Anal. Chem. 1987, 59. 2161-2164
perform this task and we will report on its performance in a future publication. One of the principal applications of our increased fluorescence detection is in antigenjantibody immunofluorescence assay. Current work (8) uses 0.1-pm fluorescent spheres for tagging the species of interest. In some cases these large tags cause steric hindrance, which inhibits the bonding and reduces the sensitivity and specificity. The use of a single chromophore for a tag should greatly reduce this problem. Even the use of a phycoerythrin molecule for a tag makes a significant improvement. The diameter for P-phycoerythrin is -100 %, ( I I ) , which is 10 times less than the fluorescent microspheres currently used or 1000 times less in volume.
ACKNOWLEDGMENT We thank Ann Perkins, summer student 1984, for her help in selection of a candidate for single molecule detection and Jimmie Parson for his assistance with the data acquisition system.
LITERATURE CITED (1) Dovichi, N. J.; Martin, J. C.; Jett, J. H.; Keller, R. A. Science 1983, 279. 845.
2161
Dovichi, N. J.; Martin, J. C.; Jett, J. H.; Trkuia. M.; Kelier, R. A. Anal. Chem. 1984, 56, 348. Nguyen, D. C.; Kelier, R. A.; Trkuia, M. J. Opt. Soc. Am. 8: Opt. Phys. 1987, 4, 138. Hirschfeld, T. Appl. Opt. 1978. 15, 2965. Kirsch, B.; Voigtman, E.; Winefordner, J. D. Anal. Chem. 1985, 57, 2007. McGuffin, V. L.; Zare, R. N. Appl. Spectrosc. 1985. 39,847. Mathies, R. A.; Stryer, L. Applications of Fluorescence in Siological Sciences; Taylor, D. L., Waggoner, A. S.,Lanni, F., Murphy, R. F., Birge, R. R., Eds.; Alan R. Liss. Inc.: New York, 1986; pp 129-140. Saunders, G. C.;Jett, J. H.; Martin, J. C. Clin. Chem. (Winston-Sa/em, N . C . ) 1985, 31, 2020. Miiby, K. H.; Zare, R. N. Am. Clin. Prod. Rev. 1984, fP(Jan.). Glazer, A. N. Mol. Cell. Biochem. 1977, 18, 125. Glazer, A. N.; Stryer, L. Trends Biochem. Sci. (Pers. Ed.) 1984, 9 , 423. Oi. V. T.; Glazer, A. N.; Stryer, L. J . CellBiol. 1982, 93,981. Seagrave, J. C.;Deanin, G. G.; Martin, J. C.; Davis, B. H.; Oliver, J. M., Cytometry 1987, 8, 287. Cooper, R. B. Intmducflon to Oueuing Theory; Macmiiian: New York, 1972; pp 43-43. Lindmo, T.; Fundingsrud. K. Cytometry 1981, 2, 151.
RECEIVED for review January 28, 1987.
Accepted April 10, 1987. This work was performed under the auspices of the Department of Energy.
Determination of Boron in Biological Tissues by Inductively Coupled Plasma Atomic Emission Spectrometry Swasono R. Tamat and Douglas E. Moore* Pharmacy Department, University of Sydney, Sydney, New South Wales 2006, Australia
Barry J. Allen Applied Physics Division, Australian Atomic Energy Commission, Lucas Heights Research Laboratories, Menai, New South Wales 2234, Australia
A method Is descrlbed for the determination of boron in Mologlcal samples based on digertlon of tissue wlth perchloric acid and hydrogen peroxide, followed by lnducthrely coupled plasma atomic emlsslon spectrometry. The method glves a linear response for boron concentratlons in the range 0.05-100 ppm and Is suitable for use in the evaluatlon of complex boronated compounds in neutron capture therapy. An altemathre approach, us4g Methykm Blue complexation, was examined but was found to be subject to interference from perchlorate. Hydrofiuorlc acld can be used, but the h e a r range is llmited to 4 ppm and a longer acld dlgestion thne Is required.
Boron neutron capture therapy (BNCT) of cancer requires selective accumulation of 'OB-containing chemicals or biochemicals in the malignant tissue before irradiation with neutrons ( I ) . The subsequent ( n p ) reaction results in highenergy deposition within cellular dimensions. Since the ultimate efficacy of BNCT depends on the ratio of tumor dose to maximum normal tissue dose, it is necessary to evaluate a B-containing agent by determining the boron concentration in the tumour and adjacent tissues or sensitive organs, after the O ' B compound is administered to a suitable laboratory animal test system. Boron determination in biological tissue samples requires an ash- or digestion treatment of the biological material with decomposition of the complex boron compound (such as 0003-2700/87/0359-2161$01.50/0
BloHlo2-)to determinable boric acid species. A number of methods have been reported for the breakdown of the biological material and the subsequent assay of boron. Some of these can be extended to the low concentration levels required in the evaluation of BNCT (e1ppm). For example, a colorimetric assay of boron in plant material involved wet digestion of the samples, followed by the use of 1,l'-dianthrimide in concentrated sulfuric acid as the colorimetric reagent ( 2 ) . This method was extended to tissue samples containing polyhedral boranes and nonvolatile carboranes, although interferences from some oxidizing agents were reported (3, 4). A radio frequency combustion with excited oxygen plasma was proposed for the decomposition of animal tissues, after which oxidative degradation of the complex boron compound was achieved with potassium permanganate, and the boron was determined by the curcumine method (5). The plasma ashing method took 17 h and some loss of boron was acknowledged during the precipitation of the manganese dioxide. Fusion with alkali was reported to decompose the biological material and simultaneously convert the complex boron compounds to borate ion (6). Treatment with hydrofluoric acid yielded tetrafluoroborate which could be extracted as a complex with Methylene Blue and determined by visible spectrophotometry. However, the alkali fusion method is time-consuming and requires extremely careful technique if the loss of boron is to be avoided. Prompt y-ray spectrometry has been used for the determination of trace amounts of loB in animal tissue (7, 8). 0 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 17, SEPTEMBER 1, 1987
Table I. ICP Operating Conditions rf forward power
induction coil optical system monochromator
27.12 MHz, 1.2 kW (max 10 W reflected) with self-contained cooling system entirely automatic, controlled at 32 "C sequentially operated 1-m Czerny-Turner, 165-800 nm using a high-performance 2400 line-mm-' holographically produced grating, bandwidth 0.013 nm set at 9 for concn 0.05-2 pg-mL-'; set at 15 for concn 0.05-100 pg.mL-'
attenuator torch and sample introduction system torch three concentric quartz tubes carrier gas welding grade argon, 99.5% coolant, 12 L-rnin-' gas flow rates plasma, 0.8 Lmmin-' carrier, 1 L-min-' gas temperature 8000 K permanently aligned pneumatic type, cross flow glass capillary nebulizer spray chamber glass, Scott-type 2.5 mL-min-' samde feed rate Although this method has the advantage of not involving pretreatment of samples, the requirement of a nuclear reactor as the neutron source restricts its application. Besides prompt y-ray spectrometry and chemical ionization mass spectrometry (9),an a-track etching technique (10, 11) has been proposed to quantitate the loB isotope in tissues following neutron irradiation, as well as to determine the microscopic distribution of loB in histological sections of the tumour. The major deficiencies of the above assay methods are their lack of sensitivity, the risk of loss of boron in the digestion step, or the need for neutron irradiation during the development process. Since the evaluation of potentially useful compounds for toxicity and tumour incorporation involves administering various doses to a number of laboratory animals, the need for an accurate and rapid boron assay is evident. This paper describes a method for the assay of the boron concentration in biological samples based on an acid digestion procedure, which reduces the amount of sample manipulation, followed by inductively coupled plasma atomic emission spectrometry (ICP-AES), which avoids the necessity to convert the complex boron compound to boric acid. Boron, being one of the more refractory elements in flame atomic absorption, is readily assayed to the 10 ppb level by inductively coupled plasma atomic emission spectrometry (ICP-AES) (12). A comparison has been made in which the acid digestion procedure has been coupled with the Methylene Blue spectrophotometric method (6). EXPERIMENTAL SECTION Apparatus. The ICP-AES used was a Bausch & Lomb Model 1350. The operating conditions, given in Table I, were optimized for multielement analysis. The routine function sequences were executed by the controlling computer (PDP 11/03) with SAS/ICP 3510 software. The characteristic wavelengths of boron are 208.893,208.959,249.678, and 249.779 nm with ratio of net analyte intensity to background intensity ( I J Z b ) 25, 30, 53, and 63, respectively. The 249.678-nm line was used throughout the experiments. Spectrophotometric measurements were made with a PerkinElmer Lambda 5 UV/visible spectrophotometer. For the solubilization of tissues a Techne horizontal shaking bath SB-4 was operated at 75 OC and 100 oscillations/min. Reagents. Decaborane B1a14(Callery Chemicals), dicesium mercaptoundecahydro-closo-dodecaborate,Cs2B12HllSH(New England Nuclear), and Methylene Blue (BDH Chemicals) were used without further purification. Dicesium N-succinimidyl 3-(undecahydro-closo-dodecaboranyldithio)propionate(SBDP) was prepared from Cs2Bl2Hl1SHand N-succinimidyl 3-(pyridy1dithio)propionate (SPDP) (13). Solutions of CszBlzHllSHand SBDP were prepared in water, while BIOHll was dissolved in methanol-water (1:9). All other reagents were of analytical grade (Merck or BDH Chemicals). Double distilled water (DD water) was used throughout the experiments.
Boric acid standard solution (Merck) contained 5000 ppm B. Dilutions were made with DD water to provide solutions containing 0-100 ppm B. Biological tissue samples were obtained by dissection of 8 week old Wistar rats. These materials were frozen until use. All solutions were kept in low-boron glass (Kimble) or polyethylene (Packard) vials. Procedure. Sample Preparation for the ICP-AES Method. The acid digestion procedure for biological tissue was adapted from the method of Mahin and Lofberg (14). To a weighed sample of rat tissue (1OC-200 mg) in a polyethylene vial, 0.3 mL of 70% perchloric acid was carefully added and the contents were swirled to mix. Then 0.6 mL of 28% hydrogen peroxide was added and the vials were placed in the shaking bath for 1 h at 75 "C. After incubation, the vial contents were clear and colorless. The samples were cooled to room temperature, diluted with 4 mL of DD water, and filtered through a 0.45-~mMillipore filter before being introduced into the ICP-AES instrument. Sample Preparation for the Methylene Blue Method. The rat tissue was acid digested for 1 h as above, using 0.35 mL of concentrated HzSO4 and 0.3 mL of 28% hydrogen peroxide, and after cooling, 0.15 mL of 50% hydrofluoric acid was added, followed by a further 3 h of incubation in the shaking bath at 75 "C. The following is a modification of the procedure of Yoshino et al. (6). The solvated tissue sample (ca. 0.8 mL) was transferred to a polyethylene separatory funnel with 9.2 mL of DD water. Then 10 mL of 0.01 M sodium carbonate solution was added, followed by 0.5 mL of 0.003 M Methylene Blue solution and 10 mL of 1,2-dichloroethane. The mixture was shaken for 3 min, and the 1,2-dichloroethane phase was transferred to another separatory funnel which contained 5 mL of silver sulfate solution (9.25 X lo4 M). After the mixture was shaken for 3 min, the organic phase was separated and its absorbance read at 660 nm vs. a blank solution which had been treated similarly. Recovery of Boron from Complex Boron Compounds. Separate solutions of B10H14, Cs2B12H11SH,NazB4O7,and SBDP in the concentration range from 0.2 to 10 ppm B were prepared containing perchloric acid and hydrogen peroxide as above, then, without the heating and filtering steps, the boron concentrations were assayed directly by ICP-AES. The tissue digestion procedure was applied to various tissue samples to which an aliquot of a standard solution of BloH1,had been added. In another experiment, rat muscle samples were weighed into two sets of seven polyethylene vials. One set acted as control. To the vials of the other set were added 4, 5, 6, 7, 8, 9, and 10 pg of B from a 20 ppm B stock B10H14solution. Both sets were treated with HC104/Hz02as described above for ICP-AES assay. A similar set of samples were treated by the oxidative decomposition method for assay with Methylene Blue.
RESULTS AND DISCUSSION ICP-AES Method. The 249.678-nm emission line of boron was chosen for the analysis rather than the 249.779-nm line, on the basis of the lesser possibility of interference from iron and phosphorus (15). The standard curve, obtained by using
ANALYTICAL CHEMISTRY, VOL. 59, NO. 17, SEPTEMBER 1, 1987
Table 11. Recovery of Boron from Complex Boron Compounds
compounds B10H14
CszBlzHllSH
SBDP
NazB407.H20
expected boron concn, pg.mL-' 0.20 0.60 0.88 1.00 1.09 1.70
no. of replicates (n)
3 3 5
3 3 3
0.20 0.40 0.80 0.99 1.19
2
0.57 0.96 1.79 1.80 3.57 4.15 9.43
2 2
0.40 0.60 0.80 1.00 1.50 2.00
2
2 2
3 2
2 2
2 2 2 2
2 2
2 2
boron concn found, pg.mL-'
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Table 111. Background Boron Levels and Recovery of Added Boron from Various Rat Tissue Samples
recovery of boron, % tissue
0.20 f 0.01 100 0.60 f 0.01 100 0.87 f 0.01 98.7 0.98 f 0.01 98 1.08 f 0.01 99.1 1.70 f 0.02 100 0.20 f 0.01 100 0.39 f 0.01 97.5 0.77 f 0.02 96.3 0.97 f 0.02 98.0 1.18 f 0.02 99.2 0.56 f 0.01 98.2 0.96 f 0.01 100 1.78 f 0.02 99.4 1.77 f 0.01 98.3 3.58 f 0.04 100.3 4.10 f 0.05 98.8 9.23 f 0.15 97.9
HC104+ HzOz brain ileum skin jejunum caecum spleen muscle bone kidney liver heart blood lungs
0.070 0.063 0.072 0.065 0.069 0.073 0.074 0.071 0.077 0.069 0.069 0.075 0.063 0.054
av
0.068 f 0.006
amt of boron
added," kg
boron concn found, pgmL-I
recovery of added
boron, %
4.61
0.896
100
4.61
0.896
100
4.61 4.61
0.890 0.875
99.3 97.7
4.61
0.925
103.2
4.61 4.61
0.886 0.855
98.9 95.4
4.61
0.908
101.3
0.891 f 0.021
99.4 f 2.3
"Boron added as BioH14 from a stock solution. Final sample volume was 5.15 mL in all cases. Double distilled water was used as solvent blank.
0.42 f 0.01 105 0.61 f 0.01 101.7 0.79 f 0.02 98.8 1.02 f 0.01 102 1.48 f 0.02 98.7 1.95 f 0.04 97.5
Table IV. Recovery of Boron Added to Rat Muscle Tissue
99.4 f
background boron concn, pLg.mL-'
1.7"
amt of boron added: pg
amt of boron recovered, pg
recovery of added boron, %
ICP-AES Method
Average recovery.
solutions diluted with DD water from the stock standard boric acid solution and constructed from the relative signal intensity (with DD water as blank), showed that the effective linear dynamic range using ICP-AES is 0.05-100 ppm B. A high concentration of inorganic salts in the sample matrix, such as phosphate-buffered saline (PBS) solution, can shift the location of the hot portion of the plasma and therefore alter the signal for specific analyte (12). However, in our experience, this occurred only when the sample solution contained sodium ion of more than 0.1 M. Most protein and tissue samples in our experiments contained about 0.01 M sodium ion. Nebulization effecta are possible due to the high acidity of the samples (16,17),but these can be eliminated by matching the standard and sample preparation as closely as possible. We found that the presence of the components of the acid digestion mixture at their final concentrations (4% HC104and 4% H,O,) in the standard solutions had no effect on the standard curve. Nevertheless, the acid content was always matched between samples and standard solutions. The relative standard deviation (RSD) for five readings of 1-s duration was less than 2% for any sample up to 100 ppm B. This short-term precision was used to dictate the long-term precision by rejection of results with RSD greater than 2%. Fairchild (7)suggested that conventional atomic absorption and emission spectrometric measurements are not well suited to the complex boron cage compounds such as B1J-I14, on the basis that these compounds are not well dissociated in the flame. We have tested several complex boron compounds which are intermediates in the synthesis of BNCT agents, with the results given in Table 11. It appears that the boron cages are completely dissociated at the high temperatures (so00 K) achieved in the argon plasma. Up to 1.7 ppm B in B10H14, 1.2 ppm B in Cs2B12H11SH,and 10 ppm B in SBDP solutions gave a linear response for increasing concentration, with slopes
no. of replicates, (n)
5.0 6.0 7.0 8.0
9.0 10.0
4 4 4 4 4 4
5.10 f 0.05 5.86 f 0.04 6.92 f 0.04 7.80 f 0.07 8.81 f 0.06 10.23 f 0.19
102 97.7 98.9 97.5 97.9 102.3 99.8 dz
2.lb
Spectrophotometric Method 0.4 0.5 0.8 1.0 1.2 1.5 2.0 4.0 6.0
0.38 0.46 0.81 0.98 f 0.01 1.14 f 0.03 1.37 f 0.05 1.80 f 0.07 3.50 f 0.21 5.30 f 0.24
95 92 101.3 98 95 91.3 90 87.5 88.3
" Boron added as BloHll from stock solution containing 20 pg of B/mL to approximately 200 mg of rat muscle. bAverage. the same as the calibration curve. The results are expressed in terms of the recovery of the expected amount of boron from each compound. In the acid digestion method described here, there is less possibility that any boron will escape, compared to the much higher temperature used in alkali fusion (6). The validity of the tissue digestion method, including the filtration step, was tested by determining the background B levels in a number of tissue samples as well as the recovery of B from B10H14 added to several of the tissues. Table I11 shows that the range of tissues registered a B content of the same order as the reagent blank, indicating the sensitivity of the method to be limited by the incipient B levels in the reagents and apparatus. The average base-line correction was applied to the B determination of spiked samples of tissues. For the different
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 17, SEPTEMBER 1, 1987
tissues, the recovery of added B was always close to loo%, with an average recovery of 99.4 f 2.3%. Similarly, Table IV shows that the recovery of a range of added B amounts to rat muscle tissue gave 99.8 f 2.1 % recovery. These results confirmed that there was no boron loss during heating or filtering. Evaporative loss of liquid during heating was less than 0.3%. Although the standard curve was applicable over the wide linear range up to 100 ppm, these tests were carried out over the more restricted range which applies for the biodistribution testing of BNCT compounds. On the basis that the ICP-AES method was successful in standard compounds, it has been applied to the determination of B in synthesized BNCT compounds, such as boronated human serum albumin conjugates, boronated poly(amino acid) conjugates, and boronated antibody conjugates. Methylene Blue Complexation Method. Several strong oxidizing agent combinations were investigated for the conversion of the complex boron compounds to tetrafluoroborate, prior to complexation with Methylene Blue. For example, the combination of perchloric acid, hydrogen peroxide, and hydrofluoric acid failed to give the expected recovery of boron on complexation. This may be due to an ion-pair complex formation of Clod-with Methylene Blue, observed as a precipitate in the aqueous phase but apparently soluble in 1,2dichloroethane. Potassium permanganate was reported to be sufficient to oxidize complex boron compounds, but some loss of boron can occur in the precipitation of MnO, ( 5 ) . The oxidizing agent used in the present work is a combination of 50% hydrofluoric acid with 28% hydrogen peroxide. At elevated temperature, solvated fluorine is produced which will oxidize readily any complex boron compound in the solution to boric acid. The fluoride ion regenerated from fluorine during oxidation of the boron compound subsequently will react with boric acid to produce hydrofluoboric acid. Excess hydrogen peroxide was removed from the mixture by heating before the dilution with water. By use of boric acid standard solutions, the linear dynamic range of the spectrophotometric method was found to lie between 0.1 Qg of B and 4 pug of B, similar to that reported previously (6)but not as extensive as for the ICP-AES method. Experiments using tissue (spiked with B,,H,, solution) containing up to 1.2 pg of B showed that two stages (tissue digestion for 1 h, followed by 3 h oxidative decomposition) were adequate for complete recovery of B by the Methylene Blue method, as shown in Table IV. Complete recovery was not achieved when more than 1.2 pg of B was used, a result that can not be explained at this stage, since an excess of the reagents was used. Furthermore, any hydrogen peroxide remaining in the solution affected the complex formation between tetrafluoroborate and Methylene Blue. In the visible spectrophotometric method, definite interferences from other species have been reported (18). We have experienced interference by Sn(II), Fe(II), nitrate ion, perchlorate ion, and hydrogen peroxide. Reducing species will
decolorize Methylene Blue, while the anions appear capable of ion-pair formation with the positively charged dye. The validity of the acid digestion method has been established for a full range of biological tissue samples. All tissues were digested completely and resulted in clear and colorless solutions, except for bone, which in digestion with sulfuric acid and hydrogen peroxide produced milky solutions, presumably due to calcium sulfate formation. Both acid digestion methods are relatively efficient. Up to 50 samples can be handled simultaneously, in 90 min by ICP-AES and in 5 h for the visible spectrophotometric method. Both are applicable to a variety of complex boron compounds. The ICP-AES has the advantage in a lesser digestion time requirement, fewer chemical interferences, and the fact that it gives a linear response over a wider range of B concentration.
ACKNOWLEDGMENT Thanks are extended to D. M. Levins, A. Reid, and C. Misfud for assistance with the ICP-AES measurements and G. R. Wellum of NEN for the gift of Cs2B12HllSH. Registry No. SBDP, 85070-11-7; B, 7440-42-8; B10H14, 17702-41-9;CS2Bl,HIlSH, 12448-23-6;Na2B,07, 1330-43-4.
LITERATURE CITED (1) Soloway, A. H. I n Progress in BOfOn Chemistry; Steinberg, H., McCloskey, A. L., Eds.; Pergamon: Oxford, 1964; Voi. 1, pp 203, 206. (2) Eiiis. G. H.; Zook, E. G.; Baudisch, 0. Anal. Chem. 1949, 21, 1345-1348. (3) Soloway, A. H; Messer, J. R . Anal. Chem. 1964, 36, 433-434. (4) Kaczmarczyk, A.; Messer. J. R.: Peirce, C. E. Anal. Chem. 1971, 4 3 , 271-272. (5) Ikeuchi, I.; Amano, T. Chem. fharm. Bull. 1978, 26, 2619-2623. (6) Yoshino, K.; Okamoto, M.; Kakihana. H.; Nakanishi, T.; Ichihashi, M.; Mishima, Y. Anal. Chem. 1984, 56,839-842. (7) Fairchild, R. G. I n Workshop on Radiobiology and Tumour Therapy with Heavy Particles, and IAEA Research Coordination Meeting, Villigen, Switzerland, April 27-29, 1982; BNL Report 31402. (8) Kobayashi, T.; Kanda, K. Nucl. Instrum. Methods 1983, 204, 525-531. (9) Cook, C. J.; Dubiel, S. V.; Hareiand, W. A. Anal. Chem. 1985, 5 7 , 337-340. (10) Mishima, Y.; Shimakage, T. P/gment Cell: Riley, V., Ed.; Karger: Basel, 1976; Voi. 2, pp 394-406. (11) Wilson, D. J.; Linklater, H.; Izard, B. E.; Allen, 9. J. Proceedings of the Third Australian Conference on Nuclear Techniques of Analysls; Australian Institute of Nuclear Science and Engineering (AINSE): Lucas Heights, 1983; pp 50-53. (12) Siavin. W. Anal. Chem. 1986, 5 8 , 589A-597A. (13) Alam, F.; Soloway, A. H.; McGuire, J. E.; Barth, R. F.; Carey, W. E.; Adams, D. J. Med. Chem. 1985, 28,522-525. (14) Mahin. D. T.; Lofberg, R. T. Anal. Biochem. 1986, 16, 500-509. (15) Nemodruk, A. A.; Karalova, 2 . K. Analytical Chemlstry of Boron, (English Translation): Ann Arbor-Humphrey: Ann Arbor, MI, 1969; p 20. (16) McQuaker. N. R.; Brown, D. F.; Kiuchner, P. D. Anal. Chem. 1979, 57,1082-1084. (17) Que Hee, S.S.: Macdonald. T. J.; Boyle, J. R . Anal. Chem. 1985. 57. 1242- 1252. (18) Isozaki, A.; Utsumi, S. Nippon Kagaku Zasshi 1967, 8 8 , 741-744.
RECEIVED for review February 2,1987. Accepted May 5,1987. Financial support for this study was provided by the Australian Institute for Nuclear Science and Engineering.
