistersorbent phases, the Twister is
Thermal desorption of the Twister in the TDU followed by cryoforemoved and transferred to thecusing of the analytes in the CIS and temperature programmed
MultiPurpose Sampler (MPS)transfer to the GC column.
sample tray. From there, it isautomatically transferred to
the Thermal Desorption Unitfrozen ground vanilla pods. The flavor com(TDU) for thermal desorption and transfer
pound profiles of two bourbon vanilla podsof the analytes to the GC column. Due towere investigated. One was a perfect pod of
the much higher phase volume of the sorgood quality and the other had been rejected
bent (Twister: 125µL / SPME: 0.5 µL), theby a customer as unacceptable due to an “alcoHSSE technique is exceptionally efficient inhol” off flavor, thought to be caused by bacextracting medium to non-polar compounds.
terial degradation products, including guaiaAn EG-Silicone Twister is now also availcol formed by degradation of vanillin underable, which enables efficient extraction ofanaerobic conditions.
polar compounds such as phenols and otheraromatic alcohols. Using HSSE, a total of 19
previously unknown compounds were foundSolid Phase Micro Extraction
and identified.

(SPME)

In his literature searches, Toth found a large
number of articles reporting on the analysis
of vanilla extracts based on the SPME technique, especially when it came to polar compounds in alcohol extracts. He ascribed thisto the multitude of SPME phases available,ease of automation as well as fast desorptionof the concentrated analytes in the GC inlet.
SPME has proven itself to be selective, efficient and highly useful in practical analysis
work when it comes to extraction of volatilecompounds from the headspace phase. The
main shortcoming of SPME lies in the limited phase volume (0.5 µL), limiting the sorptive capacity and thus the sensitivity of thetechnique. Nevertheless, SPME was used to
extract 35 compounds from vanilla, including contaminants from packaging as well aseight new compounds, which hadn’t previously been identified.

20

Dynamic Headspace (DHS)
Dynamic Headspace, as the name implies, is
not a static technique relying on equilibrium
between phases, but rather a dynamic techniquerelying on driving analytes out of the headspace,
and thus indirectly out of the sample, using carrier gas. Purged analytes are trapped and concentrated on an adsorbent trap at the purge outlet. In this case, Tenax TA adsorbent was usedin the trap. As opposed to SPME and HSSE,
Tenax TA showed no specific affinity for individual compound classes, but rather concentrated analytes over a wide polarity range. However, Toth found that compounds with threeor less carbon atoms generally were not as efficiently trapped. Using DHS, he was able toextract and identify 24 compounds from thehigh quality vanilla, including 10 compounds
that had not yet been found in vanilla.

GERSTEL Solutions Worldwide – No. 14

Direct Thermal Desorption (DTD)
For direct thermal desorption, the sample wasplaced in a suitable inert glass tube (TDUliner) between two plugs of glass wool andplaced in the MPS sample tray. The TDU
liner was transferred to the TDU by the MPS.
Thermal desorption/thermal extraction was
performed using the following temperature program: Initial temperature 30 °C –60 °C/min – 275 °C.
Desorbed analytes were trapped in the
GERSTEL Cooled Injection System (CIS)
PTV-type inlet and transferred to the GC
column using a temperature program. Direct
thermal desorption using the TDU-GC/
MS method enabled Toth to identify 74
compounds in both the “good” and “bad”
vanilla samples. After further work, he foundanother 30 flavor compounds that had not
yet been reported in vanilla pods. The mostremarkable difference between the “good”
and “bad” vanilla was indeed in the vanillin
concentration: The “good” sample contained
1.2 %, the “bad” only 0.1 %. In the accepted“good” vanilla, Toth further found high concentrations of acetic acid, 2-methoxy phenol,hydroxyl dihydromaltol, 5-(hydroxymethyl)furan-2-carbaldehyde, 4-hydroxybenzaldehyde, hexadecanoic acid and 1-octadecanol.
Toth reports: “The list of compounds identified in the good bourbon vanilla pod correlates well with previously reported data fromthe literature”. Compounds, which he was thefirst to report in vanilla pods, included acetone,
2-methyl propanal, 3-hydroxy-3-pentene-2one, 2(5H)-furanone, 2-hydroxy-2-cyclopentene-1-one, 4-hydroxy-5-methyl-3(2H)furanone, furan-2-carboxylic acid (2-furoicacid), lilial acid, 4-(4-hydroxyphenyl)-3-bu-

ten-2-one, 4-(4-hydroxy-3-methoxyphenyl)3-butene-2-one (E), two isomers of vanillin glyceryl acetal, 1-octadecanol, ethyl heptadecanoate, ethyl octadecanoate, z-12-pentacosene and z-14-nonacosene.
In the “bad” vanilla pods, Toth identified
high concentrations of compounds such as:
2-methoxy phenol, 2-methoxy-4-methylphenol, hexadecanoic acid and 1-octadecanol.
Among the biggest differences uncovered by direct thermal desorption of the“good” and “bad” bourbon vanilla pods werethe loss of vanillin, increased concentrations of 2-methoxy-4-methyl phenol and2-methoxy phenol as well as the loss ofhydroxyl dihydro maltol and hydroxymethyl
furfural, Toth writes. Unlike the results fromthe analyses performed by SPME, HSSE, and
DHS, the direct thermal desorption results
didn’t include fusel alcohols, even though therejected vanilla products did contain thesecompounds at different concentration levels.
The presence of fusel alcohols is in itself astrong indication that bacterial degradation
has taken place.
Even though each technique had clear
strengths and weakness, they complemented
each other well. When combined, they serve
to provide a comprehensive picture of thediverse chemical landscape of a complex product such as vanilla. As a flavor analyst, being
able to draw on the whole range of headspacetechniques: SPME, HSSE, Dynamic Headspace (DHS) and direct thermal desorption(DTD) provides a critical advantage in termsof being able to cover all analytes while using
solvent-free and highly sensitive extraction
techniques. The flavor chemist’s efficiency aswell as the resulting data is greatly improved
when these techniques are automated, andthe ultimate enhancement is provided by the
GERSTEL MultiPurpose Sampler (MPS),
which automates all of these techniques on asingle platform.

Sources

Total Ion Chromatogram of a Tahitian Vanilla resulting from DTD-(TDU)-GC/MS analysis. The following
compounds were found: 2-(5H)-furanone, 2-hydroxy-2-cyclopentene-1-one, 2-acetyl-2-hydroxy-gammabutyrolactone, 3,5-dihydroxy-2-methylpyrane-4-one, 3-phenyl-2-propenoic acid, 4-hydroxy-2-methoxy
cinnamic aldehyde, 4-(4-hydroxy-3-methoxyphenyl)-3-butene-2-one (E), 2 isomers of 2-(4-hydroxy3-methoxyphenyl)-1,3-dioxane-5-ol, kauren and z-12-pentacosene.

GERSTEL Solutions Worldwide – No. 14[1] Stephen J. Toth: Comparison and integration of analytical methods for thecharacterization of vanilla chemistry.
Proquest, Umi Dissertation Publishing
2012[2] Flavor, Fragrance, and Odor Analysis,
2. Edition, CRC Press, Taylor & Francis
Group 201221

Toxicology

Pain Management (PM) drugs in urine– can the analysis be fully automated?
A lot of useful information can be extracted from urine regarding medication taken and metabolism of drugs in
the human body. The technique most often used is enzymatic hydrolysis of metabolites followed by SPE clean-up
and HPLC-MS/MS determination. When performed manually, the sample preparation is labor intensive and time
consuming. If hydrolysis and clean-up are automated and dispersive SPE (DPX) is chosen instead of standard SPE,the process is accelerated as shown in the following example.

22

GERSTEL Solutions Worldwide – No. 14

The amount of work required
pletely automated solution:is significant whenever toxHydrolysis of the conjugates
icologists and clinical chemists
formed during drug metabowant to determine the concenlism in order to quantify the
tration of active pharmaceutitotal amount of drug taken.
cal ingredients (APIs) - or of
They used a Dual Head vertheir metabolites - in urine.
sion of the GERSTEL MultiOne of the time-intensive
Purpose Sampler (MPS). This
steps required is the hydrolysis
system uses one head to perof conjugated analytes to theirform the DPX based sample
original form. This transforextraction and clean-up, whilemation is typically performed
the second head performs theenz ymatically, for examinjection into the LC/MS sysple, using β-glucuronidasetem. The Dual Head configu(GUSB). To ensure that theration enables optimum samhydrolysis reaction is comple preparation and injection
plete and reproducible, contime of less than 7.5 mintrol, monitoring and optimiutes per sample following thezation of various parameters
automated hydrolysis [1]. Fredis needed. Among these are
Foster arrives at the following
the pH value, the temperatureconclusion after finishing andand the length of the hydrolyevaluating the project: “We canGraphical representation of the automated DPX urine cleanup process.
sis period, which varies fromnow offer a combined autoenzyme to enzyme. These facmated system, which performs
tors have a profound impact onhigh throughput urine analydisposable pipette tip by fixed screens at the sis, including enzymatic hydrolysis, extraction
the quality of the analysis results. The same
top and bottom of the tip. Sample is aspirated and LC-MS/MS determination.” The manis the case for matrix compounds, present in
into the DPX tip only, eliminating both the ual work required is minimal: A 1 mL sample
significant amounts in urine. In order to reachrisk of sample to sample carry-over and the of urine is manually pipetted into an autosthe very low limits of detection required forneed for extensive washing of syringes used ampler vial. The vial is capped and placed inmonitoring of API residues and metabolites,in systems based on standard SPE cartridges. the autosampler tray. All further steps in theinterfering compounds from the matrix, orWith the sample-powder mix inside the tip, sample preparation and introduction process
those generated during the hydrolysis proair is aspirated leading to turbulent mixing of are controlled by the MAESTRO software,
cess, must be eliminated.Typically, this is done
the phases resulting in highly efficient extrac- fully automated.
using a suitable extraction technique such astion. Typically, the remaining sample is dissolid phase extraction (SPE), which is also
Separation of the target analytes: Morcharged and the concentrated analytes eluted phine and its hydrolyzed conjugate morwidely used in forensic analysis. Performingwith a small volume of solvent into an empty phine-3-glucoronide, as well as oxazestandard, cartridge based SPE has a number
clean autosampler vial followed by LC/MS or pam (oxazepamglucoronide) and oxyof drawbacks when used for this type analyGC/MS determination. Key differentiators of morphone (oxymorphoneglucoronide)sis. It uses relatively large amounts of costly
DPX are: Fast extraction; high recovery rates, was performed using the following
solvent, the solvent and sample elution needand the very small amounts of solvent used. column: Poroshell 120, EC-C18
to be precisely controlled, increasing the risk
Reducing solvent use in the laboratory brings (3.0x50 mm, 2.7 µm). The detecof error and making the process slow, and themany benefits ranging from improved work tion system used was an Agilent
resulting sample dilution increases detection
environment to reduced cost for purchasing 6460 Triple Quadrupole MSlimits.
and disposing of often toxic solvents. “A fur- with Jetstream electrosprayther key benefit is the ease of automation”, source. Analyte quantifiAttractive alternative
says Fred D. Foster, Application Scientist at cation was performed
to standard SPE
GERSTEL, Inc. near Baltimore, Maryland, using deuterated
When he was initially searching for a more“and this includes automated introduction to isotopes.
efficient solid phase extraction method, Wilthe LC/MS analysis system.”liam E. Brewer from the University of South
Carolina developed Disposable Pipette
Wanted: Complete automation
Extraction (DPX), a dispersive SPE (dSPE)At GERSTEL, Inc. Fred Foster and his coltechnique. Instead of the sorbent being presleagues have long been focused on automatent as a packed bed, in DPX the sorbent is
ing the last key step on the way to a coma loose powder contained inside a standard

GERSTEL Solutions Worldwide – No. 14

23

Putting the automated system
to the test
MAESTRO Prep Sequence Scheduler for the optimization of the automated β-glucuronidase hydrolysis
procedure using 0.66M acetate buffers at different pH.

Calibration curves for Morphine and Oxazepam resulting from automated hydrolysis of Morphine-3-glucuronide and Oxazepam glucuronide, DPX clean-up, and LC/MS/MS determination.

Stacked view of Oxazepam peaks resulting from pH variation experiments. No significant difference was found when the 0.66M acetate buffer pH was varied between 4.0 (A), 4.5 (B), and
5.0 (C).

24

In order to establish that the enzymatichydrolysis of urine samples using the typical β-glucuronidase procedure could be automated successfully, triplicate urine samples
spiked at a concentration of 1000ng/mL with
Oxazepam glucuronide were hydrolyzed bothmanually and using the automated hydrolysis
procedure. Following hydrolysis, all samples
were extracted and analyzed using the DPXLC/MS/MS procedure. The results for the
manual and automated procedures matchedwell with only a 4% difference between thetwo sets of results.
The automated β-glucuronidase hydrolysis procedure was then compared to a typicalacid hydrolysis procedure in which equal parts
concentrated hydrochloric acid were added
to spiked urine samples containing either
Oxymorphone-3-β-D-glucuronide or Oxazepam glucuronide at 1000ng/mL and thenallowed to incubate at 100o C for 90 minutes.
After cooling to room temperature, the pH ofthese samples was adjusted to 4 using diluteammonium hydroxide prior to extraction
along with the automated β-glucuronidasehydrolyzed sample group using the automated
DPX-LC/MS/MS procedure. The final volumes of the samples being compared were
adjusted prior to extraction, in order to ensurethat the final concentrations would be equivalent. The native Oxymorphone and Oxazepam concentrations were found to be lower
when the acid hydrolysis procedure was used.
Since no response was observed when monitoring for Oxymorphone-3-β-D-glucuronide
and Oxazepam glucuronide, it is believed thatthe lack of response may be due to either further degradation of the native analytes orinterference with their ionization rather thanincomplete hydrolysis when using the acidhydrolysis procedure.
One of the benefits of automation is theease with which designed experiments canbe performed in order to quickly optimize orcompare various steps involved in the manual procedures being automated. An examination of the automated β-glucuronidasehydrolysis procedure using 0.66M acetate
buffers of different pH was easily set up in
MAESTRO and performed by the MPS, theMAESTRO Prep Sequence Scheduler for theexperiment is shown in the box on this page.
A stacked view of mass chromatograms resulting from Oxazepam determinations is also
shown; as can be seen, no significant difference was found when changing the pH of the
0.66 M acetate buffer from 4.0 (A) to 4.5 (B)or to 5.0 (C).
In order to ensure that the automatedhydrolysis procedure was complete and could

GERSTEL Solutions Worldwide – No. 14

MultiPurpose Sampler (MPS XL) with GERSTEL DPX Option used for the automated hydrolysis and DPX-LC/MS/MS method shown
mounted on top of an Agilent 6460 LC-MS/MS system without adding to the lab bench footprint.

be used within an automated DPX-LC/MS/
MS method for the quantitation of analytes,
standards and QC samples in urine were prepared using the glucuronide conjugated anaAutomation
Automated DPX Prep Sequence(Cleanup procedure).
1. Aspirate 750 µL acetonitrile from the fast
wash station using the 2.5 mL DPX syringe.
2. Pick up DPX tip (patented sorbent: DPX-RPS) from the DPX Tray.
3. Add 500 µL acetonitrile through the DPX tip
into the urine sample on the MPS tray.
4. Wait for 6 seconds to ensure complete wetting of the DPX sorbent with acetonitrile.
5. Aspirate the complete sample; mix sample
and sorbent turbulently by aspirating 1400
µL of air into the DPX tip.

lytes Morphine-3-β-D-glucuronide or Oxazepam glucuronide and then both the native
and the conjugated forms of the analytes weremonitored using the previously described LC/
MS/MS method. In all cases, the absence ofdetected response for the glucuronide conjugated analytes proved the successful completehydrolysis of the urine sample being analyzed.
The accuracy and precision achieved forMorphine and Oxazepam using the complete automated hydrolysis-DPX- LC/MS/
MS method were determined by extracting
replicate (n=6) QC samples at 75 ng/mL concentrations. Accuracy data averaged 102 % forMorphine and 96.3 % for Oxazepam and theprecision (% CV) was 3.52 % for Morphineand 4.70 % for Oxazepam.
Representative calibration curves forMorphine and Oxazepam are shown on

Method parameters
Analysis conditions LC
Mobile Phase:

A: 5 mM ammonium
formate in water with
0.05% formic acid
B: 0.05% formic acidin methanol
Isocratic, 50:50 (A:B)
at a flow of
0.300 mL/min
10 Minutes
2 µL (loop overflow)
55 °C

6. After an equilibration period of 5 sec: Discharge the content of the DPX tip back into
the vial on the tray.

LC Pump conditions:

7. Bring the DPX tip to the “PipWaste“ positionand discard it.

Run time:
Injection volume:
Column temperature:

8. Transfer 250 µL of the upper liquid layer inthe vial to a clean 2 mL autosampler vial;
Cap the vial.

Analysis conditions MS
Operating mode:
Electrospray, positivemode + Agilent Jet
stream
Gas temperature:
350 °CGas flow (N2):
5 L/min
Nebulizer gas pressure: 35 psi
Sheath gas temperature: 250 °C
Sheath gas flow:
11 L/minCapillary voltage:
4000 V
Nozzle voltage:
500 V

9. Dilute the extract by adding 250 µL of formic
acid 0.05% in water into the sample vial.
10. Inject 25 µL of the diluted sample into the
HPLC injection valve.

GERSTEL Solutions Worldwide – No. 14

the preceding page. Regression analysis forboth analytes resulted in R2 values of 0.99
or greater. The complete automated process
resulted in linear calibration curves with R2values 0.99 or greater achieved for glucuronide
conjugated analytes with LOQs of 1 ng/mL
for both Morphine and Oxazepam.

Conclusion and outlook
The automated system performed well; Fred
Foster and his colleagues were able to demonstrate that the described enzymatic hydrolysis and subsequent DPX cleanup methods
were successfully automated for glucuronide
conjugated analytes in urine using the dual
head GERSTEL MPS autosampler and sample preparation robot. Analytes were rapidly
and reproducibly isolated from hydrolyzed
urine samples and subsequently determinedusing the described automated DPX cleanup
procedure coupled with introduction to
LC/MS/MS based on the Agilent 6460 Triple Quadrupole Mass Spectrometer.
The automated hydrolysis method
described here can be combined with othersample preparation techniques such as SPE,liquid/liquid extraction, centrifugation, andevaporative concentration etc.
This research is currently being expanded
to facilitate the hydrolysis procedure in smaller
liquid handling containers, such as microtiterand deepwell plates, with automated heating
and shaking included. In addition to this, otherenzymatic reaction possibilities (e.g., Tryptic
Digestion) will be evaluated for full automation with the GERSTEL MPS system.

Literature[1] F. D. Foster, J. R. Stuff, E. A. Pfannkoch,
W. E. Brewer. Automated Hydrolysis, DPX
Extraction and LC/MS/MS Analysis of PainManagement Drugs from Urine, GERSTELAppNote 1/2014

25

Vortex/Shaker option for the MPS

The Vortex
and shaker
option (mVORX)
for the GERSTELMultiPurpose
Sampler (MPS) performs vortexing andagitation at up to 3,000
rpm, speeding up sample preparation steps
such as liquid-liquid extraction, dissolution, and homogenization. Them
VORX performs efficient simultaneous vortex mixing of various vial
sizes and 96 well plates. The MPS automatically loads samples placedin 2, 4 and 10 mL vials. A heating option for 96 well micro-titer plates
is also available. Mixing movements are finely controlled using linear
motors and are restricted to the horizontal plane, allowing even themost sensitive samples to be mixed efficiently without over-agitation.
Automated operation under MAESTRO software control enables reliable and flexible sample processing, the PrepAhead functionality
ensures best possible productivity and throughput.

Automatedspiking ofstandard tubes
for thermal
desorption(TDS/TDU)The GERSTEL Tube Spiking System(TSS) enables automated generationof standard tubes for Thermal Desorption analysis. Using the GERSTEL MultiPurpose
Sampler (MPS), adsorbent tubes are spiked
with a user defined volume of liquid standard
and the solvent purged with a defined flow ofcarrier gas as required for method calibrationand validation according to international standard methods. Multiple TSS units can be mounted on the MPS for higher throughput.

Automated Pyrolysis Option
The GERSTEL PYRO module for the
GERSTEL Thermal Desorption Unit
(TDU) is available for automatedpyrolysis of 14, 98, or 196 samples incombination with the GERSTEL MultiPurpose Sampler (MPS). PYRO
enables pyrolysis
of solids andliquids at up to
1000 °C. PYRO
performs the following
techniques: Standard
pulsed pyrolysis; fractionated pyrolysis; solvent
venting combined with
pyrolysis; Evolved Gas
Analysis (EGA); derivatization combined with pyrolysis. Thermal desorption prior to pyrolysis enables thedetermination of volatile compounds and provides a clean pyrolysis chromatogram that is easy to interpret. Solvent venting can remove water prior
to pyrolysis. PYRO is controlled using the MAESTRO software or integrated
with the GC/MS software.

Multi-Position Evaporation
Station (mVAP)
A six-position
evaporation station (mVAP) isavailable for the
G E R S T E L
MultiPurpose
Sampler (MPS).
Samples are concentrated at user
defined temperatureand vacuum, enabling
significantly improved
limits of detection. The
MPS
automaticallyloads samples placed in 2 mL, 4 mL or 10 mL vials. Solvent exchange toa GC or HPLC compatible solvent can be performed for improved chromatography. mVAP can be used in a stand-alone WorkStation in combination
with SPE, Dispersive SPE (DPX) or liquid/liquid extraction to evaporate
solvent from extracts. Configuration for injection into GC/MS or LC/MS isalso possible. Every step is controlled by mouse-click using the MAESTROPrepBuilder. Just one method and/or one sequence table is needed forthe entire process including GC/MS or LC/MS analysis.

Automated Filtration Option

Microwave Prep

The Filtration option for the GERSTEL MultiPurpose Sampler (MPS)
enables efficient automated clean-up of up to 98 samples or extracts
using standard filters with Luer® fittings. Filtration can be combinedwith other sample preparation steps and/or with introduction to an
LC-MS/MS- or GC/MS system. Without adding to the laboratory workload, filtration can significantly improve the reliability of the analysis
and of the analysis system. Depending on the filter size used, up to
98 filters can be placed in the MPS
tray. Liquid transfer is performed
with exact control of flow and volume for highly reproducible results.
MAESTRO software offers simple
and efficient control by mouse-click,requiring no macro programming.

The sample preparation portfolio of the GERSTEL MultiPurpose Sampler
MPS has been expanded to include advanced microwave technology foraccelerated solvent extraction and rapid chemical reactions. A recent application example isautomated saponification of fats in foodin combination withesterification
andFAME analysis by GC.
The MPS including
microwave can becoupled directly toa GC/MS or LC/MS
system or used instand-alone mode.

26

GERSTEL Solutions Worldwide – No. 14

TDS 3C: Thermal desorption
the cooler way
The
GERSTEL
Thermal Desorption System (TDS)
is an accepted
standard systemused world-wideby leading companies to perform
standardized test
methods in theautomotive, semiconductor
andToy Safety fields
and widely in thefood, flavor andfragrance
andpackaging industries among many others. The TDS 3C represents
the latest addition to the extensive GERSTEL thermal desorption
product line. It can be cooled using a cryostatic cooling device
for simpler handling when only moderate trap temperatures are
required.

GERSTEL MPS
robotic PRO
MPS robotic PRO, the newest memberof the GERSTEL MultiPurpose Sampler (MPS) family, is a highly efficient
GC/MS autosampler, optimized tohold a significantly larger number ofsamples for higher throughput. MPS
robotic PRO provides fast, reliable andhighly accurate processing of complex
tasks. Tools can be exchanged automatically, enabling automated change
between Liquid Injection, Headspace, and SPME. MPS robotic PRO
is controlled by the GERSTEL
MAESTRO software enabling PrepAhead productivity under integrated
GC/MS system control. Priority samples can be added at any
time without stopping the ongoinganalysis sequence. Fast and errorfree set-up of the daily analysis
sequence is performed using theintelligent sequence editor functions or by importing it directlyfrom your LIMS system.

Online SPE system: SPExos
GERSTEL SPEXOS performs online SPE with automated cartridge exchange.
SPEXOS uses small cartridges, which are inserted directly into the HPLC
mobile phase for elution resulting in quantitative transfer of analytes to the HPLC column. Much smaller sample sizes can beused to reach required detection limits andmuch less solvent is needed, for example,
for analyte elution, increasing the concentration factor and reducing both cost andenvironmental impact.
In combination with the GERSTEL MPS and
MAESTRO software, liquid sample preparation, clean-up and LC-MS/MS analysis canbe performed in one integrated system
operated with one integrated sequencetable. The Prep-Ahead functionality withmulti-sample overlap ensures maximum
efficiency and throughput for thecomplete system, including the
LC-MS/MS.

More than one Twister needed:
Move up to the Twicester
A cooperation between GERSTEL K.K., Tokyo, Japan and the ResearchInstitute for Chromatography (RIC) in Belgium has produced theMulti-Stir Bar Sorptive Extraction (mSBSE) technique. mSBSE has
been used successfully for the determination of a variety of flavor
compounds in aqueous matrices suchas green tea. Key elements of the technique are to use PDMS and EG-Silicone
Twisters simultaneously in one sample
vial and to desorb these together in the
TDU for combined and comprehensive
analysis. While the PDMS Twister stirs
the sample and extracts non polar to
medium polarity analytes, one or more
EG-Silicone Twisters are kept stationary
at the vial wall by a special holder. The
EG-Silicone Twisters extract polar analytes, especially compounds with -OH
or -NH functional groups. The Twicester
technique enables the determinationof a wide range of analytes of different
polarities in one extraction step and one
GC/MS run.

Controller C200
The compact GERSTEL C200 controller offers heating and cooling control
including temperature ramps for GERSTEL modules such as the Cooled
Injection System (CIS) PTV-type GC inlet, the Cryo Trap System (CTS) for
multidimensional GC, the GERSTEL Thermal Extractor (TE 2), as well as themarket leading GERSTEL Olfactory Detection Port (ODP). Multiple cycles
with temperature ramps can be performed for highly efficient conditioningof up to 50 GERSTEL Twisters or 10 adsorbent tubes in the GERSTEL Tube
Conditioner (TC). The C200 can be
operated standalone using the scroll
wheel at the front or through the
GERSTEL MAESTRO software. The
system status is clearly indicatedthrough the colored light in the scroll
wheel.

GERSTEL Solutions Worldwide – No. 14

27

Preview - coming up in the next issue

www.gerstel.com

GERSTEL online: Information on products,
applications, events and downloads, aswell as e-Newsletter subscription andgeneral information about GERSTEL andour customer focused solutions:www.gerstel.com and www.gerstelus.com

NEW

Imprint
Published by
GERSTEL GmbH & Co. KG
Eberhard-Gerstel-Platz 145473 Mülheim an der Ruhr, Germany

Pleasant flavors and fragrances applied to
products can improve consumer acceptanceand increase consumer binding, improving sales.
This makes it easy to explain the increased useof flavors and fragrances in consumer products,
food and beverages. When applied to carinteriors, fragrances can enhance the feel of thedrive supposedly making it a better, maybe evena more relaxed experience. Little wonder thenthat industry expends considerable amountsof resources to identify flavor compounds withgood properties and to extract and reproduce
them in the laboratory. It is equally clear thatthe human nose plays a central role in the
final assessment of the quality of flavors andfragrances for any practical application. To matchthe highly sensitive human nose, highly sensitive
chemical analysis is required in this field in orderto document and control the quality of odorized
products. Especially gas chromatography incombination with mass selective detection andolfactory detection GC-MS/ODP is a key tool.
How else would one be able to sniff out andprovide reliable quantitative results on odors
and their sources? Incidentally, this applies not
only to pleasant odors, but certainly equally to
off-odors, compounds that smell unpleasant oreven stink making consumers reject a product.
In the next GERSTEL Solutions worldwide
Magazine, we shed light on flavor and fragrance
analysis as well as on the determination of offodors. In addition, we report on concentration
techniques used to determine pesticides without
the risk of carry-over as well as on the efficientand sensitive determination of mycotoxins infood.

Editorial director
Guido Deussing
Uhlandstrasse 16
41464 Neuss, Germany
guido.deussing@pressetextkom.de
Scientific advisory board
Dr. Eike Kleine-Benne
eike_kleine-benne@gerstel.de
Dr. Oliver Lerch
oliver_lerch@gerstel.de
Dr. Malte Reimold
malte_reimold@gerstel.de
Translation and editing
Kaj Petersen
kaj_petersen@gerstel.com
DesignPaura Design, Hagen, Germany
www.paura.de

ISSN 1619-0076 · 05 / 2014

Focus on flavor & fragrance analysis and product safety

www.gerstel.com
www.gerstel.com

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