Sodium biphenyl method for determination of covalently bound

Fluorine in Organic Compounds and Biological Materials. Pothapragada ... organic fluorine compounds (15,16), the procedures described herein permit a ...
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Anal. Chem. 1982, 5 4 , 1132-1137

Sodium Biphenyl Method for Determination of Covalently Bound Fluorine in Organic Compounds and Biological Materials Pothapragada Venkateswarlu Commercial Chemlcals Divislon/3M, Building 236-GB, 3M Center, St. Paul, Minnesota 55 144

Sodium biphenyl reagent is used to cleave the covalent fluorine bonds in organlc compounds. The fluoride Ions so released are extracted Into a small volume of water and determined spectrophotometrlcaliy or with the fluoride ion electrode. Procedures for micro and macro analyses have been developed. Recoveries of 0.03-500 Mg of fluorine from organlc compounds are quantltative. These methods are simpler, more rapid, and more economlcal than the previously publlshed sodlum blphenyi methods for the determination of fluorine In organic compounds. The present method for determlnation of organic fluorine In blologicai materlals has been validated by recovery studles and by corroborative results of analyses based on (a) an oxygen bomb/GC technlque and (b) an approach Involving radloanalyticai techniques, whereby the dlfficultles, uncertalntles, and inaccuracies of chemical determination of organlc fluorine In a reference method are avoided.

Ever since the recognition of the presence of two forms of fluorine (inorganic and organic) in blood (1,2), there has been an interest in the determination of organic fluorine (covalently bound F) in biological materials such as blood serum. Organic fluorine in such samples is generally determined by subtracting inorganic fluoride from total fluorine. For determination of inorganic fluoride several methods are available (3). For determination of total or organic fluorine, it has been demonstrated that methods based on colorimetric analysis of fluoride in diffusates of unashed samples are inadequate; in most cases, the covalently bound fluorine is not cleaved under experimental conditions (4). The open ashing techniques are subject to errors due to loss of organic fluorine during ashing and contamination with extraneous fluoride. These problems are not encountered in techniques involving confined combustion of samples as with the oxygen bomb (5, 6) and the results so obtained are reliable. However, oxygen bomb combustion procedures are not as rapid to carry out as the other procedures. Lately there has been an active industrial hygiene interest in monitoring blood fluorine levels in large groups of plant workers exposed to fluorochemicals (7,8) and a corresponding need for a more rapid method of analysis. In this context, determination of halogens following rapid reductive cleavage of the covalently bound halogens with sodium naphthalene (9) and sodium biphenyl (10,11) reagents seemed promising. This approach was explored several years ago by this author for the determination of organic fluorine in blood plasma (3)and was found to be unsuitable because of (a) the low levels of organic fluorine in normal plasma, (b) the relatively high fluorine blank of the sodium biphenyl reagent that was then used, and (c) the uncertainties regarding quantitative extraction techniques needed for concentrating the organic fluorine to overcome the reagent blank. However, with the present availability of sodium biphenyl reagent having an adequately low fluoride blank and with increased knowledge of fluorochemical extraction techniques, the sodium

biphenyl method has been now found to be practical for the routine determination of organic fluorine in blood serum of industrial plant workers exposed to fluorochemicals. Compared to the previously published sodium biphenyl methods for the determination of fluorine in organic compounds (12, 13), in petroleum products (14), and in experimental biological samples having significantly high levels of organic fluorine compounds (15,161, the procedures described herein permit a smaller sample size, are simpler and more rapid to carry out, and are more economical. Further, the problem of low recoveries encountered by some in the analysis of certain fluorochemicalsby the sodium biphenyl method (15) has been identified and resolved in this study. The fluorine after reductive cleavage with the sodium biphenyl reagent may be determined spectrophotometrically or with the fluoride ion electrode depending on the fluorine levels of the samples, the nature of the matrix, the interferences, the available laboratory facilities, and personal preferences. These various procedures are described in this paper. EXPERIMENTAL SECTION Reagents. Reagent grade chemicals and double distilled deionized water were used throughout. Sodium biphenyl reagent, flash point (closed cup) 30 "C, was obtained from Southwestern Analytical Chemicals, Inc. Perchloric acid (Mallinkrodt) used was 70%. Sodium fluoride stock solution, 1000 ppm F, was prepared by dissolving 2.2105 g of NaF in 1 L of water. This solution was appropriately diluted t o obtain sodium fluoride solutions of desired strength covering the range of fluorine in the test samples. Sodium acetate buffer (pH 4.8) was prepared by adding 5 mL of 10 M acetic acid to 5 mL of 5 M sodium hydroxide solution. AMADAC-F solution was a 10% solution in water of "AMADAC-F" reagent for fluoride obtained from Burdick and Jackson Laboratories, Inc. This reagent is a blended mixture of a lanthanum salt, alizarin complexan, and a buffer. Apparatus. The equipment used included 5-mL polypropylene stoppered tubes (Falcon tube 2063), 10-mL polystyrene disposable tubes (Falcon tube 2001), Orion pH meter (Model 701A),Orion switch box (Model 605), Orion fluoride ion electrodes (Model 94-09) and Orion reference electrodes (Model 90-Ol),Leitz photometer (Model M), mutlitube vortexer (Scientitic Manufacturing Industries, Model 2600), and ultrasonic cleaner (Mettler Electronics). Procedures. (A) Fluorine in Organic Compounds, of F. To a 1-mL Spectrophotometry,Macro Method, 5-500 diethyl ether solution of the organic compound (5-500 pg of F), in a 5-mL polypropylene test tube (Falcon tube 2063), 1 mL of sodium biphenyl reagent was added, and the tube was stoppered immediately. Following a 10-min reaction time, 1 mL of water was added. After the hydrogen gas has escaped, the tube was restoppered and the contents were vigorously vortexed for 5 min to extract the inorganic fluoride into the aqueous layer. A multitube vortexer was used to process samples and standards. The tube was centrifuged in a clinical centrifuge for 2 min at 2500 rpm. The supernatant was aspirated and the lower aqueous layer was washed twice with ether (0.5 mL each time). The aqueous extract was made up with water to 5 mL by weight or by volume. For the latter, the csntents of the tube were made up with water to the same height as that of 5 mL of water placed in a separate tube. The limits of accuracy of making up the volume by this procedure were 5 0.02 mL.

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0003-2700/82/0354-1132$01.25/00 1982 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 54, NO. 7, JUNE 1982

Blank and inorganic fluoride standards covering the anticipated levels of fluorine in the unknown samples were also processed simultaneously and exactly as above, and made up to 5 mL starting with 1mL of ether, 1 mL of sodium biphenyl reagent, and 1 mL of the fluoride standard solutions (5-500 pg of F/mL). Aliquots of the sample and standard solutions, all of the same volume and none containing more than 4 pg of F or exceeding 500 pL, were pipetted out into 15-mL polystyrene test tubes (Falcon tube 2001). Four milliliters of water was added to each, followed by 1 mL of AMADAC-F solution. Alternatively, different aliquots of fluoride extracts of sample and standard solutions varying from 1to 500 pL were taken, but the volumes of each were made up to 500 pL with the aqueous extract of the blank solution, before addition of 4 mL water and 1 mL AMADAC-F solution for color development. After standing 1 h, the solutions were read on a spectrophotometer (620 nm) either in the above test tubes or, preferably, were transferred to and read in a standard square cuvette for more precise results. The fluoirine content of the sample was calculated from the calibration curve prepared with the fluoride standards. Organic fluorine compounds may be dissolved in an appropriate dry inert solvent which does not contain fluorine or destroy the sodium biphenyl reagent. If a compound is dissolved in a solvent other than ether, the ether in the above procedure should be replaced by the corresponding solvent in processing of the inorganic fluoride standards as well. (B) Fluorine in Organic Compounds, Fluoride Electrode, Macro Method, 1-5 pg of F. To a 1-mL ethyl ether solution of the organic compound (1-5 pg of F), in a 5-mL polypropylene test tube (Falcon tube 2063), 1mL of sodium biphenyl reagent was added; the tube was stoppered immediately. Following a 10-min reaction time, 1 mL of water was added. After the hydrogen gas had escaped, the tube was restoppered and the contents were vigorously vortexed for 5 min to extract the inorganic fluoride into the aqueous layer. The test tube was centrifuged in a clinical centrifuge for 2 min at 2500 rpm. The Supernatant was aspirated and the lower aqueous layer was washed twice with ether (0.5 mL each time). To the above water extract, 100 pL of perchloric acid was added followed by 10 p L of methyl red indicator. The excess acid was back-titrated with 2.5 M sodium hydroxide solution. A 50-pL portion of sodium acetake buffer (pH 4.8) was added, and the contents were brought to 5 mL volume with water. Inorganic fluoride standards were also processed exactly as above starting with 1mlL of fluoride standard solutions (0-5 pg of F/mL), 1mL of ether, and 1mL of sodium biphenyl reagent. The standard and test solutions were monitored with the fluoride ion specific electrode. The fluoride content of the sample was calculated from the calibration curve. (C) Fluorine in Body Fluids and Tissue Homogenates, Fluoride Electrode, Micro Method, 0.01-0.3 pg of F, F in Sample 1-30 ppm. To LO pL of the sample (blood serum), in a 5-mL polypropylene test tube (Falcon tube 2063), was added 0.25 mL of toluene or ether, followed by 2 pL of perchloric acid. The extraction of the fliaorochemical from the sample into the organic liquid phase was achieved by vigorous vortexing of the contents for 15rnin or better by placing the sample in an ultrasonic water bath for 15 min. One milliliter of sodium biphenyl reagent was added and the tube promptly stoppered. After a 10-min reaction time, the contents were vortexed for 15 min. The biphenyl reagent was destroyed by adding 0.25 mL of water. The contents were vostexed vigorously for 5 min and centrifuged at 2500 rpm for 2 min. The supernatant was aspirated and the aqueous layer washed twice with ether (0.5 mL each time). One hundred microliters of perchloric acid was added followed by 10 pL of methyl red indicator. The excess acid was back-titrated with 2.5 M sodium hydroxide (dispensed from an Eppendorf pipet). Ten microliters of sodium acetate buffer (pH 4.8) was added and the volume built to 1mL with distilled water, taking advantage of a faint circle (mold mark) at the 1 mL level. With the aid of background illumination, this faint mark was precisely aligned with a nylon thread (fish line) stretched horizontally in front of the tube. The volume was made up to 1 mL using the fish line as the graduation mark. The limits of accuracy or making up the volume by this procedure were 1 f 0.02 mL.

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Alternatively, the reaction tubes were weighed prior to use and the volume of solutions was built to 0.5 mL or any suitable constant volume by weight. Ten-microliter volumes of inorganic fluoride standards (1, 2, 4,15,20,25, and 30 ppm of F) were also processed simultaneously in the same way and brought to the same volume as the test samples. The standards and the samples were read with the hanging drop fluoride electrode (17) and the fluorine content of samples was calculated from the calibration curve. With prolonged use, as when monitoring a large number of samples for 4-8 h, the adhesion of the Parafilm to the hanging drop electrode seemed to deteriorate, on some occasions developing crevices between the Parafilm and the electrode membrane wherein submicroliter volumes of solutions applied to the electrode were entrapped. This resulted in contamination of samples and standards. To eliminate this problem, the Parafilm was no longer used in assembling the hanging drop fluoride electrode. On some occasions, perceptible sample size errors were encountered while pipelking 10-pLvolumes of blood serum samples with the Eppendorf pipet; the 10-pL samples were, therefore, weighed rapidly on an electronic balance and the exact sample volume was calculated, taking into account the specific gravity of serum (1.026). (D) Fluorine in Body Fluids and Tissue Homogenates, Following Extraction of Fluorochemicals, Fluoride Electrode, Micro Method, 0.01-0.3 pg of F, F in Sample 1 ppm and Less. Blood serum, 0.1-1.0 mL, was acidified with 20-200 pL of perchloric acid in a 5-mL polypropylene test tube (Falcon tube 2063) and extracted, by vortexing vigorously for 5 min with 3 mL of ethyl ether. The contents were centrifuged; the bulk of the ether layer was transferred to another test tube (Falcon tube 2063) and evaporated down to slightly less than 1.0 mL (never to dryness) in a water bath at 50 "C and using a N2jet. Two more extractions were carried out with 1.0 mL of ether each time. The pooled ether extracts were made up to 3.0 mL. After 0.5 g of anhydrous sodium sulfate powder (not granules) was added, the contents were vortexed 5 min. An aliquot of the ether extract (1mL) was transferred to yet another test tube (Falcon tube 2063); 1 mL of sodium biphenyl reagent was added and the tube promptly stoppered. The reaction mixture was processed further exactly as in the preceding micro method, procedure C, and the aqueous fluoride extract after neutralization and buffering was diluted to 1 mL with water. Fluoride standards were processed starting with 10 pL of standard solutions (1-30 ppm F) and 1mL of ether and following the same procedures as for the 1mL anhydrous ether extract of samples. The sample and standard solutions were read with the hanging drop fluoride electrode (17). (E) Covalent Fluorine in Body Fluids Such as Urine Which Have Significantly High Levels of Inorganic Fluoride. The urine sample was checked for pH and carefully examined for any precipitate or sediment which, if found, was dissolved in a minimum amount of dilute hydrochloric acid. The pH of the sample was adjusted to 6. Five milliliters of urine (pH 6) was boiled for 5 min with 50 mg of fluoride-low calcium phosphate (18) and cooled. The volume was restored to 5 mL by addition of an appropriate amount of water; the contents were mixed and centrifuged at 3000 rpm for 5 min. The supernatant was saved. Alternatively, 2 mL of urine (pH 6) was vortexed vigorously with 20 mg of fluoride-low calcium phosphate for 10 min and centrifuged. The supernatant was vortexed with another 20 mg of fluoride-low calcium phosphate and centrifuged. The supernatant, following the two adsorptions was saved. Ten microliters of supernatant, prepared either way, was diluted with 10 pL of TISAl3 I1 (19) and monitored with the hanging drop fluoride electrode to ensure that the sample had been completely defluoridated. The fluoride standards used in this instance were 0-01, 0.02, and 0.04 kg of F/mL. An aliquot of the supernatant was, then, analyzed for covalent fluorine by procedure C or procedure D, depending on the level of covalent fluorine in the sample. The removal of fluoride from normal urine samples by the above techniques is often complete and should require no further

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ANALYTICAL CHEMISTRY, VOL. 54, NO. 7, JUNE 1982

Table I. Recovery of Covalently Bound Fluorine in Selected Organic Compounds by the Sodium Biphenyl Method, Spectrophotometric and Fluoride Electrode Procedures procedure

compound

A. spectrophotometry

Freon E-4 (C,,F,,O,) Freon E-5 (C,,HF,,O,) FOMB YO4 (C,F,O),

B. fluoride electrode macro method

0-fluorobenzoic acid perfluorooctanoic acid ammonium perfluorooctanoate

sample size, pg

pg

429 427 460

426 r 1 8 (4) 437 f 1 0 (4) 464 i. 18 ( 4 )

(N-ethylperfluorooctane-

su1fonamido)ethyl alcohol 1H,1H,2H,2H-perfluorooctanol 1H,1H,2H,2H-perfluorodecanol

C. fluoride electrode micro method

potassium perfluorooctanesulfonate Flecainide acetate: 2,5-bis( 2,2,24rifluoroethyl)-

of F found mean

* std error ( n )

% recovery mean

*

std error ( n ) 99.2 f 4.2 (4) 102.4 i. 2.3 (4) 100.9 f 3.9 (4)

5.65 5.90 5.62 5.54

5.81 f 0.048 (4) 5.86 i: 0.243 (3) 5.96 (5.68, 6.24) 5.36 f 0.21 (10)

102.8 f 0.85 (4) 99.3 f 4.12 (3) 106.0(101, 111)(2) 96.7 + 3.79 (10)

6.72 3.88

6.79 (6.59, 6.99) 3.92 (3.92, 3.92)

101.0 (98,104) (2) 101.0 (101, 101) (2)

0.18 0.06 0.03 0.12

0.171 + 0.009 ( 5 ) 95 f 5.00 ( 5 ) 0.062 (0.06, 0.064) 103.3 (100, 107) ( 2 ) 0.028 (1) 93 (1) 0.11 (0.10, 0.12) 92 (84, 100) (2)

N-( 2-piperidylmethy1)benza-

mide acetatea

a

A therapeutic agent.

Table 11. Recoveries of Covalent Fluorine in Selected High Molecular Weight Fluorochemicals, Comparison of Results Obtained by the Procedure of Clark et al. and by the Present Method (Procedure A ) Clark et al. ( 1 5 )

compd (mol wt) Freon-E4 (784) Freon-E5 (950) FOMB YO4 (1500)

mg of compd/ mL of sodium biphenyl reagent

% recovery

-8 -8 -8

78.8 52.2 10.4

checking for inorganic fluoride. However, in the case of urine samples containing significantly higher levels of fluoride (as when experimental animals are administered selected fluorine compounds), it would be important to check for complete defluoridation of the samples prior to organic fluorine analysis by procedure C or procedure D. RESULTS AND DISCUSSION (A) Organic Fluorine Compounds. Results of fluorine analyses of selected organic fluorine compounds (mostly highly fluorinated) using the sodium biphenyl reagent and different types of analytical instrumentation for final measurement of fluoride and covering a wide range of sample sizes, from 500 pg down to 0.03 pg of F, are shown in Table I. Recoveries of fluorine in all the above cases were in close agreement with the expected values: 99-102% (430-460 pg of F, spectrophotometry, procedure A), 95-106% (3.9-5.9 pg of F, fluoride electrode, procedure B), and 92-104% (0.03-0.18 pg of F, fluoride electrode, procedure C). Using the principle of reductive cleavage of fluorine by alkali metals in organic solvents, Wheeler (12),Jones et al. (13),Clark et al. (15),and Strahm (20) have also demonstrated good recoveries of fluorine in the case of most organic fluorine compounds they tested (sample size equivalent to 7000-17000 pg of F). However, their procedures are not suitable for analysis of covalent fluorine in the microgram and submicrogram amounts in microsamples of organic compounds and biological materials. Whereas the fluorine cleaved from the test sample is diluted several-fold in each successive step in their procedures, it is extracted into as little as 250 pL of water in the present method. I t is through this unique feature of extraction into a small volume of water that we are now able to achieve our primary objective of microanalysis of organic fluorine in organic compounds and biological materials.

present method, procedure A mg of compd/ mL of sodium % recovery biphenyl reagent mean ~tstd error ( n ) 0.61 0.61 0.67

99.2 i 3.9 ( 4 ) 102.4 f 2.2 (4) 100.9 + 3.8 (41

Further, Clark et al. (15) observed that by their procedure, the recoveries in general decreased with increasing molecular weight of the test compound, e.g., they were 90.1, 100.6,91.5, 78.8, 52.2, 8.0, 8.3 and 10.4% in the case of Freon E-1 (mol wt 286), Freon E-2 (mol wt 452), Freon E-3 (mol wt 618), Freon E-4 (mol wt 784), Freon E-5 (mol wt 950), Freon E-9 (mol wt 1614), Freon E-11 (mol wt 1946) (DuPont), and FOMB YO-4 (mol wt 1500) (Montedison), respectively. Because the recoveries of some of these compounds were not quantitative, they proposed calibration curves based on recoveries for analysis of such compounds in blood and tissue homogenates. Three of the above compounds with low recoveries (1040%) were analyzed for organic fluorine by the present sodium biphenyl method (procedure A). The fluorine recoveries were quantitative. These results are shown in Table 11. A cardinal principle underlying the success of the sodium biphenyl method is that the test compound, in its entirety, is in true solution before the biphenyl reagent is added, or at any rate before the biphenyl reagent is subsequently destroyed by the addition of water or 2-propanol or otherwise. While this principle has been ardently followed in all the present procedures, it was comprised in the procedure described by Clark et al. (15). They added 10 pL of liquid fluorochemicalsdirectly to 2 mL of sodium biphenyl reagent and terminated the reaction a t the end of 2 min. It was now tested and found by us that 10 pL of the high molecular weight fluorochemicals (Table 11) do not dissolve completely in 2 mL of 1,2-dimethoxyethane or toluene (solvents in the sodium biphenyl reagent) in spite of ultrasonication for several minutes. Hence it was now inferred that the low recoveries obtained by Clark et al. were due to incomplete solubilization of the fluorochemicals in the solvent prior to treatment with the biphenyl reagent. In light of these circumstances, it is difficult to assess the value

ANALYTICAL CHEMISTRY, VOL. 54, NO. 7, JUNE 1982

Table 111. Recovery of Total Fluorine (10 ppm) in Human Plasma Spiked with Ammonium Perfluorooctanoate by Sodium Biphenyl-Fluoride Electrode Method (Procedure C, Final Volume 0.5 ML) analysis no. 1

2 3 4 5 6 7 8

9 10 11 12 13 1.4 15 16 17 18 19 20 21 22 23 24 mean

sample abs amt of fluorine size, present, pg ILL 2.4 2.8 3.0

3.7 4.6 4.7 4.8 4.9 4.9 !5.4

9.7 10.0 10.0 10.0 10.0

10.2 10.3 10.4 10.7 10.8 10.8 10.9 18.1 1l.1

* std error ( n )

0.024 0.028 0.030 0.037 0.046 0.047 0.048 0.049 0.049 0.054 0.097 0.100 0.100 0.100

0.100 0.102 0.103 0.104 0.107 0.108 0.108 0.109 0.111 0.111

Table IV. Comparison of Results of Analyses of Total Fluorine in Human :Blood Sera by Oxygen Bomb/GC Method and by the Sodium Biphenyl/FIuoride Electrode Method (Procedure C )

% recovery

sample no.

104 98 93 96 99 96 95 95 98 88 97 103 96 102 106 100 100 98 96 97 97 90 98 98 97.4

total F, ppm sodium biphenylfluoride oxygen bomb-GCa electrode 1.4 2.7 2.9 3.9

1

2 3 4 5 6 7 8 9

4.1

10 11

12 13 14

*

2.7 (24)

of calibration curves based on “percent recovery” for the determination of organic fluorine in blood and liver homogenates. In the present studies, a “clear” ether solution of a salt of a perfluorinated acid (potassium perfluoroctanesulfonate) was decomposed with the sodium biphenyl reagent, with a fluorine recovery of only about 50%. Apparently the salt was not truly and wholly solubilized. The salt was converted to the acid by the addition of 2 pL of perchloric acid to 1mL of the above ether solution and vortexed for 2 min to facilitate the solubilization of the acid in the ether, before treatment with the sodium biphenyl reagent. The fluorine recovery was quantitative; thus conversion of a salt of a fluoroorganic acid to the acid form appears to enhance the solubility of the fluorochemical component in the solvent and thereby result in improved recovery of fluorine. (B) Biological Materials. The basic principles (a) conversion of salts into acids and (b) complete solubilization of the fluorochemical prior to treatment with the sodium biphenyl reagent were incorporated in procedures C and D for the determination of organic fluorine in biological materials. A normal human blood plasma sample, spiked with ammonium perfluorooctanoate to a level of 10 ppm total fluorine, was analyzed by the fluoride electrode micro method (procedure C). Twenty-four aliquots of the spiked plasma varying from 2.4 to 11.1HLwere so analyzed. The recovery of fluorine (absolute amount of I? 0.024-0.11 pg) was 97.4 f 4.06 (standard error), n = 24 (Table 111). Twenty-four samples of blood serum from plant workers exposed to fluorochemicals in an industrial environment were analyzed for total fluorine by the sodium biphenyl method (procedure C) and by the oxygen bomb/GC method (6). The results of analyses obtained by the two methods are similar (Table IV). Serum sample 18, Table IV, was analyzed in quintuplicate by the ox,ygen bomb and the sodium biphenyl methods; the results were 8.8 f 0.4 (std dev) and 9.2 0.05 (std dev) ppm F, respectively. One milliliter of sodium biphenyl reagent, as prescribed in

*

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15 16 17 18 19 20 21 22 23 24 a Sample size, 100 p

~

1.8 2.4 2.7 3.4 5.1 4.1 5.6 4.8 7.0 8.2 5.7 5.4 6.6 7.1 7.4 5.8 9.2 7.6 10.5 9.2 10.9 10.9 12.5 13.5

4.6 4.9 5.4 6.4 6.8 7.0 7.1 7.2 7.3 7.7 8.8 8.8 8.9 9.8 10.7 11.6 13.1 13.2 14.5 . Sample size, IO p

~ .

Table V. Comparison of Results of Analysis of Organic Fluorine in Human Blood Sera by Oxygen Bomb/GC Method and by the Sodium Biphenyl/Fluoride Electrode Method (Procedure D)

sample no. 1 2 3 4 5

organic F, ppm sodium biphenyloxygen fluoride bomb-GC electrode 0.1 0.2 0.4 0.4 0.7

0.2 0.4 0.5 0.5 0.8

the present method, tolerates only 10-20 pL of water. The procedure recommends treating no more than 10 p L of the body fluids or tissue homogenates with 1 mL of sodium biphenyl reagent. With this limitation, the lower limit of the method (procedure C) is 1ppm total F in the sample, provided contamination with extraneous fluoride is carefully precluded and the fluoride electrodes are regularly cleaned and maintained in good condition, free from organic deposits on the membrane (19). If less than 1 ppm levels of organic F in biological samples are required to be measured, to obtain more reliable results, recourse is taken t o procedure D in which organic fluorine is extracted from an adequately large sample in amounts which overcome the reagent blank variations and trace fluoride contamination and which yields fluoride ions in concentrations easy to measure accurately with the fluoride electrode. Procedure D was employed for the determination of less than 1 ppm organic fluorine in a limited number of human blood serum samples. Results obtained by this procedure and by the oxygen bomb GC technique are similar (Table V).

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ANALYTICAL CHEMISTRY, VOL. 54, NO. 7, JUNE 1982

Table VI. Comparison of Results of Organic Fluorine Analyses of Body Fluids and Soft Tissues of Rats Administered Certain Radioactive Fluorochemicals by the Sodium Biphenyl Method with the Values Expected on the Basis of Radioactive Analyses of the Samples

14C-fluorochemicaladministered

body fluid or tissue

F ppm found, F ppm based sodium biphenyl on radioactive method (procedure) analysis

(N-ethylperfluorooctylsu1fonamido)ethanol

plasma kidney liver

6.9 (D) 11.8 (D) 749.6 (D)

6.4 10.8 677.3

potassium perfluorooctylsulfonate

plasma kidney liver urine

26.1 (D) 33.9 (D) 85.1 (D) 0.4 (E)

25.9 33.2 84.5 0.4

ammonium perfluorooctanoate

plasma kidney liver urine

16.0 (D) 17.0 (D) 44.9 (D) 4.8 (E)

16.0 15.0 41.4 5.0

Procedure C, in which no attempt is made to remove inorganic fluoride present in the biological material, measures organic fluorine plus inorganic fluoride. If the inorganic fluoride level is relatively insignificant, concentrations of total fluorine and organic fluorine are essentially the same. When necessary, the inorganic fluoride in the sample can be measured directly with the fluoride ion electrode (22)or, in special circumstances, after isolation and further concentration by calcium phosphate adsorption (2)or reverse extraction (17). Inorganic fluoride is subtracted from the total fluorine to obtain the value for covalent fluorine. In procedure D, organic fluorine in the biological sample is extracted into an ether layer. However, not all inorganic fluoride in the sample remains in the aqueous phase. Because of acidification of the sample prior to extraction with ether, a significant amount of the inorganic fluoride, presumably as hydrogen fluoride, also appears in the ether layer along with the organic fluorine. Therefore, unless the inorganic fluoride in the sample is too small to affect the results of organic fluorine analysis, it should be removed prior to extraction of the organic fluorine into the ether layer, as in the case of urine samples. Defluoridation of the samples is readily achieved by taking recourse to calcium phosphate adsorption techniques (2, 18) as in procedure E. The soundness of the present methodology is further demonstrated by an approach involving radioanalytical techniques. This was designed to avoid errors due to difficulties, uncertainties, and inaccuracies inherent in the chemical determination of small quantitaties of organic fluorine by a reference method. Samples of body fluids and tissues of rats that were administered certain 14C-labeledfluorochemicals were analyzed. Results obtained by the present method (procedures D and E) are similar to the values calculated from the radioactive measurements of the samples and the specific activities of the fluorochemicals involved (Table VI). Using their procedure, Stein et al. (26)obtained 36-108% recoveries of different fluorocarbons added to blood and concluded therefrom that each fluorochemical required a different standard curve (based on percent recovery). The procedure prescribes a sample size of 10 mL to measure fluorine in the range of 1-30 ppm. For measurement of fluorine in the very same range, the sodium biphenyl method described herein (procedure C) requires a sample size as small as 10 pL. Further, since the recoveries of covalent fluorine are quantitative in all cases (Tables I, 111, and IV), a single inorganic fluoride standard curve is adequate for all organic fluorine compounds, instead of different standard curves for different compounds as required in the procedure described by Stein et al. (16).

As indicated before, the final measurement of fluoride ions in the present sodium biphenyl method can be made by spectrophotometry or by potentiometry (fluoride ion electrode). In the case of organic fluorine compounds, spectrophotometry provides a fast means of analysis. If the accompanying interfering ions (e.g., as from sulfur and phosphorus if present in the organic compound) exceed the limits of tolerance of the spectrophotometric method and if the available sample size is small and the fluoride level is below the limit of accurate measurement by the method, the final measurement can be made with the fluoride electrode. For measurement of organic fluorine in biological materials, as in the case of blood serum samples, the sodium biphenyl-fluoride electrode method (procedure C) is faster than the oxygen bomb-gas chromatography technique; and 10 samples (in duplicate) per day can be analyzed for organic fluorine by procedure C. Twenty to twenty-five samples (in duplicate) and the fluoride standards are processed in a day and read on the following day with 6-12 fluoride electrodes connected to two pH meters through two electrode switch boxes. An important feature of the present method (procedures C and D) is the microsample size required for analysis. This offers an advantage in a clinical laboratory when a large number of different analyses are to be carried out on a limited serum sample. These procedures should find useful application in investigations with small laboratory animals. Since the technique requires only 20-50 HL of blood, which could be readily obtained by bleeding a superficial vein, it should no longer be necessary to sacrifice animals at selected intervals in a “time study”, as has been the practice so far, for obtaining samples large enough for fluorine analysis by the macro procedures. Trace amounts of fluorochemicals separated on TLC plates (courtesy of A. Mendel and J. E. Gagnon, 3M) have been quantitated by the present method (Table I, procedure C). This method has also been used for locating and quantitating fluorochemical metabolites on TLC plates in studies on flecainide acetate metabolism in humans (personal communication, L. I. Harrison, 3M). ACKNOWLEDGMENT I wish to express my sincere appreciation to Robert A. Prokop for his encouragement, interest, and valuable discussions. I thank Donald F. Hagen and Larry D. Winter for their interest and counsel throughout the studies. Radioactive fluorochemicals used in these studies were prepared by Fred E. Behr and characterized by Larry D. Winter and James D. Johnson. William C. McCormick and Jay T. Hewitt kindly

Anal. Chem. 1982,5 4 , 1137-1 I41

helped with radioactive animal studies. Thanks are also due to Vicki Bunnelle and Gary W. Kirsch for excellent technical help. LITERATURE CITED (1) Taws, D. R. Nature (London) 1968, 217, 1050-1051. (2) . . Venkateswarlu. P.: Slnaer. L.: Armstrona, - W. D. Anal. Biochem. 1971, 42,350-359. (3) Venkateswarlu, P. "Methods of Biochernlcal Analysls"; Gllck, D., Ed.; Wlley/Intersclence: New York, 1977;Vol. 24, pp 93-201. (4) Venkateswarlu, P. Biochern. Med. 1975, 1 4 , 368-377. (5) Venkateswarlu, P. Anta/.Blochem. 1975, 68,512-521. (6) Belisle, J.; Hagen, D. F. Anal. Biochem. 1978, 87,545-555. (7) Grlfflth, F. D.; Long, J. E. Am. Ind. Hyg. Assoc. J . 1980, 4 1 , 576-583. (8) Ubel, F. A,; borenson, S . D.; Roach, D. E. Am. Ind. Hyg. Assoc>.J . 1980, 4 1 , 5814-589. (9) Benton, F. L.; Hamill, W. H. Anal. Chem. 1948, 20,269-270.

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(10) Pecherer, B.; Gambrili, C. M.; Wilcox, G. W. Anal. Chem. 1950, 22, 3 1 1-3 15. (11) Liggett, L. M. Anal. Chem. 1954, 26, 748-750. (12) Wheeler, P. P.; Fauth, M. I . Anal. Chem. 1986, 38, 1970. (13) Jones, 8. C.; Heveran, J. E.; Senkowski, B. 2. J . Pharm. Sci. 1971, 60,1036-1039. (14) Wilson, J. N.; Marczewski, C. 2 . Anal. Chem. 1973, 45, 2409-2412. (15) Clark, L. C.; Wessler, E. P.; Miller, M. L.; Kaplan, S . Mlcrovasc. Res. 1974, 6,320-340. (16) Stein, T. P.; Robbins, W. K.; Brooks, H. B.; Wallace, H. W. J . Biorned. Mater. Res. 1975, 9 , 479-485. (17) Venkateswarlu, P. Anal. Chem. 1974, 4 6 , 878-882. (18) Venkateswarlu, P.; Sita, P. Anal. Chem. 1971, 4 3 , 758-760. (19) Orion Research "Instruction Manual: Fluoride Electrodes"; 1977. (20) Strahm, R. D. Anal. Chem. 1959, 3 1 , 615-616. (21) Venkateswarlu, P. Clln. Chlm. Acta 1975, 5 9 , 277-282.

RECEIVED for review November 25, 1981. Accepted March 1, 1982.

Sampling arid Determination of 2,4-Bis(carbonylamino)toluene and 4,4'-Bis(carbonylamino)diphenylmethane in Air Samuel P. Tucker" and James E. Arnold Natlonal Instltute for Occupational Safety and Health, Clncinnati, Ohio 45226

Separate sampllng and analytical methods for 2,4-bls(carbonylam1no)toluene (2,4-TDI) and 4,4'-bls(carbonylamino)diphenylmethane (MDI) In air were developed. The sampler for 2,4-TDI conslsts of a glass tube Containing two sectlons of glass wool coated wlth the reagent N-[(rl-nltrophenyl)methyl]propanamlne. The sampler for MDI contains a glass fiber fllter Impregnated wlth the same reagent. The dllsocyanates react wlth the reagent to form urea derlvatives whlch are analyzed by hlgh-pressure llquld chromatography. 2,4-TDI vapor at concentrations ranglng from 100 to 3500 pg/m3 can be determined for 10-L air samples. MDI can exlst In both vapor and aerosol forms and can be determined at concentrations ranglng from 80 to 1000 pg/m3 for 10-L air samples.

Exposure to 2,4-bis(carbonylamino)toluene (known as toluene 2,4-diisocyanate and 2,4-TDI) and 4,4'-bis(carbony1amino)diphenylmethane [known as 4,4'-methylenebis(pheny1 isocyanate) and MDI] in air can cause eye, nose, and throat irritation. Some people may become sensitized to TDI and MDI and develop asthmatic reactions. MDI or partly polymerized MDI can cause contact eczema ( I ) . It has been estimated that approximately 1.5 million metric tons of TDI, MDI, and polymers of MDI will be produced in the world in 1982 (2). The present OSHA standards for 2,4-TDI and MDI are 140 and 200 pg/m3, respectively, as ceiling concentrations ( 3 ) . However, NIOSH has recommended standards for TDI and MDI which may permit better worker protection. The recommendations for TDI and MDI, respectively, are 35 and 50 pg/m3 as time weighted averages for up to a 10-h shift of a 40-h workweek and 140 and 200 pg/m3 as ceiling concentrations for any 10-min period of sampling (1). A few well-known methods for determining 2,4-TDI and MDI in air involve sampling with impingers and analysis by

either colorimetry or high-pressure liquid chromatography (4-6). Impingers are cumbersome for workers to wear, and spillage can occur from many impingers. Toluene for use as a solvent for the reagent N - [(4-nitropheny1)methyllpropanamine in impingers is toxic and flammable. Also, there have been restrictions applicable to sending toluene by the U.S. Postal Service (7). A method for isocyanates in air reported by Keller and Sandridge employs a sampling tube containing a solution of N-[(4-nitrophenyl)methyl]propanamineon glass powder and thin-layer chromatography (8). Two different types of samplers have been developed which can collect TDI and MDI in air. The sampling tube for TDI vapor contains two sections of glass wool coated with the reagent N-[(4-nitrophenyl)methyl]propanamine. MDI, which can exist in both vapor and aerosol forms, may be collected inefficiently by the sampling tube. The sampler for MDI contains ti glass fiber fiter impregnated with the same reagent. 2,4-TDI and MDI react with the reagent to form urea derivatives, 2,4-TDlU and MDIU, which are analyzed by highpressure liquid chromatography (HPLC). Although N - [ (4nitrophenyl)methyl]propanamine is somewhat unstable, it reacts with 2,4-'I'DI and MDI rapidly to form urea derivatives in high yield (9). EXPERIMENTAL SECTION Reagents. N-[(4-Nitrophenyl)methyl]propanaminehydrochloride and the urea derivatives 2,4-TDIU and MDIU were synthesized (9). Two crops of 2,4-TDIU were used; mp 137-142 and 127-130 O C . MDIU was recrystallized from benzene and dried in vacuo; mp 161-162 O C . Reagent-Coated Glass Wool. A 1 N sodium hydroxide solution (15 mL) was added to a solution prepared from 300 mg (0.0013 mol) of N - [(4-nitrophenyl)methyl]propanaminehydrochloride and 25 mL of water. The mixture was shaken. N - [ ( 4 Nitrophenyl)methyl]propanaminewas extracted with 50 mL of hexane. Then 40 mL of the hexane solution was transferred to a 50-mL beaker wrapped with aluminum foil and containing 1.8 g of silanized glass wool (Alltech Associates, Arlington Heights,

Thls artlcle not subject to US. Copyrlyiit. Publlshed 1982 by the American Chemlcal Soclety