3.3 Liquid Chromatography
The biggest advantage of LC is the ability to analyze metabolites without the need to
derivatize them. Unlike GC, there is no need for additional extract cleanup before
instrumental analysis. As in the case of gas chromatography, only highly specific and
sensitive methods, i.e., using mass spectrometry, are useful in biomonitoring studies.
The sample preparation process is simplified, but it comes at a price. LC-MS
methods are susceptible to ion suppression phenomenon which can strongly affect
both sensitivity and repeatability [14, 29].
Only two published methods used HPLC with spectrophotometric detection for
the determination of synthetic pyrethroid metabolites. Smith et al. [27] developed a
HPLC-UV method for the determination of 3PBA and MPA – a metabolite of
bifenthrin in the urine of people professionally exposed to this insecticide. Bartosz
et al. [12] in turn developed HF-LPME-HPLC-DAD method for determination of
3PBA and 4OH3PBA in rat and human urine. Both methods, due to high LOD and
LOQ values, are not suitable for the determination of metabolites in the urine of
non-occupationally exposed subjects.
Separation of analytes is carried out using HPLC, UPLC, and UHPLC coupled
with mass spectrometers with various types of analyzers: triple quadrupole ESI
[14, 16, 21, 29, 31, 34], turbo ion spray (TIS) [30], Q-TOF (ESI) [32, 35, 36], and
high-resolution Orbitrap [19, 37].
Sample preparation for LC-MS analysis included offline solid-phase extraction
[16, 21, 29–32, 34–36], liquid-liquid extraction [14], online SPE [37], and the
QuEChERS [19].
The popularity of the QuEChERS methodology stems from its unique simplicity
and applicability to almost any type of matrix. Therefore, an attempt was made to
apply this methodology to the preparation of a biological sample in order to quantify
the concentration of pesticide metabolites in human urine.
5 mL of urine was hydrolyzed enzymatically (1 mL of 0.2 M acetic buffer and
10 μL of β-glucuronidase aryl sulfatase) and then subjected to simplified
QuEChERS procedure by addition of 10 mL of acetonitrile and QuEChERS salt
packet. Acetonitrile layer was then evaporated at 37
C under a stream of nitrogen
and reconstituted in 200 μL of methanol/water (10:90, v:v) containing 0.1% of acetic
acid. Extract was analyzed with the use of UHPLC-HRMS system. Five pyrethroid
metabolites were monitored: cis-DCCA, trans-DCCA, DBCA, 3PBA, and 4F3PBA.
Additionally, Plackett-Burman design was used to optimize the parameters affecting
the analytical response [19]. Unfortunately, LOQs were in the range of 2–10 ng/mL.
López-García et al. [37] developed a method for simultaneous quantification
of selected organophosphate and pyrethroid metabolites in human urine and compared three independent sample preparation protocols including offline SPE,
TurboFlow™, and online SPE. For TurboFlow™ and online SPE protocols, raw
urine sample (without hydrolysis) was filtered through a 0.2 μm nylon filter, and
0.5 mL was subjected to online extraction. The best peak shapes and recoveries
were obtained with TurboFlow™ methodology. This technique was the only one
enabling detection of cis-/trans-DCCA, since no signal was produced when offline
Analytical Methods for Determination Urinary Metabolites of Synthetic. . .
57
The biggest advantage of LC is the ability to analyze metabolites without the need to
derivatize them. Unlike GC, there is no need for additional extract cleanup before
instrumental analysis. As in the case of gas chromatography, only highly specific and
sensitive methods, i.e., using mass spectrometry, are useful in biomonitoring studies.
The sample preparation process is simplified, but it comes at a price. LC-MS
methods are susceptible to ion suppression phenomenon which can strongly affect
both sensitivity and repeatability [14, 29].
Only two published methods used HPLC with spectrophotometric detection for
the determination of synthetic pyrethroid metabolites. Smith et al. [27] developed a
HPLC-UV method for the determination of 3PBA and MPA – a metabolite of
bifenthrin in the urine of people professionally exposed to this insecticide. Bartosz
et al. [12] in turn developed HF-LPME-HPLC-DAD method for determination of
3PBA and 4OH3PBA in rat and human urine. Both methods, due to high LOD and
LOQ values, are not suitable for the determination of metabolites in the urine of
non-occupationally exposed subjects.
Separation of analytes is carried out using HPLC, UPLC, and UHPLC coupled
with mass spectrometers with various types of analyzers: triple quadrupole ESI
[14, 16, 21, 29, 31, 34], turbo ion spray (TIS) [30], Q-TOF (ESI) [32, 35, 36], and
high-resolution Orbitrap [19, 37].
Sample preparation for LC-MS analysis included offline solid-phase extraction
[16, 21, 29–32, 34–36], liquid-liquid extraction [14], online SPE [37], and the
QuEChERS [19].
The popularity of the QuEChERS methodology stems from its unique simplicity
and applicability to almost any type of matrix. Therefore, an attempt was made to
apply this methodology to the preparation of a biological sample in order to quantify
the concentration of pesticide metabolites in human urine.
5 mL of urine was hydrolyzed enzymatically (1 mL of 0.2 M acetic buffer and
10 μL of β-glucuronidase aryl sulfatase) and then subjected to simplified
QuEChERS procedure by addition of 10 mL of acetonitrile and QuEChERS salt
packet. Acetonitrile layer was then evaporated at 37
C under a stream of nitrogen
and reconstituted in 200 μL of methanol/water (10:90, v:v) containing 0.1% of acetic
acid. Extract was analyzed with the use of UHPLC-HRMS system. Five pyrethroid
metabolites were monitored: cis-DCCA, trans-DCCA, DBCA, 3PBA, and 4F3PBA.
Additionally, Plackett-Burman design was used to optimize the parameters affecting
the analytical response [19]. Unfortunately, LOQs were in the range of 2–10 ng/mL.
López-García et al. [37] developed a method for simultaneous quantification
of selected organophosphate and pyrethroid metabolites in human urine and compared three independent sample preparation protocols including offline SPE,
TurboFlow™, and online SPE. For TurboFlow™ and online SPE protocols, raw
urine sample (without hydrolysis) was filtered through a 0.2 μm nylon filter, and
0.5 mL was subjected to online extraction. The best peak shapes and recoveries
were obtained with TurboFlow™ methodology. This technique was the only one
enabling detection of cis-/trans-DCCA, since no signal was produced when offline
Analytical Methods for Determination Urinary Metabolites of Synthetic. . .
57