Assessment of headspace solid-phase microextraction (HS-SPME) for control of asymmetric bioreduction of ketones by Alternaria alternata

Article abstract of DOI:10.1080/10242422.2019.1664478

The aim of this study was to assess the effectiveness of headspace solid-phase microextraction (HS-SPME) compared to liquid–liquid extractions using with methylene chloride (CH₂Cl₂) for control of fungal biotransformation of ketones of varying volatility. The proposed method was successfully applied. The best way to extract all the components of the mixture (alcohols, aldehydes) in quantities similar to the extraction of methylene chloride was the use of fibres coated with a combination of nonpolar material. SPME fibre assembly polydimethylsiloxane/divinylbenzene (PDMS/DVB) was most suitable for the extraction of the products mixture obtained after biotransformation of acetylcyclohexane and acetophenone. On the other hand, the best results were obtained for 2-acetylthiophene, α,α,α-trifluoroacetophenone and their derivatives using divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fibre. In addition, our study showed that Alternaria alternata is a good biocatalyst for bioreduction of ketones to alcohols according to Prelog's rule.

Full text of DOI:10.1080/10242422.2019.1664478

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: https://www.tandfonline.com/loi/ibab20
Assessment of headspace solid-phase
microextraction (HS-SPME) for control of
asymmetric bioreduction of ketones by Alternaria
alternata
Rafał Ogórek & Bogdan Jarosz
To cite this article: Rafał Ogórek & Bogdan Jarosz (2019): Assessment of headspace solid-
phase microextraction (HS-SPME) for control of asymmetric bioreduction of ketones by Alternaria
alternata, Biocatalysis and Biotransformation, DOI: 10.1080/10242422.2019.1664478
To link to this article: https://doi.org/10.1080/10242422.2019.1664478
Published online: 14 Sep 2019.
Submit your article to this journal
Article views: 6
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=ibab20

BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2019.1664478
RESEARCH ARTICLE
Assessment of headspace solid-phase microextraction (HS-SPME) for control
of asymmetric bioreduction of ketones by Alternaria alternata
a
b
ꢀ
Rafał Ogorek and Bogdan Jarosz
aDepartment of Mycology and Genetics, Institute of Genetics and Microbiology, University of Wroclaw, Wroclaw, Poland;
bDepartment of Chemistry, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
ABSTRACT
ARTICLE HISTORY
Received 11 January 2019
Revised 25 March 2019
Accepted 22 August 2019
The aim of this study was to assess the effectiveness of headspace solid-phase microextraction
(HS-SPME) compared to liquid–liquid extractions using with methylene chloride (CH2Cl2) for con-
trol of fungal biotransformation of ketones of varying volatility. The proposed method was suc-
cessfully applied. The best way to extract all the components of the mixture (alcohols,
aldehydes) in quantities similar to the extraction of methylene chloride was the use of fibres
coated with a combination of nonpolar material. SPME fibre assembly polydimethylsiloxane/
divinylbenzene (PDMS/DVB) was most suitable for the extraction of the products mixture
obtained after biotransformation of acetylcyclohexane and acetophenone. On the other hand,
the best results were obtained for 2-acetylthiophene, a,a,a-trifluoroacetophenone and their
derivatives using divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fibre. In add-
ition, our study showed that Alternaria alternata is a good biocatalyst for bioreduction of
ketones to alcohols according to Prelog’s rule.
KEYWORDS
HS-SPME; liquid–liquid
extractions; fungal
biotransformation; ketones;
Alternaria alternate
Introduction
pregnant also may be more sensitive to methylene
chloride. Additionally, this chemical agent can reach
the developing foetus through the placenta, it also
can enter breast milk, and it affects the nervous sys-
tem (brain) and can cause headaches, dizziness, nau-
sea, clumsiness, drowsiness, and other effects like
those of being drunk. However, unlike the poisonings,
cancer in people caused by methylene chloride can
take years to develop and is more difficult to docu-
ment (Hall and Rumack 1990; Soden 1993; Mahmud
and Kales 1999). Therefore, it is necessary to search for
new sensitive analytical methods that do not require
the use of organic solvent. One of the ways may be
solid-phase microextraction (SPME).
Extraction is one of the most critical steps in the
chemical analysis of samples (Chen et al. 1981). Since
many years, a standard method used for sample prep-
aration after biotransformation is liquid–liquid extrac-
tions using with intera alia methylene chloride
(CH2Cl2) (Parshikov et al. 2000; Skrobiszewski et al.
2013). This solvent will form two layers in contact
with aqueous solutions if they are used in sufficient
quantities, and it is also used in pharmaceutical,
environmental, food, and chemical industries (Chen
ꢀ
et al. 1981; Mahmud and Kales 1999; Ogorek and
Jarosz 2013).
Methylene chloride is a common and an effective
extraction solvent, but this chemical agent is very
harmful to the environment and to human health,
among others, it may lead to poisoning, death or can-
cer (Leikin et al. 1990; Mahmud and Kales 1999). For
example, once inhaled or absorbed through the skin,
is converted to carbon monoxide, which interferes
with oxygen delivery. Therefore, methylene chloride
can make angina and other heart symptoms worse in
people with heart disease. People with lung condi-
tions, smokers, and people who are overweight or
SPME was developed by Pawliszyn et al. (1990) and
it is an organic solvent-free sample preparation fol-
lowed by desorption inter alia into a gas chromato-
graph (GC) or high-performance liquid chromatograph
(HPLC) for subsequent analysis (Arthur and Pawliszyn
ꢀ
1990; Gorecki and Pawliszyn 1995). This method is an
efficient and sensitive for the extraction and pre-con-
centration of samples, mainly because of the lack of
organic solvents and reduction in associated interfer-
ences (Ouyang and Pawliszyn 2006; Zhuo-Min et al.
2006). Most commonly used SPME analysis are direct
ꢀ
CONTACT Rafał Ogorek
rafal.ogorek@uwr.edu.pl; rafal-ogorek@wp.pl
Department of Mycology and Genetics, Institute of Genetics and
Microbiology, University of Wroclaw, Przybyszewskiego Street 63/77, Wroclaw, 51-148, Poland
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

ꢀ
R. OGOREK AND B. JAROSZ
2
immersion (DI-SPME) mode and headspace (HS-SPME)
mode. DI-SPME mode it used for extraction of analytes
(absorbent-type fibres), mainly non-volatile compounds
from aqueous matrices, and HS-SPME mode for the
analysis of volatile and semi-volatile compounds avail-
able in air or in the headspace volume above liquid or
4-acetylpyridine, 2-bromoacetophenone, 4-bromoace-
tophenone, 3-nitroacetophenone, 4-nitroacetophe-
none. All the substrates were purchased from Fluka.
Screening tests were aimed at identifying substrates
that were biotransformed by the fungus (A. alternata
18570) and determining the times in which the sub-
strates were not completely converted to products.
ꢀ
solid matrices (adsorbent-type fibres) (Gorecki and
ꢀ
Pawliszyn 1995; Gorecki et al. 1999; Lord and Pawliszyn
2000). Literature report also about various factors affect-
ing the efficiency of extraction of the analyte in each
mode (Risticevic et al. 2009; Gura et al. 2010).
Currently, SPME has been successfully used to rap-
idly concentrate volatile and semi-volatile organic
compounds and a wide variety of other organic com-
pounds occurring in aqueous and other matrixes and
used to elucidate the biotransformation pathways of
various chemicals by microorganism (Ghiasvand et al.
Biotransformation procedure
Erlenmeyer flasks (250 mL), each containing 50 mL of
the culture medium described earlier, were inoculated
with A. alternata 18570 and incubated for 4 days at 27
oC on a rotary shaker (190 rpm). Then, 20 mg of a suit-
able substrate dissolved in 1 mL of isopropanol was
added and incubated for 24 h (acetylcyclohexane),
30 h (a,a,a-trifluoroacetophenone) or 48 h (acetophe-
none, 2-acetylthiophene) – the times established on
the basis of screening tests. After incubation, the sam-
ple was extracted by using headspace HS-SPME or by
using liquid–liquid extractions with methylene chlor-
ide (CH2Cl2).
ꢀ
2011; Bocato et al. 2012; Kotowska and Bienczyk 2013).
Literature reported also about comparison of SPME and
liquid–liquid extraction for the analysis of various
chemical compounds (Bonadio et al. 2008; Thompson-
Witrick et al. 2015). Nevertheless, there are no reports
about comparison of HS-SPME and liquid–liquid
extraction using methylene chloride for the analysis of
biotransformation of varying volatility compounds.
Therefore, the main goal of this study was to assess
the effectiveness of headspace solid-phase microex-
traction (HS-SPME) compare to liquid–liquid extrac-
tions using with methylene chloride for control of
microbial biotransformation of ketones of varying vola-
tility. Additionally, Alternaria alternata 18570 as a
potential biocatalyst for bioreduction reaction of car-
bonyl group was also assessed.
Analytical methods
The composition of biotransformation mixtures was
established by GC on Agilent 6890 N GC instrument,
fitted with a flame ionization detector (FID) and a
chiral column Chirasil-Dex CB 25 m ꢀ 0.25 mm ꢀ
0.25 lm. Reference samples of the racemic alcohols
were prepared by reducing the ketones with sodium
borohydride (NaBH4) in methanol. All the experiments
were repeated three times.
The following temperature programmes and helium
carrier gas were used to determine the composition
and enantiomeric excesses of product mixtures, for
acetylcyclohexane (1): injector and detector (FID)
330oC, column temperature: 85–200 ꢁC (rate 5 ꢁC
minꢂ1), 85 ꢁC (hold 5 min), flow 2 ml ꢃ minꢂ1, split vent
180 mL ꢃ minꢂ1; for acetophenone (2): injector and
Materials and methods
Materials
The filamentous fungus strain A. alternata 18570 was
obtained from the collection of the Department of
Forest Pathology, Mycology and Tree Physiology,
University of Agriculture in Krakow. It was cultivated
at 27 oC on Sabouraud agar slants (Biocorp, Poland)
and stored at 4 oC. Biotransformations were con-
ducted in the medium containing 3 g of glucose (The
Industrial and Trading Enterprise “Stanlab” Co. Ltd.,
Lublin, Poland) and 1 g of peptobac (BTL sp. z o.o.,
Warszawa, Poland) in water (100 mL), pH 6.8.
The substrates acetylcyclohexane (1), acetophenone
(2), 2-acetylthiophene (3) and a,a,a-trifluoroacetophe
none (4) were used in this study and they were
selected on the basis of screening tests from other
substrates such as 2-acetylpyridine, 3-acetylpyridine,
o
detector (FID) ꢂ2130 C, column temperature: 90–200ꢂꢁ1C
(rate 6 ꢁC ꢃ min ), 90 ꢁC (hold 9 min), flow 2 ml ꢃ min
,
split vent 180 mL ꢃ minꢂ1; for 2-acetylthiophene (3):
injector 200 ꢁC, detector (FID) 2ꢂ310 ꢁC, column tempera-
ture: 90–200 ꢁC (rate 6 ꢁC min ), 90 ꢁC (hold 5 min),
flow 2 ml minꢂ1
,
split vent 180 mL ꢃ minꢂ1
;
for
a,a,a-trifluoroacetophenone (4): injector and detector
(FID) 230 ꢁC, column temperature: 80–150 ꢁC (rate 3 ꢁC
minꢂ1), 80 ꢁC (hold 3 min), flow 2 mlꢃ minꢂ1, split vent
180 mL minꢂ1. In addition, during the analysis of
HS-SPME was used 1 min desorption of SPME fibre
(Table 1), and in the case of methylene chloride

BIOCATALYSIS AND BIOTRANSFORMATION
3
Table 1. Characteristics of SPME fibres used in the experiment for headspace solid-phase microextraction (HS-SPME).
Supelco SPME fibres
Compound
Name
Matrix active group
Polyacrylate
Diameter (df)
Compatibility
*
3
PA
85 lm (partially crosslinked phase)
For analyte group polar semivolatiles
(MW 80–300)
1, 2
1, 2
2, 3, 4
ꢅ
CAR/PDMS
PDMS/DVB
Carboxen/Polydimethylsiloxane
65 lm (partially crosslinked phase)
65 lm (partially crosslinked phase)
50/30 lm
For analyte group volatiles, amines, and
nitroaromatic compounds (MW 50–300)
For analyte group volatiles, amines, and
nitroaromatic compounds (MW 50–300)
For analyte group flavours (volatiles and
semivolatiles, C3-20) (MW 40–275)
Polydimethylsiloxane/Divinylbenzene
DVB/CAR/PDMS Divinylbenzene/Carboxen/
Polydimethylsiloxane
Acetylcyclohexane (1), acetophenone (2), 2-acetylthiophene (3), a,a,a-trifluoroacetophenone (4).
(CH2Cl2) extraction was used liners for injection port,
split 50:1, and Split Flow 100 mL minꢂ1
Table 2. The retention times of acetylcyclohexane (1), aceto-
phenone (2), 2-acetylthiophene (3) and a,a,a-trifluoroaceto
phenone (4) and their products (alcohols) after bioreduction
.
The commercial SPME device (Supelco) consist of a
1 cm length fused silica fibre of ca. 100 lm diameter
coated on the outer surface with a stationary phase
fixed to a stainless steel plunger and a holder similar
to a microliter syringe, and four types of SPME fibre
coated with different stationary phases (Supelco) were
used for HS-SPME – Table 1. After biotransformation,
the SPME device was installed on Erlenmeyer flasks
(250 mL) and needle with the SPME fibre was inserted
into the stopper. Then, the SPME fibre was exposed to
headspace for 15 min at 27 ꢁC, and Erlenmeyer flasks
was still shaken on a rotary shaker (90 rpm).
obtained by using GC and
a
column Chirasil-Dex CB
25 m ꢀ 0.25 mm ꢀ 0.25 lm.
a
Compound
Products tR (min)
Substrate
Enantiomer
1b
Enantiomer
2c
a
Number
Structure
tR (min)
1
9.42
12.25
12.43
2
6.78
10.80
11.14
In the case of using methylene chloride (CH2Cl2),
the samples were withdrawn (ca. 50 mL) and extracted
three times (50 mL). The extracts were dried over
MgSO4 and concentrated in vacuo. The crude mixture
obtained this way was analysed by GC as described
earlier.
3
4
8.87
2.41
11.92
18.00
12.23
18.41
Screening procedure was similar regarding the con-
ditions for GC, but it used only methylene chloride
(CH2Cl2) procedure for the extraction of the products
mixture after biotransformation. Absolute configurations
of the products were determined by comparison of
their optical rotation values with our previous research
atR – retention time; b(R)-enantiomer for compounds 1, 2 and 3, and (S)-
enantiomer for compound 4
c(S)-enantiomer for compounds 1, 2 and 3, and (R)-enantiomer for com-
pound 4.
Results and discussion
ꢀ
(Ogorek and Jarosz 2013) – shorter reaction times were
SPME is an innovative, solvent-free sample preparation
technique that is fast, relatively inexpensive, versatile,
and compatible with analytical separation systems
inter alia GC (Lord and Pawliszyn 2000). Our research
confirmed this; however, analyte properties, and the
type of fibre coating are one of the most important
determined in (R)-enantiomers of products obtained
from reduction of acetylcyclohexane (1), acetophenone
(2), and 2-acetylthiophene (3), and (S)-enantiomers of
reduction of a,a,a-trifluoroacetophenone (4) (Table 2).
Statistical analysis
ꢀ
aspects of this technique (Gorecki et al. 1999).
Therefore, we used four types of SPME fibre coated
with different stationary phases and liquid–liquid
extractions in our study (Table 1). Generally, we found
significant differences between types of extraction in
the case of the products mixture after biotransform-
ation of acetophenone (2) and 2-acetylthiophene (3)
by A. alternata. Whereas, in other cases, there no of
such differences (Table 3).
The results were analysed by ANOVA, using Statistica
12.0 package. Means were compared using Tukey HSD
(Honest Significant Differences) test at a ꢄ 0.01.
Percentage data, before analysis of variance were
transformed to Bliss (1934) angular degrees by the for-
mula y ¼ arcsin (value%)ꢂ0.5. After transformation, the
variance is approximately constant, allowing analysis
of variance to compare particular components.

ꢀ
R. OGOREK AND B. JAROSZ
4
Table 3. Comparison of composition (in % according to GC) of the products mixture obtained using different extraction methods
(liquid–liquid and HS-SPME), after 24 h biotransformation of acetylcyclohexane (1), 30 h of a,a,a-trifluoroacetophenone (4), and
48 h of acetophenone (2) and 2-acetylthiophene (3) by Alternaria alternata 18570.
Products (alcohols)
Compound
Types of extraction
Substrate
3.3 1.71
(R)-enantiomer
(S)-enantiomer
96.7 2.81
Substrate conversion
1
CH2Cl2
a2
a
0.0 0.01
0.0 0.0
0.0 0.0
0.9 0.11
0.9 0.1
0.0 0.0
0.0 0.0
5.6 0.71
4.7 0.6
5.5 0.7
76.4 1.11
73.3 0.8
a2
a
a2
a
96.7
90.4
95.9
95.9
71.2
89.9
78.5
93.6
81.8
93.0
98.9
98.0
a2
A
CAR/PDMS fibre
PDMS/DVB fibre
CH2Cl2
CAR/PDMS fibre
PDMS/DVB fibre
DVB/CAR/PDMS fibre
CH2Cl2
PA fibre
DVB/CAR/PDMS fibre
CH2Cl2
9.6 1.6
4.1 1.9
4.1 0.21
28.8 0.4
10.1 1.1
21.5 6.5
6.3 1.61
18.2 1.9
7.0 2.1
1.4 0.11
2.0 0.2
90.4 3.4
95.9 3.0
95.0 0.21
70.3 1.5
89.9 1.9
78.5 7.1
88.0 1.01
77.1 1.2
87.5 1.4
22.2 1.41
24.7 0.9
a
a
a
A
2
3
b2
a
a2
a
a2
c
a2
C
b
b
ab
bc
a2
b
ab
bc
a2
b
a
b
b2
a
a2
a
b
a
a
a
4
a2
a
a2
a
a2
a
a2
a
DVB/CAR/PDMS fibre
1
SD (standard deviation); 2For each types of extraction, means followed by the same letter are not statistically different at the a ꢄ 0.05 level according
to Tukey HSD test; others are. Letters indicate the effect of extraction types on composition of individual components of the mixture and sub-
strate conversion.
The fibres (CAR/PDMS, PDMS/DVB) and methylene
chloride extraction as control were used to evaluate
the biotransformation of compound acetylcyclohexane
(1). All types of extraction were at a similar level and
the obtained values were not statistically significant.
However, the results of HS-SPME obtained with PDMS/
DVB fibre were most similar to the control (Table 3).
Additionally, DVB/CAR/PDMS fibre was used for aceto-
phenone (2), but also in this case, the results from
PDMS/DVB fibre were most similar to the control.
However, the values obtained using the two other
fibres were exhibited significant differences as com-
pared to control (Table 3). On the other hand, the
results of products mixture after biotransformation of
substrates 2-acetylthiophene (3) and a,a,a-trifluoro
acetophenone (4) obtained using DVB/CAR/PDMS
fibre were most similar to the control – the values
of these variants were not statistically significant
(Table 3).
(Table 3). However, extraction efficiency of individual
fibres coated with a combination of nonpolar and
polar materials can be different for substrances within
the same chemical group as demonstrated by the
ꢀ
results of this study and other researchers (Gorecki
et al. 1999; Vichi et al. 2003).
In the recent years, so called “green chemistry”
enjoys great interest and the production of various
enantiopure alcohols catalysed by enzymes, microor-
ganisms and plant cells has attracted considerable
attention due to its high efficiency, high enantioselec-
tivity, mild reaction condition, and low environmental
pollution (Ni and Xu 2012; Sheldon 2012). Literature
also report that A. alternata is a good biocatalyst for
bioreduction reaction of ketones to alcohols
ꢀ
(Kurbanoglu et al. 2007; Ogorek and Jarosz 2013). Our
study confirmed these reports. Nevertheless, we
noticed that the biotransformation process was
dependent on the structure of the substrate.
Generally, in case of acetophenone (2), 2-acetylthio-
phene (3) and a,a,a-trifluoroacetophenone (4), both
product enantiomers have been detected, whereas
biotransformation of acetylcyclohexane (1) afforded
only one enantiomer. However, the number of prod-
ucts was dependent mainly on enzyme selectivity and
probably the effect of the type of extraction and
SPME fibre was negligible. Therefore, the ratio of both
enantiomers obtained from a specific substrate was
similar for all extraction methods tested because the
enantiomers behave in a similar way when extracted
on a non-chiral phase. The apparent conversion of the
substrates amounted from 71.2 to 98.8%, and it was
also dependent on enzyme selectivity (Table 3).
However, literature report that the species of filament-
ous fungi as A. alternata may convert the substrates
Literature report that, if all the analytes to be
extracted are polar, a polar fibre coating like polyacry-
late (PA) could be used. However, in the case of
extracting a mixture of compounds with different
polarities (alcohols, aldehydes, ketones etc.) fibres
coated with a combination of nonpolar material like
polydimethylsiloxane (PDMS) and a more polar mater-
ꢀ
ial like divinylbenzene (DVB) could be used (Gorecki
et al. 1999). Our research confirmed this. Therefore, in
the case of HS-SPME, the best way to extract all the
components of the mixture in quantities similar to the
extraction of methylene chloride was the use of fibres
coated with a combination of polar and nonpolar
material. On the other hand, the use of fibre coated
with polar material as polyacrylate (PA) was the cause
of excessive extraction inter alia of a polar alcohols

BIOCATALYSIS AND BIOTRANSFORMATION
5
examined by us at the same time with a higher yield
hand, the best results were obtained for 2-acetylthio-
phene, a,a,a-trifluoroacetophenone and their deriva-
tives using DVB/CAR/PDMS fibre. In addition, our
study showed that whole-cell of A. alternata is a good
biocatalyst for bioreduction reaction of ketones to
alcohols with a high enantiospecificity and substrate
conversion. Moreover, carbonyl reductases secreted by
the A. alternata reduced ketones used in this study in
accordance with Prelog’s rule and hence with high
predominance of (S)-isomers. The exception was the
reduction of a,a,a-trifluoroacetophenone, with pre-
dominance of (R)-isomers.
ꢀ
than in our study (Ogorek and Jarosz 2013). This is
probably related to the age of the fungal culture.
Because very often microorganisms, especially micro-
scopic fungi, lose their phenotypic properties as well
as enzymatic abilities along with the number of pas-
ꢀ
sages and life time in vitro (Ogorek et al. 2016).
Stereoselective and enantiospecific reduction of
ketones to form chiral alcohols in one of the most
useful reactions in organic chemistry, and it is cata-
lysed by carbonyl reductases. Those enzymes belong
to the oxidoreductase family that often need cofactors
such as NAD(H) or NADP(H) to be functionally active
(Gao et al. 2013). However, whole cells of microorgan-
isms contain all the necessary cofactors and metabolic
pathways for their regeneration. Carbonyl reductases
secreted by the fungus used in our research reduced
simple aliphatic and aromatic ketones according to
Prelog’s rule, which means that the stereochemistry
can be determined by looking at the size of the two
groups. This rule states that the enzyme has a large
and a small pocket that makes up the active site, in
which the substrate binds, and controls the stereo-
chemistry of the product based on the geometry of
the substrate. Consequently, the product as (S)-alco-
hols is obtained from simple aliphatic and aromatic
ketones (Prelog 1964). However, this should not
always be generalized and caution should be exer-
cised in particular, when Prelog’s rule is applied to
whole cells due to the presence of multiple enzymes
inside the cells (Csuk and Glaenzer 1991). This is prob-
ably one of the reasons for obtaining, in some cases, a
product mixture rather than a single product.
Acknowledgements
We thank prof. Tadeusz Kowalski (Department of Forest
Pathology, Mycology and Tree Physiology, University of
Agriculture in Krakow) for his help in the study.
Disclosure statement
No potential conflict of interest was reported by the authors.
References
Arthur CL, Pawliszyn J. 1990. Solid phase microextraction
with thermal desorption using fused silica optical fibers.
Anal Chem. 62(19):2145–2148.
Bliss CT. 1934. The method of probits. Science. 79(2037):
38–39.
~
Bocato MZ, Simoes RA, Calixto LA, de Gaitani CM, Pupo MT,
de Oliveira ARM. 2012. Solid phase microextraction and
LC–MS/MS for the determination of paliperidone after
stereoselective fungal biotransformation of risperidone.
Anal Chim Acta. 742:80–89.
ꢀ
Bonadio F, Margot P, Delemont O, Esseiva P. 2008.
Headspace solid-phase microextraction (HS-SPME) and
liquid–liquid extraction (LLE): comparison of the perform-
ance in classification of ecstasy tablets (Part 2). Forensic
Sci Int. 182(1–3):52–56.
Conclusions
The proposed method based on SPME which is a fast
and versatile technique, using no cosolvents and com-
patible with GC was successfully applied to evaluate
the results of microbial biotransformation of ketones
of varying volatility. The best way to extract all the
components of the mixture (alcohols, aldehydes) in
quantities similar to those obtained by extracting with
methylene chloride was the use of fibres coated with
a combination of a nonpolar and a polar material.
However, the enantiospecificity was dependent mainly
on enzyme selectivity and probably the effect of the
used the type of extraction and the type of SPME fibre
was negligible. Nevertheless, the commercial PDMS/
DVB fibre was most suitable for the extraction of the
product mixture obtained after biotransformation of
acetylcyclohexane and acetophenone. On the other
Chen IS, Shen CSJ, Sheppard AJ. 1981. Comparison of methy-
lene chloride and chloroform for the extraction of fats
from food products. J Am Oil Chem Soc. 58(5):599–601.
Csuk R, Glaenzer BI. 1991. Bakers yeast mediated transforma-
tions in organic chemistry. Chem Rev. 91(1):49–97.
Gao C, Zhang LJ, Xie YJ, Hu CH, Zhang Y, Li L, Wang Y, Ma
C, Xu P. 2013. Production of (3S)-acetoin from diacetyl by
using stereoselective NADPH-dependent carbonyl reduc-
tase and glucose dehydrogenase. Bioresource Technol.
137:111–115.
Ghiasvand AR, Nasseri M, Farsizaeh S, Meshkatalsadat MH,
Sadeghi-Sarabi R, Shadabi S, Borzoei M. 2011. Chemical
characterization of cultivated Tagetes minuta L. by use of
ultrasound-assisted head space SPME and GC–MS.
Chromatographia. 73(9–10):1031–1035.
ꢀ
Gorecki T, Pawliszyn J. 1995. Sample introduction
approaches for solid phase microextraction/rapid GC. Anal
Chem. 67:3265–3274.

ꢀ
R. OGOREK AND B. JAROSZ
6
ꢀ
Gorecki T, Yu X, Pawliszyn J. 1999. Theory of analyte extrac-
tion by selected porous polymer SPME fibres. Analyst.
124(5):643–649.
Ouyang G, Pawliszyn J. 2006. SPME in environmental ana-
lysis. Anal Bioanal Chem. 386(4):1059–1073.
Parshikov IA, Freeman JP, Lay JO, Beger RD, Williams AJ,
Sutherland JB. 2000. Microbiological transformation of
enrofloxacin by the fungus Mucor ramannianus. Appl
Environ Microbiol. 66(6):2664–2667.
Prelog V. 1964. Specification of the stereospecificity of some
oxidoreductases by diamond lattice sections. Pure Appl
Chem. 9(1):119–130.
Risticevic S, Niri VH, Vuckovic D, Pawliszyn J. 2009. Recent
developments in solid-phase microextraction. Anal Bioanal
Chem. 393(3):781–795.
Gura S, Joshi M, Almirall JR. 2010. Solid-phase microextrac-
tion (SPME) calibration using inkjet microdrop printing for
direct loading of known analyte mass on to SPME fibers.
Anal Bioanal Chem. 398(2):1049–1060.
Hall AH, Rumack BH. 1990. Methylene chloride exposure in
furniture-stripping shops: ventilation and respirator practi-
ces. J Occup Med. 32(1):33–37.
ꢀ
Kotowska U, Bienczyk K. 2013. Use of direct immersion solid-
phase microextraction on polyacrylate and polydimethylsi-
loxane stationary phases for simultaneous determination
of the neutral and basic pharmaceuticals in wastewater.
Cent Eur J Chem. 11(10):1634–1643.
Sheldon RA. 2012. Fundamentals of green chemistry: effi-
ciency in reaction design. Chem Soc Rev. 41(4):1437–1451.
ꢀ
Skrobiszewski A, Ogorek R, Pla˛skowska E, Gładkowski W.
Kurbanoglu EB, Zilbeyaz K, Kurbanoglu NI, Taskin M. 2007.
Highly enantioselective reduction of acetophenone by
locally isolated Alternaria alternata using ram horn pep-
tone. Tetrahedron: Asymmetr. 18(13):1529–1532.
Leikin JB, Kaufman D, Lipscomb JW, Burda AM, Hryhorczuk
DO. 1990. Methylene chloride: report of five exposures
and two deaths. Am J Emerg Med. 8(6):534–537.
Lord H, Pawliszyn J. 2000. Evolution of solid-phase microex-
traction technology. J Chromatogr A. 885(1–2):153–193.
Mahmud M, Kales SN. 1999. Methylene chloride poisoning in
a cabinet worker. Environ Health Perspect. 107(9):769–772.
Ni Y, Xu JH. 2012. Biocatalytic ketone reduction: a green and
efficient access to enantiopure alcohols. Biotechnol Adv.
30(6):1279–1288.
2013. Pleurotus ostreatus as a source of enoate reductase.
Biocatal Agric Biotechnol. 2(1):26–31.
Soden KJ. 1993. An evaluation of chronic methylene chloride
exposure. J Occup Med. 35(3):282–286.
Thompson-Witrick KA, Rouseff RL, Cadawallader KR, Duncan
SE, Eigel WN, Tanko JM, O’Keefe SF. 2015. Comparison of
two extraction techniques, solid-phase microextraction
versus continuous liquid-liquid extraction/solvent-assisted
flavor evaporation, for the analysis of flavor compounds
in gueuze lambic beer. J Food Sci. 80(3):C571–C576.
Vichi S, Castellote AI, Pizzale L, Conte LS, Buxaderas S,
Lopez-Tamames E. 2003. Analysis of virgin olive oil volatile
compounds by headspace solid-phase microextraction
coupled to gas chromatography with mass spectrometric
and flame ionization detection. J Chromatogr A. 983(1–2):
19–33.
ꢀ
Ogorek R, Dyla˛g M, Kozak B. 2016. Dark stains on rock surfa-
ces in Driny Cave (Little Carpathian Mountains, Slovakia).
Extremophiles. 20(5):641–652.
Zhuo-Min Z, Wen-Wei W, Gong-Ke L. 2006. HSSPME-GC/MS
study of the aroma volatiles of Allium species and chemo-
metric interpretation for the aroma characteristics. J Plant
Sci. 1(4):315–323.
ꢀ
ꢀ
Ogorek R, Jarosz B. 2013. Asymetryczna redukcja ketonow w
kulturze Alternaria alternata (Asymmetric reduction of
ketones in the culture of Alternaria alternate. Przemysł
Chem. 92(9):1747–1749. [in Polish].