Laura Sabatini, Anna Barbieri, Paolo Indiveri, Stefano Mattioli, Francesco Saverio Violante
Laura Sabatini, Anna Barbieri, Paolo Indiveri, Stefano Mattioli, Francesco Saverio Violante
com
Abstract
A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and fully validated, according to U.S. Food and Drug
Administration guidance, for the simultaneous determination of phenylmercapturic acid, benzylmercapturic acid and o-methylbenzyl mercapturic
acid in human urine as biomarkers of exposure to benzene, toluene and xylenes (BTX). After solid phase extraction and LC separation, samples
were analyzed by a triple–quadrupole mass spectrometer operated in negative ion mode, using isotope-labeled analogs as internal standards (ISs).
The method meets all the validation criteria required. The limits of detection of the three analytes, ranging from 0.30 to 0.40 g l−1 , and the high
throughput make the method suitable for the routine biological monitoring of co-exposure to BTX both in the occupational and environmental
settings. The validated method was applied to assess exposure to BTX in a group of 354 urban traffic wardens.
© 2008 Elsevier B.V. All rights reserved.
1570-0232/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jchromb.2008.01.022
116 L. Sabatini et al. / J. Chromatogr. B 863 (2008) 115–122
with endogenous glutathione [10]. At low levels of exposure, purchased from Argonaut Technologies Ltd. (Mid Glamorgan,
phenylmercapturic acid (PMA) has indeed been validated as UK). PMA, BMA and MBMA were supplied by Tokyo Kasei
a more specific biomarker for benzene [7,11]. Furthermore, (Prodotti Gianni, Milan, Italy). Deuterated analogs PMAd-5 and
benzylmercapturic acid (BMA) and o-methylbenzyl mercap- BMAd-7 were custom synthesized by Alchemy s.r.l. (Bologna,
turic acid (MBMA) have been proposed as reliable biomarkers Italy) to use as internal standards (ISs). All the standard com-
of exposure to toluene [12] and xylenes [13], respectively. pounds were of the highest available purity (>99%). Stock
In order to perform biological monitoring of occupationally solutions were prepared by dissolving pure powder of each ana-
and/or environmentally exposed individuals, several analytical lyte and ISs in methanol to give a concentration of 200 mg l−1 .
methods have been developed for the determination of MAs Stock solutions were stored at −20 ◦ C. Working standard solu-
in urine, based on high performance liquid chromatography tions (500 g l−1 for PMAd-5 and BMAd-7; 50–1000 g l−1 for
(HPLC) or gas chromatography–mass spectrometry (GC–MS) PMA, BMA and MBMA) were prepared weekly by dilution of
[14]. Among published methods, HPLC/tandem mass spectrom- the stock solutions in MeOH/20 mM formic acid 1:1 (v/v) and
etry (MS/MS) is currently considered as the first choice for MAs were kept at +4 ◦ C.
determination in human urine due to its high specificity and sen-
sitivity. Several HPLC–MS/MS methods have been reported for 2.2. Apparatus
the determination of PMA and BMA in urine [13,15–20] and
Gonzalez-Reche et al. [21] described a method for the quantifi- HPLC–ESI-MS/MS analysis was performed using a series
cation of dimethylphenyl mercapturic acid (a MA derived from 1100 -HPLC system (Agilent Technologies Inc., Waldbronn,
the metabolism of o-xylene) in urine as a biomarker of exposure Germany), equipped with a thermostatted well-plate autosam-
to xylenes. However, the metabolism of alkyl aromatics mainly pler and thermostatted column compartment modules. The
occurs at the side chain [22–24], producing, in the case of xylene, HPLC system was interfaced to an API 2000 triple–quadrupole
a higher amount of MBMA. Nevertheless, to our knowledge, mass spectrometer from PE Sciex (Concord, ON, Canada)
no HPLC–MS/MS methods have been developed for the anal- equipped with a TurboIonSprayTM source. A nitrogen generator
ysis of MBMA in urine. In addition, analytical methods for the system 75-72 Whatman (Maidstone, Kent, UK) was employed
simultaneous determination of several MAs would be particu- to produce N2 , used as curtain and auxiliary gas. Instrument
larly relevant in cases of co-exposure to several solvents. In fact, control and data acquisition were performed with Analyst Soft-
many studies have demonstrated that administration of toluene ware PE Sciex (rev. 1.3.2). An EZ-2plus Evaporator (GeneVac
and xylenes to rats causes depletion of glutathione in the liver, Ltd., Ipswich, UK) was used for solvent evaporation. SPE was
influencing the metabolic routes of these and other toxicants performed on a vacuum system 96-well plated VacMaster (IST).
[6,25]. Moreover, it is important to note that none of the above-
mentioned methods was fully and rigorously validated following 2.3. Extraction procedure
specific guidelines. As a consequence, little data are available
concerning the reliability of previously obtained results. Urine samples were collected and stored at −20 ◦ C. Prior to
The aim of our study was to validate an HPLC–MS/MS analysis, each sample was thawed, vigorously mixed and then
method to simultaneously determine PMA, BMA and MBMA centrifuged at 1500 × g for 10 min to obtain clear supernatant.
in human urine for a complete and accurate monitoring of expo- The clean-up procedure was optimized by comparing three dif-
sure to BTX. For this purpose, a 96-well solid phase extraction ferent types of reversed phase SPE cartridges: Isolute® C18 ,
(SPE) procedure and a micro()-HPLC separation coupled to Isolute® Env+ and EvoluteTM ABN.
electrospray ionization (ESI)-MS/MS were used. This analyti- EvoluteTM ABN cartridges were finally chosen for their
cal method was fully validated according to the FDA [26], using higher recovery (data not shown). We loaded 1 ml of centrifuged
two deuterated analogs as internal standards (ISs) and different and diluted (1:1 with aqueous formic acid 1%, v/v) urine sam-
urine lots to assess matrix effect variability. Moreover, the vali- ple spiked with 5 l of each IS working solution. The cartridges
dated method was successfully applied to measure exposure to were previously conditioned with 1 ml of MeOH and 1 ml of
BTX in a group of 354 traffic wardens. aqueous formic acid 0.1% (v/v). The stationary phase was then
washed with 1 ml of aqueous MeOH solution (10%, v/v) and
2. Experimental the analytes were eluted with two aliquots (250 l) of methanol.
The eluate was evaporated to dryness under vacuum at +40 ◦ C in
2.1. Reagents and chemicals an EZ-2 Plus concentrator. The residue was redissolved in 50 l
of 20 mM formic acid/MeOH 1:1 (v/v) and a volume of 0.5 l
All chemicals were of analytical reagent grade. Formic acid was injected into the -HPLC system.
(98%) was purchased from Fluka (Buchs, Switzerland) and
methanol of chromatographic grade was obtained from Merk 2.4. Liquid chromatography
(Darmstadt, Germany). Deionized water (18.2 M cm) was pro-
duced with a Direct-Q Millipore Waters system (Millford, MA, HPLC analysis was performed at a flow rate of 10 l min−1
USA). Isolute® ENV+ cartridges (50 mg, 1 ml) and Isolute® using 20 mM formic acid (solvent A) and methanol (solvent B).
C18 cartridges (50 mg, 1 ml) were purchased from IST (Mid Separation was accomplished on a Synergi 4u Max-RP capillary
Glamorgan, UK); EvoluteTM ABN cartridges (25 mg, 1 ml) were column (0.5 mm × 50 mm, 4 m, 80 Å, Phenomenex® Torrance,
L. Sabatini et al. / J. Chromatogr. B 863 (2008) 115–122 117
described above) in the same day and every 50 samples. Ana- ISs). Ion suppression of PMA, BMA and MBMA signal by the
lyte peak area/IS peak area ratios were plotted against nominal urinary matrix was examined as “negative” chromatographic
concentrations (PMAd-5 was used as IS for PMA and BMAd- peaks from the elevated baseline using six different urinary
7 was used for BMA and MBMA). Concentrations were back samples.
calculated from the corresponding calibration curves.
2.9.4. Specificity
2.9. Validation procedures The specificity of the method was assessed by analysis of six
individual batches of control urine, each analyzed both unspiked
The method was fully validated according to the FDA guide- and spiked at the LOQ level. The peak heights for blank matrix
lines. Limit of detection (LOD), limit of quantification (LOQ), samples should not exceed 20% of peak heights at the LOQ
linearity, precision and accuracy, recovery, ion suppression, level and accuracy at the LOQ should be within 80–120% of the
specificity and stability of the HPLC–ESI-MS/MS method were nominal value.
determined.
Table 1
Calibration parameters obtained for PMA, BMA and MBMA against ISs from three different calibration curves prepared in triplicate and analyzed in different days,
within 2 weeks
PMA BMA MBMA
Table 2
Precision and accuracy for PMA, BMA and MBMA in urinary QC samples
Nominal concentration (g l−1 ) Intra-day Inter-day
PMA
2 9.0 −8.9 5.6 −5.7
20 3.9 0.1 6.2 2.9
50 5.0 −4.0 2.8 −2.0
Average 6.0 −4.3 4.9 −1.6
BMA
2 2.8 −4.4 14.0 2.0
20 4.7 8.8 6.0 8.2
50 2.2 10.8 2.7 5.8
Average 3.2 5.1 7.6 5.3
MBMA
Fig. 3. Chromatogram of an extracted urine sample (spiked with ISs) with post-
2 8.2 7.9 5.3 9.4
column infusion of 500 g l−1 of PMA, BMA and MBMA.
20 4.1 14.2 4.1 14.2
50 1.7 14.9 3.8 11.6
in different batches of urine has been included as part of the
Average 4.7 12.3 4.4 11.7
validation procedure.
Number of replicates = 6. The experiments performed on six different urine samples
spiked after extraction showed a high-ion suppression (mean
analytes signal was 25.3% as compared to standard solutions).
of the calibration equation. Moreover, the accuracy of quantifi- These results are confirmed by post-column infusion experi-
cation at the LOQ level should be tested in six different urine ments, which indicated that the analytes peaks fell into an ion
lots and should be between 80 and 120% for all urines. Sev- suppression region (see Fig. 3).
eral LC–MS published methods reported slightly lower LOQ In the attempt to decrease ion suppression by increasing
values for PMA or BMA in urine. However, these values were analytes retention in column, several elution gradients were
obtained by analysis of a pool of urine and not from different tested. However, when the analytes retention time changed, the
urine samples [13,15–19]. Due to the unavailability of a urine region of maximum ion suppression moved accordingly (data
sample completely free of BMA, the determination of the LOQ not shown). This may be due to the fact that ion suppression of
for BMA presented some problems. An estimated LOQ value the urinary matrix in this region is caused by other MAs, which
for BMA in urine was thus calculated to be 0.7 g l−1 . Never- are present in urine at high concentration levels. For the same
theless, the actual LOQ of BMA in urine may be lower than this reason, different extraction procedures performed using differ-
estimate and could be determined only in urine samples with ent SPE cartridges (Isolute® C18 and Isolute® Env+ ) did not
non-detectable amounts of BMA. decrease matrix effect (data not shown).
Several LC–MS analytical methods for the determination of
3.2.2. Precision, accuracy and recovery PMA or BMA have been published, but only few showed ion
Table 2 shows the intra-day and inter-day accuracy and preci- suppression data. Although obtained using a different clean-
sion values of the entire procedure for PMA, BMA and MBMA. up procedure, our results are consistent with those previously
For all analytes, accuracy was within 91.1 and 114.9% and published [7].
R.S.D. did not exceed 14.0%. This showed that each analyte The repeatability of the total procedure was evaluated ana-
met the generally accepted criteria for bioanalytical method lyzing six calibration curves constructed in six different lots of
validation at all QC concentration levels. urine, instead that in a single (pooled) urine sample. Despite the
As regards recovery obtained from the SPE procedure, the high suppression due to urinary matrix, the slopes of calibra-
mean values were 82 (±4.4) % for PMA, 71.2 (±7.8) % for tion curves measured with six different lots of urine were highly
BMA and 78.3 (±11.8) % for MBMA. precise (R.S.D. < 8%), and an identical susceptibility of analytes
and ISs to matrix effect may be inferred. This, together with satis-
3.2.3. Matrix effect and ion suppression fying inter-day precision and accuracy values obtained for LOQ
Although matrix-induced alterations (suppression or concentration levels from the six lots of urine was indicative for
enhancement) of the ESI-MS/MS signals may critically impair the absence of matrix-caused quantification errors.
the reliability of a method, little attention is often paid to
this topic during method validation. In particular, previously 3.2.4. Specificity
published methods for the determination of MAs in urine did The method showed good specificity, meeting the accep-
not test matrix effect and ion suppression [13,15–20]. Due to tance criteria of the FDA. No interfering peaks were detected
the complexity of biological fluids and to the high inter-subject in the ‘blank’ samples at the mass transitions chosen for PMA,
variability of urine in particular, assessment of matrix effects MBMA and the ISs. The signal detected for BMA in all unspiked
L. Sabatini et al. / J. Chromatogr. B 863 (2008) 115–122 121
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