cases, metabolites were released from conjugates using different deconjugation
procedures before extraction from the matrix. Glucuronides and sulfates consist
over 90% of conjugates found in human urine. Considering this, measurement of
total concentration of metabolites has to be preceded by hydrolysis. Both acidic and
enzymatic hydrolyses can be performed for quantitative release of metabolites before
their isolation from urine.
The most significant disadvantage of enzymatic hydrolysis is time consumption
since it is usually performed overnight. On the other hand, this process does not need
personnel engagement; therefore, it is virtually costless. Enzymatic hydrolysis is
considered as a mild process because strong acids used for acidic hydrolysis might
destroy labile analytes. For example, it was shown that a common metabolite of
metofluthrin and profluthrin, i.e., 2,2-dimethyl-3-(1-propenyl)-cyclopropane carboxylic acid (MCA), was significantly degraded during HCl hydrolysis [8].
Acidic hydrolysis is typically performed with concentrated hydrochloric acid
added at an average ratio of 0.2 mL per each mL of urine. Sample is then heated
at 90–100
C for 60–120 min [4, 6, 9–14].
Toshima et al. [15] observed some discrepancies between determined concentrations of 3PBA from two laboratories during cross-validation study. Authors
observed significantly lower concentrations of 3PBA following enzymatic
deconjugation in some of the urine samples. The results suggested the presence of
other conjugated species of 3PBA than glucuronide and sulfate in human urine.
Although the overall agreement between the values obtained by the deconjugation
methods was fair, it appears that urine samples should be pretreated by acidic
deconjugation for the analysis in biological monitoring of pyrethroid exposure.
Different enzymes, such as β-glucuronidase type HP-1 from Helix pomatia [16],
type HP-2 [14, 15, 17, 18], glucuronidase arylsulfatase enzyme [19] and sulfatase
from Helix pomatia, type H-1, lyophilized powder [20, 21], were used for enzymatic
hydrolysis. Incubation time with enzyme in 0.2 M acetate buffer (pH 4.5–5.0) varied
between 5 and 17 h (overnight) at 37
C.
3.1.2 Extraction
Liquid-Liquid Extraction
Liquid-liquid extraction is the simplest extraction technique commonly used for
isolation of pyrethroid metabolites from human urine. After acidic hydrolysis of
urine, no pH adjustment is needed before extraction. In contrary, when enzymatic
hydrolysis is performed, the sample should be acidified before extraction. Analytes
are usually extracted to n-hexane [3, 6, 9, 11, 14, 22–24], dichloromethane [5, 25],
isopropanol-hexane (5:95) [26], tert-butyl-methyl-ether (MTBE) [8, 10], chloroform
[27], or toluene [20]. Due to the acidic character of metabolites, re-extraction from
organic solvent to alkaline solution might be later performed for sample cleanup.
Usually NaOH solution is utilized for this purpose [3, 9, 11, 14, 24, 27]. Liquidliquid extraction is considered as difficult to automate; however, Ueda et al. [8] used
Analytical Methods for Determination Urinary Metabolites of Synthetic. . .
53
procedures before extraction from the matrix. Glucuronides and sulfates consist
over 90% of conjugates found in human urine. Considering this, measurement of
total concentration of metabolites has to be preceded by hydrolysis. Both acidic and
enzymatic hydrolyses can be performed for quantitative release of metabolites before
their isolation from urine.
The most significant disadvantage of enzymatic hydrolysis is time consumption
since it is usually performed overnight. On the other hand, this process does not need
personnel engagement; therefore, it is virtually costless. Enzymatic hydrolysis is
considered as a mild process because strong acids used for acidic hydrolysis might
destroy labile analytes. For example, it was shown that a common metabolite of
metofluthrin and profluthrin, i.e., 2,2-dimethyl-3-(1-propenyl)-cyclopropane carboxylic acid (MCA), was significantly degraded during HCl hydrolysis [8].
Acidic hydrolysis is typically performed with concentrated hydrochloric acid
added at an average ratio of 0.2 mL per each mL of urine. Sample is then heated
at 90–100
C for 60–120 min [4, 6, 9–14].
Toshima et al. [15] observed some discrepancies between determined concentrations of 3PBA from two laboratories during cross-validation study. Authors
observed significantly lower concentrations of 3PBA following enzymatic
deconjugation in some of the urine samples. The results suggested the presence of
other conjugated species of 3PBA than glucuronide and sulfate in human urine.
Although the overall agreement between the values obtained by the deconjugation
methods was fair, it appears that urine samples should be pretreated by acidic
deconjugation for the analysis in biological monitoring of pyrethroid exposure.
Different enzymes, such as β-glucuronidase type HP-1 from Helix pomatia [16],
type HP-2 [14, 15, 17, 18], glucuronidase arylsulfatase enzyme [19] and sulfatase
from Helix pomatia, type H-1, lyophilized powder [20, 21], were used for enzymatic
hydrolysis. Incubation time with enzyme in 0.2 M acetate buffer (pH 4.5–5.0) varied
between 5 and 17 h (overnight) at 37
C.
3.1.2 Extraction
Liquid-Liquid Extraction
Liquid-liquid extraction is the simplest extraction technique commonly used for
isolation of pyrethroid metabolites from human urine. After acidic hydrolysis of
urine, no pH adjustment is needed before extraction. In contrary, when enzymatic
hydrolysis is performed, the sample should be acidified before extraction. Analytes
are usually extracted to n-hexane [3, 6, 9, 11, 14, 22–24], dichloromethane [5, 25],
isopropanol-hexane (5:95) [26], tert-butyl-methyl-ether (MTBE) [8, 10], chloroform
[27], or toluene [20]. Due to the acidic character of metabolites, re-extraction from
organic solvent to alkaline solution might be later performed for sample cleanup.
Usually NaOH solution is utilized for this purpose [3, 9, 11, 14, 24, 27]. Liquidliquid extraction is considered as difficult to automate; however, Ueda et al. [8] used
Analytical Methods for Determination Urinary Metabolites of Synthetic. . .
53