Anal. Chem. 1988, 60, 1397-1400 (14) Klein, M. P.; Kramer, L. N. Symp.: Seed Proteins (Proc.) 1972, 19, 265-276. (15) Hovland, C. T. "SESCA: Scanning Electron Spectroscopy for Chemical Analysis" in Proc. 7th I n t . Vac. Congr. 1977, 3 , 2363-2366. (16) Clark, D. T.; Peeling, J.; Colling, L. 6iochim. Blophys. Acta 1976, 453, 533-545. (17) Peeling, J.; Clark, D. T.; Evans, I . M.; Boulter, D. J . Sci. Food Agric. 1976, 2 7 , 331-340. (18) Colton, R. J.; Murday, J. S.; Wyatt, J. R.; DeCorpo, J. J. Surf. Sci. 1979, 8 4 , 235-248. (19) Debies, T. P., Rabalais, J. W. J . Nectron Specrosc. Reiat. Phenom. 1974, 3 , 315-322. (20) Kiasinc, L. J . Nectron Spectrosc. Relat. Phenom. 1976, 8 , 161-164. (21) Shirley, D. A. Phys. Rev. 6: Solid State 1972, 5 , 4709-4715. (22) Wagner, C. D.: Rlggs, W. M.: Davis, L. E.; Moulder, J. F.: Muilenberg.
(23) (24) (25) (26)
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G. E. Handbook of X-Ray Photoelectron Spectroscopy; Perkin-Elmer: Eden Prairie, MN, 1978. Jolly, W. L.; Bomben, K. D.; Eyermann, C. J. At. Data. Nucl. Data Tables 1984, 3 1 , 433-493. Wagner, C. D. Faraday Discuss. Chem. SOC. 1975, 6 0 , 291-300. Wagner, C. D.; Gale, L. H.; Raymond, R. H. Anal. Chem. 1980, 52, 466-482. Brown, J. R.; Shockiey, P.; Behrens, P. Q. "Albumin: Sequence, Evolution and Structural Models" in The Chemistry and Physiology of the Human Plasma Proteins; Bing, D. H., Ed.; Pergamon: New York, 1979; pp 23-40.
RECEIVED for review October 20, 1987. Accepted March 8, 1988.
Liquid Chromatographic Separation of Phosphorus Oxo Acids and Other Anions with Postcolumn Indirect Fluorescence Detection by Aluminum-Morin Shon E. Meek and D. J. Pietrzyk*
Department of Chemistry, University of Iowa, Iowa City, Iowa 52242
Phosphorus oxo aclds lncludlng organodlphosphonates are Isolated and separated on a quaternary ammonlum anion exchange column (Hamllton PRP-X100). An alumlnum-morln solutlon was used as a postcolumn reagent for the lndlrect fluorometric detection of the analytes. The variables affecting detectlon were shown to be postcolumn reaction temperature, pH, and mlxlng. Others are postcolumn reagent solutlon organlc solvent, alumlnum:morln ratlo, buffer, and their concentratlons. When these varlbles were optlmlzed, (dlfluoromethylene)dlphosphonk acld was detected at 15 ng for a 2 1 slgnaknolse ratio; the h e a r range extended to about 800 ng. Detectlon is applicable to other dlphosphonates, condensed phosphates, sugar phosphates, nucleotides, other organophosphates, and non-phosphorus-contalnlng anlons such as fluorlde, sulfate, and polyprotlc organlc acids.
oxo acids include atomic absorption (10, 11) and flame emission (12). A conventional fluorometric method for the determination of phosphate was reported in 1965 which is based upon phosphate's ability to reduce fluorescence of the aluminummorin chelate due to an aluminum-phosphate reaction (13). The study described here was undertaken to determine (1) whether postcolumn indirect fluorometric detection (IFD) is a useful detection strategy in liquid chromatographic separations, (2) whether aluminum-morin as a postcolumn IFD reagent is applicable to the detection of trace levels of condensed phosphates, phosphonates, and related anions, and (3) if applicable, the optimum conditions for the post column detection of diphosphonates. While indirect fluorescence detection using fluorescent eluents has been shown to be practical in LC detection ( 1 4 , 1 5 ) ,little has been done with indirect fluorescence as a means of postcolumn detection (16,
17). Phosphorus oxo acids are usually determined by complexation reactions that involve the orthophosphate ion ( I ) . The condensed phosphates are converted to orthophosphate through acid hydrolysis while the lower oxo acids are oxidized. The most frequently used complexation methods are based upon the reaction of orthophosphate with molybdenum reagents to form a colored complex that can be detected spectrophotometrically (2). These methods have been adapted for use in HPLC by coupling the column effluent with flow injection analysis systems (3-5) or air-segmented automated analyzers (6, 7). A postcolumn LC detection system for the oxo acids of phosphorus has been reported that eliminates the need to convert the phosphorus compounds to orthophosphate (8). This method involves a reaction between the oxo acid of phosphorus and Fe(III), which is monitored spectrophotometrically a t 340 nm. The detection limit reported is about 0.5 pg for the lower phosphates and phosphonates (8,9). Other online detection methods that have been used for phosphorous
* To whom correspondence should be addressed.
EXPERIMENTAL SECTION Reagents. Disodium (difluoromethy1ene)diphosphonate (F,MDP) was prepared by the method of Burton and co-workers (18). The disodium (dichloromethy1ene)diphosphonate (C1,MDP) and disodium 1-hydroxyethane-1,l-diphosphonate (EHDP) were obtained from the College of Pharmacy, University of Iowa, and used as received. The biological phosphate samples were obtained from Sigma Chemical Co. Morin hydrate was obtained from Aldrich Co. Acids, bases, A1(N0J3, and 95% ethanol were obtained as analytical grade when possible. HPLC grade water was prepared by passing distilled water through a Sybron-Barnstead Nanopure water purification system. Instrumentation. The chromatographic system consisted of a Du Pont Instruments 850 gradient controller and pump, a Rheodyne Model 7125 injector, a PRP-X100 4.1 mm X 150 mm, 10-pm anion-exchange column (Hamilton Co.), a postcolumn mixing unit, and a Kratos 9000-9501 fluorescence detector. The detector was operated with a Kratos mercury lamp (FSA 113) that was used in conjunction with a Kratos blue-band excitation filter (FSA 404)for excitation wavelengths between 400 and 470 nm. A Kratos high pass filter with 50% transmission at 480 nm was used as an emission filter. The detector output was recorded on a Hewlett-Packard integrator recorder, Model 3390A; peak areas
0003-2700/88/0360-1397$01.50/00 1988 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 14, JULY 15, 1988
were recorded in relative integration units. The postcolumn unit consisted of a Waters Model 6000A pump that was used to deliver the postcolumn reagent solution. Base-line pulses generated by this pump were reduced by pumping the reagent through an empty 0.8 mm X 25 mm column, 200 cm of 0.01 mm i.d. stainless-steel tubing, and a 4.1 mm X 150 mm column packed with 40-pm glass beads and then into the mixing tee (Lee Visco mixing tee no. 344790 SN152). The mixer and postcolumn reaction coil were placed inside a Du Pont temperature-controlled oven that maintained the postcolumn reaction temperature at 70 "C. Procedures. Standard solutions of N%FzMDPwere prepared by dissolving a known weight of Na2F2MDPin a known volume of deionized water. Standard dilutions of this stock solution were made to obtain lower concentrations of NazFzMDPin the range of 3-700 pg/mL. Sample injections were 1-25 p L by a Hamilton Model 702 syringe. Mobile phases containing EtOH are expressed as percent EtOH by volume. Mobile phases containing acids and buffers were made by pipet from standardized stock solutions. Morin was pipetted from a stock solution that was prepared by dissolving a known weight of morin in 1:l EtOHHzO. Mobile-phase and postcolumn reaction solution pH was determined by pH meter. The POstcolumn mobile phase was prepared and aged overnight so that the aluminum-morin chelate was fully formed to provide maximum detection sensitivity. When freshly prepared postcolumn reagent solution was used, sensitivity was signficantly reduced. All mobile phases were degassed for 5-15 min. prior to their use. R E S U L T S AND DISCUSSION The phosphorus oxo acid detection system described here is based upon the indirect fluorescence method developed for the determination of phosphate (13). In this method phosphate complexes aluminum, thereby removing the aluminum from a standard amount of aluminum-morin complex that has been added to the phosphate analyte. The fluorescence due to the aluminum-morin complex decreases, and this decrease is correlated to the amount of phosphate present in the sample by calibration with phosphate standards. Adapting this strategy to a modern liquid chromatographic separation requires a postcolumn reagent solution of the aluminum-morin complex to be mixed with the eluent from the analytical column. The aluminum-morin complex provides a steady base line of background fluorescence that is adjusted to the desired value by the detector electronic offset. When an analyte, such as phosphate, elutes from the analytical column, the analyte and morin compete to form an A1 complex. When A1 is removed from the fluorescent aluminummorin complex by an analyte, a fluorescence decrease is produced in the analyte zone. Therefore, negative peaks that correspond to lower levels of fluorescence are obtained for analytes that are capable of removing A1 from the aluminum-morin complex. For optimum IFD the eluent should contain components that are favorable to the separation but do not interfere with the aluminum-morin fluorescence (13). Analytes. Several analyte anions will reduce aluminummorin fluorescence via a competitive reaction (13). Since we were particularly interested in a procedure that would be applicable to the determination of organo diphosphonates in biological samples, diphosphonates were used as model analytes to establish the anion-exchange chromatographic conditions and the parameters that influence postcolumn IFD. Diphosphonates, such as 1-hydroxyethane-1,l-diphosphonate (EHDP), (dichloromethy1ene)diphosphonate(C12MDP),and (difluoromethy1ene)diphosphonate (F2MDP) (18, 19) are a group of drugs that have been reported to be useful in the treatment of bone and/or Ca disorders (20). These diphosphonates are tetraprotic acids (21),and mobile-phase pH can be used to control their anionic charge and affect retention on an anion exchanger. Retention and elution can also be affected by altering the eluent counteranion and its concentration. In the separations described here, which is consistent
with anion-exchange behavior (22),aqueous 20 mM HNOBwas used as the eluent for the retention and separation of FzMDP and other diphosphonates, organic phosphates, phosphate, and other inorganic anions. Postcolumn Parameters. The major postcolumn variables requiring optimization, which were identified in preliminary experiments, are (1)reaction temperature, (2) reaction pH, (3) solvent composition, (4) aluminum-morin ratio and concentration, ( 5 ) buffer concentration, and (6) reaction volume. Fluorescence excitation and emission were reported to be pH dependent (13),and this was verified experimentally. Selection of detector emission and excitation band filters was based on these results in combination with optimization of pH effects on the analyte-aluminum-morin reaction. Temperature affects aluminum-morin fluorescence intensity (13,23) and increases the rate at which analytes compete with morin for Al. When the reaction coil and mixing tee were placed inside a temperature-controlled oven and the temperature varied from 25 to 80 "C, the average peak area for FzMDP was at a maximum a t 70 "C. Including the mixing tee in the oven not only increased analyte peak area but also reduced background noise by about 20%. The peak area maximum is probably a compromise between an accelerated reaction between F2MDP and A1 and a decrease in aluminum-morin fluorescence since the background fluorescence of the aluminum-morin complex was observed to decrease as temperature was increased, particularly above 75 "C. All subsequent experiments were carried out with the tee and coil placed inside the 70 "C oven except for a 20-cm tubing connection to the fluorescence detector. Fluorescence intensity of aluminum-morin and the fluorescence change that takes place when phosphate is present is pH-dependent (13). Our studies show that the largest F2MDP analyte peak area is obtained when the pH for the postcolumn reaction is 4.3. A OAc- buffer was used because OAc- is a poor ligand for A1 and it does not alter aluminum-morin fluorescence significantly. Since the mobile-phase pH is more acidic that pH 4.3, a OAc-/OH- mixture was used in the postcolumn solution to raise the postcolumn reaction pH to 4.3. The optimum p H probably represents a compromise between the optimum fluorescence of the aluminum-morin complex, which is between pH 4 and pH 5, and increased chelation of FzMDP and A1 that occurs as the pH is increased. The presence of alcohol affects the solubility of hydroxyflavones, the ratio of metal to ligand in the complex, and the fluorescence intensity of the complex (23). Of the alcohols tested ethanol was the most favorable as a fluorescent medium (13). Figure 1 shows that as the postcolumn EtOH concentration was increased from 20 to 80% EtOH, a maximum in average FzMDP peak area was obtained at 40% EtOH. The effect of EtOH on apparent pH of the medium is responsible for the peak area maximum. This was confirmed by normalizing the data to pH 4.3 by accounting for the pH change due to the mixed solvent. The pH-normalized area curve, shown in Figure 1, illustrates that peak area increases as EtOH increases, if the pH remains constant. The fluorescence intensity of the metal-hydroxyflavone complexes depends on metal ion:chelate ratio (23). Table I shows that the peak area of FzMDP increases dramatically as the morin:aluminum complex ratio is changed from 0.384 to 2.31 a t which point the peak area begins to level off. In this experiment FzMDP peak area was determined a t 80% EtOH and a postcolumn pH of 4.1 or conditions of maximum fluorescence change. The backround fluorescence increases steadily while detector noise decreases slightly as the morin:aluminum ratio is increased to about 7.1. Even though the background fluorescence increases as the morin:aluminum
ANALYTICAL CHEMISTRY, VOL. 60, NO. 14,JULY 15, 1988 25
6
----..-
20
'
0
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1399
peak area : noise ratio
(D
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0 x
X
0
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0 2
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Aluminum Concentration x M Flgure 2. Effect of aluminum-morin concentration on F,MDP peak area. Conditions are the same as those in Figure 1 except the postcolumn solution is 4:l EtOH:H,O, 5:l morin:aluminum, 50 mM HOAc, and 28.6 mM NaOH.
Table I. Peak Area of FzMDP as a Function of Postcolumn Mobile-Phase Morin:Aluminum Ratioo
postcolumn morin concn,mM
morin:Al
0.00388 0.0116 0.0233 0.0349 0.0466 0.0582 0.0698
0.384 1.15 2.31 3.46 4.61 5.76 6.91
av peak area
X
lo7
0.222 0.834 1.84 1.78 2.00 2.07 2.20
"Conditions are the same as those in Figure 1 except the postcolumn solution is 4:l EtOH:H20, morin, 0.0101 mM A1(NOJ3, 10.0 mM HOAc, and 24.9 mM NaOH. ratio is increased, the FzMDP analyte fails to provide a larger peak probably because of increased competition for chelation from the increased concentration of morin. Acetate in the postcolumn reagent solution provides buffering ability at pH 4-5 and is a weak ligand for Al. As the OAc- buffer concentration was increased in the postcolumn solution, the average FzMDP peak area decreased slightly. In addition background fluorescence noise also decreased. When the peak area was normalized for noise by dividing the average peak area at each buffer concentration by the corresponding recorded noise height and multiplying by the recorded noise height a t 25.0 mM OAc- buffer, a maximum peak area occurred at 25.0 mM OAc- buffer. This represents the optimum postcolumn OAc- solution concentration. Trace levels of analyte that compete with morin for the A1 should be more effective a t reducing the fluorescence when the aluminum-morin complex concentration is low. However, as the aluminum-morin concentration is reduced, the background fluorescence and the magnitude of the fluorescence change will also decrease. Figure 2 shows that as the aluminum-morin concentration increases, the average F2MDP peak area increases. In Figure 2 the ratio of morin to A1 was maintained a t 5:l as the A1 concentration was increased in the postcolumn solution. The peak area increase occurs because the background fluorescence also increases as the aluminum-morin concentration increases. However, the detector background noise also increases with an increase in background fluorescence. The data were normalized to account for the noise effects by dividing the average peak area at each
A1 concentration by the corresponding noise height and multiplying by the recorded noise height obtained at 8.6 X lo4 M Al (NO,),. These values are plotted in Figure 2 as peak area:noise ratio and indicate that a maximum peak area occurs M. a t an A1 concentration of 2.0 X Preliminary experiments proved that reaction coil volume is a major factor in affecting reaction completion. Evaluation of different lengths and diameters of postcolumn stainless-steel tubing indicated that a compromise between diameter and length was needed to obtain an optimum postcolumn reaction volume. The tube diameter/length affects base-line noise and postcolumn reaction volume and time. Large diameter, longer tubes were required for a favorable reaction time while small diameter tubes were favorable for increased efficiency. A postcolumn reaction tube internal diameter of 0.02 in. and a length of 100 cm gave an adequate reaction time and peak efficiency with a reasonable minimum amount of postcolumn pump noise. This corresponds to a reaction tube volume of about 400 pL. An increase in the postcolumn reaction temperature also gave a bigger percent increase in FzMDP peak area when reaction time was the limiting factor. However, when all factors are considered, 70 O C was an optimum temperature. IFD Calibration Curve. The calibration curve for IFD of F2MDP after retention and elution from an anion-exchange column was linear from 39 to 824 ng of F2MDP and corresponds to the equation peak area X lo7 = 0.01331 (ng of FzMDP) - 0.1745 with a correlation coefficient r = 0.998 79. Above 824 ng of FzMDP the peak area begins to level off. For the instrumentation used the minimum detection limit for FzMDP was determined to be 15 ng of FzMDP as the disodium form on the basis of a peak to noise ratio of 2 1 where peak and noise heights were measured in millimeters. This is important because studies of FzMDP and other diphosphonates and their effects in culture, embryonic bone, adult animal bone, and physiological fluids and tissue yield samples above this detection limit (19, 20). IFD Applications. IFD using aluminum-morin in LC separations is a general strategy that can be used to detect other analytes that complex Al. These analytes can be divided into those which contain only phosphorus acid functional groups and those which contain no phosphorus. Oxophosphorus acid compounds are known to have high formation constants for reaction with Al, thereby making the aluminum-morin fluorescence strategy general enough to be used for their detection after separation. Figures 3 and 4 show
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 14, JULY 15, 1988 C
I
I
I
I
0
4
8
12
rnL Figure 3. Separation and detection of organodiphosphonates. Con-
ditions are the same as those in Figure 1 except the mobile phase is aqueous 28.1 mM HNO,, the postcolumn solution is 4:l EtOH:H,O, 0.0101 mM morin, 0.00202 mM AI(NO,),, 36.5 mM HOAc, and 42.7 mM NaOH, and a reaction coil is 140 cm (0.02 in. i.d.) at 70 O C and 10 cm (0.02 in. i.d.) at ambient temperature: a, EHDP; b, CI,MDP; C, F,MDP. a. Glucose-6-Phosphate b. Fructose-l,6-Diphosphote
a. Phowhafe a. Phosphote b. Uridine-5’-Phosphate b. Acetyl Phosphate c. Cytidine-5’-Phosphate c. Phospho(eno1)pyruvote
be isolated, separated from other anions, and detected at 30-100 ng similar to F2MDP. Figure 4 demonstrates that sugar phosphates, nucleotides, and inorganic phosphates can be detected by IFD using aluminum-morin. Analytes that contain more than one phosphorus group reduce the aluminum-morin fluorescence to a greater extent than compounds with only one phosphorus functional group. Thus, fructose-1,6-diphosphate in Figure 4A, like the organodiphosphonates, has a lower detection limit than glucose-6-phosphate. In Figure 4C the acetyl phosphate sample, identified as b, gave two peaks. No attempt was made to identify which was the impurity and which was acetyl phosphate. Other non-phosphorus compounds that complex aluminum can also be detected by a aluminum-morin fluorescence change. Analytes tested include fluoride, sulfate, thiosulfate, and polyprotic organic acids such as citrate, oxalate, malate, and tartrate. The sensitivity of the fluorescence change for citrate appears to be very high, probably because citrate is a triprotic acid whereas the other organic acids are diprotic. Registry No. Na2F2MDP,84228-60-4;Na2C12MDP,2256050-5; Na,EHDP, 7414-83-7;Al(NO&, 13473-90-0;morin, 480-16-0; glucose-6-phosphate,56-73-5;fructose-l,6-diphosphate, 488-69-7; phosphate, 14265-44-2;uridine-5’-phosphate,58-97-9;cystidine5’-phosphate, 63-37-6; acetyl phosphate, 590-54-5; phospho(enol)pyruvate, 73-89-2; pyrophosphate, 14000-31-8; tripolyphosphate, 14127-68-5;fluoride, 16984-48-8;sulfate, 14808-79-8; thiosulfate, 14383-50-7;citrate, 126-44-3;oxalate, 338-70-5;malate, 149-61-1;tartrate, 3715-17-1. LITERATURE C I T E D
3
mL
mL
i;
bll ’I
0
rnt
a. Phosphate
1\
b. Pyrophosphate c.
Tripolyphosphate
I
I
I
I
10
20
30
40
mL Figure 4. Separation and detection of biological phosphates and inorganic phosphates. Conditions are the same as those in Figure 3.
several separations of oxophosphorous analyte anions including diphosphonates, condensed phosphates, sugar phosphates, nucleotides, and metabolic organic phosphates. All the separations are based on anion exchange and depend on eluting conditions that provide favorable anion-exchange selectivities between the anionic analytes. In general, about 1-5 pg of each analyte was used in Figures 3 and 4. Figure 3 illustrates a separation of three diphosphonates. Since the mobile-phase pH is about 1.7, the diphosphonates are separated and eluted as predominately monovalent anions. The elution order is consistent with the acid strength of the diphosphonates where FIMDP is the strongest acid. If the mobile phase HNOB concentration is decreased, retention increases. In physiological samples only one of the three would be present and Figure 3 indicates that any of the three can
(1) Kiso, Y.; Kobayoshi, M.; Kitaoka, Y. Analytical Cbemisfry of Pbospborus Compounds; Halmann, M., Ed.; Wiley-Interscience: New York. 1979; pp 93-147. (2) Ramesy, R. Advances in Cbromatograpby; Giddings, J., Grushka, E., Cazes, J., Brown, P., Eds.; Marcel Dekker: New York, 1986; Voi. 25, DO 219-244. (3) k a i , Y.;Yoza, N.; Ohashi, S. Anal. Cbim. Acta 1980, 775, 269-277. (4) Hirai, Y.; Yoza, N.; Ohashi, S. J . Cbromatogr. 1981, 206, 501-509. (5) Hirai, Y.; Yoza, N.; Ohashi, S. Cbem. Lett. 1980, 5 , 499-502. (6) Yamaguchi, H.; Nakamura, T.; Hirai, Y.; Ohashi, S. J . Cbromatogr. 1979, 772, 131-140. (7) Yoza, N.; Ito, Y.; Hirai, Y.; Ohashi, S. J . Cbromafogr. 1980, 796, 471-480. (8) Imanari, T.; Tanabe, S.; Toida, T.; Kawanishi, T. J . Cbromatogr. 1982, 250, 55-61. (9) Edelmuth, S. Ph.D. Thesis, University of Iowa, 1986; p 54. (10) Yoza, N.; Kouchiyama, K.; Miyajima, T.; Ohashi, S. Anal. Lett. 1975, 8. 641-653. (11) Kouchiyama, K.; Yoza, N.; Ohashi. S. J . Cbromatogr. 1978, 147. 271-279. (12) Julin, B. G.; Vanderborn, N. W.: Kirkland, J. J. J . Cbromatogr. 1975, 7 72, 443-453. (13) Land, D.; Edmonds, S. Mikrocbim. Acta 1986, 6, 1013-1023. (14) Mho, S.;Yeung, E. S. Anal. Cbem. 1985, 57, 2253-2256. (15) Rigas, P. G.; Pietrzyk. D. J., submitted for publication in Anal. Chem. (16) Werkhoven-Goewie, C. E.; Niessen, W. M. A,; Brinkman, U. A. Th.; Frei, R. W. J . Cbromatogr. 1981, 203, 165-172. (17) Torres, E. L.; van Geel, F.; Winefordner, J. D. Anal. Lett 1983, 76, 1207- 12 18. (18) Burton, D. J.; Pietrzyk, D. J.; Ishihara, T.; Fonong, T.; Flynn, R . M. J . Fluorine Cbem. 1982, 20, 617-626. (19) Rowe, D. J. Bone 1985, 6, 433-437. (20) Flaisch, H. Metab. Bone Dis. Re/. Res. 1981, 4 - 5 , 279-268. (21) Fonong, T.; Burton, D. J.; Pietrzyk, D. J. Anal. Cbem. 1983, 55, 1089-1094. ... . .. . (22) Fritz, J. S.; Gjerde, D. T.; Pohlandt, C. Ion Cbromatograpby: A. Huthig: New York. 1982. (23) White, C.; Argaver, R . Fluorescence Analysis; Marcel Dekker: New York, 1970: p 62.
RECEIVED for review October 21, 1987. Accepted March 5 , 1988. Part of this work was supported by Grant DE 8613 awarded by the National Institute of Dental Research.
