Oasis HLB being the polymeric sorbent with a hydrophilic-hydrophobic balance
is used usually in the form of cartridges [21, 29–31] as well as 96-well plate format
[16]. However, C18 sorbents are also suitable [6, 7, 32, 33].
Miniaturized format of SPE, named microextraction by packed sorbent (MEPS),
was used by Klimowska and Wielgomas to extract five metabolites from only
0.4 mL of human urine [17]. Extraction was carried out using a semiautomatic
syringe equipped with a needle with a bin filled with a small amount of C18 sorbent
(4 mg) – BIN (barrel insert and needle). The advantage of this technique is that the
sample flows through the bed twice, once when the sample is drawn into syringe and
the second time when the sample is dispensed. This technique is based on the SPE
principles, but thanks to miniaturization, it allows the extraction of very small
sample volumes and elution of analytes with microliter volume of solvent directly
to the injector. The authors, thanks to the use of large-volume injection (40 μL)
and GC-MS (LVI-GC-MS), could achieve limits of quantification in the range of
0.06–0.08 ng/mL [17].
3.1.3 Derivatization
Analytes while released during enzymatic or acidic hydrolysis and following extraction and cleanup might be directly analyzed by liquid chromatography or have to be
converted to more volatile and thermally stable products suitable for gas chromatography. Hexafluoroisopropanol (HFIP) combined with diisocarboxyldiimide
(DIIC) is the most often used derivatization reagent. The major advantage of this
reagent is that the reaction is completed at room temperature in minutes, usually,
residue after organic solvent evaporation if treated with a mixture of HFIP/DIIC in
the presence of acetonitrile or isooctane. After a few minutes, the reaction mixture is
washed with NaHCO 3 to remove excess of reagents.
Furthermore, Klimowska and Wielgomas [17] documented that hexafluoroisopropyl esters of acidic pyrethroid metabolites are formed on the solid support
(C18) during elution with hexane containing HFIP and DIIC. No byproducts, which
are harmful to GC injection liner, column, or MS detector, are formed.
Much less frequently, analytes were methylated to methyl esters by incubation
with a mixture of methanol and sulfuric acid [3, 6, 23].
Alcohol metabolites are less frequently analyzed in urine samples. Ueda et al. [8]
developed the GC-MS/MS method for determination of alcoholic metabolites
(HOCH2-FB-Al, CH3-FB-Al, CH3OCH2-FB-Al, and FB-Al) of fluorinated pyrethroids: metofluthrin, profluthrin, tefluthrin, and transfluthrin. Unfortunately, metabolites mentioned above could not be derivatized sufficiently by the HFIP/DIIC
reagent even with any modification of reaction temperature and time. On the other
hand, these metabolites were derivatized by the reagents for trimethylsilylation such
as TMSI, TMSI-TMCS, MTBSTFA, BSTFA, and BSTFA-TMCS. Of these, only
BSTFA-TMCS (99:1) showed reactivity with all hydroxyl metabolites [8].
Schettgen et al. [9, 11] and Guo et al. [26] derivatized acidic metabolites with
MTBSTFA before GC-MS/MS analysis.
Analytical Methods for Determination Urinary Metabolites of Synthetic. . .
55
is used usually in the form of cartridges [21, 29–31] as well as 96-well plate format
[16]. However, C18 sorbents are also suitable [6, 7, 32, 33].
Miniaturized format of SPE, named microextraction by packed sorbent (MEPS),
was used by Klimowska and Wielgomas to extract five metabolites from only
0.4 mL of human urine [17]. Extraction was carried out using a semiautomatic
syringe equipped with a needle with a bin filled with a small amount of C18 sorbent
(4 mg) – BIN (barrel insert and needle). The advantage of this technique is that the
sample flows through the bed twice, once when the sample is drawn into syringe and
the second time when the sample is dispensed. This technique is based on the SPE
principles, but thanks to miniaturization, it allows the extraction of very small
sample volumes and elution of analytes with microliter volume of solvent directly
to the injector. The authors, thanks to the use of large-volume injection (40 μL)
and GC-MS (LVI-GC-MS), could achieve limits of quantification in the range of
0.06–0.08 ng/mL [17].
3.1.3 Derivatization
Analytes while released during enzymatic or acidic hydrolysis and following extraction and cleanup might be directly analyzed by liquid chromatography or have to be
converted to more volatile and thermally stable products suitable for gas chromatography. Hexafluoroisopropanol (HFIP) combined with diisocarboxyldiimide
(DIIC) is the most often used derivatization reagent. The major advantage of this
reagent is that the reaction is completed at room temperature in minutes, usually,
residue after organic solvent evaporation if treated with a mixture of HFIP/DIIC in
the presence of acetonitrile or isooctane. After a few minutes, the reaction mixture is
washed with NaHCO 3 to remove excess of reagents.
Furthermore, Klimowska and Wielgomas [17] documented that hexafluoroisopropyl esters of acidic pyrethroid metabolites are formed on the solid support
(C18) during elution with hexane containing HFIP and DIIC. No byproducts, which
are harmful to GC injection liner, column, or MS detector, are formed.
Much less frequently, analytes were methylated to methyl esters by incubation
with a mixture of methanol and sulfuric acid [3, 6, 23].
Alcohol metabolites are less frequently analyzed in urine samples. Ueda et al. [8]
developed the GC-MS/MS method for determination of alcoholic metabolites
(HOCH2-FB-Al, CH3-FB-Al, CH3OCH2-FB-Al, and FB-Al) of fluorinated pyrethroids: metofluthrin, profluthrin, tefluthrin, and transfluthrin. Unfortunately, metabolites mentioned above could not be derivatized sufficiently by the HFIP/DIIC
reagent even with any modification of reaction temperature and time. On the other
hand, these metabolites were derivatized by the reagents for trimethylsilylation such
as TMSI, TMSI-TMCS, MTBSTFA, BSTFA, and BSTFA-TMCS. Of these, only
BSTFA-TMCS (99:1) showed reactivity with all hydroxyl metabolites [8].
Schettgen et al. [9, 11] and Guo et al. [26] derivatized acidic metabolites with
MTBSTFA before GC-MS/MS analysis.
Analytical Methods for Determination Urinary Metabolites of Synthetic. . .
55