2018 - Adissu Asfaw Thesis

KU Leuven
Biomedical Sciences Group

Faculty of Pharmaceutical Sciences
Department of pharmaceutical and pharmacological sciences
NEW SAMPLING TECHNIQUES IN GAS CHROMATOGRAPHY

APPLIED TO A PHARMACEUTICAL CONTEXT
Adissu Alemayehu Asfaw
Jury:
Promoter: Prof. Dr. Erwin Adams Dissertation presented in

partial fulfilment of the
Co-promoter: Prof. Dr. Ann Van Schepdael requirements for the degree
Chair: Prof. Dr. Pieter Annaert of Doctor in Pharmaceutical
Sciences
Jury members: Prof. Dr. Jef Rozenski
Prof. Dr. Eva Cuypers
Prof. Dr. Kristof Demeestere
Dr. An Adams
September 2018
0
i
Curriculum Vitae
Adissu Alemayehu Asfaw was born on 7th of October, 1981 in Mekelle, Ethiopia. He completed
his secondary school in 2000 at Atse Yohannes high school in Mekelle, Ethiopia. He graduated
as bachelor degree in Pharmacy and MSc in pharmaceutical chemistry both at Addis Ababa
University, Ethiopia in 2006 and 2009 respectively. Upon completion of his master program,
he then started working in Mekelle University, Ethiopia as academic staff and reach level of
assistance professor. In 2014, he started his PhD research at the Pharmaceutical Analysis
laboratory of KU Leuven in Belgium under the supervision of Prof. Dr. Erwin Adams and co-
promoter Prof. Dr. Ann Van Schepdael. The focus of his research was on new sampling
techniques in GC applied to a pharmaceutical context.
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Acknowledgements
Four years of PhD work would not have been accomplished without the help and support of
countless people.
First, I would like to express my sincere gratitude to my promoter Prof. Dr. Erwin Adams for
giving me the opportunity to start my PhD studies in the Pharmaceutical Analysis lab. You
always made yourself available whenever needed. Thank you for your enthusiastic supervision
and all the help and support I received from you throughout my PhD work. I am also grateful
to my co-promoter Prof. Dr. Ann Van Schepdael as head of the laboratory allowing me to start
my PhD and for her support, kindness and hospitality during my study. I would like to thank
Prof. Dr. Deirdre Cabooter as well for being a nice colleague and I wish you all the best with
your future research. I really like to thank the jury members (Prof. Dr. Pieter Annaert, Prof. Dr.
Eva Cuypers, Prof. Dr Jef Rozenski, Prof. Dr. K. Demeestere, Dr. An Adams) for reading my
thesis manuscript and for their suggestions on how to improve it as well as for their comments
while being a part of my thesis advisory committee.
I would like to express my heartfelt thanks to Kris Wolfs for providing me the theoretical and
technical explanation during the entire period of my stay in the lab, helping me to overcome
many problems around my research projects. Moreover, I always enjoyed our chats about all
kinds of topics. Special thanks to Getu, who helped me a lot from the beginning of the PhD
application process and he made my first time in Leuven very simple. I would like to thank
Zelalem (Zola) for his kind friendliness and advice at the beginning of my study.
I gratefully acknowledge the Interfaculty Council for Development Co-operation (IRO), KU

Leuven, for granting me the scholarship and Mekelle University for giving me the study leave
to pursue my PhD studies
During my PhD study, I spend my time with many different people and would like to thank all
of them for creating a pleasant environment to work in: Niels, Prasanta, Juan, Bart, Stijn,
Pranov, Karina, Huiying, Glenn, Stani, Lynch, Marwa, Shengyun, Matthias, Jasper, Stephanie,
Micheal, Maxim, Yaxin, Derick, Mélanie, Asmin and Sonia. During my time, I had the
opportunity to supervise master’s students: Chiara and Matthias. I wish both of you a very best
of luck for your future endeavors.
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A special attention definitely goes to my mam, Lemelmye. I always take you as a pillar of
strength and this was your dream, thank you for what you did till the end of your life.
My stay in Leuven was rewarding; I never missed the Ethiopian connection. In this regard, I
thank all the Ethiopian community in Leuven. Also being a part of ESAL, the football every
Sunday and the running activities were indeed the best thing I enjoyed in Leuven with the
Ethiopian community.
Last but not least, of course, is my rock, my inspiration, my world: without my gorgeous and
lovely wife, Merehawit, none of this would ever have been possible. To have a woman of such
patience and strength, put her own interest on hold to support the dreams of the man she has
married is a powerful thing, and I have striven every day to live up to that. Mar, your daily
support, understanding and encouragement helped me through the difficult moments. I would
like to thank you for everything you did for me. I appreciate my little girl Naomi, for being the
spicy of our family; I am blessed to be able to share it with such a magnificent family.
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Publications
Publications in Scientific Journals
[1] A.A. Asfaw, K. Wolfs, A. Van Schepdael, E. Adams, Determination of residual
dimethylsulphoxide in drug loaded gelatin using thermal desorber – gas chromatography,
J. Pharm. Biomed. Anal. 153 (2018) 193–198.
[2] A.A. Asfaw, K. Wolfs, A. Van Schepdael, E. Adams, Thermal desorption—gas
chromatographic methodology for the determination of residual solvents in mesoporous
silica, J. Chromatogr. A 1500 (2017) 160-166.
[3] A.A. Asfaw, M. Van Der Veken, K. Wolfs, A. Van Schepdael, E. Adams, Optimization of
reference introduction prior to calibration of thermal desorber - gas chromatography,
submitted to J. Chromatogr. A.
[4] A.A. Asfaw, K. Wolfs, A. Van Schepdael, E. Adams, Development and validation of a
thermal desorber gas chromatography method for determination of residual solvents in
drug loaded albumin, in preparation.
[5] A.A. Asfaw, J. Aspromonte, K. Wolfs, A. Van Schepdael, E. Adams, Overview of sample
introduction techniques prior to GC for the analysis of volatiles in solid materials,
submitted to J. Sep. Sci.
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Conference participations
[1] A.A. Asfaw*, K. Wolfs, A. Van Schepdael, E. Adams, Development of a thermal

desorption-gas chromatography method for the determination of residual DMSO in
gelatin, BSPS 19th Forum of Pharmaceutical Sciences, Brussels, Belgium, 17-18
October 2016: oral presentation.
[2] A.A. Asfaw*, K. Wolfs, A. Van Schepdael, E. Adams, Thermal desorption-gas
chromatography method development for the determination of residual DMSO in
gelatin, Recent developments in pharmaceutical analysis (RDPA), Rimini, Italy, 20-23
September 2017: poster presentation.
[3] E. Adams*, A.A. Asfaw, K. Wolfs, A. Van Schepdael, Determination of residual
solvents in drug loaded mesoporous silica using thermal desorption – gas
chromatography, Recent developments in pharmaceutical analysis (RDPA), Rimini,
Italy, 20-23 September 2017: oral presentation.
[4] E. Adams*, A.A. Asfaw, K. Wolfs, A. Van Schepdael, Expanding the application of
thermal desorber devices towards dynamic headspace gas chromatography fort he
determination of residual solvents, 15th international symposium on hyphenated
techniques in chromatography and separation technology (HTC-15), Cardiff, UK, 24-
26 January 2018: oral presentation.
[5] A.A. Asfaw*, K. Wolfs, A. Van Schepdael, E. Adams, Introduction of reference
solutions for thermal desorption – gas chromatography, 42nd international symposium
on capillary chromatography (ISCC), Reva del Garda, Italy, 13-18 May 2018: poster
presentation.
Supervision of postgraduate and master students
[1] Development of a headspace gas chromatography method for the analysis and stability
study of methyl salicylate in vasegreen (2016) (Name of student: Chiara Staffieri).
[2] Reference introduction prior to the optimization and calibration of thermal desorption
gas chromatography of solid samples (2017) (Name of student: Matthias Van Der
Veken).
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Table of contents
Curriculum Vitae ...................................................................................................................................ii

Acknowledgements ................................................................................................................................iii
Publications ............................................................................................................................................ vi
List of abbreviations and symbols........................................................................................................ xi
Chapter 1: General introduction ......................................................................................................... 1
1.1. Volatile organic compounds in a pharmaceutical context ................................................. 2
1.2. Gas chromatography ............................................................................................................. 3
1.2.1. Introduction [9] ................................................................................................................ 3
1.2.2. Inlets and Columns......................................................................................................... 3
1.2.3. Detectors [9]...................................................................................................................... 4
1.3. Sample introduction techniques to GC................................................................................ 6
1.3.1. Direct injection techniques ............................................................................................. 7
1.3.2. Headspace techniques .................................................................................................... 8
1.3.3. Thermal desorption (TD).............................................................................................. 14
1.4. Aim of the study ................................................................................................................... 17
1.5. References ............................................................................................................................ 18
Chapter 2: Thermal desorption - gas chromatographic methodology for the determination of
residual solvents in mesoporous silica ............................................................................................... 27
Abstract ................................................................................................................................................ 29
2.1. Introduction ......................................................................................................................... 30
2.2. Experimental........................................................................................................................ 32
2.2.1. Chemicals and reagents................................................................................................ 32
2.2.2. Instrumentation ............................................................................................................ 32
2.2.3. Standards and sample preparations ............................................................................. 33
2.2.4. Method validation ......................................................................................................... 34
2.3. Results and discussion ......................................................................................................... 35
2.3.1. Preliminary studies ....................................................................................................... 35
2.3.2. TD-GC-FID method development................................................................................ 35
2.3.3. TD-GC-FID method validation .................................................................................... 39
2.3.4. FET-HS-GC-FID method development....................................................................... 42
2.3.5. FET-HS-GC-FID method validation ........................................................................... 43
2.3.6. Applications .................................................................................................................. 44
2.4. Conclusions .......................................................................................................................... 44
2.5. References ............................................................................................................................ 45
viii
Chapter 3: Determination of residual dimethylsulphoxide in drug loaded gelatin using thermal
desorber - gas chromatography ......................................................................................................... 49
Abstract ................................................................................................................................................ 51
3.1. Introduction ......................................................................................................................... 52
3.2. Experimental........................................................................................................................ 54
3.2.1. Chemical reagents ........................................................................................................ 54
3.2.3. Standards and sample preparations ............................................................................. 55
3.3. Results and Discussion ........................................................................................................ 58
3.3.1. TD-GC method.............................................................................................................. 58
3.3.2. FET-HS-GC method..................................................................................................... 62
3.3.3. Application .................................................................................................................... 63
3.4. Conclusions .......................................................................................................................... 64
3.5. References ............................................................................................................................ 64
Chapter 4: Optimization of reference introduction prior to calibration of thermal desorber - gas
chromatography .................................................................................................................................. 67
Abstract ................................................................................................................................................ 69
4.1. Introduction ......................................................................................................................... 70
4.2. Experimental........................................................................................................................ 72
4.2.1. Chemicals and reagents................................................................................................ 72
4.2.2. Apparatus ...................................................................................................................... 72
4.2.3. Standard preparations .................................................................................................. 74
4.2.4. Offline liquid calibration .............................................................................................. 75
4.2.5. Inline liquid calibration................................................................................................ 76
4.3. Results and discussion ......................................................................................................... 76
4.3.1. Offline liquid calibration .............................................................................................. 76
4.3.2. Inline liquid calibration................................................................................................ 84
4.4. Conclusion ............................................................................................................................ 85
4.5. References ............................................................................................................................ 86
Chapter 5: Development and validation of a thermal desorber gas chromatography method for
determination of residual solvents in drug loaded albumin ............................................................ 89
Abstract ................................................................................................................................................ 91
5.1. Introduction ......................................................................................................................... 92
5.2. Experimental........................................................................................................................ 94
5.2.1. Chemical reagents ........................................................................................................ 94
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5.2.3. Standard and sample preparation ................................................................................ 96
5.3. Results and Discussion ...................................................................................................... 100
5.3.1. TD-GC method............................................................................................................ 100
5.3.2. HS-GC method............................................................................................................ 104
5.3.3. Application .................................................................................................................. 104
5.4. Conclusions ........................................................................................................................ 105
5.5. References .......................................................................................................................... 105
Chapter 6: General discussion ......................................................................................................... 109
Summary ............................................................................................................................................ 119
Samenvatting ..................................................................................................................................... 121
Scientific acknowledgements and conflict of interest statement ................................................... 123
x
List of abbreviations and symbols
A analyte
A+ ionized analyte
Ace acetone
ACN acetonitrile
AMT air monitoring trap
ATT detector attenuations
β phase ratio
BA benzyl alcohol
Bp boiling point
BSA bovine serum albumin
°C degrees Celsius
Cg concentration in the gas phase
C-H carbon-hydrogen
Co Analyte concentration in original sample
CS correction standard
Cs concentration in the sample phase
2,2-DD 2,2-dimethyl-1,3-dioxolane
DTT 1,4-dithiothreitol
DCM dichloromethane
dHS dynamic headspace
DMA N,N-dimethylacetamide
DMF N,N-dimethylformamide
DMI 1,3-dimethyl-2-imidazolidinone
DMP 2,2-dimethoxypropane
DMSO dimethylsulfoxide
e- electron
EI electron ionization
ET Equilibration temperature
EtOH ethyl alcohol
eV electron volts
FET full evaporation technique
FID flame ionization detector
xi
g gram
GC gas chromatography
h hour
H2 hydrogen molecule
HCl hydrochloric acid
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HF hydrofluoric acid
HFIP hexafluoroisopropanol
HS headspace
ICH International Council on Harmonisation
ID internal diameter
ILC inline liquid calibration
Isp isopropanol
K distribution coefficient
kPa kilopascal
L liter
LOD limit of detection
LOQ limit of quantification
M molar
m/z mass-to-charge ratio
MeOH methyl alcohol
mg milligram
MHE multiple headspace extraction
mL milliliter
MMP-2 matrix metalloproteinase-2
MPSi mesoporous silica
MS mass spectrometry
μg microgram
μL microliter
μm micrometre
ng nanogram
n-prop n-propanol
OLC offline liquid calibration
xii
PLOT porous layer open tubular
ppb parts-per-billion
ppm parts-per-million
PTFE polytetrafluoroethylene
PTV programmed temperature vaporizer
P&T Purge and trap
QF quartz filter
RS residual solvent
RSD relative standard deviation
S/N signal-to-noise
SCOT support coated open tubular
SDME single drop micro extraction
sHS-GC static headspace-gas chromatography
SIM single ion monitoring
SPME solid phase micro-extraction
T (°C) temperature in degree Celsius
t (min) time in minutes
tcrit. critical t-value for a number of degrees of freedom
TD thermal desorption
THF tetrahydrofuran
TPCK L-1-tosylamide-2-phenylethyl chloromethyl ketone
TIC total ion chromatogram
TVT total vaporization technique
Vg volume of the gaseous phase
VOCs volatile organic compounds
Vs volume of sample phase
vs. versus
Vv volume of the vial
% v/v volume per volume percentage
% w/w weight per weight percentage
WCOT wall coated open tubular
Wo amount of analyte in the sample
xiii
Chapter 1: General introduction
Based on ‘Overview of sample introduction techniques prior to

GC for the analysis of volatiles in solid materials’
Adissu Alemayehu Asfaw, Juan Aspromonte, Kris Wolfs, Ann Van Schepdael,
Erwin Adams
KU Leuven - University of Leuven, Department of Pharmaceutical and

Pharmacological Sciences, Pharmaceutical Analysis, Herestraat 49, O&N2, PB
923, 3000 Leuven, Belgium
Submitted to J. Sep. Sci.
1
1.1. Volatile organic compounds in a pharmaceutical context
Volatile organic compounds (VOCs) comprise a wide range of chemicals that can easily be
brought to the gas phase like linear and aromatic hydrocarbons, including oxygen-, nitrogen-,
sulfur- and halogen- containing compounds. VOCs are a subject of interest in different
disciplines such as pharmaceutical, food and environmental sciences either for their typical
feature or as contaminants [1]. In pharmaceuticals, VOCs can be incorporated in formulations
as active ingredients or flavoring additives, but they mainly play a role as solvents in the
production (synthesis, separation, purification) and formulation (granulation, coating). At each
of these stages, organic solvents can potentially contaminate the product and complete removal
of the organic solvents by usual manufacturing techniques is practically impossible. Therefore,
their presence in final products is inevitable where they remain as residual solvents (RS) [2].
RS do not have any therapeutic effect, but they may cause toxic effects and safety problems.
Further, RS may alter the physicochemical properties of the active substance or excipient, and
so may affect the product formulation process [3].
As a result, continuous monitoring of the quality of pharmaceuticals is needed to ensure patient
safety and meet regulatory expectations. The detection and quantification of RS in
pharmaceuticals, as required by regulatory authorities, like the European Pharmacopoeia [4]
and the International Council on Harmonization (ICH) [5], is very strict. According to ICH, RS
are classified into three classes. The limit concentration for each solvent is fixed based on their
toxicity to humans, their level of hazard to the environment and their permitted daily exposure.
Class 1 includes RS which are known or suspected human carcinogens or environmental
hazards (e.g., benzene, carbon tetrachloride, and 1,2-dichloroethane). RS under this class should
be avoided in the production of actives, excipients or pharmaceutical products, unless their use
can be tolerated in highly exceptional circumstances where they must be identified and
quantified. Limits in the range of 2–8 ppm are defined for solvents that are known to be highly
toxic. The limit of 1500 ppm applied for trichloroethane is because it is an environmentally
hazardous chemical. Class 2 includes RS which are suspected of other significant, but reversible
toxicities (e.g. acetonitrile, chloroform, hexane). Such solvents should be limited. The limit
concentrations allowed for this class have individual limits between 50 and 5000 ppm. Class 3
includes RS which are considered to be less toxic and have low risk for health when their daily
intake is below 50 mg. Solvents under this group (e.g., acetone, 2-butanol, ethanol, ethyl
acetate) need to be identified and quantified, when they are found to be >0.5% (w/w) [5].
2
Various analytical methods have been employed to determine RS in pharmaceutical materials.
One of the oldest methods relies on measuring the loss of sample weight. It is based on
measuring the weight loss of the sample during the heating process, and can be carried out at
normal pressure or under vacuum conditions. Although it is a simple and not demanding
method, it has many drawbacks, including lack of specificity, high limit of detection (about
0.1%) and also a relatively large amount of sample needed to perform the tests (about 1 - 2 g).
Moreover, atmospheric humidity can significantly modify the results obtained by the loss of
weight method [6]. This method has its successors in current trends focusing on more
sophisticated techniques like thermogravimetric analysis and differential scanning calorimetry.
Because these methods are nonspecific, only the general content of solvents can be determined,
with no indication of their identity or their number. Currently, the determination of RS in
pharmaceutical products and excipients is almost completely performed by gas chromatography
(GC). This is due to the the outstanding performance of this technique, including the substantial
separating capability of capillary columns, leading to procedures which have also been
approved by various pharmacopoeias [4, 7-8].
1.2. Gas chromatography
1.2.1. Introduction [9]
Gas chromatography (GC) is the method of choice for the analysis of volatile compounds in
various samples. In GC, components of a sample that can be brought to the gas phase are swept
through the inlet by an inert carrier gas to the analytical column. There, separation of various
analytes occurs based on their difference in affinity between the stationary phase and the carrier
gas. Since GC deals with volatile compounds, the boiling point of the analytes and the
temperature program in the oven also play an important role in the separation process. When
the separated analytes are eluted from the column, they are detected using various detectors
such as flame ionization detector, mass spectrometry, electron capture detector etc. The
response signal of the detector is recorded in function of time (retention time) in which analytes
appear as peaks. The area under an analyte peak is proportional to its concentration in the
sample.
1.2.2. Inlets and Columns

The most common sample inlet to GC is direct injection. Direct injection is a relatively simple
and reliable injection mode based on a (liquid) sample that is introduced into a hot evaporation
chamber where it vaporizes instantaneously and is then transported to the column by the carrier
gas. It is mainly used together with packed columns or wide-bore capillary columns. As such,
3
it is less suitable for narrow bore capillary columns because of their small sample capacity.
However, great efforts have been done to make direct injection compatible with the most
commonly used capillary columns. Split/splitless, on-column injection and programmable
temperature vaporization (PTV) are among the direct injection modes used frequently
nowadays [10-11]. Another popular sample introduction technique in GC analysis is headspace
(HS) based on the equilibration of the analyte between the liquid sample and the gas phase in a
vial. Sample introduction techniques will be further discussed in detail in section 1.3.
The column is the critical part where separation takes place. In general, two important types of
columns can be distinguished: packed and capillary columns. Packed columns consist of a tube
with an inner diameter of about 2 – 4 mm, which is packed with small porous particles. These
particles can be the actual stationary phase or serve as support for a liquid stationary phase.
Despite its declining popularity in favor of capillary columns, packed columns are still widely
used, especially for preparative work. Capillary columns consist of a tube with an inner
diameter of 0.10 to 0.53 mm. In contrast to packed columns, the stationary phase is coated onto
the inside wall of the column. There are three types of capillary columns commonly used in
GC: wall coated open tubular (WCOT), support coated open tubular (SCOT) and porous layer
open tubular (PLOT) columns. In WCOT columns, a thin layer of liquid stationary phase is
coated on the inner surface of the fused silica. SCOT columns have a liquid stationary phase
deposited onto a thin layer of small support particles on the inner wall of the capillary. In the
PLOT columns, solid particles are the active stationary phase [12].
1.2.3. Detectors [9]

The detector registers the elutes from the column. Detectors can be classified as either universal
or selective. Universal detectors are able to generate a signal for almost all types of sample
components that elute from the GC column. The most commonly used universal detectors are
the thermal conductivity detector (TCD) and the flame ionisation detector (FID). Selective
detectors produce a signal only for particular classes of analytes based on the response for one
or more elements in the compound of interest, such as chlorine, phosphorus or nitrogen. Some
selective detectors generate a signal for a definite physical or chemical property of the
molecules.
1.2.3.1. Flame ionization detector

FID is the most popular GC detector because of its pronounced sensitivity for organic
compounds, linear over a wide operating range and robustness. The FID operates with gases
4
(H2 as fuel and air as source of oxygen for oxidation) to form a flame and electrodes to assess
the conductivity of the flame (see figure 1.1). When only the carrier and the detector gases reach
the flame, the conductivity is almost zero. However, when an ionizable organic component
eluting from the column enters the flame, it will start to break into ions and electrons and the
conductivity of the flame increases. Two electrodes (the flame tip as cathode and a cylindrical
collector above the flame as anode) are used to measure the change in conductivity in the flame.
The resulting current is led to a recorder or a data system after amplification. The FID is
extremely sensitive for hydrocarbons and the response is directly related to the number of
carbon atoms in the sample.
Figure 1.1: Schematic overview of FID [13]
1.2.3.2. Mass spectrometer as detection system

Mass spectrometry (MS) is a spectral method used primarily for structural confirmation.
Currently, it is also frequently used as a chromatographic detector for the quantitative analysis
of various samples. Briefly, the mass spectrometer consists of three core components: ion
source, mass analyzer and detector. The role of the ion source is to transfer the neutral
compounds into charged particles (ions). The mass analyzer then separates the ions according
to their mass-to-charge ratio (m/z). Finally, the detector registers the relative abundances of
individual m/z values. GC was the first separation technique that was successfully coupled with
MS.
5
In GC-MS, an electron ionization (EI) source and a quadrupole mass analyser are the most
common combination used for routine analysis. Here, the molecules in the gaseous state under
low pressure are bombarded with a beam of high energy electrons (about 70 electron-volts; eV).
This bombardment can first dislodge one of the electrons of the molecule, producing a
molecular ion. This imparts to the molecular ion a considerable amount of surplus energy. Thus,
soon after they are formed, molecular ions could undergo fragmentation. The quadrupole then
sorts the produced ions based on their m/z [14]. A schematic overview is given in figure 1.2. In
full scan mode, which is mainly used for confirmation, mass spectral data are acquired in a
sequence at specified intervals. All measured spectra are stored in a computer to be processed.
This results in a total ion chromatogram (TIC), created by summing up intensities of all mass
spectral peaks belonging to the same scan and at each time point. When EI is used as ionization
source, the identification process is simplified by the accessibility of widespread EI spectral
libraries. In single ion monitoring (SIM) mode, where the mass spectrometer is set to measure
only one specific m/z, analytes of interest can be selectively detected for quantification.
Figure 1.2: Schematic overview of MS consisting of EI and quadrupole mass analyzer [15,16].
1.3. Sample introduction techniques to GC

Since the diameter of commonly used capillary columns is narrow, it imposed a rigorous
demand on the way a sample is introduced onto the column. As a result, in capillary GC
analysis, the main challenge is how to convert the sample into a suitable form for introduction
without degenerating it. The sample introduction techniques for a GC system depend on various
variables such as analyte type and its concentration, the sample matrix and type of column [17].
Generally, a sample cannot be introduced directly into a GC system due to the fact that, some
6
non-volatile matrix components can be present that deteriorate the GC system and disturb the
separation and/or detection of the analyte(s).
1.3.1. Direct injection techniques

1.3.1.1. Split/splitless
The split injector is the most widely used injector in combination with capillary columns. The
sample is first vaporized in a chamber. Then, the vapour is split into a small portion that is
transported to the column and a substantial portion that is vented from the system via the split
opening. In this injector mode, all steps such as evaporation, mixing with carrier gas, transport
to the column and splitting should happen without loss of proficiency [9]. Since only a fraction
of the sample enters to the separation system, it is less suitable for trace analysis.
Splitless injection is intended mainly for trace analyses (low ppm and ppb analyte
concentrations). It is carried out by injecting the sample into a hot injector to evaporate while
the split exit is closed. This way, the sample is (almost) totally transferred onto the column.
Next, the splitter is opened to vent any sample and solvent vapour present in the splitter. This
occurs after the splitless time is over.
1.3.1.2. On-column injection

Another interesting injection technique is on-column injection that compromises outstanding
quantitative results with capillary columns. Unlike the previous injection modes, the liquid
sample does not requisite to be vaporized, but is directly injected as a liquid from the syringe
onto the (cool) column. This way, discrimination due to evaporation processes in the injector
are circumvented [18-19]. Since it is difficult to find a needle which can fit the narrow capillary
column, on-column injection is frequently used in combination with a wide-bore (pre) column
in order to introduce the needle easily to the capillary column.
1.3.1.3. Programmable temperature vaporization (PTV)

PTV is a comprehensive GC injector which enables the sample to be introduced in liquid form
at cool temperatures and then to be vaporized due to its rapid heating and cooling efficiency.
Most commercially available injectors of this type are capable of working in both split and
splitless mode and, based on the liner type chosen, it can also work in on-column injection
mode. Its design is like non-programmable types, apart from the rapid heating and cooling
abilities. PTV may offer advantages including a highly efficient vaporization, large volume
injection and possibility to remove the sample solvent prior to injection. In the latter case, the
7
solvent is vaporized at the PTV cold temperature and hence already removed before the
migration of sample components starts [9, 20].
1.3.2. Headspace techniques

Headspace (HS) is a sampling method for the analysis of volatiles that has been in use for
several years already. It has proved to be a popular technique, mostly thanks to its simplicity
for automation.
1.3.2.1. Types of HS
In HS, the sample is heated in a vial for a certain time and part of the gas phase is injected in
the GC inlet port. This procedure prevents low-volatile components of the matrix from
contaminating the GC system in a simple and low cost fashion. Moreover, the procedure can
be done by means of a gastight syringe, but automation is not expensive and improves
repeatability/reproducibility while increasing the sample throughput. Mainly, three automated
systems are currently available: heated syringe, balanced-pressure and sample loop. Briefly, in
the first case a gastight syringe – with optional flushing ports – placed in an oven compartment
to avoid condensation of the sample, is used to sample the gas phase in the vial and inject it in
the GC inlet port. In balanced-pressure, the vial is pressurized to transfer part of the HS into the
GC injector, not knowing the absolute volume that is injected. In the pressure-loop case, a fixed
volume loop is filled after the pressurization step and this known volume is transferred to the
GC injector [21-22]. HS analysis can be performed both statically (sHS) and dynamically
(dHS). In sHS, the sample is usually dissolved in a suitable high boiling solvent after which a
part is transferred to a HS vial and closed with a cap having a septum. Afterwards the vial is
heated at a particular temperature for a specified time and the analytes start to release to the
vapor phase above the sample (see figure 1.3). After a thermodynamic equilibration of the
analytes between the sample and vapor phase is reached, an aliquot from the vapor phase is
transferred to the GC [23].
Kolb and Ettre [21] have explained in detail the theoretical aspects of sHS sampling. Here, only
some items relevant to the work will be briefly discussed.
8
Figure 1.3: Schematic representation of a HS vial in sHS conditions [24].
Under a specific analytical condition, the partition of a given analyte between the sample and
gaseous phase can be expressed by the partition coefficient (K):
𝐶𝑠
𝐾= (1)
𝐶𝑔
Cs is the concentration of the analyte in the sample phase and Cg is the concentration in the
gas phase.
The phase ratio β describes the ratio of the gaseous phase volume (Vg) to the sample phase
volume (Vs) in the HS vial containing the sample.
𝑉𝑔
𝛽 = (2)
𝑉𝑠
The concentration of the analyte in the gaseous phase can be calculated from the initial
sample concentration (C0):
𝐶0
𝐶𝑔 = (3)
𝐾+ 𝛽
Under a given system and conditions, both K and β are constants. Thus, if an aliquot of the
HS at equilibrium is analysed by GC, the peak area of the analyte is proportional to its
concentration in the original sample. In practice, K is very difficult to predict and it is
influenced by the composition of the sample due to any possible interaction of the analyte
with components from its environment, also known as matrix effect.
The full evaporation technique (FET) can be consider as a special type of sHS. In this method,
the analyte is completely released from the matrix to the gaseous phase while non-volatile
9
matrices are considered to remain in the vial as an inert residue. Ideally making the matrix effect
neglectable (K→ 0). As a result, the concentration of the analyte in the HS will be:
𝑊0
𝐶𝑔 = (4)
𝑉𝑣 −𝑉𝑠
In which, W0 is the total amount of the analyte in the sample and Vv the volume of the vial.
To achieve full evaporation of the analyte, the gas phase must not be saturated during
equilibration. This is practically done by using only a small amount of sample. The advantage
is that, unlike the other HS techniques, it does not suffer from matrix effects. As a consequence,
this approach allows the use of external calibration, which simplifies the application of the HS
for routine analysis. The main drawback of this technique is the loss of sensitivity caused by
the use of low amounts of sample. Another type of HS is the total vaporization technique (TVT).
Unlike FET, it is based on evaporation of the whole sample, including all matrix components,
so that there will be only a single gas (vapor) phase present in the vial. Thus, the concentration
of the analyte in the vial is:
𝑊0
𝐶𝑔 =
𝑉𝑣
TVT is often used for the preparation of vapor standards to be used for calibration.
Multiple headspace extraction (MHE) is another type of HS sampling in which a certain amount
of the sample is placed in the HS vial and a series of discontinuous extractions (the vial is vented
after each extraction and there is one chromatogram per extraction) are performed and each
successive extraction is performed after re-equilibration has occurred. The obtained peak area
of the analyte of interest will be decreasing after each extraction, following a well-known
exponential decay. After a certain number of extraction steps, the total amount of the analyte is
calculated by extrapolation from a plot of the natural logarithm of the peak area versus the
extraction number. The small amounts of sample needed and the simplicity of the calibration
are noteworthy, but MHE is also time consuming.
All the HS techniques described above can be classified as sHS. The other type of HS is dHS,
which is based on continuously removing the analyte from the sample by purging the HS vial
with a large volume of inert gas. Analytes are subsequently trapped using either cryogenic or
sorbent traps. Next, the trap is heated to thermally desorb the retained volatiles, which are then
transferred to the GC under a flow of inert gas. Since the trap is placed between the HS vial and
10
the GC column, analytes are first pre-concentrated and focused. dHS sampling removes most
of the volatile compounds, except those showing a strong affinity for the sample matrix, such
as polar compounds in aqueous samples. Purge and trap (P & T) techniques are similar to dHS
sampling, except that the gas is passed through the sample. Clearly, any apparatus used for dHS
sampling can also be employed with slight modification for P & T [25].
Despite the numerous advantages of combining HS with a trap, there are some steps that require
consideration. The time needed to completely extract and transfer the volatiles from the sample
to the cold trap must be accurately determined to avoid incomplete extraction. The hold
temperature of the trap must be as low as possible to retain the analyte(s) of interest on the trap.
The selection of the trap material or sorbent is also important, especially when aqueous samples
are analysed [26].
1.3.2.2. HS coupled with extraction techniques
In the case of particularly low analyte concentrations in complex matrices, an extraction and
concentration step may be necessary before injection. A different phase (solid or liquid) may
be used to adsorb and concentrate the analytes (sometimes selectively). Next, they can be
transferred into the GC column by different means. In the last years, both solid and liquid phase
extraction techniques for solid samples have been focusing on microextractions, mainly solid
phase microextraction (SPME) and single drop microextraction (SDME).
SPME was first proposed in the 1990’s as a single step solvent-free extraction and concentration
technique [27]. In this technique, a polymer-coated fiber or a capillary tube is used to adsorb
analytes from the HS or liquid and then transfer them to the GC column by means of a specially
conceived inlet liner [28]. This is a non-exhaustive extraction that allows low detection limits
while it is rather simple [29]. It has to be taken into account that in this approach the overall
process presents three phases: sample, HS and fiber. A clear compromise exists between the
analytes released from the matrix, their diffusion towards the adsorbent and the adsorption
efficiency of the fiber [30]. In other words, matrix effects play a central role in the development
of a SPME method. Notwithstanding it is recommended to perform the extraction under
equilibrium conditions of the three phases, it is proved that a relatively good reproducibility
may be obtained already without being under strict equilibrium if the temperature and timings
are kept constant [31]. However, maintaining only those parameters constant does not make the
method very robust, jeopardizing its routine application.
SDME was developed as a less time-consuming alternative to liquid-liquid extraction with the
great advantage of using lower amounts of solvents and no need for phase coalescence [32].
11
Moreover, its use as extraction technique from the HS fraction (HS-SDME) has also been in
use, achieving similar results as SPME [33]. In this technique, a micro-syringe is used to pierce
the sample containing vial and a micro-drop of the extraction solvent is suspended from its tip.
After a certain extraction time, the drop is withdrawn into the syringe and injected in the GC
for analysis. An important advantage of this method is the simple sample injection into the GC.
No desorption has to be considered and the extraction phase is renewed every time the
experiment is repeated, avoiding eventual carryover. In the case of HS-SDME, issues associated
to the immersion of a hanging droplet into an agitated liquid, such as loss or contamination, are
easily avoided. Nevertheless, due to its microscale, this is a non-exhaustive technique and, like
for SPME, matrix effects severely influence the results. Therefore, the use of a solvent to dilute
such samples or to enhance the release of some analytes to the HS by different treatments seems
to prevail if applicable [34-37].
1.3.2.3. Application of HS techniques for solid samples

In most officially recommended HS methods, the sample will be dissolved in an appropriate
medium and then only the volatile constituents of the sample will be extracted upon heating.
Different parameters need to be optimized in order to ensure a minimal contamination from the
matrix and a maximum extraction of the volatile analytes while achieving an equilibrium
condition between the gas and the condensed phase in the shorter time possible. The most
commonly studied parameters are the equilibration time and temperature prior to the HS
extraction. Higher temperatures can be used to help establishing an equilibrium with the gas
phase in a shorter time. On the other hand, some samples may need a lower temperature and a
longer time to prevent possible degradation of some components of the sample [38-40]. Some
auto-sampling systems include a built-in shaking mechanism that can help in reducing the
equilibration times by renewing the surface of the sample during the heating step. Special
attention has to be paid when large amounts of low-boiling point substances are present in the
sample matrix as these can harm the sensitivity of the method. Different dilution media have
been proposed to address this issue, ranging from liquid paraffin to more recent ionic liquids
[41-42]. On the other hand, the dilution of the sample will inevitably lead to a loss of sensitivity,
becoming a major issue for determining analytes at low concentrations.
When dissolution of the sample is not possible, it may be analyzed as a dry solid or with the
addition of a displacer solvent [43-45]. In both cases, it is recommended to use a MHE
experiment to verify that there is a partition system. In such system, the distribution (partition)
coefficient is considered to be constant and independent of the analyte concentration. The MHE
12
method can serve as a quick test to see whether this is the case: if the ln Ai vs. (i - 1) plot is
linear, a partition system can be assumed. On the other hand, non-linearity of this plot indicates
that the distribution coefficient is not independent of the concentration and/or that adsorption
effects are present [21].
Usually, samples having a smaller particle size or a certain porosity present shorter diffusion
times, thus shortening the equilibration time. In practice, this can be achieved by freeze grinding
the samples to avoid the loss of volatiles. However, solid samples analyzed as such tend to
present longer equilibration times than when in solution. Moreover, solids with a large surface
area are prone to present adsorption effects that will limit the linear range and eventually make
their analysis impossible by this means. For samples of this nature, i.e. highly adsorptive and
not possible to dissolve, it has been proposed to add an extra component that will displace the
analyte from the solid matrix into a liquid one, a sort of in vial liquid extraction. As the analyte
is now in the liquid phase, the system will no longer present the issues due to the adsorption.
This approach is known as the suspension approach [46].
Another interesting approach to deal with the complicated matrix effects presented by solid
samples is the use of FET [47-50]. FET has outstanding advantages in both efficiency (i.e. short
equilibrium time due to the small sample volume) and method calibration (i.e. independent of
the sample matrix). In general, a very small amount of sample is placed into a HS vial to meet
the requirement of FET. However, it is obvious that one of the major problems in FET-HS-GC
is the low sensitivity associated with the small sample size.
As mentioned before, MHE is often proposed as a verification method to ensure that the system
is behaving as a partition system. However, this method has proved to be also a very reliable
one to extract analytes from solid samples [51-53]. This method simplifies the calibration
procedure, as there is no need for standard addition or need for a synthetic matrix. The small
amounts of sample needed and the simplicity of the calibration makes this approach interesting.
Nonetheless, the need for several injections per vial and sometimes long equilibration times
make this method not very attractive to routine analysis [54].
dHS can also be used for analysis of VOCs in solid samples. In this approach, the extraction
cycles can be repeated several times leading to a none negligible increase in method sensitivity.
Compared to sHS, it provides up to 1000 times better sensitivity [25]. Nevertheless, it has to be
considered that this procedure can lead to a loss of very volatile analytes due to breakthrough
when a weak sorbent like Tenax is used or due to freezing of the cold trap. Further, there could
13
be memory effects after the analysis of samples containing a high concentration of volatiles.
Despite these limitations, this technique has been used in a wide number of applications for the
analysis of volatiles from solid samples in different fields [55-56].
For the determination of volatile analytes in solid samples, HS-SPME constitutes a very
interesting option as it can circumvent the need for a dilution medium. Analytes are then
transferred from the solid to the HS from where they are adsorbed to the SPME fiber.
Nevertheless, many methods rely on the use of a solvent to increase the recovery from the solid
matrix [57-58]. Considering the importance of the matrix effect, calibration on this type of
methods can be challenging. SDME can be easily applied for the analysis of volatiles in solid
samples by direct immersion in diluted solid samples [59] or extracting the analyte from the HS
using HS-SDME [35-37]. In this technique, the matrix effect constitutes an important drawback
when working with solid samples. Working with a diluted or pretreated sample allows using an
external calibration, simplifying the method implementation. Nevertheless, many samples may
not fully dissolve or some analytes may not be easily extracted in the pretreatment. In addition,
the extra handling of the sample increases the risk of losing part of the analytes, mostly when
they are highly volatile. Besides, these are time and solvent consuming methods, compromising
the main advantages of this method.
1.3.3. Thermal desorption (TD)
TD is a comprehensive sample preparation and introduction technique to GC. The history of
TD began in the early 1970s by primeval alterations of conventional GC injectors by packing
standard GC injector liners with sorbent material. However, the many and obvious limitations
of these (air ingress, volatile losses, contamination, single stage,…) adaptations revealed the
necessity of working out a better instrument. The first dedicated general-purpose TD
technology was consistently based on desorption of a single tube or badge. However, it did not
took long to realize that it had serious drawbacks such as huge amounts of gas that are required
for complete extraction of a standard tube while resolution and analytical sensitivity are
compromised [60]. A cryofocusing device between the sorbent tube and the GC analytical
system was applied to solve the problem. However, this did not last long as soon as the
limitations of capillary cryofocusing became apparent like ice blockage, incomplete retention
of very volatile compounds, low recovery of high-boilers and high running costs. As a result,
in most modern TD a two stage desorption is applied [61].
14
1.3.3.1. Principles of two stage TD
TD is a technique originally developed for the analysis of airborne chemicals. A sample tube
loaded with one or more sorbents collects by diffusion or forced transport the analytes from the
area to be monitored. The tube is then placed in the TD apparatus and ready for extraction. A
two stage TD involves thermal extraction of volatiles from the sample in the tube under a flow
of inert gas. The extracted analytes are then transported to and retained on a cold trap containing
one or more sorbents. This step is called the primary desorption. Next, the trap is rapidly heated
to release all the retained analytes into a flow of carrier gas, so that they are transferred to the
column as a small, discrete and concentrated volume of vapour [62]. This is known as the
secondary desorption. A schematic overview of two stage TD is given in figure 1.4. The TD
process starts by executing a leak test on a tube after being placed in the desorption position.
After passing the leak test, air is purged from the tube using inert gas to reduce the risk of
adsorbent or sample oxidation during desorption. Next, the sample tube is heated by the oven
and the desorption time begins. The efficiency of the TD method can be enhanced by optimizing
different TD parameters such as desorption temperature, time and flow and the type of sorbents
used. For instance, increasing desorption temperature will enhance the extraction of analytes.
However, the thermal stability of the sample and of the sorbent material in the sample tube
should be considered to avoid degradation. Another important parameter is the selection of the
sorbent. If the sorbent is too weak, very volatile compounds will break through, while the less
volatile ones are retained. On the other hand, if the sorbent is too strong, it will become more
difficult later to drive off (desorb) the less volatile compounds. For this reason, modern tubes
are often made with two or more beds where a weaker is placed first to the desorption flow and
a stronger bed next. This enables to retain the high volatile compounds on the strong sorbent
and the less volatile ones on the weak sorbent, making the desorption of the analytes from the
trap easier and complete since the analytes enter and leave the trap from the weaker sorbent
side as the flow direction is opposite during the sample and the trap desorption step [63]. TD
was originally developed to circumvent problems of solvent extraction such as solvent
interference, low sensitivity, automation, health and safety issues related to the solvent used. It
granted the potential of an alternative high sensitivity/ solvent-free gas extraction process that
could be fully automated. In TD, it is often possible to quantitatively retain analytes of interest
during the trapping stages while unwanted interferences are selectively eliminated to the vent
[64]. TD is mainly used for the analysis of airborne chemicals using a sample tube loaded with
an adsorbent that collects by diffusion or forced transport the analytes from the area to be
15
monitored [65-68]. Alternatively, some attempts have been done to bring samples directly into
empty TD sample tubes for direct thermal desorption [69-72].
Figure 1.4: Schematic overview of two-stage TD [73].
1.3.3.2. Calibration of TD methods

TD procedures are generally calibrated using external standards with the option to add a gas-
phase standard. Ideally, gas-phase standards are used for calibrating TD applications [74]. The
two well-known approaches to produce gas or vapor phase standards are by a static or dynamic
system [75]. The static approach performs by adding an appropriate volume of gas to a known
volume of air in a container. It is an easy, simple and inexpensive technique, but it has also
serious limitations like certain compounds that can interact with the wall of the container
resulting in loss and memory effects limiting reuse of the container [76]. So, it is challenging
to generate reliable gas standards this way [77]. Dynamic gas standard generation is much more
reliable and works by mixing a concentrated stream of gas or vapor with a diluting air stream
to consciously produce the desired concentration. However, only very few laboratories in the
world have sufficiently sophisticated apparatus to produce low-concentration (low ppm)
standard gases that are truly traceable to primary standards [78]. So, most TD methods therefore
describe external calibration using liquid standard solutions [79-80]. However, proper
16
introduction of standard solutions to the TD tube is not straightforward due to loss of volatile
analytes during the introduction step and required matrix matching.
1.4. Aim of the study

Solid materials like mesoporous silica (MPSi), gelatin and albumin are used as drug carrier
following a loading procedure that implies the use of organic solvents. This thesis comprises
the determination of RS in those solid samples which are practically impossible to dissolve or
distribute uniformly in water or common organic solvents. As RS are traces of solvents that
were used during the production, but were not completely removed, sensitive analytical
techniques are required. Although HS-GC is mainly developed for liquids or solutions of the
sample, solids can in principle be processed as well. However, when adsorption phenomena are
encountered, the distribution (partition) coefficient is no longer independent of the
concentration of the analyte. Thus, the relationship between the original concentration of
analyte in the sample and the equilibrium concentration in the HS gas phase becomes
unpredictable and thus unsuitable for quantitative work. The use of the FET approach under HS
conditions as such is also not an option since complete evaporation of the RS from those
materials cannot be guaranteed due to the presence of the solid phase. So, the above mentioned
HS approaches are not suitable in the case of insoluble porous solid samples like MPSi and
some solid samples such as gelatin and albumin, which form a precipitate upon heating in the
HS vial during equilibration.
The main objective of this project is to explore the applicability of TD as alternative sample
introduction technique to GC to determine RS in the pharmaceutical solid forms mentioned
above. Its ease of use is evaluated as well as the sensitivity that can be obtained. The latter is
an important aspect in pharmaceutical analysis, certainly when RS have to be determined.
Accordingly, chapters 2, 3 and 5 are devoted to TD-GC method development, validation and
application for the determination of RS in MPSi, gelatin and albumin respectively. Attention
was also paid to the verification of the proposed TD-GC method using orthogonal, independent
HS-GC approaches following complicated sample pretreatment making the analysis expensive,
time consuming and less obvious from a practical point of view.
Chapter 4 is dedicated to reference introduction in TD-GC. In principle, a gas-phase standard

should be used for calibrating TD-GC methods. However, this can be expensive and/or difficult
to obtain the desired concentration range. In practice, mostly a micro syringe is used to inject
a liquid standard solution directly on the TD cartridge. This is based on the assumptions that
17
the adsorption-desorption process is quantitative and that no sample is lost in manipulating the
cartridge. However, there is no easy way to verify that the starting assumptions are met. In
chapter 4, first the limitations of the actual approach are illustrated. To allow calibration without
the need of an adsorption step, direct injection of reference into the primary desorption gas
stream of the TD would be interesting. However, such a TD-GC apparatus is not commercially
available. Therefore, a TD-GC had to be modified with an injection port.
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25
26
Chapter 2: Thermal desorption - gas chromatographic
methodology for the determination of residual solvents in
mesoporous silica
Adissu Alemayehu Asfaw, Kris Wolfs, Ann Van Schepdael, Erwin Adams

J. Chromatogr. A 2017, 1500, 160–166
27
28
Abstract
In this work, thermal desorption-gas chromatography-flame ionization detection (TD-GC-FID)
was adapted to enable the determination of residual solvents (RS) in mesoporous silica (MPSi).
MPSi is often utilized in various pharmaceutical formulations or drug delivery systems and the
accurate determination of RS is an important part of pharmaceutical quality control. Seven
commonly used solvents (methanol, ethanol, acetone, isopropanol, dichloromethane,
tetrahydrofuran and hexafluoroisopropanol) were evaluated in combination with 3 types of
MPSi having pore sizes of 2-3, 15 and 25 nm. Validation results showed general recovery
values > 98% and good linearity over the concentration ranges studied. The limits of detection
(LOD) and limits of quantification (LOQ) for the different solvents ranged from 0.03 to 0.08
µg and from 0.1 to 0.2 µg per tube, respectively. Verification of the accuracy of the TD method
was investigated by using an alternative method based on complete dissolution of MPSi in
hydrofluoric acid (HF) followed by full evaporation headspace-GC (HS-GC). The results
obtained from both procedures were not statistically different (p > 0.05) when applied to actual
experimental drug samples consisting of itraconazole loaded on MPSi.
Key words: thermal desorption-gas chromatography, mesoporous silica, residual solvents,

pharmaceutical application
29
2.1. Introduction
A mesoporous material is a substance containing pores with diameters between 2 and 50 nm.
MPSi has recently emerged in many scientific fields because of its combined advantages of
strong adsorption, large active surface area and high carrying capacity [1]. A silica surface has
a high density of silanol groups (Si-OH) which can act as the center of molecular adsorption
due to their ability to form hydrogen bonds either as donor or acceptor [2]. MPSi is widely used
in environmental, analytical, synthesis and pharmaceutical fields as adsorbent, in catalysis and
in drug delivery systems [3-6]. The application in drug delivery often involves loading of the
drug by immersion of the MPSi in a drug solution followed by removal of the solvent [7].
Unfortunately, it is practically not possible to remove the solvent completely, which leads to
the presence of RS.
sHS-GC is usually applied for the analysis of RS [8]. The principal modes of sHS-GC are
especially useful for the analysis of homogeneous solutions. Single extraction, the standard
implementation of sHS, and multiple headspace extraction (MHE) rely on a partition driven
equilibrium between the liquid phase and the vapour phase. This equilibrium can be described
by the following equation:
Cg = Co/(K+β) (1)
where Cg is the concentration of the analyte in the vapour phase, Co is the original analyte
concentration in the sample, K is the partition coefficient of the analyte between the two phases
and β is the phase ratio as the relative volume of the gas phase compared to the volume of liquid
phase (i.e. sample) in the HS vial [9]. As K and β are supposed to be constant within the
boundaries of one experiment, Cg only depends on Co.
The full evaporation technique (FET) and the total vaporization technique (TVT) both avoid
the formation of an equilibrium by transferring all the analytes to the vapour phase, with or
without (in case of FET or TVT, respectively) an inert remainder of the other sample
constituents.
Solid samples can be processed by both approaches as long as the basic assumptions (partition
driven equilibrium or full transfer) are fulfilled [10,11]. However, MPSi which is known for its
adsorptive properties, will make the K-term a function of the analyte concentration. Moreover,
in the presence of other analytes there will be competition for the active sites, adding another
dependency to the K-term. So, the resulting relationship is not useful for analytical purposes.
The mesoporous structure can also give rise to capillary condensation, contributing once more
30
to the K-term. This will slow down mass transfer so that even for non-adsorbed analytes very
long times are required to transfer them to the gas phase. These phenomena can be studied using
MHE, where the curve will deviate from the theoretical model if the distribution conditions are
not fulfilled [12]. In case the analyte is retained on the MPSi, complete transfer can never be
achieved in a static system, rendering FET or TVT not applicable.
TD-GC involves dynamic sampling and is a technique developed for the analysis of airborne
chemicals. A sample tube loaded with an adsorbent collects by diffusion or forced transport the
analytes from the area to be monitored. Alternatively, material samples can be weighed directly
into empty TD sample tubes for direct thermal desorption [13,14]. Next, the tube is placed in
the TD-GC where it is heated under an inert gas flow. This step is called the primary desorption
[15,16]. The flow is directed over a cold trap which collects the analytes. After completing this
first step, the cold trap is abruptly heated performing a second desorption [17]. The small
volume and the low thermal mass of the cold trap make it possible to use this step as an efficient
injection mechanism for GC [18]. As the primary desorption process is dynamic and the
desorption temperatures can be much higher compared to sHS-GC, this technique could prove
to be a better approach for the determination of RS from MPSi.
As a last resort, one could consider to destroy the MPSi with HF and perform HS-GC-FID on
the remaining solution. However, working with HF poses its own problems and risks. Indeed,
HF is quite toxic, highly corrosive and before transferring to a glass recipient, solutions need to
be neutralized. Moreover, the aqueous environment limits the number of VOCs that can
possibly be analyzed [9] and the reaction heat together with the extra manipulations increase
the risk to lose the most volatile compounds. Nevertheless, this approach may be useful to
confirm the results obtained with the TD-GC-FID method.
In this work a TD-GC-FID methodology for the determination of seven RS on MPSi with three
different pore sizes has been developed. Immobilization of the MPSi in the sample tube was
not straightforward and required special attention. After optimization, the method was
successfully validated. A HF digestion based FET-HS-GC-FID method for the determination
of DCM on MPSi was also developed and validated. Finally, both methods were applied to
experimental samples (itraconazole loaded on Davisil LC150 silica using DCM) in order to
determine the residual DCM content. The results from the proposed TD-GC-FID method were
compared with the results from the FET-HS-GC-FID method. This is the first study
31
representing validated TD-GC-FID analysis of RS from a drug loaded on MPSi with its specific
adsorption properties.
2.2. Experimental
2.2.1. Chemicals and reagents
o-Xylene (99%), methanol (MeOH) (99.99%), toluene (99.8%) and hexafluoroisopropanol
(HFIP) (99.5+ %) were purchased from Acros Organics (Geel, Belgium). Isopropanol (Isp)
(99.5%), ethanol (EtOH) (99.5%), acetone (Ace) (99.6%) and dichloromethane (DCM) (99.5%)
were acquired from Fisher Scientific (Loughborough, UK). 1,4-Dioxane was obtained from
Sigma Aldrich (St. Louis, MO, USA) and THF from Merck (Darmstadt, Germany). HF (40%)
was supplied by Riedel-de-Haën (Seelze, Germany) and sodium carbonate by Chem-Lab
(Zedelgem, Belgium). MPSi (Syloid AL-1 FP, Syloid XDP3050, Syloid 244FP EU and Davisil
LC150) with different pore sizes (2-3, 15 and 25 nm) were obtained from Grace Davison
(Worms, Germany). Samples of itraconazole loaded on Davisil LC150 using DCM and dried
at room temperature or at 40 °C were prepared by the division Drug Delivery and Disposition,
KU Leuven (Leuven, Belgium).
2.2.2. Instrumentation
2.2.2.1. TD-GC-FID/MS
TD-GC analyses were executed on a Clarus 680 GC coupled with a SQ8T MS and equipped
with a Turbomatrix ATD 350 thermal desorber. Empty stainless steel TD tubes (89 mm x 6.35
mm o.d.) with built-in gauze were used to carry the samples. Tenax TA and Air Monitoring
Trap (AMT) were used as trap material. Tubes, traps and equipment were all purchased from
Perkin Elmer (Waltham, MA, USA). Separations were carried out on ZB-624 column (30 m x
0.53 mm, df = 3 µm) from Phenomenex (Torrance, CA, USA). The effluent from the column
was split to the FID and mass spectrometer (MS) in a 3:1 ratio. A 10-microliter syringe with an
80 mm needle (Hamilton, Reno, NV, USA) was used to apply reference and calibration
solutions. Quartz filter paper (MN QF-10, Ø = 50 mm) from Macherey-Nagel (Düren,
Germany) was used to immobilize the sample.
2.2.2.2. HS-GC-FID/MS
HS-GC analyses were performed using an HS40 autosampler (balanced pressure system)
connected to an Autosystem XL GC equipped with an FID and a Turbomass Gold MS. HS vials
(20 mL) with PTFE-Sil caps were used as sample container. All equipment, vials and caps were
purchased from Perkin Elmer. Separations of RS were carried out on an AT-Aquawax column
32
(30 m x 0.53 mm, df = 0.50 µm) from Grace (Columbia, MD, USA). The effluent from the
column was split towards the FID and MS in a 3:1 ratio.
2.2.3. Standards and sample preparations

2.2.3.1. Standards and sample preparation for TD-GC-FID
For optimization of the TD parameters, a reference mix of the seven RS under investigation
(MeOH, EtOH, Ace, Isp, DCM, THF, HFIP) was made by adding 100 mg of each RS to a 100
mL volumetric flask containing already about 10 mL of o-xylene to reduce loss by evaporation.
Subsequently the content of the flask was brought to 100 mL with o-xylene. This procedure of
adding the reference to a partially filled volumetric flask is used for all RS containing solutions
in this work.
For method validation, a 100 mg/mL standard stock solution of the different RS in o-xylene
was prepared. A 10 mg/mL solution of toluene in o-xylene was used as correction standard
(CS) to correct the peak areas for differences in volumes applied on the quartz filter paper. From
the standard stock solution, calibration solutions ranging from 0.1 mg/mL to 80 mg/mL were
prepared by an appropriate dilution scheme. Before the calibration solutions were brought to
volume (10 mL) with o-xylene, 1 mL of CS was added. Finally, 2 µL of the reference solutions
was applied to the inlet side of the tube.
For analysis of real samples where DCM was used in the production, a similar procedure was
followed, but calibration was performed for DCM only.
Concerning sample preparation, 20 mg of MPSi was brought as a plug between two double
layers of quartz filters in the TD tube (see 2.3.2.2). Recovery was checked by applying 2 µL of
1, 5 and 10 mg/mL RS solutions to the inlet quartz filters of the plug with unloaded MPSi.
2.2.3.2. Standards and sample preparation for FET-HS-GC-FID

A stock solution of DCM in dioxane was prepared by dissolving 200 mg of DCM in 20 mL of
dioxane. Calibration solutions were prepared by diluting appropriate volumes of the stock
solution to obtain solutions in the range from 1 µg/mL to 30 µg/mL. Finally 30 µL was
transferred to a HS vial.
For sample analysis, a dispersion was made by shaking 1 mL of dioxane with 10 mg of MPSi
(loaded with itraconazole) in a polypropylene Falcon tube. Subsequently, the silica was
destroyed by adding 200 µL of HF to the dispersion, closing the tube and sonicating it for 30
min. This solution was brought up to 5 mL with dioxane. Next, a spoonful (~ 2.5 g) of
33
anhydrous sodium carbonate was added. After 10 min of stirring, the sodium carbonate was
allowed to precipitate and the supernatant was decanted to a centrifuge tube which was
centrifuged at 4500 g for 10 min. 30 µL of the centrifuged solution was transferred to a 20 mL
HS vial and sealed immediately with a PTFE-Sil cap prior to FET-HS-GC-FID analysis [19].
A blank was prepared from 10 mg of Davisil LC150 silica to which 15 mg of itraconazole was
added in a Falcon tube followed by the above described sample treatment. Recovery and
interference were checked using the blank to which appropriate amounts of DCM (5, 25 and 50
µg) in solution were spiked prior to HF addition and making up to volume.
2.2.4. Method validation

2.2.4.1. Method validation of TD-GC-FID
Selectivity, recovery, repeatability, sensitivity and linearity of the optimized procedure were
verified as validation criteria on 3 different types of MPSi with pore sizes of 2-3 nm, 15 nm and
25 nm respectively.
Selectivity was monitored by evaluating the separation of the seven RS and the peak purity
through MS as well as by checking the possible interference of compounds extracted from the
blank quartz filter paper or unloaded MPSi.
For recovery experiments, before loading the 2 µL of reference solution, the sample tubes
(containing MPSi as plug between the quartz filters) were conditioned for 20 min at 320 °C
under a helium flow of 30 mL/min to ensure that they were free from volatiles. Recovery was
assessed from the chromatographic response following the application of 2 µL of RS at 3
concentration levels brought on tubes filled with the three types of MPSi, versus a calibration
line obtained by spiking wide pore MPSi. Completeness of the process was verified by
performing a second TD-GC-FID run on each sample. Each experiment was performed in
triplicate.
Repeatability was tested by analyzing six times 2 µL of a 1 mg/mL solution and it was expressed
as relative standard deviation (%RSD). The LOD and LOQ were calculated by the signal-to-
noise (S/N) ratio measurement: S/N = 3 for LOD and S/N = 10 for LOQ.
Linearity was evaluated from the regression coefficient obtained from triplicate calibration
curves. For the three different pore sizes of MPSi, a five point calibration curve for each of the
seven RS was constructed. The amounts of RS brought on the tube were 2, 8, 12, 16 and 20 µg.
34
Eight point extended calibration curves (ranging from 0.2 to 160 µg/tube) were recorded for
MPSi with the smallest pore size.
2.2.4.2. Method validation of FET-HS-GC-FID

The linearity was evaluated on the basis of the regression coefficient from a five point
calibration curve (30 to 900 ng per vial) where each point was recorded in triplicate. LOD and
LOQ values were also retrieved by the S/N approach. The accuracy of the HS method was
determined in triplicate on three levels by calculating the recovery of DCM from a spiked blank.
These data were also used to calculate the repeatability in terms of RSD-values.
2.3. Results and discussion

2.3.1. Preliminary studies
This section contains a synopsis of some preliminary studies (without experimental details).
First, common sHS for the seven RS from MPSi (as a suspension in o-xylene) was explored.
Recovery values of less than 50% and high RSD-values (> 20%) for some RS led to the
assumption that the condition of a partition driven equilibrium was not fulfilled. This was
confirmed by the MHE plot which was nonlinear, indicating that the partition requirements
(like the absence of adsorption effects) are not respected. This implies that the sHS approach is
not applicable and causes calibration problems. In some cases it is possible to suppress the
adsorption effects [12], but for samples like MPSi with a very high specific surface and a
complex pore structure, this becomes impractical.
Also FET experiments yielded some low recoveries (< 60%), demonstrating that the transfer
from MPSi is not complete for all analytes. So, it was concluded that an alternative technique
like TD should be investigated.
2.3.2. TD-GC-FID method development

2.3.2.1. Selection of solvent to prepare the reference solution
The first step is the search for a suitable solvent to dilute the analytes, in this case the RS that
have to be included in the reference solution. Water, dimethylformamide (DMF),
dimethylacetamide (DMA) and dimethylimidazolidinone (DMI) are mostly used as diluents in
HS procedures. However, they are not always directly compatible with the RS that are traced.
For example, water is not compatible with DCM, while DMF, DMA and DMI are heating up
when HFIP is added. The temperature rise causes loss of DCM and the interaction of the diluent
with HFIP results in low sensitivity and high RSD-values for the latter. o-Xylene is compatible
35
with the RS of interest and did not generate heat upon mixing. Preliminary sHS-GC-FID
experiments confirmed the usability of o-xylene for this study.
2.3.2.2. Filling of the TD tubes

In order to immobilize the MPSi in the sample tube, a heat resistant inert material is needed that
allows transport of gas, but holds the MPSi. Quartz filter paper QF-10 has the chemical
inertness of quartz and is thermally stable up to 900 °C which is far above the TD working
range. QF-10 can easily be punched into shape. A suitable punch was made by increasing the
internal diameter of a commercial TD sample tube by about 0.4 mm and sharpening the
resulting edge. The slightly oversized punched out QF-10 disks were transferred directly from
the punch to the sample tube with a glass rod to avoid contamination. Two of those disks on
each side of the sample were sufficient to immobilize the MPSi. A schematic overview is shown
in figure 2.1.
Figure 2.1: Sample immobilization in TD tube.
2.3.2.3. Optimization of TD parameters
As TD-GC is a two stage desorption process, the list of parameters involved (Table 2.1) is quite
long. As a consequence of the steady state nature of mass and heat transfer processes in the
system, most parameters are influencing each other, making optimization rather complex.
36
Table 2.1: Optimized TD parameters
Parameter Optimized settings Range explored
Desorption temperature 300 °C 200 – 350 °C
Desorption time 80 min 30 – 100 min
Desorption flow 140 mL/min 35 – 180 mL/min
Sorbent in tube Quartz filter paper 1 – 4 layers
Trap low temperature -30 °C 4 to -30 °C
Trap high temperature 280 °C
Trap hold time 10 min 2 – 20 min
Trap material AMT AMT / Tenax
Inlet split 40 mL/min 10 – 60 mL/min
Outlet split 40 mL/min 10 – 60 mL/min
Column flow 4 mL/min
GC oven program 35 °C held 9 min;

35–260 °C at 20 °C/min,
held 10 min
GC/MS interface temperature 180 °C
The first step is the selection of a suitable cold trap. Apart from the option to construct a custom-
made version, the manufacturer leaves the choice between a single bed Tenax trap or a dual bed
AMT packed with carbonaceous sorbents. Performance of the Tenax trap was inferior to the
AMT, especially for MeOH and DCM. With the selected cold trap installed, inlet and outlet
split settings were optimized towards peak shape and carry over. Trap selection and split
optimization were performed using 2 µL of the RS reference mix solution injected on a blank
tube (i.e. 2 µg of each RS on tube). To investigate the influence of the remaining parameters, a
set of experiments was performed in which the FID response for each RS was monitored in
function of a single parameter. This was done for three types of MPSi with different pore sizes
and for the blank, applying each time 2 µL of the RS reference mix solution. The resulting data
were visualized in graphs expressing the relative peak area in function of the respective
37
parameter whereby the lowest area in each dataset was arbitrarily chosen as reference point
(100%). Some examples of these graphs can be seen in figure 2.2. The parameters giving the
highest stable response for all RS/MPSi combinations were considered to be the optimum
conditions which are given in Table 2.1.
a) MPSi 25nm b) MPSi 15n

170 135
160 130
125
150
MeOH 120
Response %
140
Response %
EtOH 115
130 Ace
110
Isp
120
105
DCM
110 THF 100
100 HFIP 95
90 90
0 20 40 60 80 100 120 200 250
Prim.des. Time (min) Pr
b)MPSi
c) MPSi2-3nm
15nm
135 a) MPSi 25nm b) MPSi 15nm
170
170 135
130
160 130
160
125
150 125
150 MeOH
MeOH 120
MeOH
Response%%
MeOH 120
140 EtOH
Response %
EtOH 140
%
115 EtOH
EtOH
Response
Ace 115
Ace 130
130
110 AceAce
Isp Isp 110
IspIsp
120
120
105
DCM DCM 105
DCMDCM
THF 100
110
110 THF
THF 100
THF
HFIP HFIP
100
10095 HFIP
HFIP 95
9090 90
90 200 250
120 0200 20 250 40 300
60 80 350 100 400
120
0 50 100 150 200
Prim.des.
Prim. Des. Time
Temp(min)
(°C) Prim
Prim. des. flow (ml/min)
c) MPSi 2-3nm
170
160
150
MeOH
140
Response %
MeOH EtOH
EtOH 130 Ace
Ace Isp
120
DCM
Isp
110 THF
DCM
100 HFIP
THF
HFIP 90
0 50 100 150 200
Prim. des. flow (ml/min)
200
Figure 2.2: Charts illustrating the influence on the detector response of (a) the TD primary
desorption time, (b) the TD primary desorption temperature and (c) the TD primary
desorption flow. Each time a different type of MPSi was chosen as example.
38
2.3.3. TD-GC-FID method validation
All tubes with untreated silica immobilized between the quartz filters were conditioned before
injecting the RS reference solution to remove moisture and eventually some volatile residuals.
TD-GC-MS analysis of a blank tube (a tube containing unloaded MPSi) revealed that under
these conditions water could not be completely removed. It is possible that the small amount of
water found in the tubes containing MPSi after conditioning is absorbed from the atmosphere
in the short manipulation time between conditioning and analysis. No other peaks from the
blank were found. Full release of the RS from the blank was confirmed by performing a second
TD-GC-FID run. On all tested levels, the signals for RS and diluent in this second run, were
below the detection limit (figure 2.3).
Figure 2.3: TD-GC-FID chromatogram of first desorption (upper chromatogram) and second
desorption on the same tube (lower chromatogram).
The results from the recovery experiments in the presence of MPSi are represented in Table
2.2. As a general observation, it can be stated that a larger pore size gives a slightly better
recovery. The somewhat lower average recovery on MPSi 2-3 nm can be attributed to the
behavior of Isp. For DCM the recovery on the different types of MPSi is comparable. Although
39
recoveries are acceptable (apart from Isp on MPSi 2-3 nm), it is advisable to verify the recovery
for each new RS/MPSi combination. A possible explanation for the somewhat inferior behavior
of Isp can be its spatial configuration. Isp can probably enter the pores and adsorb to the surface,
but it experiences difficulties to diffuse out of the pores (mainly when they are small) during
desorption. This assumption was supported by the complete recovery of n-propanol. Further,
the good recovery of Isp from the large pore size silica indicates that Isp can drive off more
easily from large pores during desorption.
Table 2.2: Recovery of RS from different MPSi following TD-GC-FID.
MPSi 2-3 nm MPSi 15 nm MPSi 25 nm average

RS RS (µg) Recovery RSD Recovery RSD Recovery RSD per RS
on tube (%) (%) (%) (%) (%) (%) (%)
2 97.5 1.8 98.2 1.5 98.9 1.2
MeOH 10 98.8 0.6 98.7 1.6 98.6 0.5 98.4
20 97.7 1.1 98.2 1.3 98.8 0.5
2 97.8 1.8 98.5 1.5 98.5 1.5
EtOH 10 98.3 1.4 97.9 1.5 98.8 1.0 98.2
20 97.9 2.0 97.7 1.7 98.5 0.5
2 98.8 1.0 98.4 1.4 98.7 0.4
Ace 10 99.5 2.1 98.9 1.2 98.6 1.2 99.0
20 98.2 1.4 99.8 1.4 99.8 0.3
2 92.1 2.8 97.9 2.3 98.5 1.1
Isp 10 91.7 0.6 98.3 1.4 98.8 1.7 96.1
20 91.5 2.8 97.9 1.6 97.8 1.3
2 99.7 1.8 99.5 1.2 99.2 1.5
DCM 10 99.3 1.6 99.1 1.5 99.1 1.7 99.4
20 99.1 1.2 100.0 1.4 99.8 1.3
2 98.8 2.8 98.8 1.9 99.8 1.1
THF 10 97.5 2.3 98.7 0.5 98.3 0.6 98.4
20 97.7 1.3 98.1 1.7 98.2 1.8
2 98.1 1.7 98.9 1.5 98.4 1.0
HFIP 10 98.0 1.4 97.9 2.1 98.1 1.3 98.1
20 97.5 1.6 98.2 1.2 98.2 1.2
average per
MPSi (%) 97.4 98.6 98.7
average
without Isp 98.3 98.6 98.8
40
Repeatability data for the seven RS on three MPSi are represented in Table 2.3 as range of
RSD-values. These are all lower than 3%, which is more than acceptable for the determination
of RS.
The good sensitivity of the method was illustrated for the different RS by the LOD and LOQ
values which were found to be 0.03 – 0.08 µg and 0.1 – 0.2 µg on tube, respectively. When 20
mg of MPSi was weighed, this corresponds to 0.00015 – 0.0004 % and 0.0005 – 0.001 %
respectively.
For each of the MPSi used in this part of the study, the range of determination coefficients
obtained from the five point calibration curves for the different RS can also be found in Table
2.3. All R2 were not lower than 0.998. Moreover, it was noted that the 95 % confidence interval
of the intercept included zero. In addition, a residual plot was produced for each analyte and
the residuals were distributed randomly around the horizontal axis. So, it was concluded that
linear regression is appropriate to fit the data.
Table 2.3: Validation results for the RS on different silicas using the proposed TD-GC-FID
method. Linearity and repeatability are expressed as ranges within which the values lie for the
seven RS.
MPSi pore size Linearity Repeatability

(nm) R2 RSD (%) (n=6)
2-3 0.998-0.999 0.5-2.9
15 0.998-0.999 0.7-2.8
25 0.998-0.999 0.3-1.9
In order to expand the calibration range of the method using a single curve it would be necessary
to switch between different detector attenuations (ATT) (amplification factor) while recording
the calibration data. Otherwise peak areas could not be measured properly. Adding the same
amount of toluene to each solution would allow to compensate for variations in injection
volume. Since this is the same for all experiments, the area of toluene should always be
measured with the same ATT. An eight point calibration curve (0.2, 1, 2, 8, 20, 60, 120 and 160
µg on tube) was constructed: the first four points were recorded at ATT -4, the last five points
at ATT 0. The calibration point at 8 µg was thus recorded with both ATTs yielding two peak
41
areas. The ratio of those two areas was used to transpose the values obtained with ATT 0 to the
ATT -4 range. As the experiment was performed in triplicate, 24 areas for the toluene peak
were available to calculate the mean. The areas of the RS were normalized towards this mean
toluene area to reduce the influence of variations in injection volume. As the proposed
mechanism is only used to compensate the calibration curve without any relationship to the
sample (silica is introduced as a solid), the term internal standard was avoided as it may be
misleading. Since the MPSi with the smallest pores can be considered as the most critical, it
was selected for this experiment. The R2-values for the seven RS varied between 0.997 and
0.999 (with RSD-values < 3%) indicating that also in this range good linearity was obtained.
2.3.4. FET-HS-GC-FID method development

To 20 mg of each MPSi, 1 mL of dioxane and increasing amounts (in steps of 50 µL) of HF
40% were added. An amount of 200 µL was sufficient to transform the MPSi dispersion into a
clear solution after 30 min of ultrasonic treatment. Anhydrous sodium carbonate was used to
neutralize the remaining acid and to remove the introduced water. The FET-HS boundary
conditions for dioxane were calculated [10] and the conditions used are represented in Table
2.4.
42
Table 2.4: Used FET-HS-GC-FID parameters.
Parameter Settings
Equilibration temperature (ET) 160 °C
Equilibration time 15 min
Needle temperature ≥ 5 °C above ET
Transfer line temperature ≥10 °C above ET
Pressurization time 1.0 min
Injection time 0.04 min
Needle withdrawal time 0.4 min
Injection port temperature ≥ 15 °C above ET
Carrier gas pressure 130 kPa
35 °C held 9 min; 35–70 °C at 20 °C/min, held

GC oven
3 min; 70-160 °C at 40 °C/min, held 10 min
Split ratio 1:5
Sample volume in vial 30 µL
Detector type FID
Detector temperature FID 220 °C
2.3.5. FET-HS-GC-FID method validation

Four Falcon tubes, representing the different stages of the sample treatment (DCM/dioxane,
DCM/dioxane/itraconazole, DCM/dioxane/HF neutralized and DCM/dioxane/MPSi/HF
neutralized) containing the same amount of DCM were prepared and subjected to FET-HS-GC-
MS evaluation. DCM levels were found to be similar and no interference was noticed.
The linearity of the FET-HS-GC-FID method was determined by constructing five point
calibration curves and the R2-values were found to be equal to or higher than 0.997, indicating
that a good linear relationship was obtained for the analytes. In addition, residual plots indicated
that the residuals were randomly distributed around the horizontal axis and the 95 % confidence
interval of the intercept included zero. Repeatability of the method was reported as RSD-values.
They were all lower than 3% which is acceptable for analysis of such low amounts by GC. LOD
43
and LOQ values amounted to 10 and 30 ng/vial respectively. When 20 mg of MPSi was
weighed, this corresponds to 0.00005 % and 0.00015 % respectively. Recovery was
investigated by adding known amounts of DCM to a blank sample. Data for recovery are given
in Table 2.5.
Table 2.5: Recovery and residual amount of DCM in MPSi loaded with itraconazole using
FET-HS-GC-FID and TD-GC-FID.
Method Recovery Residual DCM in MPSi

Dried at room
average RSD (%) temperature Dried at 40 °C
% of (n=9)
DCM % of RSD (%) % of RSD (%)
(3 levels) DCM (n=6) DCM (n=6)
FET-HS-GC-FID 100.6 1.5 0.68 2.5 0.072 2.9
TD-GC-FID 99.7 2.2 0.67 2.3 0.074 2.7
2.3.6. Applications
Both methods were applied to two samples of an experimental drug formulation (itraconazole
loaded on Davisil LC150 silica using DCM) in order to evaluate the difference in drying
conditions (room temperature vs. 40 °C). As indicated in Table 2.5, a higher amount of DCM
(0.67% w/w) was found in the sample dried at room temperature than in the sample dried at
40 °C (0.074% w/w). In both cases the value is above the ICH acceptance limit (0.06 %). The
results obtained by FET-HS-GC-FID and TD-GC-FID are similar. Statistical comparison of the
two methods was performed at a 95% confidence level which indicated that there was no
significant difference (p=0.65) between the FET-HS-GC-FID and TD-GC-FID approach where
the latter was clearly more user friendly.
2.4. Conclusions
Results show that the developed TD-GC-FID method is an excellent approach for the analysis
of RS in MPSi loaded with drugs, especially when the conventional HS-GC-FID methods are
not convenient due to the unique surface chemistry of porous silica. The use of HF to dissolve
the silica prior to FET-HS-GC-FID proved to be a useful approach to confirm the results
obtained by TD-GC-FID, but it is less obvious from a practical point of view.
44
The proposed method is simple and provides good recovery for a set of analytes with different
polarity and volatility as well as good linearity over a wide range. Moreover, unlike
conventional HS-GC-FID, the proposed method does not require sample pretreatment and also
no effort to select a suitable solvent. This has the advantage that there is neither interference
from a large solvent peak nor from impurities eventually present in the solvent. In addition,
since the amount of sample that is introduced in the TD can still be increased, it is possible to
further improve the sensitivity of the method. Another possibility is to adapt the split. The
reference solution can easily be applied to the inlet quartz filters keeping the blank MPSi in its
place. Finally, analysis of residual DCM in a research sample by the proposed method yielded
quantitative results which showed no significant difference (p > 0.05) with those obtained by a
conventional method.
2.5. References
[1] M. Van Speybroeck, V.R. Barillaro, T.D. Thi, R. Mellaerts, J. Martens, J. Van
Humbeeck, J. Vermant, P. Annaert, G. Van den Mooter, P. Augustijns, Ordered
mesoporous silica material SBA-15: A broad-spectrum formulation platform for poorly
soluble drugs, J. Pharm. Sci., 98 (2006) 2648-2658.
[2] L.T. Zhuravlev, The surface chemistry of amorphous silica. Zhuravlev model, Colloid
Surface A, 173 (2000) 1-38.
[3] F. Twaiq, M. Nasser, S. Al-Hajri, M. Al-Hasani, Adsorption kinetics of alcohols over

MCM-41 materials, International Journal of Chemical, Molecular, Nuclear, Materials
and Metallurgical Engineering, 6 (2012) 673-677.
[4] I.I. Slowing, J.L. Vivero-Escoto, B.G. Trewyn, V.S.Y. Lin, Mesoporous silica
nanoparticles: structural design and applications, J. Mater. Chem., 20 (2010) 7924-
7937.
[5] K. Cha, K. Cho, M. Kim, J. Kim, H. Park, J. Park, W. Cho, J. Park, S. Hwang,
Enhancement of the dissolution rate and bioavailability of fenofibrate by a melt
adsorption method using supercritical carbon dioxide, Int. J. Nanomedicine, 7 (2012)
5565-5575.
[6] S. Kwon, R.K. Singh, R.A. Perez, E.A. Abou Neel, H. Kim, W. Chrzanowski, Silica-
based mesoporous nanoparticles for controlled drug delivery, J. Tissue Eng., 4 (2013)
http://doi.org/10.1177/2041731413503357.
45
[7] T. Limnell, H. A. Santos, E. Mäkilä, T. Heikkilä, J. Salonen, D.Y. Murzin, N. Kumar,
T. Laaksonen, L. Peltonen, J. Hirvonen, Drug delivery formulations of ordered and
nonordered mesoporous silica: comparison of three drug loading methods, J. Pharm.
Sci., 100 (2011) 3294-3306.
[8] European Pharmacopoeia, 9th ed., Identification and Control of Residual Solvents.
(Section 2.4.24), European Department for the Quality of Medicines, Council of
Europe, Strasbourg, 2017.
[9] D.M. Kialengila, K. Wolfs, J. Bugalama, A. Van Schepdael, E. Adams, Full

evaporation headspace gas chromatography for sensitive determination of high boiling
point volatile organic compounds in low boiling matrices, J. Chromatogr. A, 1315
(2013) 167-175.
[10] Y. Yu, B. Chen, C. Shen, Y. Cai, M. Xie, W. Zhou, Y. Chen, Y. Li , G. Duan, Multiple
headspace single-drop microextraction coupled with gas chromatography for direct
determination of residual solvents in solid drug product, J. Chromatogr. A, 1217 (2010)
5158-5164.
[11] J. Li, S. Shao, M. Solorzano, G. J. Allmaier, P. T. Kurtulik, Determination of the

residual ethanol in hydroalcoholic sealed hard gelatin capsules by static headspace gas
chromatography with immiscible binary solvents, J. Chromatogr. A, 1216 (2009) 3328-
3336.
[12] B. Kolb, L.S. Ettre, 2006. Static Headspace Gas Chromatography: Theory and Practice,
John Wiley and Sons, New Jersey, p. 191-204.
chromatography, Elsevier Inc., Waltham, 2012, p. 272-273.
[14] K. Ahshimoto, K. Urakami, Y. Fujiwara, S. Terada, C. Watanabe, Determination of

residual solvents in pharmaceuticals by thermal desorption-GC/MS, Anal. Sci., 17
(2001) 645.
[15] E. Crespo, S. Devasena, C. Sikkens, R. Centeno, S.M. Cristescu, F.J.M. Harren,

Proton-transfer reaction mass spectrometry (PTRMS) in combination with thermal
desorption (TD) for sensitive off-line analysis of volatiles, Rapid Commun. Mass
Spectrom., 26 (2012) 990-996.
46
[16] E. Woolfenden, Optimising Analytical performance and extending the application
range of thermal desorption for indoor air monitoring, Indoor Built Environ., 10 (2001)
222-231.
[17] N. Ramírez, A. Cuadras, E. Rovirab, F. Borrulla, R.M. Marcéa, Comparative study of

volatile organic compounds in ambient air, Talanta, 82 (2010) 719-727.
desorption-capillary GC analysis: summary of data and practical guidelines, J. Air &
Waste Manag. Assoc., 47 (1997) 20-36.
[19] N. van Boxtel, K. Wolfs, A. Van Schepdael, E. Adams, Application of acetone acetals
as water scavengers and derivatization agents prior to the gas chromatographic analysis
of polar residual solvents in aqueous samples, J. Chromatogr. A, 1425 (2015) 62-72.
47
48
Chapter 3: Determination of residual dimethylsulphoxide
in drug loaded gelatin using thermal desorber - gas
chromatography
Adissu Alemayehu Asfaw, Kris Wolfs, Ann Van Schepdael,
Erwin Adams

Pharmacological Sciences, Pharmaceutical Analysis, Herestraat 49,
O&N2, PB 923, 3000 Leuven, Belgium
J. Pharm. Biomed. Anal., 2018, 153, 193–198.
49
50
Abstract
Traditional headspace - gas chromatography (HS-GC) methods for the determination of
residual solvents (RS) start from a homogenous sample solution. Subsequently, it is
challenging to determine RS using HS-GC techniques from insoluble solid samples like gelatin
which is practically impossible to dissolve or distribute uniformly in water and common organic
solvents. In this study, a thermal desorber combined with capillary gas chromatography and
flame ionization detection/mass spectrometry (TD-GC-FID/MS) was used for quantitative
determination of residual dimethylsulfoxide (DMSO) in gelatin without sample pretreatment.
A sample of gelatin was sandwiched between two quartz filter double layers in a
polytetrafluoroethylene insert which was then placed in its entirety into a thermal desorption
tube. Factors affecting the performance of TD-GC including desorption time, desorption
temperature, desorption flow and type of adsorbent were studied by applying a standard solution
of DMSO in methanol on a blank gelatin bed. Validation results of the proposed method showed
good linearity with an R2-value higher than 0.999 for a wide concentration range and good
sensitivity with a limit of detection and limit of quantification of 0.1 µg and 0.2 µg on tube,
respectively. The proposed method shows recovery values close to 100 %. In addition, a
conventional HS-GC method following enzymatic degradation of gelatin was developed to
verify the proposed TD-GC method. Both methods were applied for the determination of
residual DMSO in gelatin that was loaded with an experimental drug. Results were comparable,
but the enzyme assisted HS-GC method was more time consuming and expensive.
Keywords: thermal desorber - gas chromatography, gelatin, residual DMSO
51
3.1. Introduction
Gelatin is derived from collagen by hydrolytic degradation which breaks crosslinks between
strands and to some extent hydrolyzes the strands into fragments. Gelatin has widespread
application in food as a stabilizer, thickener and texturizer. In pharmaceuticals it is mainly used
to make capsules, as a binder in tablet formulations, as drug carrier and also as a coating to ease
swallowing or mask unpleasant tastes [1]. Due to its biodegradability it is also often applied in
the biomedical field as sealant for vascular prostheses, wound dressing and adsorbent pad for
surgery [2]. If gelatin is heated, it undergoes structural and physicochemical transformations.
When gelatin is treated with hot water (40 to 50 °C), it dissolves and takes a coil conformation.
Further heating results in partial or complete loss of solubility in water due to crosslinking [3].
One of the most important properties of gelatin is its gelation in water which is concentration
dependent and due to H-bonds linking peptide groups and hydrophobic interactions between
apolar side-groups of the gelatin macromolecules. Addition of some solvents like DMSO will
result in a disruption of both the H-bonds and the hydrophobic interactions in the gel network
[4,5].
For the loading of drugs on carriers like gelatin, solvents are used that are removed afterwards.
However, this removal is not complete and some traces will remain as residual solvents (RS).
The detection and quantification of RS in pharmaceuticals is very strict and requires sensitive
analytical techniques to avoid potential human health risks. Moreover, RS can influence the
physicochemical characteristics of drugs such as dissolution properties and morphology [6].
GC is the most appropriate method to analyze RS from different materials [7]. It is often
combined with static headspace (sHS) injection which has the advantage that the sample matrix
is not introduced into the GC system [8]. Due to the fact that RS are present in very small
quantities, their sensitive detection using sHS-GC is challenging and not always successful [9].
sHS-GC also suffers from difficulties to determine high boiling analytes (like DMSO) in low
boiling matrices where the matrix will act as a dilute of the gas phase and can create
overpressure in the vial. Matrix matched calibration is mandatory in sHS, but this is not always
possible. The above mentioned problems can be partially solved by the full evaporation
technique (FET) which is based on complete evaporation of the analyte(s) from the sample and
so will overcome matrix effects [10]. However, traditional HS methods for the determination
of RS start from a homogenous sample solution. As a result, it is challenging to determine high
boiling RS using these techniques for solid samples (like gelatin) which are practically
impossible to dissolve or distribute uniformly in water or common organic solvents.
52
When performing HS techniques with aqueous samples, typical HS oven temperatures are only
around 85 °C to ensure that the HS pressure does not exceed the limitations of the instrument
[11]. On the one hand this temperature is too low to volatilize DMSO (which has a boiling point
of 189 °C) while on the other it causes a rigid gelatin gel. To solve this problem, Li et al.
performed a preceding binary solvent extraction to determine residual ethanol used to seal hard
gelatin capsules [12]. However, this approach is not applicable for the determination of residual
DMSO which has different physicochemical characteristics.
Direct quantification by HS-GC from solid samples could be a solution for the above mentioned
problems, but it is difficult to compensate for matrix effects during calibration. This is certainly
true for DMSO which can interact with gelatin. Multiple headspace extraction (MHE) prior to
GC has also been applied to remove matrix influences. In combination with single drop
microextraction (SDME), it has been described for the determination of residual ethanol and
methanol in drug products [13]. However, MHE is time consuming and the low volatility of
DMSO and heat instability of gelatin remain problematic. Hashimoto et al. applied thermal
desorption (TD)-GC using a pyrolyzer and on-column cryofocusing for the determination of
RS from solid pharmaceuticals [14]. The method showed some limitations, mainly related to
the cryofocusing including ice blockage, incomplete retention of very volatile compounds, loss
of high-boiling solvents due to aerosol formation and high running costs (systems consumed
up to 6 L of liquid nitrogen per hour in operation). Moreover, because the cryofocusing device
was connected directly to the GC column, it was difficult to implement essential pre-desorption
checks such as leak testing [15]. Nonetheless, a modified set-up of TD instruments creates
interesting possibilities. In its basic form, a tube with an adsorbent used to collect samples
(typically for air/environmental monitoring) is brought in the TD and heated, involving the
dynamic extraction of volatile compounds in a flow of inert gas. This is known as primary
desorption. The gas flow is transported to a cold trap which contains one or more sorbents and
works at a lower temperature. Next, it is rapidly heated so that all retained analytes are released
in a very short time (a few seconds) and transferred to the GC through a heated transfer line
[16]. The simplicity of direct TD relative to conventional sHS methods is that no additional
dissolution or salting-out steps are required and that it does not rely on partition coefficients or
equilibria. Furthermore, complete (or nearly complete) extraction is often possible in one run
[17].
In this work, TD followed by GC has been applied in an unconventional way using modified
sample introduction. The developed TD-GC method allowed quantitative determination of
53
residual DMSO in gelatin samples without any sample pretreatment. Factors affecting the
performance of TD-GC including desorption time, desorption temperature, desorption flow and
adsorbents were studied. The method was applied to an experimental sample of gelatin loaded
with a drug using DMSO. To verify the newly developed method, results were compared with
an alternative, time consuming and expensive method based on the complete enzymatic
digestion of gelatin combined with dehydration and FET-HS-GC analysis.
3.2. Experimental
3.2.1. Chemical reagents
Active human recombinant matrix metalloproteinase-2 (MMP-2, 0.1 mg/mL) was provided by
Calbiochem (Merck, Darmstadt, Germany). Methanol (99.99 %), N,N-dimethylformamide
(DMF, 99.8 %) and 2,2-dimethoxypropane (DMP, 98+ %) were obtained from Acros Organics
(Geel, Belgium). Benzyl alcohol (BA, > 99 %), DMSO (> 99.9 %) and LC-MS grade water
were purchased from Sigma Aldrich (St. Louis, MO, USA). Hydrochloric acid (37.5 % w/w)
was bought from VWR International (Fontenay-sous-Bois, France). Sodium carbonate was
from Chem-Lab (Zedelgem, Belgium).
The same instrument as in chapter 2 was used here. A polytetrafluoroethylene (PTFE)
desorption tube inserts were used to place the samples in the TD tubes. Quartz filter (MN QF-
10, Ø = 50 mm) from Macherey-Nagel (Düren, Germany) was used to immobilize the sample
in the PTFE insert and also to apply the reference solution for method validation. Separations
were carried out on an AT-Aquawax column (30 m × 0.53 mm, df = 0.50 µm) from Grace
(Columbia, MD, USA). The TD-GC parameters are listed in Table 3.1. The effluent from the
column was split to the FID and MS in a 3:1 ratio. A 10-microliter syringe with an 80 mm
needle (Hamilton, Reno, NV, USA) was used to apply 2 µL of calibration solutions. Tubes with
empty PTFE insert were conditioned at 250 °C for 20 min with a constant helium flow (30
mL/min) prior to introduction of both standards and samples.
54
Table 3.1: Used TD-GC parameters.
Parameter Optimized setting Range explored
Desorption temperature 230 °C 180 – 280 °C

Desorption time 90 min 20 – 120 min
Desorption flow 120 mL/min 20 – 200 mL/min
Trap low temperature 4 °C -30 to 10 °C
Trap hold time 6 min 2-20 min
Trap material Tenax TA AMT/Tenax TA
Inlet split 40 mL/min 10-60 mL/min
Outlet split 80 mL/min 10-100 mL/min
GC oven program 80 °C held 1 min; 80–160 °C at 40
°C/ min, held 5 min; 160-250 °C at
40 °C/min, held 2 min
3.2.2.2. HS-GC-FID
HS-GC-FID analyses were performed on a Perkin Elmer Clarus 480 equipped with a
Turbomatrix 40 HS autosampler (balanced pressure system). All HS parameters are given in
Table 3.2. HS vials and PTFE/Sil caps were purchased from Perkin Elmer as well. The column
and GC oven temperature program were the same as used for TD-GC. A thermomixer
“comfort” from Eppendorf (Hamburg, Germany) was used during digestion of gelatin.
3.2.3. Standards and sample preparations

3.2.3.1. Standards and sample preparations for TD-GC
For method optimization a 10 mg/mL solution of DMSO in methanol was prepared. For
validation, a stock solution of 200 mg/mL of DMSO in methanol was prepared. Calibration
solutions of seven concentrations between 0.1 and 160 mg/mL were prepared by diluting
appropriate volumes of the stock solution in methanol. A 80 mg/mL solution of DMF in
methanol was used as correction standard (CS) to correct the peak areas for differences in
volumes applied in the TD tube. Before the calibration solutions were brought to volume (10
mL) with methanol, 2 mL of CS was added. For method optimization and validation, 10 mg of
55
blank gelatin plugged between two double layers of quartz filter in a PTFE insert was placed in
a TD tube. Finally, 2 µL of the reference solutions were applied on the quartz filters on the inlet
side of the tube. Regarding sample analysis, 10 mg each of two experimental drug loaded
gelatin samples dried under different conditions were immobilized by sandwiching them
between two double layers of quartz filter in a PTFE insert which was placed in an empty
desorption tube prior to TD-GC analysis. Since the amounts of residual DMSO were higher
than expected (see section 3.3.3), the amount of gelatin was reduced to 2 and 5 mg to avoid
overloading.
Table 3.2: Used HS-GC parameters.
Parameter Settings

Transfer line temperature ≥ 10 °C above ET
Injection port temperature ≥ 15 °C above ET
GC oven program
80 °C held 1 min; 80–160 °C at
40 °C/ min, held 5 min; 160-250
°C at 40 °C/min, held 2 min
Split ratio 1:5

FID temperature 220 °C
3.2.3.2. Standards and sample preparations for FET-HS-GC

3.2.3.2.1. Digestion of gelatin using MMP-2 prior to HS-GC analysis
First, 50 mM HEPES buffer of pH 7.5 was prepared by dissolving 0.65 g of HEPES sodium
salt in water and the pH was adjusted with concentrated hydrochloric acid before bringing to a
volume of 50 mL with water. Next, 50 µL of MMP-2 solution (0.1 mg/mL) was diluted to 500
µL with HEPES buffer. The amount of enzyme and duration required to digest the gelatin were
56
calculated on the basis of the gelatinolytic activity [18]. The following preparations were
digested by continuously mixing 10 mg of sample in 1 mL of HEPES buffer with 100 µL of the
diluted MMP-2 solution in a thermomixer at 37 °C for 125 hours: (i) two gelatin samples dried
at different conditions after drug loading, (ii) blank gelatin for testing interference and (iii) two
spiked blank gelatins for evaluation of recovery. In the latter case, the 1 mL of HEPES buffer
was replaced by 1 mL of 0.1 and 1 mg/mL respectively of DMSO in HEPES buffer. When the
digestion procedure was complete, each solution was diluted to 5 mL with water.
3.2.3.2.2. Solutions for FET-HS-GC analysis

A 20 mg/mL stock solution of DMSO in water was prepared. Calibration solutions were
prepared by diluting appropriate volumes of the stock solution to obtain solutions in the
concentration range from 0.05 to 2 mg/mL. To improve the sensitivity, a method developed and
validated by van Boxtel et al. was applied, based on the use of an acetone acetal as water
scavenger prior to FET-GC for the analysis of high boiling analytes in aqueous samples [11].
So, 4 mL of each of the digested solutions (from section 3.2.3.2.1) or the calibration solution
were transferred into a volumetric flask of 50 mL and 40 mL of DMP was added to react with
water. Reactions were catalysed by adding 10 and 20 µL of hydrochloric acid (37.5 % w/w) to
the volumetric flask for the calibration solution and the digested solution, respectively. The
double volume of hydrochloric acid added to the digested solution was to compensate for the
buffer used during digestion. The solution cools down significantly during the hydrolysis
reaction which was considered to be complete when the flask was again at room temperature.
Before completing to volume with methanol, 1 mL of the appropriate internal standard solution
(benzyl alcohol in methanol, 4 mg/mL) was added. Samples were neutralised afterwards by
addition of a spoonful (~ 5 g) of sodium carbonate to obtain a saturated solution. Since sodium
carbonate does nearly not dissolve in the mixture, solutions were stirred for 10 min with a
magnetic stir bar to improve contact. Supernatant was decanted in a centrifuge tube and
centrifuged at 4500 g for 10 min. Next, 1 mL of centrifuged solution was transferred into a HS
vial and the open vials were placed in a vacuum oven (≤ 0.1 kPa) at room temperature for 5.5
min to remove the excess of methanol, acetone and DMP. After evaporation, the vials were
capped with a PTFE/Sil cap to be analysed with FET-HS-GC [11].

3.2.4.1. Method validation for TD-GC
Selectivity was monitored by evaluating the separation of analytes and the peak purity through
MS, as well as by checking the possible interference of compounds extracted from the blank
57
quartz filter, blank gelatin or the loaded drug. The linearity was tested by analysing calibration
solutions with the described TD-GC method at six concentrations in a range from 1 to 160
mg/mL, each injected in triplicate. After plotting the peak areas vs. the concentrations, the
regression coefficient was calculated. The limit of detection (LOD) and limit of quantification
(LOQ) were determined using the signal-to-noise (S/N) ratio: S/N = 3 for LOD and S/N = 10
for LOQ. To exclude any interference from contaminants, blank gelatin was analysed as
described above. Repeatability was tested by analyzing six tubes of 20 mg/mL standard solution
in 1 day and expressed as percentage relative standard deviation (RSD). Before loading the 2
µL of reference solution, the sample tubes (containing blank gelatin sandwiched between the
quartz filters) were conditioned for 20 min at 250 °C under a helium flow of 30 mL/min to
ensure that they were free from volatiles. The accuracy of the method was determined by
comparing the detector response after spiking 2 µL of standard solutions at three concentration
levels of 1, 20 and 160 mg/mL (corresponding to 2, 40 and 320 µg respectively) on quartz filter
in the presence and absence of 10 mg of blank gelatin. The recovery without the blank gelatin
was considered to be 100 %. Complete extraction of the analyte during the first run was
confirmed by performing a second run on the same tube.
3.2.4.2. Method validation for FET-HS-GC

Linearity was assessed by the regression coefficient of a five point calibration curve in the range
from 0.05 to 2 mg/mL. Repeatability was tested by analyzing the 1 mg/mL concentration six
times. The recovery was evaluated by spiking blank gelatin with two concentrations (10 mg of
blank gelatin spiked with 0.1 and 1 mg of DMSO) and then treated with the procedures
described in section 3.2.3.2.1. Finally, all the calibration and recovery solutions were treated
with the procedure mentioned in section 3.2.3.2.2 and then analysed by the FET-HS-GC
method.
3.3. Results and Discussion

3.3.1. TD-GC method
3.3.1.1. TD-GC method development
Quartz filter paper was used as support material to apply the reference solution and to
immobilize the gelatin because it is inert, has a high thermal stability (above 900 °C) and is
easy to cut to fit into the thermal desorption tube [19]. A sample or blank gelatin was brought
into a PFTE insert with a double layer of quartz filter at both ends (see figure 3.1) before it was
put in the desorption tube for TD analysis. This was done to ensure that molten sample does
not move out of the tube and can easily be removed after analysis. Spiking was performed on
58
the inlet side of the tube so that the spiked compounds of interest should pass through the gelatin
bed during the primary desorption. This imitates adsorption/desorption of real samples as
closely as possible.
Figure 3.1: Sample introduction to TD tube.
An important consideration during the primary desorption is whether the volatile components
released from the sample are being properly retained on the cold trap sorbent bed. With a single
sorbent any breakthrough will result in the loss of compounds, the extent of which will be
unknown. If the sorbent is too weak, the very volatile compounds will break through, while the
less volatile ones are retained. On the other hand, if the chosen sorbent is too strong, the very
volatile compounds will now be retained, but it will be more difficult later to desorb the less
volatile ones. For this reason most traps are composed of two beds, a weaker and a stronger
[20]. In this study, first an Air Toxic trap was tried which has two types of adsorbents, carbon
black as weak adsorbent and carbon molecular sieve as strong adsorbent. However, the peak
for DMSO was broad and showed tailing due to the problematic desorption of DMSO from the
strong adsorbent. Therefore, a Tenax TA trap which is weak, inert and hydrophobic, was used
and the obtained chromatograms showed sharp peaks and no interference.
To investigate the influence of the TD parameters, a set of experiments was performed in which
the FID response was monitored in function of a single parameter. An overview of the ranges
explored and final settings is given in Table 3.1. For each experiment, 2 µL of standard solution
was applied on a blank gelatin plug between quartz filter inside a PTFE insert. The resulting
data for the optimization were visualized in graphs expressing the relative peak area in function
59
of the respective parameter whereby the lowest area in each dataset was arbitrarily chosen as
reference point (100 %).
Desorption at high temperature may result in decomposition of analyte, while a low temperature
could lead to incomplete desorption of the residual solvent. Figure 3.2(a) depicts that desorption
of DMSO reached a plateau between 220 and 240 °C. So, 230 °C was selected as optimum
primary desorption temperature. No decrease in the FID peak area of DMSO or no extra peak
in the MS chromatogram was noticed till the temperature reached 240 °C.
In order to minimize analysis time and optimize sample throughput, the optimum desorption
time was investigated. As indicated in figure 3.2(b), around 80 min complete desorption was
obtained, but after 100 min a decrease was observed. This breakthrough in function of time is
caused by the increasing gas volume (= time × flow) and might be influenced by the fact that
the lower temperature could not be maintained in the centre of the trap because of the hot gas
flowing through it for a long time. Since only the compartment around the trap is temperature
controlled, the low temperature of the trap itself may not always be guaranteed during
desorption. So, 90 min was selected as desorption time.
a) Desorp ti on temp . b ) Desorp ti on ti me

2500 800
Response %
Response %
2000 600
1500 400
1000
200
500
0 0
150 200 250 300 0 50 100 150
Time (min)
T (°C)
c) Desorp ti on f l ow
250
Response %
200
150
100
50
0
0 100 200 300
Flow (ml/min)
Figure 3.2: Charts illustrating the influence on the detector response for DMSO of (a) the TD
primary desorption temperature, (b) the TD primary desorption time and (c) the TD primary
desorption flow.
60
Carrier gas flow rates may be increased to enhance the desorption process from the tube and
the trap, and to facilitate transfer to the cold trap. Implementation of sample splitting often
benefits the thermal desorption process by allowing higher tube and/or trap desorption flows
than (and independent of) conventional GC column flows. Increasing the flow can be very
useful to lower the temperature and to decrease the time required for desorption. As shown in
figure 3.2(c), the peak area of DMSO increases with the tube desorption flow till 120 mL/min.
Above this value a small decline in response was observed. This might be due to the increased
gas volume as explained above, resulting in loss of analyte before injection.
3.3.1.3. TD-GC method validation
Analysis of blank gelatin revealed the presence of a few compounds. Some could originate from
the degradation of gelatin itself. Anyhow, they did not interfere with the compound of interest.
To confirm complete extraction of DMSO and DMF during the first run, a second run was
performed on the same tube. As a result, no DMSO or DMF peak was observed in the
chromatogram of the second run (figure 3.3).
The calibration curve was constructed by repetitive injections (n = 3) of each calibration point.
The R2-value was found to be higher than 0.999, indicating that a good linear relationship was
obtained. Besides, the residual plot showed random scatter of the residuals around zero and the
95 % confidence interval of the intercept also included zero, indicating that a fit by linear
regression was suitable. The LOD and LOQ of the method were 0.05 and 0.1 mg/mL
respectively (corresponding to 0.1 and 0.2 µg on tube respectively). When 10 mg of gelatin was
brought in the tube, this corresponds to 0.001 % and 0.002 % respectively. The repeatability of
the method was good with RSD values not exceeding 3 %. Recoveries were found to be 99.8
%, 99.7 % and 99.8 % for spiking with 2, 40 and 320 µg, respectively.
61
Figure 3.3: Chromatogram of first desorption (upper chromatogram) and second desorption
on the same tube (lower chromatogram). Degradation products are related to gelatin.
3.3.2. FET-HS-GC method

3.3.2.1. FET-HS-GC method development
An attempt was made to prepare a solution of a gelatin sample for conventional HS injection to
obtain comparative quantitative data for the DMSO peak obtained by TD-GC. However, the
sample was insoluble in most of the common organic solvents available in the laboratory such
as tetrahydrofuran, DMF, acetone and methylene chloride. On the other hand, it would have
been convenient to have gelatin dissolved in a suitable solvent to perform conventional HS-GC.
An aqueous gelatin solution was obtained after digesting the gelatin according to the procedure
described in section 3.2.3.2.1. Completeness of the digestion procedure was confirmed by
leaving the solution for more than 24 h. No coagulation of the gelatin was observed.
Sensitive analyses of high boiling polar compounds like DMSO in aqueous samples is not
evident [21]. The use of FET-HS-GC can be beneficial, but still a pressure limit has to be
respected, meaning that the introduced sample volume and therefore the sensitivity is limited
for aqueous samples [22]. To overcome the abovementioned problems, DMP was used as water
62
scavenger prior to FET-HS-GC analysis enabling sample enrichment leading to 10-fold
improvement in sensitivity [11].
3.3.2.2. FET-HS-GC method validation

When blank gelatin was analysed, no peaks from the sample matrix were noticed in the
chromatogram. A calibration curve with 5 different concentration levels of DMSO was
constructed in the range from 0.05 to 2 mg/mL and an R2-value of 0.998 was obtained. Further,
the 95 % confidence interval of the intercept included zero and the residual plot showed random
scatter of the residuals around zero. The results for recovery were found to be acceptable (100.5
% and 99.2 % for blank gelatin spiked with 0.1 and 1 mg of DMSO, respectively) and revealed
that there was no effect of the digestion procedure or the matrix. RSD values (n = 6) were below
1.5 %.
3.3.3. Application
The two methods were applied to analyze residual DMSO in experimental drug loaded gelatin
samples to investigate the difference between two drying approaches (freeze drying alone
versus freeze drying followed by vacuum oven). The samples were treated according to the
protocols mentioned in section 3.2.3.1 for TD-GC and section 3.2.3.2 for FET-HS-GC. The
results are shown in table 3.3. When using 10 mg of sample for the TD-GC method, results fell
out of range because they were higher than expected (> 3 %). To avoid overloading, the amount
of sample was reduced to 2 and 5 mg for the “freeze dried” and “freeze and oven dried” sample,
respectively. For both samples the amount of DMSO is above the ICH acceptance limit (0.5
%) [6] so that further improvement of the drying process is mandatory. A statistical comparison
using a t-test at 95 % confidence level was performed and a p-value of 0.5 was found. This
indicated that there is no significant difference between the results obtained by FET-HS-GC
and TD-GC. In case of samples with a low residual amount of DMSO, the sample size in the
desorber tube can easily be increased to obtain a more sensitive determination.
63
Table 3.3: Residual amount of DMSO in drug loaded gelatin samples using FET-HS-GC and
TD-GC.
Method Residual DMSO (RSD, n = 6) in drug loaded gelatin
freeze dried at 60 °C freeze dried at 60 °C +

vacuum oven at 80 °C
FET-HS-GC 13.7 % (0.7 %) 3.6 % (0.2 %)
TD-GC 13.6 % (2.2 %) 3.7 % (1.4 %)
3.4. Conclusions
In this study, the application of TD-GC for the determination of residual DMSO in solid gelatin
samples was investigated. The use of enzymatic digestion of gelatin prior to more conventional
FET-HS-GC was also applied for comparison, but it is less obvious from a practical point of
view since the TD-GC method does not require long lasting sample pretreatment and also no
effort to select a suitable solvent. As a result, the TD-GC chromatogram shows no interference
from a large solvent peak nor from impurities present in the solvent. In addition, since larger
sample sizes can be brought in the TD tube, it is possible to further increase the sensitivity if
necessary. Quantitative results of the proposed method showed good agreement with the
conventional HS-GC method. The results also indicated that TD-GC has a great potential for
the quantitative determination of RS directly from solid products due to its ease of operation,
sensitivity and reliability.
3.5. References
[1] M. Marques, Enzymes in the dissolution testing of gelatin capsules, AAPS
PharmSciTech. 15 (2014) 1410-1416.
[2] A. Bigi, S. Panzavolta, K. Rubini, Relationship between triple-helix content and
mechanical properties of gelatin films, Biomaterials 25 (2004) 5675.
[3] M. Djabourov, J. Leblond, P. Papon, Gelation of aqueous gelatin solutions I. Structural
investigation, J. Phys. 49 (1988) 319-332.
[4] V.A. Grigor'eva, L.Z. Rogovina, G.L. Slonimskii, The rheological properties of solutions
and gels of gelatin in different solvents, Polymer Science U.S.S.R. 17 (1975) 165-171.
[5] P.V. Kozlov, G.I. Burdygina, The structure and properties of solid gelatin and the
principles of their modification, Polymer 24 (1983) 651-665.
64
[6] Guidelines for Residual Solvents, International Council on Harmonization (ICH),
Geneva, Switzerland, 2009.
[7] Identification and Control of Residual Solvents (Section 2.4.24), European
Pharmacopoeia, 9th ed., European Department for the Quality of Medicines, Council of
[8] Y. Sitaramaraju, A. Van Hul, K. Wolfs, A. Van Schepdael, J. Hoogmartens, E. Adams,
Static headspace gas chromatography of (semi-)volatile drugs in pharmaceuticals for
topical use, J. Pharm. Biomed. Anal. 47 (2008) 834-840.
[9] Y. Sitaramaraju, A. Riadi, W. D'Autry, K. Wolfs, J. Hoogmartens, A. Van Schepdael,
E. Adams, Evaluation of the European Pharmacopoeia method for control of residual
solvents in some antibiotics. J. Pharm. Biomed. Anal. 48 (2008) 113-119.
[10] D.M. Kialengila, K. Wolfs, J. Bugalama, A. Van Schepdael, E. Adams, Full evaporation
headspace gas chromatography for sensitive determination of high boiling point volatile
organic compounds in low boiling matrices, J. Chromatogr. A 1315 (2013) 167.
[11] N. van Boxtel, K. Wolfs, A. Van Schepdael, E. Adams, Application of acetone acetals
as water scavengers and derivatization agents prior to the gas chromatographic analysis
of polar residual solvents in aqueous samples, J. Chromatogr. A 1425 (2015) 62-72.
[12] J. Li, S. Shao, M. Solorzano, G.J. Allmaier, P.T. Kurtulik, Determination of the residual
ethanol in hydroalcoholic sealed hard gelatin capsules by static headspace gas
chromatography with immiscible binary solvents, J. Chromatogr. A 1216 (2009) 3328-
3336.
determination of residual solvents in solid drug product, J. Chromatogr. A 1217 (2010)
5158-5164.
[14] K. Hashimoto, K. Urakami, Y. Fujiwara, S. Terada, C. Watanabe, Determination of
residual solvents in pharmaceuticals by thermal desorption-GC/MS, Anal. Sci. 17 (2001)
645-648.
[15] E. Woolfenden, Thermal desorption for gas chromatography, in: Gas Chromatography,
C.F. Poole (Ed.), Elsevier Inc., Waltham, 2012, pp. 245-247.
[16] N. Ramírez, A. Cuadras, E. Rovira, F. Borrull, R.M. Marcé, Comparative study of
volatile organic compounds in ambient air, Talanta 82 (2010) 719–727.
65
[17] E. Woolfenden, Optimising analytical performance and extending the application range
of thermal desorption for indoor air monitoring, Indoor Built Environ. 10 (2001) 222-
231.
[18] J.L. Seltzer, K.T. Akers, H. Weingarten, G.A. Grant, D.W. McCourt, A.Z. Eisen,
Cleavage specificity of human skin type IV Collagenase (Gelatinase): Identification of
cleavage sites in type I gelatin, with confirmation using synthetic peptides, J. Biol. Chem.
265 (1990) 20409-20413.
[19] A.A. Asfaw, K. Wolfs, A. Van Schepdael, E. Adams, Thermal desorption-gas
silica, J. Chromatogr. A 1500 (2017) 160–166.
desorption-capillary GC analysis: Summary of data and practical guidelines, J. Air &
Waste Manage. Assoc. 47 (1997) 20-36.
[21] K. Ui, M. Abo, A. Okubo, S. Yamazaki, An improved method for the analysis of
dimethyl sulfoxide in water samples, Anal. Sci. 20 (2004) 223.
[22] H.C. Hu, X.S. Chai, C.H. Wei, D. Barnes, Increasing the sensitivity of headspace
analysis of low volatility solutes through water removal by hydrate formation, J.
Chromatogr. A 1343 (2014) 42.
66
Chapter 4: Optimization of reference introduction prior to
calibration of thermal desorber - gas chromatography
Adissu Alemayehu Asfaw, Matthias Van Der Veken, Kris Wolfs,
Ann Van Schepdael, Erwin Adams*
Submitted to J. Chromatogr. A
67
68
Abstract
The golden standard for calibration of thermal desorption - gas chromatography (TD-GC)
involves gas-phase loading of cartridges from a synthetic atmosphere, in a similar way as for
sample analysis. As the analytical paths for calibration and analysis are identical, all adsorption
side effects are ruled out and the exact amount of analyte reaching the detector becomes
irrelevant. In practice however, reference introduction in a TD-GC system is mostly performed
by offline liquid calibration (OLC) using a micro syringe to inject a liquid standard solution
directly on the cartridge. This is based on the assumptions that the adsorption-desorption
process is quantitative and that no sample is lost in manipulating the cartridge. Unfortunately,
these are not always fulfilled. For analytical procedures involving direct desorption, OLC is
preferred over the gas-phase loading as knowledge of the injected amount is needed and the
adsorption-desorption processes differ anyway. However, there is no easy way to verify that
the starting assumptions are met.
In this work, first the OLC approach was evaluated on a TD tube containing only quartz filters
(QF) and QF filters enclosing a bed of mesoporous silica (MPSi). Concerning the latter, loading
was performed with and without a flow of N2 gas. Based on the results, incomplete transfer of
reference was confirmed when only QF disks were used. This improved when MPSi was added
as sorbent and the presence of a sweep gas during injection boosted the transfer of analytes.
Next, an inline injection system based on a splitless GC injector was installed on the TD
instrument in order to perform inline liquid calibration (ILC). This could be used as a sorbent
free, independent tool for system validation and it allowed to verify the boundary conditions of
the OLC approach. For most of the investigated compounds, the latter is outperformed by ILC
in terms of transfer ratio.
Keywords: thermal desorption - gas chromatography, standard solution introduction,

mesoporous silica.
69
4.1. Introduction
TD-GC was originally developed as a collection, pre-concentration and analysis system for the
offline monitoring of airborne volatile organic components (VOCs). A sampling TD tube filled
with sorbent material is used to collect the VOCs from the area to be monitored by diffusion or
active sampling [1]. Subsequently, the loaded TD tubes are brought in the TD-GC and subjected
to a thermal desorption cycle under a flow of inert gas. This gas flow transports the VOCs to a
cold trap where refocussing takes place [2]. When the desorption of the sample tube is
considered to be complete, the cold trap undergoes ballistic heating to desorb the focused
VOCs, which are then transferred to the GC in a narrow volume through a heated transfer line
[3]. This ballistic heating is referred to as the second desorption step. Modern devices provide
options to split the gas flow before and after trapping as well as the option to recollect VOCs at
those splitting ports.
Calibration of TD-GC applications is ideally performed using gas-phase standards [4].

Preparation of these standards implies the loading of a known amount of gaseous analytes on a
sorbent (the same type as used for the sample) in a TD tube and can be performed in a dynamic
or static way [5]. Dynamic gas standard generation is considered to be the golden standard and
works by mixing a concentrated stream of gas or vapor with an inert gas stream to consciously
produce a lower concentration. However, there are only very few laboratories in the world that
have sufficiently sophisticated apparatus, including continuous monitoring equipment, to
produce low-concentration (low ppm) gaseous reference samples that are truly traceable to
primary standards [6]. The practical uncertainty of this approach is estimated to be 6% of which
3% is related to gas parameters (flow, pressure settings, etc.) [7]. In the static approach, an
appropriate volume of gas is added to a known volume of air in a closed vessel. After
homogenization, this mixture is then led through a TD tube which is used as reference. It is a
simple and inexpensive technique, but it has also some serious limitations. Dilution to lower
concentrations is often a source of errors: certain compounds can interact with the wall of the
vessel resulting in loss over time and memory effects limiting reuse of the container [8]. This
technique is also prone to analyte losses through condensation and dissolution into any water
adsorbed on the vessel wall. So, it is challenging to generate reliable gas standards in a static
way [9].
From practical and economic considerations, most TD-GC procedures use liquid standard
solutions as an alternative to gas-phase calibration [10-12]. In OLC, the VOCs are diluted in a
suitable diluent and microliter size amounts of these solutions are brought on clean sorbent
70
tubes with the aid of a micro syringe. Subsequent TD-GC processing of the loaded tubes
generates calibration curves. The transfer of the reference solution from the syringe to the
sorbent bed can be facilitated by the flow of an inert gas [13]. Theoretically, it is possible to
remove selectively the diluent from the sorbent by carefully choosing the diluent/sorbent
combination and flow conditions. This way, diluent side effects and overload of the TD-GC
can be avoided [14].
As the concentration and volume of reference loaded on the TD tube are known, it is possible
to calculate the theoretical amount of reference released in the TD-GC. Of course this assumes
that the adsorption and desorption of the VOCs are complete and that nothing is lost in the
process of manipulating the TD tubes. In case of gas-phase calibration, this is only a minor
concern. Indeed, as sample and reference are treated identically, systematic deviations due to
adsorption/desorption equilibria will be compensated, as well as most of the losses due to
manipulations. So, a direct relationship between the gas-phase and the chromatographic
response is established. For the OLC approach nonetheless, the situation is less obvious. The
evaporization of the liquid, which is a mix of diluent and (eventually multiple) volatile analytes,
creates a flow and temperature dependent time-concentration profile. This may vary depending
on the different analytes present and affect the adsorption equilibria. Moreover, the presence of
a ‘relatively’ large amount of diluent may provide alternative pathways through the sorbent,
resulting eventually in reduced breakthrough volumes. Removing the diluent by a drying step
may cause loss of analyte, while not removing the diluent can affect desorption properties and
jeopardize chromatographic performance. As a consequence, TD-GC procedures relying on
OLC should be validated against an alternative analytical method or dynamic gas-phase
standards. Simply optimizing the loading and desorption parameters to a maximal response
gives no guarantee for complete transfer of the calibrant.
Direct desorption of samples is an alternative use of TD-GC [15-16]. The sample to be

examined is immobilized in an empty TD tube and subjected to the two-step desorption process
without pre-concentration on a sorbent. For this type of TD-GC, calibration has to refer to the
absolute amount of reference released in the device, not to the gas-phase concentration.
In this work, first the limitations of OLC are illustrated. To allow calibration without the need
of an adsorption step, direct injection of reference into the primary desorption gas stream of the
TD would be an interesting option. This would avoid the drawbacks related to gas-phase
standards and OLC. However, such a TD-GC apparatus is not commercially available.
Therefore, a TD-GC was equipped with an injection port to allow ILC. Although intended for
71
calibration, the new implementation can also be used as a diagnostic tool to verify the
completeness of the calibrant transfer during loading required in the classical OLC method. As
illustration, both techniques are compared for the use of mesoporous silica (MPSi) as sorbent.
4.2. Experimental
4.2.1. Chemicals and reagents

Acetone (Ace) (99.6%), dichloromethane (DCM) (99.5%), ethanol (EtOH) (99.5%) and
isopropanol (Isp) (99.5%) were bought from Fisher Scientific (Loughborough, UK).
Acetonitrile (ACN) (99.5%), methanol (MeOH) (99.9%), dimethyl sulfoxide (DMSO) (99.9%)
and n-propanol (n-prop) (99+%) were obtained from Acros Organics (Geel, Belgium).
Chloroform (Chlor) (99.9%) and toluene (99.8%) were from Sigma Aldrich (Steinheim,
Germany). MPSi (Syloid AL-1 FP and Syloid XDP3050 with pore sizes of 2–3 nm and 25 nm
respectively) were provided by Grace Davison (Worms, Germany).
4.2.2. Apparatus
4.2.2.1. General instrumentation
TD injection was performed using a modified Turbo Matrix ATD 350 thermal desorber coupled
to a Perkin Elmer Clarus 680 GC (Waltham, MA, USA). An Air Monitoring Trap (AMT) was
used as trap material. Experiments were carried out using empty stainless steel ATD tubes (89
mm x 6.35 mm o.d.) with a built-in gauze. The tubes were closed with polytetrafluoroethylene
(PTFE) caps containing an O-ring as sealing element or with brass caps using a PTFE
compression ferrule. Tubes and caps were obtained from Perkin Elmer. Separation was
performed on a ZB-624 GC column (30 m x 0.53 mm, df = 3 µm) from Phenomenex (Torrance,
CA, USA). The effluent from the GC was split into a Clarus SQ 8T mass spectrometer and a
FID detector in a ratio 1:3. The finally used TD-GC parameters are shown in Table 4.1. QF-10
filters (Ø = 50 mm) from Macherey-Nagel (Düren, Germany) were used as bed and to
immobilize the MPSi in the tube. Application of reference solutions in the tubes was performed
using a 10 µL micro syringe with 80 mm needle from Hamilton (Reno, NV, USA).
72
Table 4.1: Used TD-GC-FID parameters.
Parameter Optimized settings
Desorption temperature 300 °C
Desorption time 80 min for MPSI
20 min for QF
Desorption flow 140 mL/min
Trap low temperature -30 °C
Trap hold time 10 min
Trap material AMT
Inlet split 40 mL/min
Outlet split 40 mL/min
GC oven program 45 °C held for 12 min,

45–230 °C at 40 °C/min,
held for 5 min
H2 flow 45 mL/min
Air flow 450 mL/min
4.2.2.2. Thermal desorber modification

A packed column septum port (Pye Unicam, Cambridge, UK) was mounted on a TD tube
partially filled with silanized wool (Sigma Aldrich) to form a simple splitless injector without
septum purge. A heated transfer line made from 1/16 in × 0.02 in stainless steel tubing (Sigma
Aldrich) and RD 100/03 resistance wire (Block, Verden, Germany) connects the injector with
the TD. The temperature of the injector was regulated using a K-type thermocouple and a PID
controller. The transfer line was heated by DC current control and monitored with a K-type
73
thermocouple. The complete injector assembly is inserted in the desorption flow circuit after
the pressure control module and just before the sample tube (figure 4.1). Eventual leaks or
septum failures will be diagnosed by the TD firmware as a tube seal leak.
Figure 4.1: Thermal desorber without (left) and with (right) inline injector installed, oven
compartments are omitted.
4.2.3. Standard preparations

To prepare a stock solution (20 mg/mL) of a mix of the solvents to be analysed, a small amount
of toluene was brought as solvent in a 100 mL volumetric flask to prevent the analytes from
evaporating. Next, two grams of each of the solvents (EtOH, Ace, Isp, DCM, n-prop, ACN,
Chlor, DMSO) were introduced into the volumetric flask. After thoroughly mixing, it was
completed to volume with toluene. Standard working solutions of 10 mg/mL and 2 mg/mL were
prepared by appropriately diluting the stock solution with toluene.
74
4.2.4. Offline liquid calibration
4.2.4.1. Influence of tube manipulation
Eight sets of sample tubes containing each 4 QF disks were prepared. Before each experiment
the tubes were reconditioned (320 °C, 30 min, He at 140 mL/min). To each tube, 2 µL of
standard was transferred with a micro syringe with the needle touching the upper QF layer after
which the tubes were immediately sealed. The following experiments have been carried out
(’batch mode’ is explained in 4.3.1.1):
- 10 tubes, 4 QFs, 2 µL of 2 mg/mL, brass caps, batch mode

- 10 tubes, 4 QFs, 2 µL of 10 mg/mL, brass caps, batch mode
- 10 tubes, 4 QFs, 2 µL of 10 mg/mL, brass caps, batch mode, reversed order
- 10 tubes, 4 QFs, 2 µL of 10 mg/mL, PTFE caps, batch mode
- 10 tubes, 4 QFs, 2 µL of 10 mg/mL, PTFE caps, batch mode, reversed order
- 5 times 1 tube, 4 QFs, 2 µL of 2 mg/mL, PTFE caps, run immediately after capping
- 5 times 1 tube, 4 QFs, 2 µL of 10 mg/mL, PTFE caps, run immediately after capping
- 5 times 1 tube, 4 QFs, 2 µL of 2 mg/mL, brass caps, run immediately after capping
The analytical response for each experiment was judged towards repeatability and signal height.
4.2.4.2. Influence of tube loading

Different amounts (15, 30, 50 and 100 mg) of Syloid XDP3050 were immobilized in TD tubes
between two double QF layers. For each quantity, 5 tubes were filled. Besides, 5 mixed bed
tubes containing two QF layers, 50 mg of Syloid XDP3050, one QF layer, 50 mg of Syloid AL-
1 FP and again two QF layers were prepared. The XDP3050 was located before the AL-1 FP
with reference to the loading gas stream (see figure 4.2). Before each experiment, the tubes
were reconditioned (320 °C, 30 min, He at 140 mL/min). For calibrant loading, the tubes were
connected to an injector port similar to the one used in the modification and the required flow
of N2 was established for 1 min before injection. Standard solution (2 µL of 2 mg/mL) was
transferred to the tube with a micro syringe, maintaining in the meantime the gas flow and
keeping the needle in contact with the stainless steel gauze. Hereafter, the tubes were
immediately sealed. Concerning the loading gas stream, for each combination of flow and time,
five measurements were performed in batch mode. The following settings have been examined:
a N2 flow of 0, 4 and 6 mL/min and a transfer time of 1, 2, 4 and 6 min. A N2 flow of 0 mL/min
represents experiments without the application of a loading gas. Each tube was analysed a
second time without reloading. The analytical response for each experiment was evaluated for
repeatability and signal height.
75
syringe closing cap with septum
TD tube
← gas inlet
Gas flow
needle
…
Gas flow
septum
…
gauze
double QF QF double QF
primary secondary
headspace area weak adsorbing strong adsorbing headspace area
MPSi bed MPSi bed
(Syloid XPD 3050) (Syloid AL-1 FP)
Figure 4.2: Configuration of OLC on mixed bed tubes with possibility to use a loading gas
stream.
4.2.5. Inline liquid calibration

A TD tube containing four layers of QF to give some back pressure was used to allow normal
operation of the TD during ILC. First, the influence of the injector temperature and the syringe
holding time were optimized. At the optimum conditions, 5 injections (2 µL) of 2 mg/mL
standard solution were performed. The temperature for the injector and the transfer line were
monitored before starting each run. Injections were timed 2 min after the desorption cycle had
started. The analytical response for each experiment was judged towards repeatability and
signal height.
4.3. Results and discussion

4.3.1. Offline liquid calibration
At first instance, the repeatability of this technique was determined. Unfortunately, the
sequences of 10 tubes showed an outrageous loss of signal between the first injection and the
remainder of the sequence. As there were no indications of TD-GC malfunction, the only
possible explanation is loss of analyte before the TD tube is locked in the desorption stage as
the TD firmware performs leak checks on the tube and cold trap before starting the desorption.
Possible causes for sample loss can be grouped in two areas. The first area considers the
manipulation of the loaded tubes. This includes capping, decapping and leaks caused by
damaged caps. A second source for sample loss can be sought in non-optimal loading
parameters. It was decided to investigate both areas.
76
4.3.1.1. Influence of tube manipulation
Different experiments in this part were performed in ‘batch mode’. This means that the ten
tubes were loaded with 2 µL of standard solution and placed in the TD’s storage carousel after
which a sequence of ten runs was initiated. As a consequence, the first tube had a storage time
of a few minutes while this time increased with 50 min for each following run. In the reversed
order batches, the tube cap combination was kept the same, but the order of processing was
inversed: tube 1 became tube 10, 2 became 9 and so on. The results were expressed relative to
the area obtained from the first run in a sequence and in function of storage time (figure 4.3).
Only DCM, Chlor and DMSO are shown as representative examples. The general appearance
is the same for all obtained graphs. DMSO, which has a much higher boiling point than the
other 7 solvents, shows a slow, almost linear decay. The seven other solvents exhibit a sharp
drop in signal during the first 100 min. The PTFE capped tubes reach some plateau while the
brass capped tubes show a new decay starting at 250 min. The signal drop is more profound
when PTFE caps are used. As there was no difference in behaviour between the original and
reversed order, misaligned tube-cap combinations can be neglected as origin of the sharp drop.
The linear decay of the DMSO signal could be explained as leakage due to imperfect sealing,
but the plateaus seen for the other compounds are contradicting this. At first impression, the
brass caps are performing better as the signal drop is lower, but combining the results from both
caps and recalculating the relative signal towards the highest first value (figure 4.4) reveals that
the initial signal is slightly higher for the PTFE caps, indicating a better sealing. This better
sealing together with the decapping process provides a possible explanation for the observed
effects. When the liquid standard solution is applied on the QF stack, part of it starts to evaporate
causing a local drop in temperature which slows down the process. It is obvious that the
compound with the lowest boiling point will contribute most to this process. In the meantime
the tube is capped and the process continues in a closed environment until a thermal equilibrium
is achieved in a similar way as in a headspace vial. The decapping prior to analysis is done in
an automated way by the TD. This action creates a pressure wave inside the tube. The more
performant the tube seal will be, the stronger this effect will be, which will result in more analyte
loss for the better sealing caps. If the decapping happens before thermal equilibration is reached,
less analyte will be lost.
77
10 mg/mL, brass caps 10 mg/mL, brass caps,
120,00 reversed order
100,00 120,00
100,00
Response (%)
80,00
Response (%)
80,00
60,00
60,00
40,00
40,00
20,00 20,00
0,00 0,00
0 100 200 300 400 500 0 100 200 300 400 500
Time (min) Time (min)
DCM DMSO Chlor DCM Chlor DMSO
2 mg/mL, brass caps 10 mg/mL, PTFE caps

120,00 120,00
100,00 100,00
Response (%)
Response (%)
80,00 80,00
60,00 60,00
40,00 40,00
20,00 20,00
0,00 0,00
0 100 200 300 400 500 0 100 200 300 400 500
Time (min) Time (min)
DCM Chlor DMSO DCM Chlor DMSO
Figure 4.3: Relative signal drop (in function of tube storage time) of some representative
compounds after OLC of 2 µL of two concentrations of standard solutions on QF disks in a
TD tube with two types of caps.
78
The experiments performed immediately after loading, express the achievable repeatability
with liquid injection on QF stacks (Table 4.2). The higher initial value (up to 32% for Ace)
when using the PTFE caps is confirmed. For the remainder of the experiments, the PTFE caps
were used.
120,00
Higher initial signal for PTFE caps
100,00
80,00
DCM PTFE
Response (%)
DCM brass
60,00
Chlor PTFE
Chlor brass
40,00 DMSO brass
DMSO PTFE
20,00
0,00
0 100 200 300 400 500
Tube storage time (min)
Figure 4.4: Comparison of signal loss between PTFE and brass caps after OLC of 2 µL of
standard solution on QF disks in a TD tube.
79
Table 4.2: Repeatability (n = 5) and signal difference for different caps when using OLC
applying 2 mg/mL solutions on QF and immediate runs.
Brass caps PTFE caps
Mean area RSD (%) Mean area RSD (%) ∆ area (%)
EtOH 2252591 1.7 3045875 1.7 26
Ace 2535564 1.9 3754216 2.1 32
Isp 1260212 1.5 1756324 1.4 28
ACN 3478322 1.8 4215632 1.5 17
DCM 767114 2.0 891166 2.7 14
n-prop 5004767 1.2 5963254 1.1 16
Chlor 676240 1.7 766888 1.3 12
DMSO 7928876 1.4 8274556 1.1 4
4.3.1.2. Influence of tube loading

In the previous experiments a stack of 4 QF filters was used as reservoir for the solvents. Adding
an adsorptive material to the stack would lower the concentration of analytes in the formed
headspace, ultimately leading to an undetectable signal loss. A weak sorbent will leave too high
headspace concentrations, while a strong one might result in incomplete desorption. Because
of its chemical resistance, long-term stability, adsorptive capacity and high temperature
tolerance, MPSi was chosen as adsorptive material in this experiment. Furthermore, the sorbent
bed will split the tube in sections with a small primary headspace and a larger secondary one
(figure 4.2). This compartmentation will prohibit the propagation of a pressure wave through
the tube, but the smaller compartment should see a greater pressure incident due to the
decapping action. A disadvantage of adding a sorbent bed to the system is the need for longer
desorption times.
At first, the batch experiment from the previous section was repeated. A typical result is
presented in figure 4.5. There is no longer a sharp drop in signal between the first point and the
80
second one. Instead, a signal increase is noted for the more volatile compounds. This can be
explained by taking into account the passive diffusion of the VOCs into the adsorptive bed and
so reduces the amount available for headspace formation. However, the diffusion process is
time consuming and not complete yet in the beginning.
130,00
120,00
110,00
Response (%)
100,00 Isp
DCM
90,00 DMSO
Chlor
EtOH
80,00
Ace
ACN
70,00 n-prop
60,00
0 50 100 150 200 250 300 350 400 450
Figure 4.5: Relative response for OLC of 2 µL of a 2 mg/mL standard solution on 30 mg of

Syloid 3050 without loading gas.
Adding a vector in the form of an inert gas flow to transport the VOCs from the dropping point
into the sorbent would be beneficial since insufficient transport may lead to lower adsorption
and so to higher headspace concentrations. On the other hand, a too strong vector may cause
breakthrough. The influence of a vector is illustrated in figure 4.6 where a sweep volume of 12
mL shows a considerable improvement in signal variation over time. Although vector flow and
time are orthogonal experimental parameters, expressing the vector as a volume yields a better
representation of the vector influence. A logical next step is to optimize the sweep volume and
the sorbent. However, both parameters are entangled: the point where a vector becomes too
strong depends on the adsorptive power of the bed and is often referred to as breakthrough
volume. This risk may be reduced by the mixed bed approach which increases the capacity.
81
130,00
120,00
Relative area (%)
Isp
110,00
DCM
100,00 DMSO
Chlor
90,00
EtOH
80,00 Ace
ACN
70,00
n-prop
60,00
0 100 200 300 400 500
Figure 4.6: Relative response for injection of 2 µL of a 2 mg/mL standard solution on 30 mg

of Syloid 3050 with a sweep volume of 12 mL.
Figure 4.7 presents per compound the influence of the sweep volume on the response for tubes
filled with different amounts of MPSi, including mixed bed tubes. The highest response for a
compound over the complete dataset is taken as 100%. The profile of the curve for each bed
illustrates the influence of the vector while the vertical dispersion of the datasets represents the
effect of the adsorptive capacity of the bed. For ACN and DMSO, the data are grouped in a
zone between 80% to 100%. This means that no benefit has to be expected from larger beds.
The horizontal alignment of the data indicates that the sweep volume is in the optimal range.
Indeed, larger beds contain only the risk of incomplete desorption, while stronger vector flows
would only approach the breakthrough volume. For compounds which are less easy to trap, like
Chlor and DCM, the situation is different. The signal decay seen with the 15 mg bed is an
obvious illustration of breakthrough by the vector action. For the other amounts, the results are
more on a horizontal line, which indicates that the vector is no longer the primal actor. For those
compounds, the bed construction is more important, expressed by the vertical spread of the
data. As the capacity of the beds increases, the data are converging towards the maximum (see
for example DCM with 100 mg and 50/50 mg MPSi in figure 4.7). This implies that no further
improvement can be expected with this type of adsorbents. For all compounds in this study, the
mixed bed is the best solution. The small upslope seen in the results for DCM indicates that a
sweep volume of at least 10 mL should be chosen. So, in practice a volume above this value
and below the breakthrough volume should be selected.
Although this representation of results provides an interesting tool to judge and optimize the
sorbent bed and loading conditions, the formation of the headspace cannot be excluded.
82
Verification of maximal desorption under the current conditions was done by reanalysing the
tubes without reloading [15]. In all cases, the response for the verification run was below the
detection limit. However, for compound-bed combinations requiring severe desorption
conditions, a difference may be expected between ‘maximal desorption’ and ‘total desorption’
and complete transfer of calibrants is still not guaranteed. To evaluate this, ILC was used.
Chlor DCM
120 120
100 100
Response (%)
Response (%)
80 80
60 60
40 40
20 20
0 0
0 10 20 30 0 10 20 30
Sweep volume (ml) Sweep volume (ml)
ACN DMSO
120 120
100 100
Response (%)
Response (%)
80 80
60 60
40 40
20 20
0 0
0 10 20 30 0 10 20 30
Sweep volume (ml) Sweep volume (ml)
15mg 30mg 50mg 100mg 50/50mg
Figure 4.7: Influence of the sweep volume and tube filling on the response of 4 compounds.
Different amounts (15, 30, 50, 100 mg) of Syloid XDP3050 were present in the TD tubes. In
addition, mixed bed tubes containing 50 mg of Syloid XDP 3050 and 50 mg of Syloid AL-1 PF
were used.
83
4.3.2. Inline liquid calibration
The major challenge to install the injector was the limited space available inside the TD. After
checking gas tightness and thermal stability, the flow impedance of the different beds was
verified. The tubes used for ILC contain only a layer of 4 QFs and no MPSi to exclude
adsorption. When comparing OLC and ILC, the tubes for OLC containing MPSi may show a
significant difference in impedance that could alter the inlet split and desorption flow, requiring
a separate TD flow tuning for each group of tubes. Fortunately, there was no significant
difference between the tubes, probably due to the loose packing of the MPSi beds.
The first ILC experiments showed a large variation in analytical response. When reviewing the
setup, it became clear that the carrier gas reaching the injector was not preheated. Leaving the
injection needle in the injector for at least 1 min after injection solved this issue. The influence
of the injector temperature on the signal was examined from 200 °C to 320 °C. The transfer
line was kept at its maximum temperature, being 300 °C. For DCM, Ace, ACN and Chlor a
stable response was achieved at 260 °C and higher. DMSO, Isp, n-prop and EtOH required a
minimum temperature of 280 °C. So, 300 °C was chosen as working temperature.
The results obtained with ILC were compared with those obtained from the OLC experiments
with mixed bed tubes (Table 4.3). In general, the RSD values for the results obtained with ILC
are somewhat higher than those obtained with OLC. Adding an internal standard to compensate
for the (manual) injection variations would lower the RSD. For instance, recalculating the ILC
data for DCM with ACN as internal standard results in a RSD of 1.0%. Such a correction is
however not possible for OLC due to the specific nature of each adsorbent-analyte combination,
making the effect of an internal standard rather unpredictable unless deuterated analogues are
used. As expected, the response for OLC is slightly lower for all compounds except DMSO.
The considerably lower recovery of Isp from MPSi confirms the strong affinity between those
compounds as already described in previous work [15]. Concerning DMSO, a lower response
for ILC was noticed, indicating that the transfer of DMSO was incomplete. The OLC result is
probably correct since the low volatility of DMSO reduces the chance of loss due to the
headspace mechanism. Closer examination of the ILC chromatograms revealed the presence of
small amounts of decomposition products of DMSO (dimethylsulfide and dimethylsulfone).
Thermal decomposition of DMSO above 190 °C has been reported [17]. Including a large
volume quartz liner for the injector and a fused silica tubing as inner lining for the transfer line
would reduce the tendency of compounds to adsorb on the wall, resulting in shorter transfer
times and less exposure to high temperatures, reducing the formation of decomposition
products.
84
Table 4.3: ILC vs. OLC on mixed bed tubes and 24 mL sweep flow.
Compounds ILC OLC

Average peak RSD Average peak RSD % peak area
area (n = 5) (%) area (n = 5) (%) relative to ILC
EtOH 4233097 3.2 4151115 2.6 98.1
Ace 8864455 2.3 8856539 3.7 99.9
Isp 4016806 3.5 3836106 1.5 95.5
ACN 10310131 3.5 10109914 1.1 98.1
DCM 2984347 3.3 2936329 2.0 98.4
n-prop 11970965 3.2 11857455 2.4 99.1
Chlor 2392059 2.6 2348914 2.5 98.2
DMSO 9684264 2.7 10038328 1.1 103.7
4.4. Conclusion
Whenever the reference and analyte are not following the same trajectory in GC, extreme care
should be taken in interpreting the results. The chance on incomplete transfer of reference using
OLC, due to tube manipulation or too strong adsorption, is confirmed by the presented data.
For the investigated VOCs, apart from DMSO, ILC performs better for most of the compounds
than OLC in terms of transfer ratio. Adding an autosampler to the ILC injector seems to be the
next logical step. This would result in an easy to use and quick tool for TD calibration.
For direct desorption TD, the use of ILC has to be preferred over OLC as the complete transfer
of reference compounds is a key condition. Regarding normal gas-phase analysis with TD, one
could argue that the incomplete transfer following OLC is comparable for sample and reference.
However, the determination of the 100% signal level for a certain VOC using ILC facilitates
the correct selection of adsorbent bed and loading conditions in OLC. Furthermore, ILC can be
used as a sorbent free, independent tool for system validation.
MPSi showed to be an interesting material for sorbent beds. Its thermal and chemical stability
combined with a wide range of properties allow the construction of application tailored sample
tubes. The resulting low impedance tubes allow a straightforward integration with OLC
yielding similar results as ILC.
85
4.5. References
[1] E. Woolfenden, Optimising analytical performance and extending the application range
of thermal desorption for indoor air monitoring, Indoor Built Environ. 10 (2001) 222-23.
desorption-capillary GC analysis: Summary of data and practical guidelines, J. Air &
Waste Manage. Assoc. 47 (1997) 20-36.
Chromatography, Elsevier Inc., Waltham, 2012, pp. 272–273.
control in quantification of volatile organic compounds analysed by thermal desorption–
gas chromatography–mass spectrometry, J. Chromatogr. A 1186 (2008) 348–357.
[5] E. Massold, C. Bähr, T. Salthammer, S.K. Brown, Determination of VOC and TVOC in
air using thermal desorption GC-MS – practical implications for test chamber
experiments, Chromatographia 62 (2005) 75-85.
[6] J. Namiesnik, Generation of standard gaseous mixtures, J. Chromatogr. A 300 (1984)
79-108.
[7] Markes international, Thermal desorption technical support note 30: Certified reference
materials for analysis of VOCs in air by thermal desorption (TD)/GC.
[8] M. Gautrois, R. Koppmann, Diffusion technique for the production of gas standards for
atmospheric measurements, J. Chromatogr. A 848 (1999) 239–249.
application-oriented overview of methods in a nutshell, Int. J. Chem. Eng. 2012 (2011) 1-
6.
[10] A. Ribes, G. Carrera, E. Gallego, X. Roca, M.J. Berenguer, X. Guardino, Development
and validation of a method for air-quality and nuisance odors monitoring of volatile
organic compounds using multi-sorbent adsorption and gas chromatography/mass
spectrometry thermal desorption system, J. Chromatogr. A 1140 (2007) 44-54.
[11] C. Rodriguez-Navas, R. Forteza, V. Cerda, Use of thermal desorption–gas
chromatography–mass spectrometry (TD–GC–MS) on identification of odorant emission
focus by volatile organic compounds characterization, Chemosphere 89 (2012) 1426–
1436.
86
[12] E. Rosier, E. Cuypers, M. Dekens, R. Verplaetse, W. Develter, W. Van de Voorde, D.
Maes, J. Tytgat, Development and validation of a new TD-GC/MS method and its
applicability in the search for human and animal decomposition products, Anal. Bioanal.
Chem. 406 (2014) 3611–3619.
[13] Markes international, Application note 007, Calibration: preparing and introducing
thermal desorption standards using sorbent tubes.
[14] N.A. Martin, N.L. Barber, J.K. Black, R.P. Lipscombe, C.A. Taig, A comparison of gas-
and liquid-loaded sorbent standards for the calibration of measurements of volatile organic
compounds, Atmos. Environ. 41 (2007) 7666–7671.
[15] A.A. Asfaw, K. Wolfs, A. Van Schepdael, E. Adams, Thermal desorption-gas
silica, J. Chromatogr. A 1500 (2017) 160–166.
dimethylsulphoxide in drug loaded gelatin using thermal desorber – gas
chromatography, J. Pharm. Biomed. Anal. 153 (2018) 193–198.
[17] X. Yang, X. Zhang, Z. Guo, W. Bai, L. Hao, H. Wei, Effects of incompatible substances
on the thermal stability of dimethyl sulfoxide, Thermochim. Acta 559 (2013) 76– 81.
87
88
Chapter 5: Development and validation of a thermal
desorber gas chromatography method for determination of
residual solvents in drug loaded albumin
Adissu Alemayehu Asfaw, Kris Wolfs, Ann Van Schepdael, Erwin Adams

To be submitted to J. Chromatogr. B
89
90
Abstract
The conventional approach for residual solvent (RS) analysis is headspace-gas chromatography
(HS-GC). This starts from a homogenous sample solution and is based on the equilibrium of
the analyte between the sample and the gas phase. Unfortunately, an aqueous solution of
albumin forms irreversible hydrophobic aggregates when it is heated above 50 °C.
Consequently, the use of HS-GC for RS analysis in albumin becomes problematic due to the
presence of an additional solid phase in the HS vial.
In this work, a method using a thermal desorber (TD) combined with GC was developed for the
determination of RS in drug loaded albumin. Samples were immobilized between two double
layers of quartz filter (QF) in a polytetrafluoroethylene (PTFE) insert which was placed in an
empty desorption tube prior to TD-GC analysis. The liquid standard mix consisted of ethanol
(EtOH), acetone (Ace), dichloromethane (DCM) and chloroform (Chlor) dissolved in toluene.
Offline liquid calibration (OLC) was applied by introducing 2 µL of the standard mix under
counter flow of an inert gas into the TD tube containing a mixed bed of mesoporous silica
(MPSi) immobilized between two double layers of QF. The OLC results were verified using
the inline liquid calibration (ILC) approach based on a heated GC injector installed on the TD.
The validation results revealed that the proposed method has good recovery (> 98 %). R2-values
(> 0.998) indicated good linearity over a wide range. The limit of detection (LOD) and limit of
quantification (LOQ) were found to be 0.01 and 0.04 µg on tube, respectively. Repeatability of
the method was reported as RSD-values and they were lower than 3 %. A method based on the
complete enzymatic digestion of albumin combined with conventional HS-GC was developed
to verify the completeness of release of the RS from the albumin. Both the TD-GC and HS-GC
were applied for the determination of EtOH and DCM in two different albumin samples loaded
with experimental drugs. Statistical comparison indicated that there was no significant
difference (p > 0.05) between the two methods. However, the HS-GC method following
enzymatic degradation is much more expensive and time consuming.
Keywords: Thermal desorber – gas chromatography, Albumin, Residual solvents
91
5.1. Introduction
Albumin is the most abundant plasma protein to which a number of drugs and endogenous
molecules can bind [1]. Since albumin is biodegradable and lacks toxicity, it is an ideal
candidate for drug delivery and it can serve as a depot and transporter protein, especially in
nano-sphere and nano-capsule preparations [2]. This nano level albumin has well-defined sizes
and reactive functional groups (thiol, amino and carboxyl) on its surface, which enable to bind
ligands and allow surface modification [3]. Moreover, the release of drugs from albumin occurs
naturally by protease digestion. Albumin can be obtained from egg white (ovalbumin), bovine
serum (bovine serum albumin or BSA) and human serum (human serum albumin or HSA) [4].
Several advances in albumin-based drug delivery have led to some commercially available
products [5]. Several techniques to load drugs on albumin have been described taking into
account the solubility of the drugs. Passive loading is the most common one. Here, drug and
carrier are co-dispersed in the same medium, which requires organic solvents [6, 7, 8]. Seen
their health risk and influence on the physicochemical properties of the drug, it is important to
remove the organic solvents as completely as possible from the final product before human use.
However, it is practically not possible to remove them completely. As a result, some remain as
RS and their presence and amount should be determined [9]. GC is the method of choice for
RS analysis from diverse types of materials [10]. It is often coupled with HS, which only
transfers the volatile components of the sample to the column and avoids introduction of non-
volatiles, resulting in a simpler and cleaner analytical approach. Static HS-GC (sHS-GC) is the
preferred method for RS in pharmaceuticals. It is based on the equilibration of the analyte
between the sample and the gaseous phase above it in the HS vial [11]. Although sHS-GC has
its merits, it also shows some serious limitations in terms of applicability in some conditions
like for example difficulties to determine high boiling analytes in a low boiling matrix where
the latter acts as a diluent of the gas and minimizes sensitivity [12]. Due to possible matrix
effects, matrix matched calibration is mandatory in sHS, but this is not always achievable,
especially for insoluble samples. The full evaporation technique (FET) is a HS method based
on complete transfer of the analyte from the sample to the gaseous phase and thus can overcome
the limitation of sHS in terms of matrix effects [13]. However, there is a temperature and
pressure limitation of the HS instruments so that the FET approach can not always be fully
exploited. Especially matrices with large volume expansion like aqueous samples could lead to
double injections or over-pressurized vials [14].
Albumin is freely soluble in water, but it readily coagulates when the aqueous solution is heated
above 50 °C and it does not revert to monomers upon cooling. Coagulation of protein solutions
92
by heating seems not a pure temperature effect since water as such or in the form of steam is
also crucial for this process. For instance, albumin crystals can be heated to 150 °C under dry
conditions without change [4, 15, 16]. So, when the conventional approach for RS analysis is
followed and an aqueous solution of albumin is heated in the HS oven during equilibration,
coagulation will occur. The effect of the presence of this additional solid phase on the nature of
the equilibrium is not well known. An option to overcome the abovementioned problems is to
perform a direct quantification from solid samples using HS-GC. However, this approach is
also not free from difficulties because of matrix effects and lack of certified reference materials
for the calibration step. Multiple headspace extraction (MHE) techniques have been developed
to remove the influence of matrix effects for direct quantitative determination of analytes from
solid matrices in combination with single drop microextraction (SDME) [17]. However, MHE
is time consuming and needs equilibration of the analyte between the two phases in HS.
Moreover, heat instability of albumin due to the presence of water absorbed from the
atmosphere in the short manipulation time between preparation of the sample and analysis make
MHE problematic too. Recently, a thermal desorber (TD) coupled with GC has been used in an
alternative way for the analysis of RS from gelatin and MPSi [18, 19].
In this work, the applicability of TD-GC has been explored for the quantitative determination
of RS in albumin. Factors affecting the performance of the method such as desorption time,
temperature and flow during primary desorption and adsorbents in the trap were studied. For
calibration, RS were loaded on a mixed bed (50 mg Syloid XDP3050 and 50 mg Syloid AL-1
FP) of MPSi sandwiched between 2 double layers of QF in a TD tube using OLC [20].
Completeness of reference transfer from this calibration tube to the GC was verified by ILC
using a heated GC injector installed on the TD instrument [20]. The proposed method was
applied for the determination of residual EtOH and DCM in two different drug loaded albumin
samples. A second method, based on the complete enzymatic digestion of albumin combined
with HS-GC in FET mode, was developed to verify if the release of the RS from the albumin
samples in the TD was complete.
93
5.2. Experimental
5.2.1. Chemical reagents
Ace (99.6 %), DCM (99.5 %) and EtOH (99.5 %) were bought from Fisher Scientific
(Loughborough, UK). n-Propanol (99+ %) was obtained from Acros Organics (Geel, Belgium).
Chlor (99.9 %), toluene (99.8 %), L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)
treated trypsin from bovine pancreas, 1,4-dithiothreitol (DTT) ( > 98 %) and iodoacetamide
were delivered by Sigma Aldrich (Steinheim, Germany). Tris base (99.9 %) was obtained from
AppliChem GmbH (Darmstadt, Germany). MPSi (Syloid AL-1FP (pore size 2-3 nm) and
Syloid XDP3050 (pore size 25 nm)) was provided by Grace Davison (Worms, Germany).
Hydrochloric acid (37.5 % w/w) was from VWR International (Fontenay-sous-Bois, France).
The same instrument as in chapter 2 was used here. All consumable TD accessories were
purchased from Perkin Elmer. Separations were carried out on a ZB-624 column (30 m x 0.53
mm, df = 3 µm) from Phenomenex (Torrance, CA, USA). The TD-GC parameters are listed in
Table 5.1. The effluent from the column was split to the FID and MS in a 3:1 ratio. A 10-
microliter syringe with an 80 mm needle (Hamilton, Reno, NV, USA) was used to apply 2 µL
of calibration solutions. Tubes with empty PTFE inserts were conditioned at 250 °C for 20 min
with a constant helium flow (30 mL/min) prior to introduction of both standards and samples.
According to an in-house design [20], a GC injector (300 °C) was connected to the sampling
end of the TD tube by a heated transfer line. This enabled to introduce the standard solution
directly into the TD during the desorption step. This guarantees no loss of calibrants and
complete transfer to the GC column.
94
Table 5.1: Used TD-GC parameters.
Parameter Optimized setting

Desorption temperature 180 °C
Desorption time 60 min
Desorption flow 100 mL/min
Trap low temperature -30 °C
Trap hold time 8 min
Trap material AMT
Inlet split 30 mL/min
Outlet split 60 mL/min
GC oven program 45 °C held 12 min; 45–230 °C at
40 °C/ min, held 5 min
5.2.2.2. HS-GC-FID
The HS-GC-FID analyses were carried out on an Agilent HP6890 GC equipped with a Perkin
Elmer Turbomatrix 40 HS autosampler (balanced pressure system). All HS parameters are
given in Table 5.2. HS vials and PTFE/Sil caps were purchased from Perkin Elmer. The column
and GC oven temperature program were the same as used for TD-GC.
95
Table 5.2: Used HS parameters.
Parameter Settings
Transfer line temperature ≥ 10 °C above ET
Injection port temperature ≥15 °C above ET
45 °C held 12 min; 45–230 °C at
GC oven program
40 °C/ min, held 5 min
Split ratio 1:5
5.2.3. Standard and sample preparation

5.2.3.1. Standard and sample preparation for TD-GC
A 2 mg/mL reference solution mix of EtOH, Ace, DCM and Chlor was prepared by adding 200
mg of each in a 100 mL volumetric flask, which contained already a certain volume of toluene
to minimize loss due to evaporation. After bringing to volume with toluene, it was used for
method optimization. Starting from a stock solution (20 mg/mL of each of the RS, prepared in
a similar way as above) calibration solutions of six concentrations between 0.02 and 18 mg/mL
were made. A 40 mg/mL solution of 1-propanol in toluene was used as correction standard (CS)
to correct the peak areas for small differences in volumes when 2 µL was applied to the TD
tube. Before the calibration solutions were brought to volume (10 mL) with toluene, 1 mL of
CS was added.
Method optimization was carried out by bringing 10 mg of blank albumin in a PTFE insert,
which was placed in its entirety between two double layers of QF in a TD tube (Fig. 5.1). Next,
2 µL of the reference solution was introduced by the application of OLC using a microliter
syringe. Loading was performed by placing the needle on the stainless steel gauze of the TD
96
tube under an inert gas flow (30 mL/min) so that the solution is directly taken up by the tube
cartridge [20].
Concerning sample analysis, 10 mg each of two experimental BSA samples loaded with
experimental drugs were immobilized by sandwiching them between two double layers of QF
in a PTFE insert which was placed in an empty desorption tube prior to TD-GC analysis.
For calibration, the standard solution was introduced on MPSi sandwiched between QF in the
TD tube on which RS were loaded by the OLC approach described above. The MPSi was a
combination of 50 mg Syloid XDP 3050 and 50 mg of Syloid AL-1 PF silica. The silicas were
placed in such a way that the large pore size MPSi was positioned first in the direction of the
loading flow and a layer of QF was applied between the two MPSi to prevent mixing.
Figure 5.1: Sample introduction to TD tube.
5.2.3.2. Standard and sample preparation for HS-GC

5.2.3.2.1. Reagent preparations
A 0.4 M Tris stock solution was prepared by dissolving 12.1 g of Tris base in 200 mL of water.
Next, the pH was adjusted to 7.8 with 5 M HCl before bringing to the final volume (250 mL)
with water. The solution was further stored at 4 °C. A buffer solution was made by adding 5
mL of the Tris stock solution to 8 g of urea in a 20 mL volumetric flask, brought to volume
with water. A reducing agent was prepared by dissolving 150 mg of DTT in 3.75 mL of water
followed by adding 1.25 mL of the Tris stock solution. An alkylating reagent was made by
dissolving 36 mg of iodoacetamide in 750 µL of water and adding 250 µL of the Tris stock
solution. Solution were mixed by gentle vortexing.
97
5.2.3.2.2. Digestion of albumin using trypsin prior to HS-GC analysis
A 1 mg/mL solution of trypsin was prepared by dissolving 20 mg of TPCK treated trypsin from
bovine pancreas in 20 mL of 1 mM HCl (pH 3) and stored at –20 °C. This was used for 10 mg
each of (1) the two drug loaded albumin samples, (2) three blank albumin samples spiked with
5, 800 and 1500 µg of each EtOH and DCM for evaluation of the recovery and (3) blank
albumin for the interference test. Next, suspensions (10 mg/mL) in buffer solution were
prepared and subjected to the digestion procedure mentioned below. First, 50 µL of reducing
agent was added to the suspensions (sample, blank and spiked blank) and left for 1 h. Next, 200
µL of alkylating agent was added, which was left again for 1 h. This was followed by adding
200 µL of reducing agent again to consume any unreacted iodoacetamide and to allow the
reaction to proceed at room temperature for another 1 h. Then, the reaction mixture was diluted
by adding 7.75 ml of water in order to reduce the urea concentration to about 0.6 M, a
concentration at which trypsin retains its activity. Finally, 1 ml of trypsin solution was added
to the resulting preparation and the digestion was carried out overnight at 37 °C. This gave a
protease-to-substrate ratio of 1-to-20 [21]. At each step, the solution was mixed by gentle
vortexing.
5.2.3.2.3. Solutions for HS-GC analysis
After the digestion procedure was complete, the digested solution was cooled to room
temperature and the pH of the solution was adjusted to < 6 by adding concentrated acetic acid.
Next, 2 mL of internal standard (IS) (40 mg/mL of 1-propanol) was added to the digested
solution before bringing it to volume (20 mL) with water. Then the whole preparation was
centrifuged at 4500 g for 10 min. Finally, 10 µL of the solution was transferred to a HS vial and
sealed with a PTFE/Sil-cap, ready for HS-GC analysis under FET conditions.
A stock solution of 20 mg/mL reference mix of EtOH and DCM in water was prepared.
Calibration solutions were prepared by diluting appropriate volumes of the stock solution to
obtain solutions in the concentration range from 0.2 to 160 µg/mL. Also here IS was added
before bringing to volume and finally 10 µL was brought in a HS vial.

5.2.4.1. Method validation for TD-GC
The method validation was done by evaluating selectivity, linearity, LOD, LOQ, accuracy and
precision as per the ICH guideline [22]. The selectivity of the method was demonstrated by
monitoring the separation of the analytes, the peak purity as well as possible interference from
98
blank albumin through MS. The LOD and LOQ were determined using a signal-to-noise (S/N)
ratio of 3 and 10 respectively. The linearity was tested by analysing calibration solutions with
the described TD-GC method at six concentrations in a range from 0.02 to 18 mg/mL, each
injected in triplicate. The regression coefficient was used to evaluate the linearity. In order to
properly measure the peak area and to produce a single calibration curve covering a wide range
for all the samples, switching between different detector attenuations (ATT) (amplification
factor) was applied while recording the calibration data [19]. To exclude any interference from
contaminants, blank albumin was analyzed before every sample analysis. Repeatability was
tested by analyzing six tubes with 2 µL of 2 mg/mL standard solution on MPSi in 1 day and
expressed as percentage relative standard deviation (% RSD). The accuracy of the method was
determined by comparing the detector response of 2 µL of the standard solutions at three
concentration levels (0.02, 2 and 18 mg/mL) spiked on 10 mg of blank albumin and the detector
response of the ILC of the same amount of the standard solution. The recovery from the ILC
was considered to be complete. Maximal extraction of the analytes during the first run was
confirmed by performing a second run on the same tube.
To verify the results of OLC, ILC was used. In the ILC approach, the standard solution was
injected into an in-house designed heated (300 °C) GC injector, which was installed on the
sampling end of the TD tube by a heated transfer line [20]. This was used to introduce the
standard solution directly to the TD tube during the primary desorption stage.
5.2.4.2. Method validation for HS-GC

LOD and LOQ of the method were also determined by the signal-to-noise ratio. For each
compound, linearity was checked in a concentration range from LOQ (0.2 µg/mL) to 160
µg/mL and evaluated by the regression coefficient of a five-point calibration curve. Method
accuracy was evaluated by carrying out recovery experiments at three concentration levels
obtained by spiking 5, 800 and 1500 µg of each reference on 10 mg of blank albumin, followed
by treating them with the digestion procedure as described in sections 5.2.3.2.2 and 5.2.3.2.3.
Instrument repeatability was assessed by evaluating the RSD of the peak areas obtained by
analyzing six vials with a concentration of 20 µg/mL.
99
5.3. Results and Discussion
5.3.1. TD-GC method
5.3.1.1. TD-GC method development
The sample and blank introduction into the TD tube was the step that involved a modification
of the original application of the TD instrument, which normally mainly focuses on the
collection of volatiles from air/environment using a tube with an adsorbent. In order to
introduce a solid sample in the TD tube, a supporting material on both ends of the tube is
required to keep the sample inside the tube and to prevent contamination of the system flow
path. This supporting material should be inert, thermally stable and able to fit into the TD tube.
QF-10 was found to fulfill the above-mentioned requirements and was used throughout the
experiment. All experiments were performed by sandwiching a sample, blank albumin or a
mixed MPSi bed between two double layers of QF-10 as explained before.
One of the critical steps in TD is that the packing material (adsorbent) on the cold trap must be
strong enough to retain the analytes released during tube desorption and weak enough to allow
desorption of all the retained components during trap desorption. AMT and Tenax TA were
assessed for the cold trap as sorbent and AMT was found superior for the compounds examined
here. This could be due to the fact that it contained a weak and a strong sorbent which enable
to retain the high volatiles on the strong sorbent and the less volatiles on the weak sorbent,
making the desorption of the analytes from the trap easier and complete. A PTFE insert was
used to avoid contamination of the TD tube by the heated albumin so as to allow reuse without
a thorough cleaning step.

It is possible to enhance the extraction efficiency of the TD method by optimizing different
parameters of the TD such as desorption temperature, time and flow. To examine the influence
of these parameters, a set of experiments was performed in which the detector response for each
RS was supervised in function of a single parameter. The resulting data are shown in graphs
expressing the relative peak area in function of the respective parameter where the lowest area
in each dataset was arbitrarily chosen as reference point (100 %). To obtain maximum primary
desorption, the sample tube temperature needs to be set as high as possible. However, the range
of possible temperatures is restricted by the packing/sample/matrix stability, the lability of the
components of interest and the temperature limitations of the system. To avoid overcrowding
of the graphs, EtOH and DCM were selected as representatives to show the pattern of parameter
100
effects. Although it is recommended to extend the tube desorption time first and raise the
temperature in a second stage when experiencing incomplete desorption, this can seriously
lengthen the GC run. So, first the tube desorption temperature was optimized. As indicated in
Fig. 5.2a, the relative peak area increases as the temperature for tube desorption increases till it
reaches 180 °C, after which a slight decrease was noticed, probably due to degradation of
albumin that blocks release of the RS. So, 180 °C was selected as optimum primary desorption
temperature.
Next, in order to minimize the analysis time and optimize sample throughput, the optimum tube
desorption time was studied. Complete extraction of all compounds studied was obtained
around 60 min and gradually declined later on (Fig. 5.2b). A possible explanation of the latter
may be the breakthrough volume that is reached for the investigated compounds at the chosen
conditions for desorption and trapping. Thus, 60 min was selected as optimum desorption time.
To speed up desorption and reduce the primary desorption time and temperature, the carrier gas
flow rate may be increased. The option of using higher flow rates to speed up desorption is
particularly useful during the analysis of labile components when high temperatures should be
avoided. Increasing carrier gas flow rates could enhance the desorption process from the tube
and the trap, but at the same time it is also important to lower the flow through the trap during
primary desorption to avoid loss of analytes. This can be achieved by changing the split on the
inlet to the cold trap, which enables to set a high flow through the tube and a lower through the
trap. The investigation for an optimum desorption flow through the tube during primary
desorption indicated that the peak area increased with the desorption flow until 100 mL/min
(see Fig. 5.2c). Above this flow rate the response starts to decline slowly. This could be due to
the increased hot gas volume flowing through the cold trap, making that the trap is not
maintained at its lower temperature during the primary desorption step and resulting in
breakthrough.
101
a ) D e s o r p t i o n t e mp . b ) D e s o r p t i o n t i me
270
250 250
230 230
210
210
Response (%)
Response (%)
190
190
170
170
150 EtOH
150
130
130 110
EtOH DCM
110 90
90 DCM 70
70 50
50 0 50 100 150
150 200 250 300 Time (min)
T (°C)
c) Desorption flow
190
170
Response (%)
150
130
110
90 EtOH
70
DCM
50
0 50 100 150 200

Flow (mL/min)
Figure 5.2: Charts illustrating the influence on the detector response of (a) the TD primary
desorption temperature, (b) the TD primary desorption time and (c) the TD primary desorption
flow.
5.3.1.3. TD-GC method validation

TD-GC-MS analysis of blank albumin revealed that no peaks from the blank interfered with
the compounds of interest. Maximal extraction of all studied RS from the albumin was
confirmed by performing a second desorption under the optimum conditions on the same tube
on each recovery level and none of the analyte peaks in the chromatogram of the second run
was above the detection limit (Fig. 5.3). The calibration curves were constructed using two
different approaches: OLC and ILC. In order to avoid overloading of the peaks of high
concentration points in the calibration curve, in both approaches it was needed to switch
between different detector attenuations (ATT) (amplification factor) while recording the
calibration data. This was performed by adding the same amount of 1-propanol to each solution
as CS [19]. A six point calibration curve (0.04, 0.4, 4, 16, 28 and 36 µg on tube) was constructed
and each concentration was injected in triplicate. The R2-values for the four RS varied between
102
0.998 and 0.999 (with RSD-values < 2.8 %) indicating that good linearity was obtained.
Moreover, statistical analysis of the linearity data showed that the 95 % confidence interval of
the intercept for all the compounds did include zero. Further, the residual plot showed random
scatter of the points around the horizontal axis. The results from the recovery experiments
indicated that good recoveries were obtained between 98.2 and 99.1 % with RSD-values not
exceeding 3 % for all the compounds at the three levels. The good sensitivity of the method
was illustrated for the different RS by the LOQ values, which were found to be 0.04 µg on tube
and the LOD found to be 0.01 µg on tube. When 10 mg of albumin was introduced in the tube,
the LOQ and LOD corresponded to 0.0004 % and 0.0001 %, respectively. The repeatability of
the method was found to be acceptable with RSD values < 3 %. The completeness of release
from the reference tube in OLC was verified by ILC and the obtained results for the two
approaches were similar as the analyte release at the three concentration levels was found to be
between 98.4 and 99.9 % for OLC relative to ILC.
Figure 5.3: Chromatogram of first desorption (upper chromatogram) and second desorption
on the same tube (lower chromatogram). Degradation products are related to albumin.
103
5.3.2. HS-GC method
5.3.2.1. HS-GC method development
When analyzing aqueous solutions of albumin with conventional HS, aggregations are formed
upon heating in the HS oven during equilibration. Since the effect of the presence of this third
solid phase on the results of analysis is unknown, it was very important to assure the complete
transfer of RS from the albumin into solution and to prevent the formation of a precipitate
during the entire HS analysis. This was achieved by digesting the albumin with trypsin to obtain
an aqueous sample. A homogenous solution without precipitate was observed when performing
this optimized HS method. The small amount of sample brought into the HS vial allowed to
work under FET conditions [13].
5.3.2.2. HS-GC method validation

A blank albumin treated with the digestion procedure was analyzed with the optimized HS-GC-
MS method and the results showed that no interference was found. The results of the accuracy
experiments (in a range of 98.1 to 99.3 %) indicated that neither the digestion procedure nor
the matrix had an effect on the recovery. The linearity of the five point calibration curves was
determined by evaluating the R2-values which were found to be equal to or higher than 0.999.
The residual plot confirmed that linear regression was appropriate to fit the data. Further, the
95 % confidence interval of the intercept also included zero. The LOD and LOQ of the method
were found to be 0.8 and 2 ng in vial respectively. Precision of the method was reported as RSD
and it was lower than 2 %.
5.3.3. Application
Both the TD-GC and HS-GC methods were applied to determine residual DCM and EtOH in
two different samples of albumin loaded with experimental drugs. The samples were subjected
to the procedures mentioned above which were required for the TD-GC and HS-GC methods
before analysis. As the amount of EtOH in the sample was higher than expected and fell out of
range with the TD-GC method, the sample amount in the tube was reduced with a factor 5. As
indicated in Table 5.3, the amount of DCM was lower compared to EtOH. This could be due to
the higher volatility of DCM. The amount of DCM in the sample (0.048 %) is below the ICH
acceptance limit (0.06 %) whereas the amount of EtOH (1.5 %) is above the acceptance limit
(0.5 %). This indicates that a more intense drying approach is needed to remove this solvent
from the samples. The results obtained by HS-GC and TD-GC are similar and statistical
104
comparison performed at a 95 % confidence level indicated that there was no significant
difference (p = 0.12) between the two approaches where the TD-GC method was easier, shorter
and more user friendly.
Table 5.3: Amount of RS found in drug loaded albumin samples using HS-GC and TD-
GC
Method RS in drug loaded albumin
Albumin loaded with Albumin loaded with
Drug 1 Drug 2
DCM (RSD, n = 6) EtOH (RSD, n = 6)
HS-GC 0.048 % (1.7 %) 1.49 % (0.3 %)
TD-GC 0.047 % (1.3 %) 1.47 % (1.4 %)
5.4. Conclusions
The presented results demonstrated that the proposed TD-GC method for the determination of
RS from solid albumin loaded with drug is simple, precise, linear and accurate. To verify the
proposed method, a conventional HS-GC method was also developed based on the complete
enzymatic digestion of albumin. However, this approach is less obvious for routine analysis as
it is time consuming and rather expensive. Unlike the HS-GC method, the proposed method
does not require complicated sample pretreatment steps and there is no need to search for a
suitable solvent. This avoids solvent related problems like interference of solvent impurity
peaks with the peak(s) of interest. Both methods were applied for the determination of residual
DCM and EtOH in two different experimental drug loaded albumin samples. The proposed TD-
GC method yielded quantitative results, which showed no significant difference (p > 0.05) with
those obtained by the HS-GC method.
5.5. References
[1] A. Loureiro, N.G. Azoia, A.C. Gomes, A. Cavaco-Paulo, Albumin-based nanodevices as
Drug Carriers, Curr. Pharm. Des. 22 (2016) 1371–1390.
[2] A.O. Elzoghby, W.M. Samy, N.A. Elgindy, Albumin-based nanoparticles as potential
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controlled release drug delivery systems, J. Control. Release 157 (2012) 168–182.
[3] X. Tao, S. Jin , D. Wu, K. Ling, L. Yuan , P. Lin , Y. Xie, X. Yang, Effects of particle
hydrophobicity, surface charge, media pH value and complexation with human serum
albumin on drug release behavior of mitoxantrone-loaded pullulan nanoparticles,
Nanomaterials 6 (2015) 2.
[4] W. Lohcharoenkal, L. Wang, Y.C. Chen, Y. Rojanasakul, Protein nanoparticles as drug
delivery carriers for cancer therapy, Biomed. Res. Int. 2014 (2014) 1-12.
[5] A. Sethi, M. Sher, M.R. Akram, S. Karim, S. Khiljee, A. Sajjad, S.N. Shah ,G. Murtaza,
Albumin as a drug delivery and diagnostic tool and its market approved products, Acta
Pol. Pharm. Drug Res. 70 (2013) 597–600.
[6] K. Nidhi, S. Indrajeet, M. Khushboo, K. Gauri, D.J. Sen, Hydrotropy: A promising tool
for solubility enhancement: A review, Int. J. Drug Dev. Res. 3 (2011) 26–33.
[7] J. Park, B. Sun, Y. Yeo, Albumin-coated nanocrystals for carrier-free delivery of
paclitaxel, J. Control. Release 263 (2017) 90–101.
[8] S.Y. Mina, H.J. Byeona, C. Leea, J. Seoa, E.S. Leeb, B.S. Shinc, H. Choid, K.C. Leea, Y.
S. Youna, Facile one-pot formulation of TRAIL-embedded paclitaxel-bound albumin
nanoparticles for the treatment of pancreatic cancer, Int. J. Pharm. 494 (2015) 506–515.
[9] Guidelines for Residual Solvents, International Council on Harmonization, Geneva,
Switzerland, 2009.
[10] Identification and Control of Residual Solvents (Section 2.4.24), European
Pharmacopoeia, 9th ed., European Department for the Quality of Medicines, Council of
[11] C. Cheng, S. Liu, B.J. Mueller, Z. Yan, A generic static headspace gas chromatography
method for determination of residual solvents in drug substance, J. Chromatogr. A 1217
(2010) 6413–6421.
[12] W. D’Autry, C. Zheng, J. Bugalama, K. Wolfs, J. Hoogmartens, E. Adams, B. Wang,
A.Van Schepdael, Liquid paraffin as new dilution medium for the analysis of high boiling
point residual solvents with static headspace-gas chromatography, J. Pharm. Biomed.
Anal. 55 (2011) 1017–1023.
[13] D.M. Kialengila, K. Wolfs, J. Bugalama, A. Van Schepdael, E. Adams, Full evaporation
headspace gas chromatography for sensitive determination of high boiling point volatile
organic compounds in low boiling matrices, J. Chromatogr. A 1315 (2013) 167.
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[14] N. van Boxtel, K. Wolfs, A. Van Schepdael, E. Adams, Application of acetone acetals as
water scavengers and derivatization agents prior to the gas chromatographic analysis of
polar residual solvents in aqueous samples, J. Chromatogr. A 1425 (2015) 62-72.
[15] H. Chick, C.J. Martin, On the “heat-coagulation” of proteins, Part II. The action of hot
water upon egg-albumen and the influence of acid and salts upon reaction velocity, J.
Physiol. 43 (1911) 1–27.
[16] V.A. Borzova, K.A. Markossian, N.A. Chebotareva, S.Yu. Kleymenov, N.B. Poliansky,
K.O. Muranov, V.A. Stein-Margolina, V.V. Shubin, D.I. Markov, B.I. Kurganov, Kinetics
of thermal denaturation and aggregation of bovine serum albumin, PLoS One 11 (2016) p.
e0153495.
determination of residual solvents in solid drug product, J. Chromatogr. A 1217 (2010)
5158-5164.
dimethylsulphoxide in drug loaded gelatin using thermal desorber – gas chromatography,
[19] A.A. Asfaw, K. Wolfs, A. Van Schepdael, E. Adams, Thermal desorption—gas
silica, J. Chromatogr. A 1500 (2017) 160-166.
[20] A.A. Asfaw, M. Van Der Veken, K. Wolfs, A. Van Schepdael, E. Adams, Optimization of
reference introduction prior to calibration of thermal desorber - gas chromatography,
submitted to J. Chromatogr. A
[21] H. Kolsrud, H. Malerod, S. Ray, L. Reubsaet, E. Lundanes, T. Greibrokk, (2012). A
critical review of trypsin digestion for LC-MS based proteomics, Integrative proteomics,
Hon-Chiu Leung (Ed.), InTech, pp 73-92.
[22] International Council on Harmonization. Draft Guideline on Validation of Analytical
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108
Chapter 6: General discussion
109
110
6.1. General discussion
GC is considered to be the golden standard for the analysis of volatiles in various samples from
many different fields such as pharmaceutical, forensic, environmental and food samples. In
pharmaceuticals, GC is mainly used for the determination of RS in raw materials and final
products. In addition, it is also applied for the identification, impurity profiling and
quantification of volatile active ingredients in different formulations. Although there has been
no real progress in the GC separation process since the introduction of capillary columns, some
interesting improvements in sample preparation and introduction techniques have been made
during the last years.
The most common sample introduction to GC is direct injection, which introduces and
vaporizes a portion of sample (liquid) in a hot chamber with a liner. Although it is simple and
cheap, it suffers from limitations due to accumulation of non-volatile components of the sample
matrix that cannot be vaporized in the injector liner. Repetitive injections of such a sample will
finally cause inlet blockage and contamination of the GC column. To solve this issue, isolation
of the non-volatile components from the sample matrix before introduction into the GC is
usually performed using sample preparation procedures. However, this approach has also some
serious drawbacks as it is often a multi-step procedure that may result in analyte losses and long
processing times. Moreover, toxic organic solvents are frequently used and it is not always easy
to automatize the process for routine analysis. Another constraint of direct injection is the
impossibility to analyse solid samples that cannot be dissolved in a suitable solvent.
Later, HS techniques were introduced to circumvent the shortcomings of direct injection. HS is

based on transferring a part of the vapor phase containing the analytes to the GC column, while
keeping non-volatile sample residues in the HS vial. This enables HS to avoid contamination
of the GC inlet and column. However, this could only be achieved with a drop in sensitivity
due to the fact that only a small portion of the analyte in the gaseous phase is transferred to the
GC. This sensitivity issue could be improved using dHS. However, this approach is also not
free of problems due to the adsorbents used in the trap, which may lead to unpredictable
quantitative results because of breakthrough or freezing of the cold trap. Further, there could be
a memory effect after the analysis of samples with concentrated volatiles [1].
Static HS-GC is the method of choice for the determination of RS in pharmaceuticals.

Conventional HS methods for RS analysis normally start from a homogenous sample solution.
The main challenge in sHS is the influence of matrix effects on the recovery of the method.
111
This problem could be avoided by using matrix matched calibration. However, this is not
always possible. A possible solution is MHE, which is hypothetically based on complete
extraction of the analytes from the sample. As a result, there is no matrix effect on the recovery.
However, its time-consuming aspect makes it not very attractive for routine analysis. Another
option to exclude matrix effects is HS in FET mode [2]. This approach is very useful on
condition that complete release of the analytes can be guaranteed.
For solid samples which are not soluble in water or most common organic solvents, the scenario
is not straightforward. Although sHS is mainly applicable to sample solutions, it is in principle
also possible to analyse samples as a solid. However, the solid has to behave as a partition
system, which means that the distribution (partition) coefficient is considered to be constant
and independent of the analyte concentration [3]. Solids with a large surface area and active
functional groups like MPSi are prone to present adsorption effects and make that the
equilibrium is no longer purely partition driven. As a result, the relationship between the
original concentration of analyte in the sample and the equilibrium concentration in the HS gas
phase is difficult to model and thus unsuitable for quantitative work. For samples of this type,
it has been suggested to add a liquid that can alter the surface properties of the solid and will
displace the analyte from the solid matrix into the liquid. As the analyte is now in the liquid
phase, the issues due to the adsorption effect will be no longer present [4]. However, this
approach will only work if the analyte is superficially adsorbed and accessible to the displacer
liquid, which is not the case when it is enclosed in the structure of the solid and cannot be
completely released under HS conditions by diffusion like in MPSi, gelatin and albumin. For
those samples, a more drastic approach is necessary. Destruction of the matrix is an option, but
this is usually not simple, time-consuming, expensive and/or difficult to automatize using
standard HS equipment. For instance, HF can be used to destroy MPSi and to ensure the release
of the analytes from the MPSi. However, HF reacts with glass, which can result in partly
dissolving the HS vial and so making this approach unsuitable. Although it is possible to do HS
analysis with diluted HF after neutralization in a PTFE vial, its toxicity and complicity still
prevent its use for routine analysis. For analysis of the solid samples mentioned above, the use
of the FET approach under HS conditions is also not an option since complete evaporation of
the RS from those samples cannot be assured. In a preliminary study, we have proved that for
one or another reason all HS approaches failed to determine RS in the examined solid samples.
However, from the experiments we observed that the extraction of analytes from those samples
increased with higher extraction temperature (better recovery by FET vs. sHS) and extraction
112
number (MHE vs. sHS). Consequently, an alternative technique that works in a continuous
sampling mode at high temperature should be tried. In the case of MPSi it is difficult to prove
that RS were completely released from the matrix and for gelatin and albumin, the matrix causes
difficulties in the HS instrument since they form a precipitate when heated in the vial during
equilibration. The effect of the presence of this precipitate on the partition of the analytes is
unknown and makes quantitative work unpredictable.
In this project, we have shown that TD is an efficient and simple sample introduction technique
to GC for the determination of RS in solid samples without complicated sample preparation
steps. TD is a dynamic system, which involves thermal extraction of analytes from the sample
into a flow of inert gas. This flow, in combination with a desorption temperature much higher
than HS, provides the TD a good efficiency to extract the RS from the matrix. This technique
is very applicable, especially when conventional HS-GC methods are not convenient. It
circumvents problems such as adsorption effects related to the surface chemistry and structure
of MPSi, insolubility in most common solvents and formation of a precipitate due to heating
during equilibration like in case of gelatin and albumin samples. Another advantage of the TD
technique is that, unlike HS-GC, there is no exertion required to select a suitable solvent and
subsequently no interference of the solvent peak nor from impurities present in the solvent.
Moreover, it is also possible to improve sensitivity by increasing the amount of sample in the
TD tube or adapting the split flow.
TD is normally designed for air monitoring applications by collecting samples from the
environment in a TD tube with sorbents. It was necessary to modify the original sample
introduction mode of the TD for our purpose. The first step was the selection of a suitable
supporting material to keep the sample in the TD tube. QF was selected for this purpose due to
the fact that it is inert, thermally stable (up to 900 °C) and it is easy to cut and fit the TD tube.
As a result, in this study sample introduction was performed by sandwiching the sample
between two double layers of QF. In the case of gelatin and albumin also a PTFE insert was
used to facilitate cleaning and reuse of the tube. The efficiency of the extraction of the analytes
can be enhanced by optimizing the TD parameters such as desorption temperature and time and
the flow of the inert gas. The RS studied are commonly used in pharmaceutical technology for
drug loading.
Following method development, the final objective of any analytical measurement is to obtain
consistent, reliable and accurate data. Validated analytical methods play a major role in
113
achieving this goal. The results from method validation can be used to judge the quality,
reliability and consistency of analytical results, which is an integral part of any good analytical
practice. Validation of analytical methods is also required by most regulations and quality
standards that impact laboratories. Official methods do not need to be validated as long as they
are not changed, but the laboratory should demonstrate that it is capable of successfully running
the method. When a new analytical method has been developed, it needs to be fully validated
by performing validation experiments. Comparing the results with a standard method is
appropriate. As a result, for the proposed TD-GC methods it was always important to verify the
obtained results using another method.
In this thesis, the applicability of TD-GC towards three cases has been demonstrated. They are
all related to the determination of RS in drug loaded samples. This analysis is not always evident
as (very) tiny amounts of analytes have to be determined in a relatively large amount of sample.
Moreover, interactions like adsorption effects can occur between the RS, drug and carrier so
that the release of the analytes can be hampered. First the determination of 7 RS (varying in
polarity and volatility) in drug loaded MPSi was studied. Different types of MPSi with pore
sizes ranging from 2-3 to 25 nm were applied and a good recovery (>98%) was found for most
of the analytes, which was much better than that obtained with the HS approaches (not more
than 60%). Generally, large pore size MPSi provides a (slightly) higher recovery than the
narrow size indicating that it is more difficult for RS to diffuse out of smaller pores. In addition,
the spatial configuration of the solvent plays a role as illustrated by the difference between
isopropanol and n-propanol. As a result, it is desirable to validate properly the recovery for each
new RS/MPSi combination. The methodology was completely validated for the quantitative
determination of DCM in drug loaded MPSi samples. A FET-HS-GC method after dissolving
the MPSi in HF was used to compare the results. Those were found to be similar for both
methods, but it is clear that the newly developed TD-GC method is much more user friendly.
Using TD-GC, care must be paid to the filters on the fixed and mobile seal of the TD instrument
when very fine MPSi is used. This is because these very fine particles can pass through the
filters and clog the transfer line, which is a similar scenario that occurs with particles dislodged
from PLOT type capillary columns when used with MS.
A second and third application were related to the determination of RS in gelatin and albumin.
When gelatin is treated in hot water above 50 °C, this results in partial or complete loss of
solubility due to crosslinking. DMSO is frequently used to dissolve drugs prior to loading on
carriers like gelatin. The determination of DMSO as RS in gelatin is quite challenging: DMSO
114
has a high boiling point (189 °C) and gelatin is almost impossible to dissolve in common
solvents. The lower temperature to keep gelatin in aqueous solution is not compatible with the
higher temperature to analyse DMSO by HS-GC. Indeed, at high temperature gelatin becomes
insoluble and the influence of an additional solid phase on the analysis results is not known.
Direct quantification by HS-GC from solid gelatin is difficult at the temperatures that are
reached in HS and DMSO can interact with gelatin. Albumin is a similar case. It is freely soluble
in water at room temperature, but it forms an irreversible precipitate upon heating. This means
that an aqueous solution of an albumin sample cannot be subjected to HS analysis. The presence
of this solid phase together with the liquid and gaseous phase in the vial could have different
effects on the equilibration of the analytes than when it is absent. As a result, the analysis of RS
in albumin with HS-GC is not straightforward. The applicability of TD-GC for the
determination of DMSO in gelatin and RS in albumin without any sample preparation
procedure was explored. To verify the proposed TD-GC method, an alternative method based
on complete enzymatic digestion of the gelatin and albumin sample followed by FET-HS-GC
was applied. In case of DMSO, to improve the sensitivity, water was completely removed from
the sample using 2,2-dimethoxypropane (DMP) under acidic catalysis prior to FET-HS-GC
analysis. A 10-fold increase of sensitivity was obtained compared to standard FET of aqueous
samples. However, this procedure is only applicable for high boiling compounds like DMSO
since there could be loss of low boiling compounds like Ace, DCM, Chlor during the vacuum
drying steps. Both the proposed TD-GC and the HS-GC methods were applied for the
determination of RS in drug loaded samples for comparison and proved to be similar. However,
the enzymatic degradation is laborious, expensive and needs a longer analysis time. TD can
work at a high temperature, which is an advantage for efficient desorption, but it could end up
with condensation of degradation products of unstable samples in the split line which can
eventually be clogged. However, this can mostly be overcome by heating the split line.
Another important issue is the calibration stage, which is the most critical step of the TD
procedure, especially when the RS are directly desorbed from the solid sample while the
calibration is performed using a standard liquid mixture. Ideally, TD procedures are calibrated
using gas phase standards generated either dynamically or statically, but those can be expensive
and/or difficult to obtain in the desired concentration range [5]. As a result, calibration of a TD-
GC system is mostly performed by syringe injection of a reference solution directly on the tube
bed [6,7]. Ultimately, transport can be assisted by an inert gas flow. In this approach, it is very
important to emphasize that there should be no difference in the adsorption and desorption
115
phenomena between the sample and the calibration. Unfortunately, such difference is likely to
happen in our case due to the presence of diluents for the calibration and direct desorption for
the solid sample. It is also assumed that both the adsorption and desorption of the analyte are
exhaustive and that there is no loss of analytes during the introduction steps. In chapter 4, the
fulfillment of those assumptions for OLC is evaluated and an alternative ILC by direct injection
of reference into the primary desorption gas stream of the TD was proposed. A possible source
of analyte loss with the OLC technique was identified as incomplete transfer of reference,
especially when only QF disks were used, to the TD-GC system was demonstrated. This was
found to be due to tube manipulations resulting in loss of analytes during injection or before
the desorption cycle started, especially for very volatile analytes like DCM and Chlor. This was
improved when MPSi was added as sorbent since it has good retention capacity, even for the
very volatile analytes. This was further developed by application of a sweep gas during injection
as it enhanced the transfer of analytes to the tube bed. To verify the complete desorption of all
the analytes, a second desorption was performed on the same tube and it was confirmed that no
peak of the investigated analytes was observed in the second chromatogram. Although it was
possible to confirm maximal desorption, it was still necessary to check the completeness of the
release of all the analytes from the MPSi bed to the TD-GC system. This was done by ILC,
which guaranteed no loss of analytes during reference solution introduction or decapping of the
tube. For the investigated VOCs, apart from DMSO, ILC was better than OLC in terms of
transfer ratio. The use of ILC has to be favored over OLC in case of a direct desorption TD
where assuring the complete transfer of the reference compounds is very important for
quantitative purposes. The approaches were applied and worked well for the determination of
RS in albumin. Furthermore, the ILC approach can be used as an adsorption-desorption free,
independent tool for the validation of the TD-GC system. The only drawback is that for ILC no
standard equipment can be used since such an apparatus is not commercially available.
6.2. Future prospectives

In this project, OLC utilized MPSi beds as sorbent in the tubes. MPSi is a good choice as sorbent
because it is inert, stable and can provide a wide range of adsorption properties, which enable
to prepare a tailored sample tube for specific analytes. This requires validating the recovery of
every analyte from a given MPSi as differences between different types of MPSi were noticed.
This can be considered as a drawback when a large group of analytes and MPSi with different
pore sizes are used since it is mandatory to evaluate each analyte-MPSi combination for
recovery. In the future, an all-inclusive study can be executed to evaluate the recovery of a large
116
group of analytes in combination with different types of MPSi. This would enable to create one
general OLC approach for different analyte-MPSi combinations.
Another area where more efforts can be put is the improvement in the ILC design. In the actual
setup, the transfer line, which connects the installed GC injector with the sampling end of the
TD instrument, is made from stainless steel. This could result in adsorption of some high boiling
polar compounds like DMSO, resulting in a lower recovery. A large volume quartz liner for the
injector and a fused silica tubing as inner liner for the transfer line may bring a solution, but an
analytical laboratory like ours lacks the feasibility to construct such liners.
In addition, ILC is performed manually by injection with a micro syringe. So, the applied
volume is vulnerable for variation making the use of an internal standard advisable. A possible
upgrade would be to incorporate an autosampler in order to make it more repeatable and suitable
for routine analysis and to increase the sample throughput.
Further, ILC could be a very useful tool to study gas phase adsorption on different types of
sorbents by placing them in the TD tube. ILC could be beneficial for surface studies of solid
materials in the same way as inverse GC. The importance of solid dosage forms and the rapid
development of novel and complex approaches to solid-state drug delivery have fueled
characterization of solid pharmaceutical materials. The ILC approach could be used for this
purpose by packing the material in the TD tube and study for instance the surface energy of
pharmaceutical powders as a function of relative humidity. Such data are also important for
understanding stability behavior and practical storage of pharmaceutical materials. Porous
materials like MPSi are one of the most complex materials to analyse by most techniques. This
complexity is due to the combination of heterogeneous surface sites and an intricate system of
internal and external surfaces. ILC has the potential to differentiate solute adsorption due to
micropore filling versus surface adsorption as solutes can enter or not, inside the pores of MPSi
based on their molecular size and configuration. Accordingly, it is possible to select a solute
that cannot enter into the pore and determine the surface adsorption. This could allow to
estimate the true external surface area. Another application of the ILC approach could be
measuring the adsorption of (gases of) solutes to packaging materials by placing the packing
material in the TD tube.
6.3. References
[1] Snow, N. H., Bullock, G. P., Novel techniques for enhancing sensitivity in static headspace
extraction gas chromatography. J. Chromatogr. A 12172 (2010) 726-2735.
117
[2] Brault, A., Agasse, V., Cardinael, P., Combret, J., The full evaporation technique: A
promising alternative for residual solvents analysis in solid samples. J. Sep. Sci. 28 (2005)
380–386.
[3] Kolb, B., Ettre, L. S., Static Headspace Gas Chromatography: Theory and Practice, John
Wiley and Sons, New Jersey, 2006, pp. 191–204.
[4] Kolb, B., Auer, M., Analysis of water in liquid and solid samples by headspace gas
chromatography. Fresenius. J. Anal. Chem. 336 (1990) 297–302.
application-oriented overview of methods in a nutshell, Int. J. Chem. Eng. 2012 (2011) 1-
6.
[6] Ribes, A., Carrera, G., Gallego, E., Roca, X., Berenguer, M. J., Guardino, X., Development
and validation of a method for air-quality and nuisance odors monitoring of volatile organic
compounds using multi-sorbent adsorption and gas chromatography/mass spectrometry
thermal desorption system. J. Chromatogr. A 1140 (2007) 44-54.

control in quantification of volatile organic compounds analysed by thermal desorption–
gas chromatography–mass spectrometry, J. Chromatogr. A 1186 (2008) 348–357.
6.4.
118
Summary
The determination of small amounts of RS in solid samples that are difficult to dissolve like
MPSi, gelatine and albumin is not evident. Conventional HS-GC has difficulties to deliver
reliable results for this type of samples. In this study, the application of TD-GC in a modified
mode has been investigated. So, samples were sandwiched between 2 double layers of QF in a
TD tube.
In chapter 2, an easy-to-apply TD-GC method has been developed and validated for the
determination of 7 RS commonly used for drug loading of MPSi. MPSi displays some specific
properties in terms of adsorption and active surface area. The method showed excellent
sensitivity, linearity, recovery and repeatability and was finally applied on drug loaded MPSi.
An independent HS-GC method following HF treatment to destroy the matrix has also been
applied to compare with. There was no significant difference in the results obtained from the
two methods. However, the HS-GC method needs complicated sample preparation steps and is
less suitable from a practical point of view.
In chapter 3, the application of TD-GC for quantitative determination of residual DMSO in
solid gelatin has been investigated. The proposed method was successfully optimized and
validated. Another approach based on the complete enzymatic digestion of gelatin combined
with dehydratation followed by HS-GC was developed and applied to verify the proposed
method. Quantitative results were in good agreement. Unlike HS-GC, TD-GC does not require
to select a suitable solvent to dissolve the sample. As a result, there is no interference in the
chromatogram from a large solvent peak nor from impurities present in the solvent. In addition,
since the sample size in TD can be eventually further increased, it is possible to improve the
sensitivity.
In chapter 4, the ILC system – based on a modified GC injector installed on the TD instrument
has been proposed. This allowed to evaluate properly the OLC approach by injecting standard
solutions on sample tubes containing each 4 QF disks, a single size MPSi bed or a mixed MPSi
bed immobilized in a TD tube. For the MPSi beds, loading was performed with and without
sweep gas during injection by connecting the tube to an injector port with a flow of N2. Based
on the obtained results, it was possible to confirm that there was incomplete transfer of reference
substances during OLC due to tube operation, especially when only QF was used. This
improved when MPSi was added as sorbent and the presence of a sweep gas minimized the
differences with ILC. For the investigated volatile organic compounds, apart from DMSO, ILC
surpasses OLC in terms of transfer ratio.
119
Finally, in chapter 5, work around the use of TD-GC for the determination of RS in albumin
has been presented. Calibration was performed with OLC (tube with a mixed MPSi bed loaded
under a sweep gas flow) as well as ILC to check if the transfer of the calibrants with OLC was
complete. An independent method based on the complete enzymatic digestion of albumin
combined with HS-GC was applied to verify the complete release of analytes from the albumin.
Both the proposed TD-GC and HS-GC methods were applied for the determination of residual
EtOH and DCM in two drug loaded albumin samples. The results obtained by the two methods
were comparable. However, the TD-GC method is easier and more practical, illustrating once
more its great and interesting potential.
120
Samenvatting
De bepaling van kleine hoeveelheden residuele solventen (RS) in vaste, moeilijk oplosbare
monsters zoals mesopore silica (MPSi), gelatine en albumine is niet vanzelfsprekend.
Conventionele headspace gaschromatografie (HS-GC) slaagt er niet altijd in om voor dit type
monsters betrouwbare resultaten af te leveren. In deze studie werd de toepassing van GC in
combinatie met thermische desorptie (TD) in een aangepaste vorm onderzocht. Zo werd het
monster in de TD tube gefixeerd tussen twee dubbele lagen kwartsfilter.
In hoofdstuk 2 werd een gemakkelijk toe te passen TD-GC methode ontwikkeld en gevalideerd
voor de bepaling van zeven RS die courant gebruikt worden voor het beladen van MPSi met
geneesmiddelen. MPSi vertoont enkele specifieke eigenschappen wat betreft adsorptie en
actieve oppervlakte. De methode vertoonde een uitstekende terugvindbaarheid, gevoeligheid,
lineariteit en herhaalbaarheid en werd finaal toegepast op beladen MPSi monsters. Om te
kunnen vergelijken werd er, na afbraak van de matrix met waterstoffluoride, eveneens een
onafhankelijke HS-GC methode uitgevoerd. Er werd geen significant verschil vastgesteld
tussen de resultaten bekomen met de 2 methoden. De HS-GC methode vereist echter een
ingewikkelde staalvoorbereidingsstap en is dus minder geschikt vanuit praktisch oogpunt.
In hoofdstuk 3 werd de toepassing van TD-GC onderzocht voor de bepaling van residueel
dimethylsulfoxide (DMSO) in gelatine. De voorgestelde methode werd succesvol
geoptimaliseerd en gevalideerd. Een andere benadering, gebaseerd op de volledige
enzymatische afbraak van gelatine gecombineerd met een dehydratatiestap gevolgd door HS-
GC werd eveneens ontwikkeld en toegepast om de voorgestelde methode te verifiëren. De
kwantitatieve resultaten vertoonden een goede overeenkomst. In tegenstelling tot HS-GC,
vereist TD-GC niet de selectie van een geschikt solvent om het monster in op te lossen.
Bijgevolg is er in het chromatogram ook geen interferentie van een grote solventpiek, noch van
kleine piekjes die eventueel te wijten zijn aan onzuiverheden in het solvent. Bovendien is het
mogelijk om de gevoeligheid van TD-GC nog te verbeteren aangezien er zonder probleem een
grotere hoeveelheid monster in de tube kan gebracht worden.
In hoofdstuk 4 werd een kalibratiesysteem voorgesteld waarbij de referentievloeistof
rechtstreeks in de TD wordt gebracht door middel van een aangepaste GC injector. Hiervoor
werd de term inline liquid calibration (ILC) gebruikt. Dit liet een grondige evaluatie toe van de
gebruikelijke kalibratie die gebeurt door een referentievloeistof op het sorbens in de tube aan
te brengen en waarnaar gerefereerd wordt als offline liquid calibration (OLC). Als sorbens werd
gekozen voor kwartsfilters, enkelvoudige MPSi of een mix van MPSi, al dan niet in combinatie
121
met een stroom van stikstofgas om verlies tijdens het injecteren van de referentievloeistof te
beperken. Op basis van de resultaten kon besloten worden dat de transfer van
referentiesubstanties bij de OLC benadering onvolledig was, vooral wanneer enkel kwartsfilters
gebruikt werden. Dit verbeterde aanzienlijk wanneer MPSi werd toegevoegd als sorbens en de
verschillen met ILC waren miniem wanneer ook nog bijkomend een gasstroom werd aangelegd.
Desalniettemin presteerde ILC nog altijd ietwat beter dan OLC, uitgezonderd voor DMSO.
Tenslotte werd in hoofdstuk 5 werk voorgesteld omtrent het gebruik van TD-GC voor de
bepaling van RS in albumine. Kalibratie werd zowel uitgevoerd door middel van OLC (tubes
met een MPSi mix en referentievloeistof aangebracht onder een inerte gasstroom) als ILC om
na te gaan of de transfer van referentiestoffen bij OLC volledig was. Daarnaast werd nog een
onafhankelijke methode – op basis van de enzymatische afbraak van albumine gevolgd door
HS-GC – aangewend om de complete vrijstelling van de analieten uit albumine te controleren.
De voorgestelde TD-GC en HS-GC methoden werden vervolgens toegepast voor de bepaling
van residuele hoeveelheden ethanol en dichloormethaan in albuminemonsters beladen met een
geneesmiddel. De resultaten bekomen met de verschillende methoden waren vergelijkbaar. De
TD-GC methode was echter gemakkelijker en praktischer, hetgeen eens te meer haar grote en
interessante potentieel illustreert.
122
Scientific acknowledgements and conflict of interest statement
The authors gratefully acknowledge the support of the Interfaculty Council for Development
Co-operation (IRO, KU Leuven).
All the authors involved declare no conflicts of interest.
123

2018 - Adissu Asfaw Thesis

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KU Leuven

Biomedical Sciences Group

NEW SAMPLING TECHNIQUES IN GAS CHROMATOGRAPHY

Adissu Alemayehu Asfaw

Promoter: Prof. Dr. Erwin Adams Dissertation presented in

I gratefully acknowledge the Interfaculty Council for Development Co-operation (IRO), KU

[1] A.A. Asfaw*, K. Wolfs, A. Van Schepdael, E. Adams, Development of a thermal

Supervision of postgraduate and master students

Curriculum Vitae ...................................................................................................................................ii

Based on ‘Overview of sample introduction techniques prior to

KU Leuven - University of Leuven, Department of Pharmaceutical and

Submitted to J. Sep. Sci.

1.2.2. Inlets and Columns

1.2.3. Detectors [9]

1.2.3.1. Flame ionization detector

Figure 1.1: Schematic overview of FID [13]

1.2.3.2. Mass spectrometer as detection system

1.3. Sample introduction techniques to GC

1.3.1. Direct injection techniques

1.3.1.2. On-column injection

1.3.1.3. Programmable temperature vaporization (PTV)

1.3.2. Headspace techniques

1.3.2.3. Application of HS techniques for solid samples

Figure 1.4: Schematic overview of two-stage TD [73].

1.3.3.2. Calibration of TD methods

1.4. Aim of the study

Chapter 4 is dedicated to reference introduction in TD-GC. In principle, a gas-phase standard

KU Leuven - University of Leuven, Department of Pharmaceutical and

J. Chromatogr. A 2017, 1500, 160–166

Key words: thermal desorption-gas chromatography, mesoporous silica, residual solvents,

2.2.3. Standards and sample preparations

2.2.3.2. Standards and sample preparation for FET-HS-GC-FID

2.2.4. Method validation

2.2.4.2. Method validation of FET-HS-GC-FID

2.3. Results and discussion

2.3.2. TD-GC-FID method development

2.3.2.2. Filling of the TD tubes

Figure 2.1: Sample immobilization in TD tube.

2.3.2.3. Optimization of TD parameters

Parameter Optimized settings Range explored

Desorption temperature 300 °C 200 – 350 °C

Desorption time 80 min 30 – 100 min

Desorption flow 140 mL/min 35 – 180 mL/min

Sorbent in tube Quartz filter paper 1 – 4 layers

Trap low temperature -30 °C 4 to -30 °C

Trap high temperature 280 °C

Trap hold time 10 min 2 – 20 min

Trap material AMT AMT / Tenax

Inlet split 40 mL/min 10 – 60 mL/min

Outlet split 40 mL/min 10 – 60 mL/min

Column flow 4 mL/min

GC oven program 35 °C held 9 min;

GC/MS interface temperature 180 °C

a) MPSi 25nm b) MPSi 15n

EtOH 130 Ace

Table 2.2: Recovery of RS from different MPSi following TD-GC-FID.

MPSi 2-3 nm MPSi 15 nm MPSi 25 nm average

MPSi pore size Linearity Repeatability

2-3 0.998-0.999 0.5-2.9

2.3.4. FET-HS-GC-FID method development

Equilibration temperature (ET) 160 °C

Equilibration time 15 min

Needle temperature ≥ 5 °C above ET