A routine gas chromatographic (GC) method is described for the analysis of ... DEP free. 250. 750. 1250. DETP-K. 100. 300. 500. DEDTP-K. 100. 300. 500.
Journal of Analytical Toxicology,Vol. 25, April 2001
Routine Gas ChromatographicDetermination of DialkylphosphateMetabolites in the Urine of Workers Occupationally Exposedto Organophosphorus Insecticides A.N. Oglobline 1, G.E. O'Donnell 1, R. GeyeP, G.M. Holder 2, and B. Tattam 2 1LaboratoryServices, WorkCoverNew South Wales, Thornleigh, NSW, 2120 Australia and 2Facullyof Pharmacy, Universityof Sydney, NSW, 2006 Australia
Abstract
]
A routine gas chromatographic (GC) method is described for the analysis of dialkylphosphate metabolites in the urine of workers occupationally exposedto organophosphorusinsecticides. The procedure involves derivatizing a freeze-dried urine sample with pentafluorobenzyl bromide and then determining the metabolites using dual capillary column GC with flame photometric detection.
not found a suitable method to date. Several of the published methods either have lengthy sample preparation protocols or require labor-intensive,highly skill-dependent steps in order to complete the analysis. The method described in this paper combines the sample preparation approach of one published paper and the derivatization/analysis approach of another. For the sample preparation step, the technique of freeze drying was chosen because of its suitability for large batches of samples.
Introduction In recent yearsthe restricted use of organochlorineinsecticides in Australia has resulted in an increased utilization of organophosphorus insecticides (OPs). As a consequence, more interest has been shown in the biological monitoring of OPs in occupationally exposed individuals. The main method of monitoring workers exposed to OPs in Australia has been the wellknown bloodcholinesteraseenzyme assay.Biologicalmonitoring via urine testing has been limited to specific OPs such as parathion and chlorpyrifoswhere assays have existed. Literature referencesto analyticalmethods for determining the dialkylphosphate (DAP) metabolites of OPs in the urine of occupationally exposed workers go back for approximately 30 years. Recently Moate et al. (1) and previously Aprea et al. (2) and Nutley and Cocker (3) publishedmethods for the analysisof DAPmetabolites in urine and they reviewedthese analyticalmethods which were published over the last three decades. The six main rnetabolites that have been tested in the past are DMP (dimethylphosphate), DMTP (dimethylthiophosphate), DMDTP (dimethyldithiophosphate), DEP (diethylphosphate), DETP (diethylthiophosphate), and DEDTP(diethyldithiophosphate).Figure I shows the structures of the six metabolites. Our laboratory,which specializesin biologicalmonitoring and industrial hygiene testing, has been looking for a method in the literature that could be applicablefor routine urine monitoring of large numbers ofworkers occupationallyexposedto OPs.We have
c%o,,p:.o
%%0 ,p;o
CH30
C2H50
OH
OH
DMP
DEP
CH~O~ ~S /p CH30 "OH
C~H.O~ ~S ~ o ,p, C2H50 OH
DMTP
DETP
CH30,,S
C~H.O, L o /p,~S
CH30"P'sH DMDTP
C2H50 SH
DEDTP
Figure 1. Chemical structuresof the six dialkylphosphatemetabolites.
Table I. Freeze Dryer Operating Conditions Temperature
Vacuum
Stage
(~
(mT)
Time (min)
Initial stage Stage 1 Stage2 Stage3 Stage4
NA -40 -20 0 +16
NA 10 10 10 5
30 60 420 360 300
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Journal of AnalyticalToxicology,VoL 25, April 2001
instrument for analysis.This derivatizationapproach was used by Nutley and Cocker (3) after the urine samples underwent azeotropic distillation.
Freeze drying was used by Peterson (4) for preparing urine samples before they underwent derivatizationwith a triazene reagent and subsequent sample cleanup. For the derivatizationand analysis of the DAP metabolites, pentafluorobenzylbromide was selectedas the derivatizationagent and gas chromatographywith flame photometric detection (GC-FPD) was selected as the
Experimental
Table II. Concentrationsof the Six Working Standard Solutions
DMP-Na DMTP-Na DMDTP-K DEPfree DETP-K DEDTP-K
L.1
L.2
(pg/L)
(pg/L)
500 100 100 250 100 100
1500 300 300 750 300 300
c:~Ctltotal-1~OOnov17~ub1.001.ChenntllA
Equipment The GC used consisted of a model 17A instrument from L.3 Shimadzu Australia Manufacturing (Melbourne, Australia) (pg/L) equipped with two flame photometric detectors with phosphorus filters. The split/splitless injection port was connected to a 1-m 2500 retention gap of deactivated phenylmethyl capillary tube (0.53500 ram i.d.) from SGE,InternationalPry Ltd. (Melbourne,Australia). 500 The two capillarycolumns used were a BPX5(30-m length, 0.321250 mm i.d.,0.5-mm filmthickness) and a BPXS0(30-m length, 0.32500 500 mm i.d., 0.25-mm film thickness) from SGE, Australia. A model AOC-20iShimadzu autosamplerwas used to make the injections onto the GO. A A React-Therm TM Dry Block Sample Derivatization System (Pierce,Rockford,IL) was used to derivatize the DAP metabolites, and a microproBPX5 Column cessor-controlledfreeze-dryinginstrument, DuraTopTM from FTS Systems, Inc. (Stone Ridge,NY) was used to freezedry the urine samples. An Eppendorf MultipettePlus pipettor was used for transferring small aliquots of liquids and 16e mL fiat-bottom Wheatonglass vials (21 x 70 ram) from Alltech AssociatesPty Ltd. (Cat. No. 988661, Baulkham Hills, Australia) were used for the urine samples.
L 5
M 10
15
20
Time (rain)
c:'~chrOma-l',OOnovI 7~Jhi.O01.ChannelB
B BPX50 Column
Z 10
7s
.... 15
Time (rain)
Figure 2. Typicalchromatogramsof a blankurinesampleon BPX5(A) and BPX50(B) columns. 154
Chemicals Acetonitrile (0.006%water content), OmniSolv brand, was obtainedfrom EM Science (Gibbstown, NJ). Anhydrouspotassiumcarbonatewas obtained from Fluka ChemieAG (Buchs, Switzerland),and pentafluorobenzylbromide (PFBBr) in 5-mL ampoules was obtained from Merck (Darmstadt, Germany). Five of the DAPmetaboliteswere difficult to obtain locally,so we had them synthesized at the Moscow State University, Department of Chemistry. They were DMP and DMTP as the sodium salts (both 98% purity) and DMDTP, DETP,and DEDTPas the potassium salts (all three with 98% purity). The sixth metabolite, DEP,was obtained in the free form from ChemService,Inc. (WestChester, PA)with 98.1% purity,and dibutylphosphoric acid (DBP),the internal standard, was also obtained from ChemService with 97.4% purity. GC operating conditions The oven temperature was programmed to start at 120~ and increase at a rate of 7~ to 290~ The injector port was set at 240~ with 2-pL aliquots of samples and standards being
Journal of Analytical Toxicology, Vol. 25, April 2001
r162
roll- 1~C0nr
A
1, Channel A
BPX5 Column
injected onto the GC. The top parts of the flame photometric detectors were set at 240~ and the bottom parts at 190~ Helium (ultra high purity grade from BOC Gases, Sydney, Australia) was used as the carrier gas at a head pressure of 146 kPa. This is equivalentto a flowrate of 3.0 mUmin or an average linear velocity of 60 cm/s. The detector gases used were air at 100 mL/min and hydrogenat 100mL/min.Nitrogenwas used as the makeup gas at 80 kPa.
Freeze dryer operating conditions The Dura-Topfreeze dryer was operated in the 'program' mode with four stages of operation as set out in Table I. lO
PFBBrderivatization reagent working solution
T i m e (min)
PFBBrampouleswere stored in the refrigerator. Before making up the PFBBr derivatizationsolution, the ampoule was allowed to equilibrate to room temperature and then opened carefully in the fume cupboard. (Caution: PFBBr is a potent lachrymatorand should only be handled in a fume cupboard.) The 5 mL of PFBBr from the ampoule was mixed with 15 mL of acetonitrile, and this working solution was stored in a glass vial with a Teflon liner in the refrigerator.The solution was found to be stable for six months.
c:~=hn:~ma- l'~OOnovl 7~2.001, Channel e 751
BPX50 Column
o;
,
I
u
5
10
15
0
T i m e (min)
Figure 3. Typical chromatogramsof a blank urine samplespiked at the following levels: 1500 pg/L DMP, 750 pg/]- DEP,and 300 pg/L each of DMTP, DMDTP, DETP,and DEDTPon BPX5(A) and BPX50(B) columns.
(;:~hroma-1 ~00novl 7~240g. Channel A
N
BPX5 Column
o ,4 to
o
5
10
Time
15
(rain)
Figure 4. Typicalchromatogram of an occupationally exposedworker's urine sample.
Preparation of standard solutions Stock standard solutions. Individual1000-mg/L stock standard solutions of the DAP metabolites were prepared by weighing out 25 mg of each of DMTP, DMDTP, DEP, DETP, and DEDTP, dissolving in acetonitrile,and making up to 25 mL in a volumetric flask with acetonitrile. DMP-Na, which has a lower solubility in acetonitrile, was made up byweighingout 10 mg and making up to 100 mL with acetonitrile. The internal standard (ISTD) stock solution was made up by weighing out 25 mg of dibutylphosphoricacid, dissolvingit in acetonitrile,and then making up to 25 mL in a volumetric flaskwith acetonitrile. Mixed standard solution. A mixed standard solution that had the following concentrations was prepared: 50 mg/L DMP,25 mg/L DEP,and 10 mg/L DMTP, DMDTP, DETP and DEDTP. One milliliterof the stock standard solutions of DMTP, DMDTP,DETP,and DEDTPand 2.5 mL of the DEP stock standard solution were transferred by pipette to a 100-mLvolumetric flask.Also, 50 mL of the DMP stock standard solution was transferred to the same 100-mLvolumetric flask and the contents made up to 100 mL with acetonitrile. Internal standard solution. A 10-mg/Lsolution of DBP was prepared by taking a 1-mL aliquot of the ISTD stock solution and making it up to 155
Journal of Analytical Toxicology, Vol. 25, April 2001
100 mL with acetonitrile in a volumetric flask. Working standard solutions. Approximately 5-mL aliquots of blank urine from unexposed persons with no detectable amounts of DAP metabolites were pipetted into each of three 10-mL volumetric flasks. To this urine was added 500 IJL of the 10-mg/L DBP internal standard solution. To each of the three flasks was then added 100 lJL, 300 pL, or 500 IJL of the mixed standard solution to give three levels of working standard solutions as shown in Table II. The solutions were made up to the mark with the same blank urine, capped, and mixed thoroughly. These working standard solutions were freshly prepared with every batch of urine samples to be tested. Duplicate 2-mL aliquots of these three working standard solutions were placed into the 16-mL fiat bottom glass vials ready for freeze drying. Sample preparation One-hundred microliters of the 10-mg/L DPB internal standard solution and a 2-mL aliquot of the urine sample were added to a 16-mL glass vial in an appropriate rack. Once all of the urine samples had been prepared in this way, the racks containing the sample vials and the standard solutions were placed inside the freeze-dryer chamber. Thermocouples nos. 1 and 2 were placed inside two blank urine vials, and the freeze dryer chamber was closed and operated according to the parameters set out in Table I and the manufacturer's instructions. The freeze drying of samples, blanks, and standards was done overnight without any vial caps on and was completed in 19.5 h.
all the freeze-dried samples. This was followed by the addition of 30-50 mg of anhydrous potassium carbonate to each vial using a spatula. Next, 100 IJL of the PFBBr derivatization reagent solution was added to all vials using the Eppendorf Multipette Plus Pipettor. The vials were all sealed using Teflon-lined screw caps and placed into a preheated dry heating block for 4 h at 60~
Sample analysis After the heating step was completed the vials were removed from the heating block, shaken vigorously for 10 s and then placed into the vial racks and allowed to come to room temperature (about 30 rain). Using a Pasteur pipette, the top layer of each vial was transferred into a 1-mL glass GC vial, taking care not to disturb the sediment on the bottom of each vial. The GC vials were capped, and an aliquot of the solution was injected onto the GC according to the conditions described in the Equipment section. (Caution: Care should be taken when disposing of any waste PFBBr reagent, any urine or any vials/caps that have come into contact with this reagent.)
Results and Discussion
Quantitation of the DAP metabolites was performed using a three-point calibration for each analyte using the three levels of working standard solutions with DBP as the internal standard. Results were reported as the mean of the two simultaneous deterSample derivatization minations from the BPX5 and BPX50 columns. The method was The following steps had to be performed as soon as possible as found to be linear up to 5000 pg/L for DMP, 2500 lJg/L for DEE and 1000 tJg/L for DMTP, DMDTP,DETE and DEDTP. lyophilized urine samples are very hygroscopic and can easily become 'soggy' or 'gummy' when exposed to normal atmospheric The method detection limits (MDL) were calculated based on conditions, which prevents the derivatization process from the level of noise plus three standard deviations of that noise, in occurring properly. Two milliliters of acetonitrile were added to blank urine samples (n = 6 over 5 months). The levelsfound were 50 tJg/L DMP, 10 IJg/LDEE 101Jg/LDMTP,10 tJg/L Table III. Four Months Precision and Reproducibility Data of Spiked Urine DMDTP,5 IJg/L DETP, and 5 1Jg/Lfor DEDTP. The Samples (n = 5) limits of reporting (LOR) for the metabolites were 200 ]Jg/L DMP, 100 lJg/L DEE and 25 IJg/L for DMP DEP DMTP DMDTP DETP DEDTP DETE DEDTP, DMTP,and DMDTP.The LOR were (,g/L) (,g/L) (,g/L) (,g/L) (,~/L) (Ig/t) set at a level to prevent the reporting of environmental background levels of the DAPmetabolites. Level 1 100 In our laboratory, urinary results with creatiTruevalue 500 250 100 100 100 103 nine levels of between 0.004 and 0.027 mol/L are Determination 463 241 118 102 93 24 creatinine corrected (5), and the DAP metabolites SD 28 13 28 20 20 23.0% are reported as micromoles per mole creatinine. CV 6.1% 5.4% 23.4% 19.6% 21.8% Results falling outside this creatinine range are Level 2 reported uncorrected as micromoles per liter. Truevalue 1500 750 300 300 300 300 Typical chromatograms of a blank, a spiked, and Determination 1376 747 296 293 295 304 an occupationally exposed worker's urine are SD 154 18 39 41 42 37 shown in Figures 2-4. Figure 2 shows typical CV 11.2% 2.4% 13.1% 13.9% 14.2% 12.0% chromatograms of a blank urine sample run on the BPX5 and BPX50 columns. Although a small Level 3 5oo peak corresponding to DEDTP in retention time Truevalue 2500 1250 500 500 500 5o4 was detected on the BPX50 column, this was not Determination 2675 1332 479 485 511 52 confirmed on the BPX5 column. An even smaller SD 170 45 50 60 62 10.4% peak also corresponding to DMTP in retention CV 6.4% 3.4% 10.5% 12.4% 12.2% time was detected on both columns. However,this 156
Journalof AnalyticalToxicology,Vol. 25, April 2001
background level was below the LOR for this method. This high* lights the benefits of running the samples simultaneously down two columns for confirmation of positive results. Figure 3 shows a blank urine sample spiked at the following levels: 1500 IJg/L DMP, 750 IJg/L DEP, and 300 lJg/L for DMTP, DMDTP, DETP, and DEDTP on both the BPX5 and BPX50 columns. Figure 4 shows a chromatogram from an occupationally exposed worker's urine. The sample was collected at the end of the shift from the worker who had been using chlorpyrifos on that day. The metabolites DETP and DEP were both found in the urine. DETP was found at a level of 520 IJg/L, and DEP was below the LOR for this method. DMTP was also detected at a similar background level to the blank urine sample. The novelty of this method is that it combines two approaches or techniques that have been used by past workers and consolidates them into one simple procedure that can be used for routine analysis. The problem of isolating the polar DAP metabolites from water so that derivatization can readily occur is solved by the use of freeze drying. This enables the samples to be processed overnight and ready for analysis the next day. Lengthy extraction and cleanup procedures are avoided. Use of the freeze-drying technique to produce a dry urine extract also makes the technique amenable to a derivatization reaction occurring in the preferred solvent, acetonitrile. The PFBBr derivatization was chosen over the benzyl derivatization procedure described by Peterson (4) because the latter technique produced multiple derivatives of some of the DAP metabolites and hence made the analysis less sensitive. Although the detection limits of this method are not as low as those of Moate et al. (1), Aprea et al. (2), Nutley and Cocker (3), and Hardt and Angerer (6), the method has still been found to be very useful in our laboratory when monitoring workers exposed to organophosphorus insecticides. The current test used to monitor exposure to organophosphorus insecticides is the cholinesterase enzyme activity assay in blood. This test is routinely performed in our laboratory and often gives enzyme activity levels where there are no significant differences between the pre- and postexposure results. However, appreciable levels of DAP metabolites in the urine of the exposed workers are detected. The precision data for the test method are shown in Table III. This was determined by the analysis of spiked urine samples at three concentration levels. The samples were analyzed in dupli-
cate on different days (n = 5) over a four-month period by different analysts using the same equipment.
Conclusions The main advantage of this method is the simplicity of the sample preparation side of the procedure. This has been mainly due to the use of a freeze dryer, which greatly reduces the amount of time spent by analysts in preparing the urine samples for analysis. The method was developed for the purpose of routine analysis of urine from workers occupationally exposed to organophosphorus insecticides. We have found that the level of reporting of this method has been sufficient for determining a wide range of exposures.
References 1. T.F. Moate, C. Lu, R.A. Fenske, R.M.A. Hahne, and D.A. Kalman. Improved cleanup and determination of dialkyl phosphates in the urine of children exposed to organophosphorus insecticides. J. Anal. Toxicol. 23:230-236 (1999). 2. C. Aprea, G. Sciarra, and L. Lunghini. Analytical method for the determination of alkylphosphates in subjects occupationally exposed to organophosphorus pesticides and in the general population. J. Anal ToxicoL 20:559-563 (1996). 3. B.P. Nutley and J. Cocker. Biological monitoring of workers occupationally exposed to organophosphorus pesticides. Pestic. Sci. 38: 315-322 (1993). 4. J.C. Peterson. Improved analysisofthe alkylphosphate metabolitesof organophosphate pesticides in human urine. Paper presented at the International Symposium on Biological Monitoring in Occupational and Environmental Health, 11-13 September 1996, Espoo, Finland. 5. Threshold Limit Values for Chemical Substances and Physical Agents. Biological Exposure Indices. American Congress of Governmental Industrial Hygienists, Cincinnati, OH, 2000. 6. J. Hardt and J. Angerer. Determination of dialkyl phosphates in human urine using gas chromatography-mass spectrometry.J. Anal Toxicol. 24:678-684 (2000). Manuscript received March 2, 2000; revision received December 7, 2000.
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