Anal. Chem. 1983, 55, 91-94
It is interesting to note that there is no correlation between published data for relative oscillator strengths of Sm lines (9) and the relative intensities of the Sm RIMS peaks. Because of the power level of our laser system and the optical arrangement necessary to transmit the laser beam into the mass spectrometer, optical saturation of the ionization was not realized, and one might assume that the RIMS signals would be some function of oscillator strength of the respective transitions involved. There are certain deficiencies in our intensity monitoring procedure. With time, the concentration of the thermal sample changes. Our evaluation of laser dye intensity over the three dye ranges is only qualitative. Even within a given dye range there is, perhaps, 30% variation possible in signal strength measurement caused mainly by statistical variation in the small number of RIMS ions generated. As a result of the above reasons, or because of presently undefined reasons, there is little if any correlation of relative oscillator strength and RIMS signal. Compare, for example the strong intensity of a RIMS peak at 435.6 nm and a weak RIMS peak at 438.0 nm with reported relative oscillator strengths of 28.8 and 330, respectively (9). This observation is interesting, but until more precise control of the
91
experimental parameters is possible, no real conclusions can be drawn. LITERATURE C I T E D Hurst, G. S.; Payne, M. G.; Kramer, S. D.; Young, J. P. Rev. Mod. Phys. 1979, 5 1 , 767. Youno. J. P.: Hurst. G. S.: Kramer. S. D.: Pavne. M. G. Anal. Chem. 1979y51, 1050A. Worden, E. F.; Conway, J. G. ACS Symp. Ser. 1980, No. 131, 381-425, and references therein. Donohue, D. L.; Young, J. P.; Smith, D. H. I n t . J. Mass Spectrom. Phys. 1982, 4 3 , 293. Martin, W. C.; Zalubas, R.; Hagen, L. Natl. Stand. Ref. Data Ser. ( U . S . , Natl. Bur. Stand.) 1978, NSRDS-NBS 60. Meggers, W. F.; Corllss, C. H.; Scribner, B. F. NBS Monogr ( U . S . ) 1975, No. 145. Blaise, J.; Wyard, J. F.; Hockstrd, R.; Kruiver, P. J. G. J. Opt. SOC. Am. 1971, 61, 1335. Parr, A. C.; Inghram, M. G. J. Opt. SOC.Am. 1975, 6 5 , 613. Kamarovskli, V. A,; Perkin, N. P.; Nikiforova, G. P. Opt. Spectrosc. 1970, 2 9 , 116.
RECEIVED for review July 2, 1982. Accepted September 27, 1982. Research sponsored by the U.S. Department of Energy, Division of Chemical Sciences and the Office of Health & Environmental Research, under Contract W-7405-eng-26with Union Carbide Corp.
Determination of Organic-Bound Chlorine and Bromine in Human Body Fluids by Neutron Activation Analysis dames D. McKinney" National Institute of Environmental Health Sciences, Research Trlangle Park, North Carolina 27709
Adel Abusamra and John H. Reed Science Applications, Inc., 4030 Sorrento Valley Blvd., San Diego, California 9 2 12 1
The levels of organic-hound chlorine and bromlne In human milk and serum are determlned by neutron activation analysis. Desalted milk and seruim fractions are Irradiated wlth neutrons In a nuclear reactor and the resultlng y-rays of 38CIand "Br are measured. The dosaltlng procedure, achieved by using Blo-Gel molecular sleves, virtually removes all lonlc chlorlde and bromides from milk and serum. Radloactlve tracer studles wlth polychlorinated blphenyl-14CIndicate a recovery of 90% through the Blo-Gel column. The total organlc chlorlne In 2,2-(4-chlorophenyl)-l,l-dlchloroethene spiked milk and heptachlor spiked milk, determlned after being desalted and lrradlated accordlng to thls procedure, substantlates a good recovery of the added spike. The lower limits of detection of organlc-bound chlorine and bromine In milk or serum are 50 and 5 parts per bllllon (ppb), respectlvely.
The level of organic-bound chlorine (TOCl) and bromine (TOBr) compounds has been rising in the environment due to the growing commercial use of large quantities of halogenated hydrocarbons a n d the previously unregulated dumping of organic waiik. Halogenated hydrocarbon residues in breast milk or serum are normally measured by a combination of solvent extraction and gas chromatography with electron capture detection (GC/EC) (2). Identification and measurement of the full spectrum of halogenated organic 0003-2~00/83/0355-0091$01.50/0
compounds become a difficult task. A value for TOCl and TOBr can be produced by using neutron activation analysis on desalted milk and serum fractions. This information is valuable in answering questions regarding the total amount of such compounds present in human milk or serum. For determination of TOCl and TOBr in milk or serum, ionic species such as sodium chloride (NaC1) and sodium bromide (NaBr) must be removed completely by a desalting process before neutron activation is performed. Neal and Florini used Sephadex G-25 as a molecular sieve to remove NaCl from serum, utilizing batch extraction with a centrifuge (2). Uziel and Cohen used gel filtration for desalting certain nucleotides (3). Ludkowitz and Heurtebise determined protein-bound iodine in serum, through neutron activation, after desalting with ion exchange resins ( 4 ) . Fritz and Robertson determined protein-bound trace metals in serum by using neutron activation after gel filtration with Bio-Gel P-6 ( 5 ) . The nuclear activation process does not differentiate between inorganic chlorides and chlorine bound to organic molecules. Also neutron capture can recoil organically bound chlorine into free atoms through the Szilard-Chalmers reaction (6). Preirradiation separations of ionic chlorides and bromides from organic-bound chloride and bromine species are therefore necessary. The separation of inorganic sodium chloride and other ionic species in milk and serum from the organic-bound halogens associated with the protein and lipid fractions can be accom0 1982 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 1, JANUARY 1983
plished with Bio-Gel filtration chromatography. Bio-Gel P-2, a product of the Bio-Rad Corp., Richmond, CA, is neutral, hydrophilic, porous, poly(acry1amide) bead material with the property of retarding the movement of small ionic species relative t o the movement of large globular protein macromolecules. In effect, the larger molecules are eluted first and isolated from the ionic fractions, such as Na+ and C1-. The separation is virtually complete as verified by tracer experiments. EXPERIMENTAL SECTION Apparatus. The chromatography column consists of a 1.1 X 25 cm glass column with a Teflon stopcock. A glass-wool plug is placed a t the bottom of the column to prevent Bio-Gel from slipping through. Bio-Gel P-2, hydrated for 4 h in a 0.1 N NH4N03 solution, is slurried into the glass column to fill a height of 15 cm. Columns are eluted with 100 mL of 0.1 N “,NO3 prior to loading the sample. Prior to being desalted, human milk is emulsified with a 3/4-in. titanium probe of an ultrasonic generator unit operated a t 100 W. The Model 1510 ultrasonic unit used is produced by Braunsonic Corp., San Francisco, CA. The probe of the unit is washed with acetone and distilled water after each operation. Nuclear Reactor. The nuclear reactor of the University of Missouri at Columbia, with a neutron flux of 5 X 1013(n/cm3)/s was used for neutron activation analysis (NAA). At the reactor, the radiochemistry laboratories are equipped with a pneumatic tube system which permits the irradiation of samples for a preset length of time and then returns them to the laboratory for further analysis. The y-ray spectrometer consists of a 45-cm3 Ge(Li) detector used with a 4096-channel pulse height analyzer for counting and measuring the radioactivity. Reagents. Bio-Gel P-2 (size 100-200) mesh was used throughout the work. Reagent-grade ammonium nitrate was obtained from Malinkrodt Chemicals, St. Louis, MO bovine serum albumin (BSA) Cohn fraction V from Sigma Chemicals, Inc., St. Louis, MO and ultrapure water from Arrowhead Water Co., Los Angeles, CA. The water was distilled, deionized, treated by reverse osmosis, and bottled in 5-gallon clean polyethylene bottles. Polyethylene irradiation vials of 6 mL capacity were obtained from Olympic Plastics Inc., Los Angeles, CA. Cleaning of Glassware and Vials. Glass beakers and columns were cleaned with 6 N “OB, high-purity water, reagentgrade acetone, and high-purity water and then dried under a laminar flow hood. Traces of chlorides and bromides were cleaned from the polyethylene irradiation vials by placing them on a mechanical shaker inside a polyethylene bottle and washing once with 6 N HN03 for 6 h, three times with high-purity water for 3 h, once with reagent-grade acetone for 6 h, and three times with high-purity water for 3 h. Washed vials were placed in a clean glass tray inside a drying oven set a t 70 “C for 6 h to dry. Dry vials were stored inside closed polyethylene bags until time of use and were handled with clean tongs. General Procedure, One and two-tenths milliliter of human milk was transferred to a clean 10-mL glass beaker and 100 pL of 20% bovine serum albumin (BSA) and 30 pL of 3 N “,OH were added. The mixture was emulsified for 10 s with the titanium probe. One milliliter of the emulsified mixture was passed through a 1.1X 15 cm Bio-Gel P-2 column, with 0.1 N NH4N03as eluant. The eluted milky fraction (2.5 mL) was collected and sealed in a precleaned polyvial and irradiated for 4 min at a neutron flux of 5 x 1013(n/cmz)/s. After irradiation, the outside of the vial was washed with a 7 M HN03 solution and rinsed with water. A needle syringe containing a 2 mL solution of 1%NaOH, NaC1, and KBr was injected into the vial containing the irradiated milk. The added NaCl and KBr act as carriers for any recoiled C1 and Br atoms while NaOH reacts chemically with any chlorine or bromine in the free state. The total content was transferred to a counting vial and counted for 3 min. The 1642-keV y-ray of 38Cland the 617-keV y-ray of 80Br are measured and compared to a standard solution containing 5 pg of C1- and 0.54 pg of Br-, irradiated, and counted under similar conditions. Milk substitutes such as Similac and Enfamil were treated the same as milk. However, BSA was not added to human serum, and it was not emulsified with an ultrasonic unit before desalting.
BSA, added to milk, acts as a binder to water-soluble herbicides of chlorine-bound organic structure. The process of eluting milk or serum through Bio-Gel P-2 columns takes 15 min; three milk or serum samples can be desalted simultaneously. Tracer Experiments with Polychlorinated B ~ p h e n y l - ~ ~ C , 2,4,5-Trichloropheno~yacetic-~~C Acid, and Sodium Chloride-38CI.Fifty milliliters of milk was placed inside a 100-mL beaker and was emulsified with the probe for 3 min. A I .25-pCi portion of polychlorinated biphenyls (PCB-14C,31 mCi/mmol) dissolved in 170 pL of dimethyl sulfoxide was added. The milk was mixed and emulsified again for 3 min more. The spiked milk containing 25 nCi/mL was desalted on Bio-Gel P-2 as described in the procedure. The eluted milk fraction was collected in a liquid scintillation vial containing Riafluor liquid scintillation cocktail and was p counted. By cornparisan of the results to reference standards, 92% of the original spike was found to be recovered. Similar tracer experiments were performed with a spike of 2,4,5-trichlorophenoxyacetic-14C acid (2,4,5-TJ4C). Carbon-14 tracer experiments, carried out in quadruplicate, were extended to serum and Enfamil. It was found in preliminary experiments that the addition of 100 pL of 20% BSA to human milk prior to desalting is necessary to aid the binding of 2,4,5-T. One millicurie of Na3*C1in 2 mg of NaCl was mixed with 1.2 mL of human milk and desalted according to the procedure previously outlined. The desalted milk fractions were y-counted for 38Cl content and were compared to the 38Clvalues of the original spike. Recovery of 2,2-(4-Chlorophenyl)-l,l-Dichloroethene (p,p’-DDE)and Heptachlor from Spiked Milk. Milk was also spiked with p,p’-DDE. The spiked milk was measured with NAA to contain 33 pg p,p’-DDE/mL. Aliquots of spiked milk were diluted with unspiked milk in duplicate to contain p,p’-DDE equivalent to ll3, ‘I6,‘Il,,, 1/30, and of the original spike per milliliter of milk. Each of these spiked milk samples were desalted on Bio-Gel P-2 columns and analyzed with neutron activation for total organic-bound chlorine content. For quality assurance, external (blind) heptachlor spiked milk samples containing the equivalent of nominally 0.5, 1.0, 1.5, 2, and 3 Kg of TOCl were introduced as a part of total samples supplied for analysis. Five samples of each spike level and 25 nonspiked controls were analyzed for TOCl content. 7-ray Spectral Analysis. The computer program GRPANI, was employed to process the magnetic tapes containing the y-ray spectral data (7). GRPANL is a general purpose peak-fitting program that validates the y-ray energy and calculates the intensities for the y-ray lines of interest. In addition, GRPANL estimates the errors associated with the calculated intensities. An iterative least-squares procedure is used in the fitting process since the peak position and peak-shape parameters enter nonlinearly into the peak fitting algorithm. The y-ray intensities were then corrected for the various experimental parameters such as: chemical yield of the preirradiation procedure, mass of irradiated sample, duration of the neutron irradiation, delay between irradiation and sample counting, count time, analysis “dead time”, and blank (vials and reagents) contributions. The corrected intensities were then compared to intensities of y-ray lines from standard solutions of chlorine and bromine irridiated along with the unknown sample. RESULTS AND DISCUSSION Because of the low levels of TOCl and TOBr being analyzed, extreme care must be exercised to keep blank values low. Irradiation vials and pipet tips were handled with clean metallic tongs; clean surgical gloves were worm during laboratory operations. Table I indicates the range of chlorine and bromine in nanograms contributed from polyethylene irradiation vials, ammonium nitrate buffer solution, and (20%) BSA added t o each gram of milk sample analyzed. Blank values were used t o correct the results from each analysis. Internal laboratory quality controls and external quality assurance samples were analyzed as part of laboratory procedures t o check accuracy and consistency of results. With each 350 samples of milk or serum, five quality control rep-
ANALYTICAL CHEMISTRY. VOL. 55, NO. 1. JANUARY 1983
200
400
600
800
1000
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o
Y
0
-
izno
MOO
1600
1800
2000
Channel Number
Flgure 1. y-ray spectrum of desaited human milk.
Table I. Chlorine and Bromine Levels in Blanks and Reagents amt, ng blanks CI 12-15 15-30 0.1 N ",NO, irradiation vials 10-22 BSA (20%) solution 61
w
Br 3-5 4-7 2-4 56
Table 11. TOCl Level in Pooled Human Milk and Serum pooled human serum pooled human milk amt of amt of amt of amt of TOCI, TOBr, TOCI, TOBr, ppm ppm ppm ppm 0.063 0.12 0.92 0.64 0.93 0.061 0.66 0.15 0.79 0.062 0.76 0.15 0.66 0.056 0.56 0.15 0.97 0.063 0.61 0.13 av 0.65 t 0.07 0.14 f 0.014 0.69 t 0.07 0.062 * 0.03 licates of each of the following were analyzed: pooled human milk, pooled human serum,heptachlor-spiked pooled human milk, heptachlor-spiked pooled human serum, sample duplicates of human milk, sample duplicates of human serum, bovine serum albumin, 20% solution, clean, empty polyethylene irradiation vials, ammonium nitrate eluting buffer solution, and ultrapure water. Tables I1 and 111show replication of results of some of the quality controls cited above. Tracer experiments with PCB-"C and 2,4,5,-T-"C were performed to measure the percent recovery of organic-hound chlorine compounds through the d d t i n g process on Bio-Gel P-2. A PCB-"C mixture containing approximately 54% by weight chlorine to simulate Aroclor 1254 was used to spike pooled human milk. A recovery of greater than 90% was demonstrated for both PCB and 2,4,5-T in human milk and milk suhstitutea carried through the desalting procedure. The recovery data for p,p'-DDE shown in Table IV also demonstrates the reproducibility of the recovery over a wide concentration range. The experiments with N a W l demonstrated the ability of the Bio-Gel P-2 column to separate inorganic chloride ions in milk from the TOCl fractions. The eluted milk fraction
Table 111. TOCl Level in Heptachlor Spiked Milk and Serum pooled milk pooled serum amt of TOCI, ppm amt of TOCI, ppm 6.45 5.26 6.39 4.92 6.82 4.84 6.53 5.52 6.56 4.40 av 6.55 t 0.16 5.0 f 0.42 Table IV. Recovery of p,p'-DDE from Spiked Milk IIg of CI due to P.P'-DDE ratio of TOCl measd calcd measd to calcd S 14.70 14.70 1.0 7.35 1.13 s/2 8.35 s/3 4.92 4.90 1.0 2.45 1.19 S/6 2.93 S/lO 1.56 1.47 1.06 S115 1.08 0.98 1~1n ., .... 5/20 0.73 0.73 1.0 S/30 0.50 0.49 1.02 S/40 0.36 0.94 0.34 1.05 + 0.08 ~
~
~
Table V. Separation Factonr of Cl- from Desalted Fluids fraction of eluted fraction spike sample 1x 10-5 human desalted milk 1st mL followed desalted milk 1X 10.' milk 1.6 X 2nd mL followed desalted milk 3rd mL followed desalted milk 2.1 X desalted milk 3.3 x 10.6 cows 1X milk 1st mL followed desalted milk 2 x 10.6 Enfamil desalted Enfamil 1 s t mL followed desalted Enfamil 3 X 10.' 2nd mL followed desalted Enfamil 5 X 10.' 5 x 10.6 human desalted serum 7X serum 1st mL followed desalted serum 2nd mL followed desalted Serum 5X contained only 0.001% of the original N a W 1 spike. In addition, the first eluted milliliters that followed the desalted milk fraction also contained less than 0.001% of the original
94
Anal. Chem. 1083,
Table VI. Chlorine Levels in Eluted Milk Fractions amt of C1, pg/mL fraction (mL) column 1 column 2 1 0.034 0.034 2 0.019 0.015 0.038 3 0.026 4 0.03 0.030 5 & 6* 0.280 0.294 7 0.066 0.064 8 0.3 11.0 9 149.0 23.6 234.0 10 216.0 11 170.0 38.0 12 lost 3.52 13 0.082 0.208 a Desalted milk, Table VII. Recovery of Heptachlor Spike Erom Blind Study p g of TOCl as fig of TOCl as heptachlor heptachlor spike added spike recovered 0.5 0.48 t 0.11 1.0 0.87 f 0.13 1.5 1.36 t 0.14 2.0 1.78 t 0.25 3.0 2.80 t 0.2 Na38C1spike. With NAA, human milk has been found experimentally to contain an average of 0.43 mg of Cl-/g; hence, less than 5 ng of C1- can be expected to carry over to the desalted milk fraction. This amount is considered within the blank values of the operating procedures. This experiment was run in duplicate on human milk, cows milk, Enfamil, and human serum. Results are summarized in Table V. When human milk was desalted according to the described procedure, the results shown in Table VI are typical. The fractions of interest contained 0.294 mg/mL chlorine and were preceded by a low chlorine fraction (0.03 pg) and followed by another low chlorine fraction (0.066 pg). Eluted fractions
55,94-98
containing NaCl appear later in fractions 10 and 11. A second column separation does not substantially improve the desired result further supporting the efficiency of the Bio-Gel column techniques. Figure 1 exhibits a y-ray spectra of neutron-irradiated human milk after desalting on Bio-Gel P-2. The heptachlor-spiked milk samples were submitted to the analyzing laboratory in a blind study to truly test the accuracy and reproducibility of the method. The method showed good reproducibility and accuracy as shown by the results gives in Table VII. TOCl from control milk (0.28 f 0.045 pg) was used to correct the results. CONCLUSIONS This work describes a method which can be automated to a large extent and applied to large numbers of human body fluids for TOCl and TOBr analysis. These levels should be useful in continuing epidemological studies of the exposure of human populations to halogenated hydrocarbons in the environment. The method might also be useful as an ancillary method for determining fluid concentrations of halogenated hydrocarbons in toxicological studies with laboratory animals. ACKNOWLEDGMENT The authors thank Phillip Albro and his staff of the National Institute of Environmental Health Sciences for their help and assistance in this work. The experimental portion of this project was carried out by Science Applications, Inc., San Diego, CA. Registry No. p,p’-DDE, 72-55-9; heptachlor, 76-44-8. LITERATURE CITED (1) . . Biorseth. A,: Lunde, G.: Dvbina. . - E. Bull Environ. Contarn. Toxicol. 1977, 18 (9,581-587. (2) Neal, M. W.; Florlni, J. R. Anal. Blochem. 1973, 55, 328-330. (3) Uzlel, M.; Cohen, W. E. 8/0Chern. 8lophys. Acta 1865, 103, 539-541 -. - . . . (4) Ludkowitz, J. A.; Heurteblse, M. IAEA-SM-157/51, 437-447. (5) Frltz, K.; Robertson, R. J. Radioanal. Chern. 1968, 1 , 463-473. ( 6 ) Szilard, L.; Chalmers R. A. Nature (London) 1834, 132, 462. (7) Gunnink, R.; Ruhter, W. D. GRPANL Lawrence Llvermoore Laboratory: UCRL-52917: Jan 1980.
RECEWED for review February 1,1982. Accepted July 19,1982.
Mathematical Model for Concentric Nebulizer Systems Anders Gustavsson Department of Analytical Chemistry, The Royal Institute of Technology, Fack,
A mathematlcal model for concentric nebullzer systems Is developed. The model Is usable for the calculation of the cutoff dlameter of the nebullzer system, the normal dlstrlbutlon parameters of the aerosol (the droplet dlstrlbutlon) generated by the nebulizer, the efflclency of the nebullzer system, and the aerosol concentration. The model also allows the optlmization of nebullzer systems. The mathematlcal model Is shown-by experlmentslo be In agreement with practlce.
There are very few articles of earlier date that describe theoretical work on nebulizers. But during the past years there has been an increased interest in nebulizers, nebulizer systems, and theory describing the way in which a nebulizer works.
S-100 44
Stockholm, Sweden
This increased interest has resulted in an increased number of papers during later years. Scientists working in spectroscopy have in the last years accepted the fact that it is impossible to investigate excitation sources (e.g., plasmas, flames) without a thorough knowledge of the nebulizer system. If this knowledge is missing it is not possible to take into consideration the influence of the nebulizer system on the measured properties of the excitation source. Thus, it w ill not be possible to separate the properties of the nebulizer system from the properties of the excitation source. Work on the measurement of aerosol dispersions has been published by Browner, Cresser, and Novak (1-4) and by Mohamed, Fry, and Wetzel (5). A paper on the effect of sample temperature in analytical flame spectroscopy has also been published by Browner and Cresser (6),where they have
0003-2700/83/0355-0094$01.50/00 1982 American Chemlcai Society
