Monitoring of pyrethroid metabolites in human urine using solid-phase extraction followed by gas chromatography-tandem mass spectrometry

Analytica Chimica Acta 401 (1999) 45–54

Monitoring of pyrethroid metabolites in human urine using solid-phase extraction followed by gas chromatography-tandem mass spectrometry F.J. Arrebola a,∗ , J.L. Mart´ınez-Vidal a , A. Fernández-Gutiérrez b , M.H. Akhtar c a

Department of Analytical Chemistry, University of Almer´ıa, 04071 Almer´ıa, Spain Department of Analytical Chemistry, University of Granada, 18071 Granada, Spain The Southern Crop Protection and Food Research Centre, Food Research Program, Agriculture and Agri-Food Canada, 43-McGilvray Street c/o University of Guelph, Guelph, Ont, Canada, N1G 2W1 b

c

Received 18 March 1999; received in revised form 15 June 1999; accepted 25 June 1999

This article is dedicated to Dr. Antonio Arrebola Ram´ırez of the University of Granada, in memorium. Abstract A method was developed for the determination of cis-, and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Cl2 CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Br2 CA cis-, trans), 2-(4chlorophenyl)-3-methylbutyric acid (2-ClBA), and 3-phenoxybenzoic acid (3-PBA) in human urine. These compounds are major metabolites of commonly used synthetic pyrethroids permethrin, acrinathrin, cypermethrin, fenvelarate, deltamethrin, cyfluthrin and cyhalothrin. The method involves extraction of metabolites using solid-phase extraction, additional liquid–liquid extraction clean-up and derivatization followed by detection and quantification by sensitive gas chromatography-tandem mass spectrometry. 3-(2-methoxyphenoxy) benzoic acid was used as an internal standard. Recoveries of spiked compounds ranged between 92.3 and 113.3%, and 87.4 and 121.1% at fortification levels of 100 and 10 ng ml−1 , respectively. The relative standard deviation was lower than 14.6% in all cases. The method was employed to detect and quantify pyrethroid metabolites in the urine of pest control operators and non-occupationally exposed subjects. The concentration of metabolites ranged from 269 pg ml−1 for one of the non-exposed subjects to 2.8 ng ml−1 for one of the exposed pest control operators. ©1999 Elsevier Science B.V. All rights reserved. Keywords: Synthetic pyrethroids; Metabolites; Gas chromatography; Tandem mass spectrometry; Monitoring

1. Introduction Synthetic pyrethroids are widely used as pest control agents in agricultural production and protection of human health (used in hospitals and in homes). They are effective against a wide spectrum of pests [1]. In a formulation they may be present as a single isomer or as a mixture of several stereo-, and optical isomers ∗ Corresponding author. Tel./fax: +34-950-21-54-83; E-mail address: [email protected] (F.J. Arrebola)

[2]. These insecticides are absorbed via the skin, by inhalation and from the gastrointestinal tract. They impart neurotoxicity by blocking sodium and potassium channels of the neuronal membrane. Some pyrethroids affect the peripheral nervous system, while the others attack the central nervous system [3–8]. On absorption, they are rapidly metabolized by warm-blooded animals by hydrolysis of the ester bond or by oxidation. Both acid and alcohol moieties, on hydrolysis, are converted into their corresponding acid, partially conjugated and eliminated in urine. Thus a rapid, reli-

0003-2670/99/$ – see front matter ©1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 9 9 ) 0 0 5 1 9 - X

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Fig. 1. General structure of synthetic pyrethroids and their main metabolic pathway in mammals.

able, and sensitive method is essential to protect population and especially pest control operators from undue exposure to synthetic pyrethroids [3–26]. Fig. 1 shows the general structure of pyrethroids and the major initial metabolic pathway. Several methods have been published for the analysis of pyrethroid metabolites in urine using gas chro-

matography-mass spectrometry (GC-MS). Liquid– liquid extraction (LLE) is the most commonly used sample preparation technique [27–33]. A few methods involve extraction of urine on C18 solid-phase extraction (SPE) cartridges [34,35]. In general, SPE extract is cleaner than LLE and provides a better chromatographic profile. For GC analysis extracts were derivatized by various derivatizing agents. Methyl derivatives, in general, were not easily separated from each other, and were also less sensitive to mass spectrometer detector. However, 1,1,1,3,3,3-hexafluoroisopropyl derivatives of carboxylic acids have shown excellent chromatographic properties including high sensitivity in mass spectrometry. The hexafluoroisopropyl derivatives are separated and detected either by gas chromatography-electron capture detector (GC-ECD) or by gas chromatographymass spectrometry (GC-MS). Since the concentration of target analytes in a real sample is extremely low, there is a need to increase the sensitivity of detection. One of the approaches is to use the selected ion monitoring (SIM) technique, which provides higher sensitivity but reduced qualitative information than the full scan spectra. The use of negative chemical ionization on a high resolution double focusing mass spectrometry (NCI−HRMS) has been recommended, but is not easily available in many laboratories. Recently, ion trap mass spectrometry has shown great promise for the determination of very low levels of pesticides and metabolites in biological samples using tandem mass spectrometry [36,37]. This paper describes a new, simple, and sensitive method for the quantitative determination of cis-, and trans-Cl2 CA, cis-Br2 CA, 2-ClBA and 3-PBA, the major metabolites of commonly used pyrethroids, in human urine using a combination of SPE and GC-MS/MS. The method was validated with real field urine samples from six pest control operators and two non-occupationally exposed individuals in Almer´ıa (Spain).

2. Experimental 2.1. Chemicals Pesticide grade n-hexane, acetone and methanol (Merck, Darmstadt, Germany) were used as received.

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Organic free water was prepared by distillation and then by passing through Milli-Q SP column (Millipore, USA). Analytical grade sulfuric acid (98% purity), sodium chloride and potassium carbonate were purchased from Panreac (Barcelona, Spain). 1,1,1,3,3,3-Hexafluoroisoproanol (HFIP) and diisopropylcarbodiimide (DIC) were from Adrich (Steinheim, Germany) and were used as received. Pure cis-, and trans-Cl2 CA, cis-Br2 CA, 2-ClBA and 3-PBA were available from previous studies [18–21]. Stock solution of an individual metabolite at 400 ␮g ml−1 was prepared in acetone and stored in a freezer (−30◦ C). The working solutions were obtained by appropriate dilution of the stock solution with the same solvent, and stored in a refrigerator (4◦ C). 3-(2-methoxyphenoxy) benzoic acid (3-MPBA) was used as an internal standard (IS) at 3 ␮g ml−1 in acetone. Sigmasil A was purchased from Sigma (St. Louis, MO. USA). Sep-Pak cartridge for solid-phase extraction packed with 500 mg C18 was from Waters (Milford, MA, USA). 2.2. Equipment A Saturn 2000 gas chromatograph-ion trap mass spectrometer (Varian Instruments, Sunnyvale, CA, USA) was used. The gas chromatograph was fitted with a 8200 autosampler, a split/splitless temperature programmable injector 1078 and operated in splitless mode. The column used was a DB5-MS 30 m × 0.25 mm I.D. × 0.25 ␮m film thickness (J & W Scientific, Folsom, CA, USA). The ion trap mass spectrometer was operated in the electron ionization mode (EI, 70 eV) and the MS/MS option was used. The data handling system had an EI-MS/MS library specially created for the target analytes under our experimental conditions. In addition, other EI-MS libraries were also available. The carrier gas used was helium (purity N50). A test tube shaker with a variable speed controller was purchased from Ika-Works (Wilmington, NC, USA). 2.3. Sample collection Urine from volunteer pest control operators (six) and non-occupationally exposed (two) individuals

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were collected and stored in sterilized containers. Samples were frozen immediately and kept −30◦ C until analyzed. Information on pesticide exposure was obtained by evaluation of a questionnaire, personal interview and medical examination. 2.4. Analytical procedure 2.4.1. Urine extraction procedure 3 ml aliquot of urine was placed in a screw capped glass centrifuge tube to which was added 100 ␮l IS and 0.5 ml conc. H2 SO4 and heated at 90◦ C in a water bath for 1 h. The sample was cooled to room temperature, diluted with 3 ml distilled water, and passed through the C18 cartridge previously conditioned with 7.5 ml methanol and 5 ml distilled water in that order (Note: cartridge was not allowed to dry during conditioning). To carry out a clean-up step, 6 ml of distilled water were added. The last drops of liquid from cartridge were withdrawn with a vacuum pump. Analytes from the cartridge were eluted with 10 ml acetone, which was removed by a gentle stream of nitrogen at room temperature. 2 ml of saturated aqueous NaCl solution and 5 ml n-hexane were added and shaken vigorously for 3 min. The organic layer was removed by a Pasteur pipette. The aqueous phase was re-extracted twice more with 5 ml n-hexane each. The combined extract was evaporated under a stream of nitrogen without heat and the residue dissolved in 1 ml n-hexane. For derivatization, 10 ␮l HFIP was added with gentle mixing followed by addition of 15 ␮l DIC. After shaking for 3 min, the extract was washed with 1 ml of 5% aqueous potassium carbonate solution to neutralize the excess derivatizing agent. The organic layer was transferred to a 2 ml autosampler vial for analysis. 2.4.2. GC-MS/MS conditions A 1 ml aliquot of the extract was injected into a temperature-programmed injector in a split closed mode for 1 min. Injector temperature was programmed from 90◦ C (hold 0.1 min at 90◦ C) to 280◦ C at the rate of 200◦ C min−1 and held at 280◦ C for 15 min. The oven temperature was modified from 60◦ C, (hold for 1 min) to 270◦ C at the rate of 20◦ C min−1 and held there for 5 min. The mass spectrometer was calibrated weekly. The operating conditions are summarized in Tables 1 and 2.

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Table 1 Mass spectrometer operating conditions Ionisation mode Multiplier voltage Multiplier gain A/M amplitude voltage Trap temperature Manifold temperature Transfer-line temperature Emission current Automatic Gain Control (AGC) AGC prescan ionization time AGC target

by filling the trap with target ions. The AGC target was fixed at 2000 counts because higher values caused electrostatic interactions among ions in the ion trap chamber. For analysis, a parent ion (m/z and its relative abundance) was chosen for each analyte. An isolation window of 2u was used for all compounds. A non-resonant wave form (collision-induced dissociation) was selected for all compounds except cis-, and trans-Cl2 CA-HFIP. Non-resonant excitation is often useful with parent ions that fragment by the breakage of a single weak chemical bond to form highly stable ions containing functional groups that do not undergo significant rearrangements. The resonant excitation method is an alternative when the parent ions require the breakage of multiple chemical bonds or that undergo significant rearrangements. The excitation amplitude was studied to obtain a spectrum with parent ions as molecular peaks (between 10 and 20% of relative abundance). The count threshold was set at 1 count for improving the sensitivity and avoiding background noise when analyzing for a low concentration of analytes. The EI-MS/MS spectra of various derivatives are shown in Fig. 2. The base peak was selected for all quantification.

EI 1700 V 1 × 105 4.0 V 200◦ C 45◦ C 260◦ C 80 ␮A on 1500 ␮s 2000 counts

MS/MS was performed in a non-resonant mode for all compounds except cis- and trans-Cl2 CA-HFIP, which was done in a resonant mode.

3. Results and discussion 3.1. Effect of experimental variables 3.1.1. Instrumental variables Several derivatizing agents were investigated and the chromatographic properties of various derivatives were studied. Under our chromatographic conditions methyl esters of 2-ClBA and cis-Br2 CA were not separated from each, but their trimethylsilyl and HFIP derivatives did. HFIP derivatives of all metabolites investigated eluted in less than 10 min and were well separated from each other. The retention times of HFIP derivatives were: cis-Cl2 CA, 5.633 min; trans-Cl2 CA, 5.682 min, 2-ClBA, 6.654 min; cis-Br2 CA, 6.860 min, 3-PBA, 8.557 min, and 3-MPBA (IS), 9.484 min. In order to optimize sensitivity for mass detection, the Automatic Gain Control (AGC) was switched on

3.1.2. Sample preparation Acidic hydrolysis of the conjugated carboxylic acids was performed by adding sulfuric acid and heating. Thus free metabolites are obtained prior to the extraction. On the basis of previous experiences it was decided to use SPE C18 cartridge for sample preparation since it was found that eluents from the SPE cartridges were better suited for direct GC analysis and they seldom required clean-up prior to GC analysis. On the other hand, eluents from LLE must involve a clean-up step

Table 2 MS/MS characteristics of various derivativesa Compound

Activation time (min)

Range (m/z)

Parent ion (m/z)

Cl2 CA-HFIPb 2-ClBA-HFIP Br2 CA-HFIP 3-PBA-HFIP 3-MPBA-HFIP (IS)

5.0 6.0 6.8 8.0 9.1

110–330 80–330 80–380 100–380 100–410

323 320 369 364 394

a b

Excitation time = 40 ␮s; isolation window = 2 u. Resonant waveform.

Ass defect (␮/100 u) +27 −81 0 +68 +70

Excitation amplitude (V)

Excitation storage level (m/z)

0.4 64.0 56.0 65.0 68.0

110 110 80 100 100

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Fig. 2. EI-MS/MS spectrum and structural formulas: (a) cis- and trans-Cl2 CA-HFIP; (b) 2-ClBA-HFIP; (c) Br2 CA-HFIP; (d) 3-PBA-HFIP; (e) 3-MPBA-HFIP.

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to avoid reduction in column efficiency and contamination of injector and ion trap. Several solvents (acetone, n-hexane, diethyl ether, methanol, ethyl acetate, and dichloromethane) were tested for elution efficiency. The maximum elution was found with 10 ml acetone. For a quantitative recovery of the analyte, the eluent from SPE C18 must be treated with 2 ml saturated aqueous sodium chloride and extracted thrice with n-hexane. Methyl, trimethylsilyl and hexafluoropropyl derivatives of acids were prepared and their chromatographic properties investigated. Methylation was performed by treating the eluent with methanol and concentrated H2 SO4 , followed by extraction with n-hexane. It is a long, cumbersome and tedious process. Methyl derivatives were volatile and need addition of toluene to avoid loss during concentration. Methyl derivatives were also less sensitive to the mass detector. The trimethylsilyl esters were obtained by a reaction of dried eluent with of Sigmasil A. Although, trimethylsilyl derivatives were much more sensitive than methyl derivatives, the excess of the derivatizing agent and its by-products produced several interfering peaks. HFIP derivatives which are produced by a rapid coupling of the eluent with HFIP in the presence of DIC reaction, are highly sensitive and do not produce interfering substances.

number of peaks. A slightly improved chromatogram (Fig. 3(b)) was obtained when the quantification ion (m/z 323) was selected. Fig. 3(c) is an EI-MS/MS chromatogram of the same sample. With MS/MS, if a co-eluted interferent has the same identification ion as the analyte, such an interferent can be avoided using special experimental conditions for the collision-induced dissociation and quantifying with a specific ion from the analyte. The S/N values obtained for cis- and trans-Cl2 CA in the MS/MS chromatogram improved about 25 times the results reached when SIM mode was used. The instrument was calibrated using a clean sample fortified with each metabolite in the range of 0.05 to 100 ng ml−1 . An internal standard was used at 100 ng ml−1 urine. The choice of 3-(2-methoxyphenoxy)benzoic acid as an internal standard was based on its structural and chromatographic compatibility with the compounds being analyzed. Calibration curves and calculations were performed using a quantification menu of the Data System and the results are shown in Table 3. The lack-of-fit test was used for the three replicates of each concentration in order to check the linearity of the calibration graphs. The values found for the test (Plof (%)) demonstrated good linearity for all the compounds in the ranges studied.

3.2. Identification and quantification of target analytes

3.3. Recoveries and limits of detection

GC-MS/MS analysis of a working standard solution consisting of derivatives of interested metabolites was performed and a library was created. This acted as our resource/reference library. A data handling system was used for search and identification. For example, a target analyte was searched by its retention time (±10 s window), and confirmed by comparison with the EI-MS/MS spectra in the library. A positive confirmation required a minimum spectral fit of >700 and the signal-to-noise level (S/N) > 3 (for quantification ions). For quantification S/N was set at >10. Fig. 3 represents EI-MS and EI-MS/MS chromatograms of cis- and trans-Cl2 CA-HFIP obtained following derivatization of the recovered spiked Cl2 CA at 1 ng ml−1 . The EI-MS total ion chromatogram is shown in Fig. 3(a) which exhibited large

To determine detection and quantifiable limits, control urine was spiked with known amounts of each compound prior to extraction, and the procedure was followed. Recoveries were excellent (>90%) at the spiking levels employed (10 and 100 ng ml−1 ), except

Table 3 Information on calibration curvesa,b Compound

a

b

R2

Plof (%)

cis-Cl2 CA-HFIP trans-Cl2 CA-HFIP 2-ClBA-HFIP Br2 CA-HFIP 3-PBA-HFIP

0.444 3.718 10.562 1.166 7.775

2.197 3.085 9.818 0.715 5.121

99.50 99.11 99.66 96.36 99.46

0.99 0.62 0.11 0.93 0.40

a n = 3. a, intercept; b, slope; R2 , correlative coefficient, P (%), lof Probability level.of lack-of-fit. b All the intercepts were statistically non-significant.

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Fig. 3. Chromatograms of Cl2 CA in human urine at the fortification level of 1 ng ml−1 . (a) Total ion chromatogram using EI-MS; (b) mass chromatogram using EI-MS and selecting the quantification ion; (c) mass chromatogram using EI-MS/MS.

for cis- and trans-Cl2 CA which was slightly lower than 90% at the 10 ng ml−1 level (Table 4). The repeatability expressed as relative standard deviation (RSD) was smaller than 14.6%. The limits of detection (LOD) and quantification (LOQ) for each compound are given in Table 4. The calculated LOD for an individual compound is in the low parts per trillion (ppt) level, a good indication of the sensitivity of EI-MS/MS. High sensitivity may, in part, be attributed to the complete elimination of undesirable matrix information and the considerably high response of HFIP esters. Repeatability of the method and low LOD and LOQ are sufficient for

monitoring exposure of pyrethroids among the pest control operators and the population at large and have improved the results obtained in previous methods.

3.4. Applications of method The method was validated with real samples obtained from pest control operators and non-occupationally exposed individuals. The operators indicated the use of pyrethroids in their formulations. Urine samples were obtained from males between the ages of 18 and 54 years. The operators had

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Fig. 4. Mass chromatograms of a pest control operator urine using EI-MS/MS and selecting the quantification ions. (a) cis- and trans-Cl2 CA; (b) 2-ClBA; (c) Br2 CA; (d) 3-PBA.

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Table 4 Recoveries (R%), relative standard deviations (RSD), limits of detection (LOD) and quantification (LOQ) in urine. (n = 5) 10 ng ml−1

Compound

cis-Cl2 CA-HFIP trans- Cl2 CA-HFIP 2-ClBA-HFIP Br2 CA-HFIP 3-PBA-HFIP

100 ng ml−1

R%

RSD%

R%

89.9 87.4 98.6 104.1 121.1

14.5 14.6 14.3 13.7 13.5

96.1 102.6 104.5 92.3 113.3

LOD (pg ml−1 )

LOQ (pg ml−1 )

6 5 2 19 3

20 17 7 62 10

RSD% 13.7 12.1 6.2 8.5 4.8

Table 5 Concentration (pg ml−1 ) of various metabolites in occupationally (OEP) and non-occupationally (NOEP) exposed people Sample No.

Age

Experience (years)

cis-Cl2 CA

trans-Cl2 CA

2-ClBA

Br2 CA

3-PBA

Total

1 2 3 4 5 6

34 33 36 44 18 54

14 12 18 8 4 40

156 358 176 519 227 238

205 337 376 823 278 337

– – – – 58 –

– – – 69 – –

930 1115 785 1404 986 828

1291 1810 1337 2815 1549 1403

23 23

– –

54 –

123 –

– –

– –

320 269

497 269

1 2

OEP

NOEP

4–40 years experience in handling pest control products. Fig. 4 represents EI-MS/MS chromatograms of urine extracts of pest control operators following derivatization with HFIP. Chromatograms were recorded under various SIM conditions. The chromatogram in Fig. 4(a) is for urine analysis carried out selecting the quantification ion for HFIP derivatives of cis-, trans-Cl2 CA (Fig. 4(a)). Chromatograms in Fig. 4(b–d) are recorded in the same way as for HFIP derivatives of 2-ClBA, cis-Br2 CA and 3-PBA, respectively. Concentrations of various metabolites in an individual operator’s and non-occupationally exposed individual’s urine, as determined by the application of EI-MS/MS, are shown in Table 5. The data show that all subjects eliminated cis-, and trans- Cl2 CA and 3-PBA in urine, but much higher levels were found in pest control operators. 2-ClBA and Br2 CA were found in one individual each suggesting that fenvalerate and deltamethrin were not in common use in that region. One explanation for higher concentration of pyrethroid metabolites in operators’ urine is that most did not use any protective apparel. The questionnaire and individual interview further revealed that they did

not follow commonly recommended safety practices such as washing hands, entry or re-entry etc. However, none of the operators showed any overt sign of abnormality.

4. Conclusions The method described above using SPE as the extraction media followed by GC-MS/MS was suitable for the assay of metabolites of commonly used synthetic pyrethroids in human urine. The method was simple, reproducible, highly sensitive and required HFIP derivatization before analysis. The method has been validated with actual urine samples obtained from six pest control operators and two non-occupationally exposed individuals. Analysis of data from pest control operators clearly showed that exposed individuals eliminated metabolites in urine. The total concentration of various pyrethroid metabolites in the urine of pest control operators ranged between 1291 and 2815 pg ml−1 , which was much higher than those found in non-occupationally exposed subjects (269–497 pg ml−1 ).

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Acknowledgements This research was supported by the European Union Project SMT4-CJ96-2048 (DG-12-RSMT) and by the Comision Industrial de Ciencia y Tecnologia (CICYT) Project No. AMB97-1194-CE.

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