tecnicas analiticas

1. In Vivo Solid-Phase Microextraction with in Vitro Calibration:
Determination of Off-Flavor Components in Live Fish
Ziwei Bai,† Alexandre Pilote,‡ Pallab Kumer Sarker,‡ Grant Vandenberg,‡ and Janusz Pawliszyn*,†
†Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada
‡Department des Sciences Animales, Université Laval, Pavillon Paul-Comtois, Québec, Québec G1K 0A6, Canada
ABSTRACT: The presence of off-flavor compounds in fish
represents a significant economic problem encountered in
aquaculture production. The off-flavor compounds are due to
the absorption of substances produced by a range of
microorganisms. Currently, a number of strategies have been
used to prevent or limit the growth of these microorganisms.
Therefore, it is important to evaluate the effectiveness of
strategies via monitoring the concentrations of off-flavor
compounds in the recirculating aquaculture system. In vivo
solid-phase microextraction (SPME), a rapid and simple
sample preparation method, will allow monitoring the
concentration of off-flavor compounds in live fish. In this
research, geosmin and 2-methylisoborneol (2-MIB) produced
by cyanobacteria and actinomycetes, which are the major sources for “earthy” and “muddy” flavors in fish, were selected as
representatives. In order to accurately quantify these compounds in fish muscle, two kinetic calibration methods, on-fiber
standardization and measurement using predetermined sampling rate, were used as quantification methods, which were both
validated by traditional methods. The detection limit of in vivo SPME in fish muscle was 0.12 ng/g for geosmin and 0.21 ng/g for
2-MIB, which are both below the human sensory thresholds.
Recirculating aquaculture systems (RAS) are fish culture
systems in which water is largely reused after undergoing
treatment.1 Currently, commercial RAS production systems
typically recirculate over 99% of their water usage, thus
significantly reducing water consumption.2 In addition, RAS
improves opportunities for waste management and nutrient
recycling,3 thus providing better hygiene and disease manage-ment,
4 as well as biological pollution control.5 However, one
major disadvantage of producing fish in RAS is the presence of
off-flavor compounds. Among those flavors, the “earthy” and
“muddy” odors constitute more than 80% of the off-flavor
problems found in farm-raised fish.6 Such flavors come from
the absorption by fish of substances including geosmin and 2-
methylisoborneol (2-MIB), which are produced by a broad
group of microorganisms in water.7−9 The presence of
undesirable off-flavors in products raised in RAS may cause a
major reduction in the consumption of the products, or make
them unsuitable for sale. The presence of off-flavors therefore
represents a major hurdle for the wide-scale adoption of RAS
technologies as a production technique.
Currently in RAS research, there is focus on developing
strategies to prevent or limit the development of micro-organisms
that produce the off-flavor compounds found in fish.
Therefore, in order to evaluate the effectiveness of these
strategies on microorganisms, a method needs to be
implemented that can monitor the level of target compounds
by repeatedly sampling the individual living fish in RAS over
time. Recently, solid-phase microextraction (SPME) has been
widely used as a rapid, inexpensive, and solvent-free sample
preparation method, which combines sampling, analyte
isolation, and enrichment into one step.10
Successful use of in vivo SPME depends on the selection of
an appropriate calibration method. The on-fiber standardization
method is one of the most widely used calibration methods for
SPME in vivo sampling,11−15 which uses the symmetry between
the desorption of deuterated standards from the extraction
phase to the sample and the absorption of analytes from sample
to the extraction phase.16 Recently, another calibration method
using predetermined sampling rates of the analytes has been
reported by Ouyang et al.17 It assumes that the rate of mass
transfer or sampling rate remains constant throughout the
duration of sampling within the linear range. The relationship
between the concentration of target analytes in the sample
matrixes (C0) and the extracted amount of analytes at time t(n)
can be expressed with eq 1:
C0 = n/Rst (1)
where Rs is the sampling rate for the target analyte and t is the
sampling time. Use of eq 1 assumes that the intersample matrix
differences in semisolid tissues (such as fish muscle) are slight
between individuals of the same species. Consequently, the
Received: November 15, 2012
Accepted: January 18, 2013
Published: January 18, 2013
Article
pubs.acs.org/ac
© 2013 American Chemical Society 2328 dx.doi.org/10.1021/ac3033245 | Anal. Chem. 2013, 85, 2328−2332

2. Analytical Chemistry Article
sampling rate of the SPME fiber can be predetermined under
laboratory conditions and directly used for real sample
analysis.18 With this method, no distribution coefficient (K)
determination and deuterated standard uploading prior to
sampling are needed any longer.
The objective of this study was to develop an effective and
simple method to determine geosmin and 2-MIB in fish using
in vivo sampling technique. In order to accomplish this goal,
two kinetic calibration methods were investigated and verified:
(1) on-fiber standardization and (2) measurement using
predetermined sampling rates of analytes. Finally, the
developed methods were applied for on-site determination of
off-flavors. These results were compared to those obtained from
the traditional methods of analysis using lethal sampling and
microwave distillation prior to SPME.
■ EXPERIMENTAL SECTION
Chemicals and Materials. Geosmin and 2-MIB were
purchased from Wako Chemicals U.S.A., Inc. Deuterated MIB
(MIB-d3) was obtained from CDN Isotopes Inc. (Pointe-Claire,
Quebec, Canada), and deuterated geosmin (geosmin-d3) was
purchased from Sigma-Aldrich (Oakville, Ontario, Canada),
with the purity of 99%. All chemicals purchased were of the
highest possible purity and were used without further
purification. Chloroform for fat content measurement was
purchased from EMD Chemicals Inc. (Damstadt, Germany).
HPLC (high-performance liquid chromatography) grade
methanol was purchased from Fisher Scientific (Unionville,
Ontario, Canada). HPLC grade acetone was obtained from
Caledon Laboratories Ltd. (Georgetown, Ontario, Canada).
Pump oil was purchased from Varian Vacuum Technologies
(Lexington, MA). Nanopure water was obtained using the
Barnstead Nanopure water system. The 1 cm metal core
commercial PDMS fibers were obtained from Supelco
(Bellefonte, PA). Taint-free rainbow trout fillets were
purchased from Sobeys Inc. (Ontario, Canada). Rainbow
trout fillets contaminated with geosmin and 2-MIB were
obtained from Alma Aquaculture Research Station (University
of Guelph, Ontario, Canada). All fish tissue experiments were
performed in accordance with protocols approved by our
institutional Animal Care Committee (AUP no. A-12-01) in the
Chemistry Lab Facility at the University of Waterloo. All in vivo
fish experimental procedures were approved by the Animal
Care Committee at University of Guelph (AUP no. 12R066).
Symmetry of Geosmin and 2-MIB on SPME under in
Vitro Condition for the On-Fiber Standardization
Calibration Method. Desorption time profiles of deuterated
standards and absorption time profiles of analytes were
performed simultaneously to verify the symmetry. Deuterated
standards preloaded onto the fiber prior to the extraction were
prepared by using headspace sampling from deuterated
standards pump oil solution under 25 °C. Both homogenized
and nonhomogenized fish tissue samples were utilized.
Contaminated homogenized fish muscle was obtained by
spiking specific amounts of geosmin and 2-MIB methanol
solution into taint-free homogenized fish tissue. Nonhomogen-ized
fish tissue with off-flavors was prepared by cutting each
sample in 4 g size. Sampling was conducted by directly inserting
the SPME fibers in the homogenized and nonhomogenized
fish. All the experiments were performed under 8.5 °C, the
same temperature as in vivo sampling. The absorption time
profile was obtained by extracting from geosmin and 2-MIB
contaminated samples for different sampling times. Meanwhile,
the desorption time profile was drawn by preloading same
amounts of standards and desorbing in the samples for different
times.
In Vivo Sampling System Setup. For this study, in vivo
sampling experiments were performed at Alma Aquaculture
Research Station, University of Guelph. The source of water in
the station was from the underground wells nearby and was
proved to contain no detectable 2-MIB or geosmin by using a
headspace SPME analysis technique.19 The water temperature
was kept constant at 8.5 °C. Rainbow trout (Oncorhynchus
mykiss) were of marketable table size at 900 ± 154 g (n = 48).
Before experiments, fish were acclimated with the diet in a flow-through
system 1 week prior to experimentation. The system
was located indoors and consisted of 16 (729 L) tanks. Each
tank held four fish, three for in vivo sampling and one for lethal
sampling. Before exposure to tainted water, the fish were
deprived of food for 48 h. This was in order to minimize
variations in metabolic rates and ventilation rates between
individual fish with different feeding histories.20 During the
exposure time, the fish were deprived of food as well. The
uptake of off-flavor compounds from contaminated water by
fish as well as the in vivo sampling experiments were both
performed in a static, albeit oxygen offered system (no water
flow rate). The water inside each static system (round tank)
was transferred from flow-through system, and the volume was
90 L.
The procedure for operating in vivo sampling was as follows:
First, fibers were prepared by uploading deuterated geosmin
standard from the headspace of pump oil solution. Before each
SPME sampling, fish were anesthetized in a 80 μg/mL MS-222
water solution for the duration of approximately 1 min. After
that, an 18 gauge needle was used to pierce the fish skin, where
it is below the anterior point of the dorsal fin (as shown in
Figure 1), and a 21 gauge long stainless steel wire was
Figure 1. SPME fiber inserting position for in vivo sampling in fish
muscle.
introduced into the same perforation and penetrated to the
same length as the SPME fiber, because the needle was not as
long as the SPME fiber. Then the SPME fiber was inserted, and
the hub was pushed to the end. Following fiber placement, fish
were placed back in the static tank. After 30 min of SPME
extraction, the fish was anesthetized again. At last, the fiber was
removed, gently washed with Nanopure water, wiped with a
Kimwipe, sealed with a Teflon cap, wrapped with aluminum
foil, and stored in the dry ice until gas chromatography mass
spectrometry (GC/MS) analysis.
2329 dx.doi.org/10.1021/ac3033245 | Anal. Chem. 2013, 85, 2328−2332

3. Analytical Chemistry Article
Table 1. Time Constant Results and Conclusions for 2-MIB and Geosmin under Both in Vitro and in Vivo Conditionsa
2-MIB geosmin
desorption a (min−1) (R2) absorption a (min−1) (R2) desorption a (min−1) (R2) absorption a (min−1) (R2)
homogenized fish tissue 0.012 ± 0.002 (0.946) 0.011 ± 0.001 (0.984) 0.008 ± 0.001 (0.987) 0.008 ± 0.001 (0.984)
nonhomogenized fish tissue 0.012 ± 0.003 (0.844) 0.012 ± 0.006 (0.780) 0.007 ± 0.001 (0.884) 0.010 ± 0.004 (0.837)
live fish 0.010 ± 0.003 (0.852) 0.013 ± 0.006 (0.790) 0.012 ± 0.003 (0.747) 0.011 ± 0.007 (0.839)
aCorrelation coefficients are given in parentheses.
Instrumentation. A Varian 3800 GC coupled with a Varian
4000 electron ionization ion-trap mass spectrometer (GC/EI-IT-
MS) (Varian, Inc. Walnut Creek, CA, U.S.A.) was used for
analysis. The GC was equipped with a capillary column (RTX-
5, 30 m × 0.25 mm i.d., 0.25 μm film thickness) (Restek
Corporation, Bellefonte, PA, U.S.A.). The oven temperature
program was 60 °C (0.5 min) increasing at 40 °C/min to 110
°C, increasing at 1 °C/min to 117 °C, increasing at 5 °C/min
to 156 °C, increasing at 40 °C/min to 250 °C (10 min). The
split/splitless injector of the GC was equipped with a Merlin
microseal injector adapter (Merlin Instrument Company, Half
Moon Bay, CA, U.S.A.) for metal fiber desorption. For both
liquid injection and SPME, splitless mode was used, and split
was opened after 5 min of desorption. The temperature of the
transfer line was 280 and 240 °C for the ion trap. The column
flow rate of the helium carrier gas was 1 mL/min. The 1079
injector was equipped with a programmed temperature
vaporizer. The temperature of the injector was 250 °C. When
using selected ion storage (SIS) scan mode, m/z 112, m/z 150,
m/z 115, and m/z 138 were selected for quantification of
geosmin, 2-MIB, geosmin-d3, and MIB-d3, respectively. As
qualifier ions, m/z 126 and 182 were for geosmin, m/z 168 was
to represent 2-MIB, m/z 129 and 185 were for geosmin-d3, and
m/z 171 were selected for MIB-d3. When performing the in
vivo sampling analysis, 2-MIB required using MS/MS mode,
and geosmin kept using SIS mode. Under MS/MS mode, the
parent ion of 2-MIB m/z 95 yielded daughter ions m/z 67 and
m/z 91. The excitation amplitude was set as 1.0 V applied with
resonant waveform. The emission current and maximum
ionization time were set as 80 μA and 65 000 μs, respectively.
■ RESULTS AND DISCUSSION
Preliminary Experiments. Selected ion storage mode was
utilized for symmetry determination. To determine geosmin,
geosmin-d3 was used as an internal standard. In this case, the EI
spectrum of the labeled compound produced a base peak at m/
z 115, which contained three deuterium atoms, in the
agreement with the fragmentation proposed by Lloyd and
Grimm.21 The quantification of geosmin and geosmin-d3 was
achieved using the ion pair at m/z 112 and 115, and
qualification was established by using the ions m/z 126 and
182 for geosmin and m/z 129 and 185 for geosmin-d3.
However, for 2-MIB and MIB-d3, the base peak ion at m/z
95 for both was not a deuterated fragment. Finally, only m/z
150 and 138 could be decided to represent 2-MIB and MIB-d3.
However, in that case, the limit of detection for 2-MIB was as
high as 10 ng/g in fish tissue, which was more than 10 times
higher than the human sensory threshold.22 Consequently, the
on-fiber standardization calibration method is not suitable for
determination of low contamination of 2-MIB in fish.
Therefore, measurement using the predetermined sampling
rates method, which does not require the use of the internal
standard, was placed.
Fouling issues due to the adsorption of macromolecular
compounds on the SPME coating surface, such as lipids and
protein,23 was found after the fiber was desorbed in the GC in
the form of 2-MIB and geosmin peak intensity enhancement.
To address this, following the desorption, the fiber was gently
wiped with an acetone-soaked Kimwipes and rinsed with large
amounts of water, followed by conditioning in the GC injector
for 10 min at 250 °C. As a result, the repeatability of the fiber
was kept acceptable.
Symmetry of Desorption and Absorption Verification
under in Vivo and in Vitro Conditions. The kinetics of
absorption and desorption was performed in homogenized fish
tissue, nonhomogenized fish tissue, and live fish. Time profiles
were used for this verification. Because this calibration method
could not be used to determine a low concentration of 2-MIB
in fish tissue, the concentrations of 2-MIB in all these three
systems were high. The details about the concentration for both
analytes were as follows: homogenized fish tissue 0.5 μg/g for
2-MIB and 0.025 μg/g for geosmin; nonhomogenized fish
tissue and live fish were around 0.3 μg/g for both analytes in
these two systems. The time constants of absorption and
desorption were very similar for both 2-MIB and geosmin in all
three systems, which means the symmetry was kept all through
the desorption and absorption processes (as shown in Table 1).
Different Concentrations of Analytes in Homogen-ized
Fish Tissue. A various range of concentrations (0.5, 2.5,
5.0, 12.5, 25.0 ng/g) of 2-MIB and geosmin in homogenized
fish tissue were prepared and followed by a 30 min extraction.
Equation 2 describes the entire absorption kinetics of the
analyte from the sample matrixes to SPME liquid, in which n is
the amount of extracted analyte at time t, ne is the amount of
analyte extracted at equilibrium, and a is a absorption time
constant.24 According to eq 2, the extraction amount should be
proportional to the initial concentration, if all the parameters
keep constant. The linearity displayed in Figure 2 indicates that
the extraction time constant for both analytes remained
consistent in this concentration range. In addition, the value
of the extraction time constant was obtained in Table 1. This
discovery was important for both calibration methods. For the
on-fiber standardization calibration method, it needs the
Figure 2. Different concentrations of analytes in homogenized fish
tissue.
2330 dx.doi.org/10.1021/ac3033245 | Anal. Chem. 2013, 85, 2328−2332

4. Analytical Chemistry Article
symmetry of desorption and absorption during the sampling
time. For the calibration method using a predetermined
sampling rate, it requires the constant sampling rate.
= − − = − −
fs f s
fs +
s
n at n at
K VV
K V
[1 exp( )] e [1 exp( )] C
0
(2)
Determination of Sampling Rate for Calibration Using
a Predetermined Rate. Different concentrations (0.5, 2.5,
5.0, 12.5, 25.0 ng/g) of 2-MIB and geosmin in homogenized
fish tissue were prepared to obtain the extraction amount n
under 30 min of sampling time, and all the other conditions
were performed the same as in vivo sampling SPME. Plotting
C0t versus n, the slope obtained was Rs (as shown in Figure 3).
As a result, the Rs value was determined to be 1.6 × 10−4 g/min
for 2-MIB and 7.8 × 10−5 g/min for geosmin.
Validation of in Vivo SPME Sampling Using Micro-wave
Distillation SPME (MD-SPME). Nine fish were exposed
to a low concentration of geosmin and 2-MIB (0.05 ng/mL) in
water. Preloading was performed by headspace extracting from
50 μg/g geosmin-d3 pump oil solution with agitation, and the
extraction time was 10 s under 25 °C. The on-fiber
standardization (OFS) calibration method was applied for the
quantification of geosmin, and the predetermined sampling rate
(PDSR) approach was used to determine both 2-MIB and
geosmin. Fat content of rainbow trout was assessed according
to Zhang et al.’s method.25 According to the results, there is no
obvious relationship between mass, fat, and concentration of
analytes. Fish from different tanks do not exhibit a significant
difference. To confirm the accuracy of results, a traditional
method, microwave distillation SPME, was used for validation.
Microwave distillation and SPME headspace extraction
procedures were executed in accordance with Lloyd and
Grimm’s publication.21 The same instrument parameters were
used as in vivo sampling analysis of fish. Table 2 displays the
results of off-flavor compounds concentrations of nine low-contaminated
fish using two sample preparation methods. The
Student t test was applied to confirm that the on-fiber
standardization calibration method and the measurement using
a predetermined sampling rate calibration method or in vivo
SPME and MD-SPME yield the same results, which validates
the use of the in vivo sampling SPME technique with both
calibration methods.
■ CONCLUSION AND FUTURE DIRECTIONS
An in vivo SPME sampling technique to measure the
concentration of off-flavor compounds in fish was developed.
When compared with traditional methods, it has the advantages
of time efficiency, simplicity of procedure, and most important,
the capability of monitoring target compound concentration
changes when sampling from the same individual fish. Kinetic
calibration methods were used for quantification. The on-fiber
standardization method was used to measure geosmin, while
predetermined kinetic rates were employed to determine the
concentration of both 2-MIB and geosmin in live fish. The
symmetry character of desorption of internal standards from
fiber and absorption of analytes onto fiber for these two
compounds was confirmed.
The future application of this method could assist in real-time,
nonlethal monitoring of key sensory qualities and drug/
pesticide or other contaminants residues of fish and other food
products prior to harvesting. To facilitate the on-site analysis, a
portable GC or other instrumentation could be utilized.
■ AUTHOR INFORMATION
Corresponding Author
*Phone: +1-519-888-4641. Fax: +1-519-746-0435. E-mail:
janusz@uwaterloo.ca.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
The authors thank the Natural Sciences and Engineering
Research Council (NSERC) of Canada for financial support.
The authors acknowledge Alma Research Station of University
of Guelph for their support in providing the in vivo sampling
equipment.
■ REFERENCES
(1) Rosenthal, H.; Castell, J. D.; Chiba, K.; Forster, J. R. M.; Hilge,
V.; Hogendoorn, H.; Mayo, R. D.; Muir, J. F.; Murray, K. R.; Petit, J.;
Wedemeyer, G. A.; Wheaton, F.; Wickins, J. EIFAC 1986, 100.
(2) Verdegem, M. C. J.; Bosma, R. H.; Verreth, J. A. J. Int. J. Water
Resour. Dev. 2006, 22, 101−113.
(3) Piedrahita, R. H. Aquaculture 2003, 226, 35−44.
(4) Summerfelt, S. T.; Sharrer, M. J.; Tsukuda, S. M.; Gearheart, M.
Aquaculture 2009, 40, 17−27.
Figure 3. Determination the Rs of 2-MIB and geosmin in
homogenized fish tissue.
Table 2. Results Comparison between MD-SPME and in
Vivo SPME
2-
MIB
(ng/
g)
geosmin
(ng/g)
fish
no.
mass
(g) sex
fat
content
(%) PDSR
MD-SPME
(ng/g) OFS PDSR
MD-SPME
(ng/g)
1 1013.0 F 3.4 3.4 3.0 2.6 2.6 3.0
2 883.3 M 3.0 3.9 3.9 3.0 3.3 3.7
3 855.4 M 2.7 2.8 3.6 2.1 2.2 2.9
4 1003.2 M 3.7 2.6 4.4 2.0 1.6 3.0
5 894.1 M 1.6 3.3 3.3 3.4 5.0 4.6
6 985.6 M 2.1 3.4 4.6 2.2 2.6 3.7
7 772.6 M 2.9 4.9 3.7 6.1 5.9 5.9
8 958.3 M 2.7 3.8 5.4 3.0 3.9 5.1
9 896.0 M 3.7 4.5 4.4 5.2 5.2 5.0
2331 dx.doi.org/10.1021/ac3033245 | Anal. Chem. 2013, 85, 2328−2332

5. Analytical Chemistry Article
(5) Zohar, Y.; Tal, Y.; Schreier, H. J.; Steven, C.; Stubblefield, J.;
Place, A. Urban Aquacult. 2005, 159−171.
(6) Grimm, C. C.; Lloyd, S. W.; Batista, R.; Zimba, P. V. J.
Chromatogr. Sci. 2000, 38, 289−296.
(7) Gerbek, N. N. J. Antibiot. 1969, 22, 508−509.
(8) Rosen, A. A.; Mashni, C. I.; Safferman, R. S. Water Treat. Exam.
1970, 19, 106−119.
(9) Yurkowski, M.; Tabachek, J. L. J. Fish. Res. Board Can. 1976, 33,
25−35.
(10) Arthur, C. L.; Pawliszyn, J. Anal. Chem. 1990, 62, 2145−2148.
(11) Zhou, S.; Zhao, W.; Pawliszyn, J. Anal. Chem. 2008, 80, 481−
490.
(12) Togunde, O. P.; Oakes, K.; Servos, M.; Pawliszyn, J. Anal. Chim.
Acta 2012, 742, 2−9.
(13) Zhang, X.; Oakes, K. D.; Hoque, M. E.; Luong, D.; Metcalfe, C.
D.; Pawliszyn, J.; Servos, M. R. Anal. Chem. 2011, 83, 3365−3370.
(14) Zhou, S.; Zhang, X.; Ouyang, G.; Es-haghi, A.; Pawliszyn, J.
Anal. Chem. 2007, 79, 1221−1230.
(15) Zhou, S.; Oakes, K. D.; Servos, M. R.; Pawliszyn, J. Environ. Sci.
Technol. 2008, 42, 6073−6079.
(16) Chen, Y.; Pawliszyn, J. Anal. Chem. 2004, 76, 5807−5815.
(17) Ouyang, G.; Vuckovic, D.; Pawliszyn, J. Chem. Rev. 2011, 111,
2784−2814.
(18) Ouyang, G.; Oakes, K. D.; Bragg, L.; Wang, S.; Cui, S.; Servos,
M. R.; Dixon, D. G.; Pawliszyn, J. Environ. Sci. Technol. 2010, 45,
7792−7798.
(19) Lloyd, S. W.; Lea, J. M.; Zimba, P. V.; Grimm, C. C. Water Res.
1998, 32, 2140−2146.
(20) Robertsona, R. F.; Jaunceyb, K.; Beveridgec, M. C. M.; Lawton,
L. A. Aquaculture 2005, 245, 89−99.
(21) Lloyd, S.W.; Grimm, C. C. J. Agric. Food Chem. 1999, 47, 164−
169.
(22) Robertson, R. F.; Jauncey, K.; Beveridge, M. C. M.; Lawton, L.
A. Aquaculture 2005, 245, 89−99.
(23) Pawliszyn, J. Anal. Chem. 2003, 75, 2543−2558.
(24) Pawliszyn, J. Handbook of Solid Phase Microextraction; Chemical
Industry Press: Beijing, China, 2009.
(25) Zhang, X.; Oakes, K. D.; Cui, S.; Bragg, L.; Servos, M. R.;
Pawliszyn, J. Environ. Sci. Technol. 2010, 44, 3417−3422.
2332 dx.doi.org/10.1021/ac3033245 | Anal. Chem. 2013, 85, 2328−2332