14SS
Anal. Chem. lSS2, 64, 1499-1501
CORRESPONDENCE
Determination of Monofluorophosphate, Orthophosphate, and Polyphosphates by High-Performance Liquid Chromatography with a Photodiode Array Detector Norimasa Yoza,' Sachiko Nakashima, Tetsuya Nakazato, Nobuyuki Ueda, Hiroki Kodama, and Akira Tatedaf Department of Chemistry, Faculty of Science, Kyushu University, Hakozaki, Fukuoka 812, Japan Monofluorophosphate (MFP, P03F2-),rather than sodium fluoride, has been extensively used in commercial dentifrices as a fluorine donor as reported in a special issue on Monofluorophosphate Perspectives.' MFP provides a cariespreventive effect that is generally considered to involve the conversion of hydroxyapatite to more caries-resistant fluoroapatite by the action of the fluoride ion produced by the hydrolysis of MFP to orthophosphate (PI, Pod3-). MFP + H,O
-,F- + P,
(1)
The fluoride ion has usually been monitored by an ionselective electrode24 to characterize the reactions of MFP. In both fields of basic investigationsand clinical applications, however, it has been increasingly reported that MFP reactions are not likely to be as simple as in eq 1 and more advanced analyticaltechniques, such as HPLC and NMR spectroscopy, are needed to analyze not only inherent contaminantsin MFP chemicals but also all reaction products of MFP, including po1yphosphates.l-3 It has been suggested that pyruvate kinase catalyzes anonphysiological "fluorokinase reaction" in eq 2.6-9 The reverse reaction is also likely to proceed. Careful characterization and removal of contaminants in MFP have been required in the investigation of such enzyme reactions.7
+
F- + ATP s MFP ADP (2) According to our current and preliminary experiment, MFP is rapidly hydrolyzed in the presence of alkaline phosphatase and is so reactive even in the absence of catalysts that it reacts with orthophosphate to form polyphosphates. Thus, advanced analytical methods for determination of inorganic and biochemical phosphorus compounds are necessary to characterize the unpredictable reactivity of MFP in metabolic s y ~ t e m s , l possibly ~ ~ - ~ S NreactionslO ~ as exemplified by the known reactions in eqs 1 and 2, and to understand the fate + Laboratory of Chemistry, College of General Education, Kyushu University, Ropponmatsu, Fukuoka 810, Japan. (1)Gron, P.; Ericsson, Y. Caries Res. 1983,17,Suppl. 1, 1-136. (2) Lindahl, C. B. Caries Res. 1983,17,Suppl. 1, 9-20. (3)Duff, E.J. Caries Res. 1983,17,Suppl. 1, 77-90. (4) Venetz, W.P.;Mangan, C.; Siddiqi, I. W. Anal. Biochem. 1990, 191,127-132. (5) Farely, J. R.;Tarbaux, N. M.; Lau, K.-H. W.; Baylink, D. Calcif. Tissue Int. 1987,40, 35-42. (6) Pearce, E. I. F. Caries Res. 1983,17,Suppl. 1, 21-35. (7) Nowak, T.Arch. Biochem. Biophys. 1978,186,343-350. (8)Mildvan, A.S.;Leigh, J. S.; Cohn, M. Biochemistry 1967,6,18051818. (9) Nowak, T.; Maurer, P. J. Biochemistry 1981,20,6901-6911. (10) Yoza, N.; Okamatsu, M.; Tokushige, N.; Miyajima, T.; Baba, Y. Bull. Chem. Soc. Jpn. 1991,16-20.
of MFP in environmental systems.1l The present paper describes the application of highperformance liquid chromatography (HPLC) and 31PNMR spectroscopy1° to the rapid and sensitive analysis of orthophosphate (PI,Po&, diphosphate (P2,P207"), triphosphate (P3, P30105-), and cyclo-triphosphate (cP3, P30g3-) in MFP samples. A photodiode array detector13 was particularly useful for identification and quantitation of various contaminants in MFP chemicals. 31PNMR spectroscopy was employed to give supporting evidence for the conclusion provided by HPLC experiments.
EXPERIMENTAL SECTION MFP chemicals (Na2P03F)from different sources were used as samples to indicate the methods of analyzing MFP and contaminants. No standard MFP sample of guaranteed purity has been available. Sample I (MFP-I),commercially available from Aldrich Chemical Co. (Milwaukee, WI), was used as a reference in analyzing sample I1 (MFP-11) that was not commercially available but was supplied as a gift from an industrial company. An HPLC systemlW13 using an anion-exchangecolumn (TSKgel SAX, 4-mm i.d. X 25 cm) was operated in the same manner as in previous papers.loJ3 A postcolumn detection systeml3 consisted of a high-temperature reactor (140-150 "C) using a molybdenum(V)-molybdenum(V1) reagent and a photodiode array spectrophotometric detector (Shimadzu SPD-MGAS,200800 nm) with a flow cell (10-mm light path, 8 pL). The detector was useful not only to quantitate all phosphorus compounds but also to provide an absorption spectrum of the so-calledheteropoly blue complex that was passing through the flow cell. 3lP NMR measurements by employingat JEOL-GX-400 (162 MHz for 31P) were made with MFP-I, MFP-11, and other reference samples: sodium salts of PI,Pz,P3 and cP3. Chemicalshifta, 6 values in ppm, were measured relative to an external reference of 85% H3P04,with positive values being downfield of the reference.lO
RESULTS AND DISCUSSION Separated phosphorus compounds were allowed to react with a Mo(V)-Mo(V1) reagent in a postcolumn reaction coil that was maintained at 140-150 "C to accelerate the formation of a colored heteropoly complex.l3 All spectrophotometric information over 200-800 nm of the colored complex that '
(11)Yoza, N.; Sagara, Y.; Morioka, H.; Handa, T.; Hirano, H.; Baba, Y.; Ohashi, S. J. Flow Injection Anal. 1986,3, 37-46. (12) Yoza, N.; Hirano, H.; Baba, Y.; Ohashi, S. J. Chromatogr. 1985, 325,385-393. (13) Yoza, N.;Akazaki,I.;Nakazato,T.; Ueda, N.; Kodama,H.; Tateda, A. Anal. Biochem. 1991,199,279-285. (14) Yoza,N.;Tokushige,N.;Nakazato,T.;Takehara,K.;Ide,Y.;Baba, Y. Chem. Lett. 1990,1487-1490.
0003-2700/92/0364-1499$03.00/0 0 1992 American Chemical Society
1500
ANALYTICAL CHEMISTRY, VOL. 64, NO. 13, JULY 1, 1992 0.080
""V
7
0
14
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35
28
21
7
14
21
Flgure 1. Three-dimensionalHPLC profile for a mixed solution of MFP, P1, PPI and P3 obtained by a photodiode array detector. Conditions: samples each 0.10 mM except for 0.13 mM P3, 100 pL; eluent, 0.18 M KCI 0.1 % (w/w) EDTA(4Na);12.13recording 0.4 AUFS, 400-800 nm; peak assignment (A) P1, (6) MFP, (C) P2, (D) PI.
+
I
I
I
I
35
TR/mi n
JR/mi n
-I
28
1
I
1
1
I
Flgure 3. Three-dimensional HPLC profile for an MFP-I solution. Conditions: sample 1.0 mM MFP-I, 100 pL; eluent, the same eluent as in figure 1; recording 0.080 AUFS, 400-800 nm; peak assignment, the same as in Figure 2.
T O-020
I
ii
Bii t jI ;C 1 1
a
h
0
7
14
21
28
35
TR/mi n
b
Flgure 4. Threedimensional HPLC profile for an MFP-I1 solution. Conditions: sample 1.0 mM MFP-11, 100 pL; eluent, the same eluent as in Figure 1; recording 0.020 AUFS, 400-800 nm; peak assignment, the same as in Figure 2.
0
26
52
7a
104
130
TR/mi n Flgure 2. Two-dimensional HPLC profiles for MFP-I and MFP-I1 solutions. Conditions: samples (a) 1.0 mM MFP-I and (b) 1.0 mM MFP-11, 100 pL; recording (a) 0.1 AUFS and (b) 0.05 AUFS, 800 nm; eluent, the same eluent as in Figure 1; peak assignment (A) P1, (6) MFP, (C) P2, (D) Ps, (E) cP3, (a) unknown.
was passing through a flow cell was computer-savedand could be reproduced 3-dimensionally (Figure1)and 2-dimensionally at any wavelength (Figure 2). Figure 1shows a 3-dimensional HPLC profile, retention time (TR)-wavelength (A)-absorbance (AU),for a mixed solution of MFP, P1, P2, and P3. All species are well resolved and give the same absorption spectrum. MFP, as well as polyphosphates,l3are not likely to react directly with the Mo(V)-Mo(V1) reagent but are hydrolyzed to P1that reacts directly with the Mo(V)-Mo(V1) reagent to form the heteropoly blue complex with absorption maxima at around 335 and 825 nm.11J3 The peak for cP3, appearing at 115 min (not shown), was also confirmed to provide a spectrum identical to that of PI. The measurement at any wavelength between 600 and 820 nm is recommendedto quantitate each phosphorus compound in a linear dynamic range, 1X 10-6to 5 X 10-4 M. A variablewavelength detector may be used, rather conveniently, for routine analysis. The sensitivities relative to that at 800 nm are approximately 100 (800 nm), 66 (750 nm), 49 (700 nm), 40 (650 nm), and 29 (600 nm). Relative standard deviations
of the measurement at 800 nm are 5% at the 10-6 M level and 2 % a t the M level. As has been discussed in the previous paper concerning the detection limit of Pz in the presence of 20 000-fold excess of P1,13 the detection limit of each component is variable depending on the relative peak intensities of neighboring components. Detailed experiments and extensive discussionare needed to formulate the detection limit of MFP and its contaminants and will be reported elsewhere. Two-dimensional HPLC profiles for MFP-I and MFP-I1 recorded at 800 nm are shown in Figure 2. The top portions of the major peaks for MFP have to be outside the recording scales of 0.1 and 0.05 AUFS (absorbance unit full scale) to highlight the small peaks of contaminants: PI, Pa, P3, cP3, and an unknown species. The two profiles are different in that the total amount of the contaminants in MFP-II(6.3 % phosphorus) is less than that in MFP-I (12.5%), but remarkably high amounts of the cP3 (3.9%) and the unknown species Q (0.9%) are observed in MFP-11. Figures 3 and 4 show 3-dimensionalpresentations for MFPI and MFP-I1 samples. The unknown species Q, as well as PI, Pz, P3, and cP3, gave the spectrum identical to that of P1 in Figure 1. Sincetetraphosphate and higher polyphosphates have been confirmedto appear behind P3,10-12J5the unknown species might be a fluorine-containingphosphorus compound, such as difluorophosphate, that is converted to P1 in the reactor. The relative amounts of six species in Figures 2-4 remained unchanged at least for 1 week when the sample solutions of MFP-I and MFP-I1 were allowed to stand at room temperature (about 25 "C). Hence, the contaminants are likely not the reaction products of MFP in the solutions, but the inherent contaminants in the MFP chemicals. (15) Baba, Y.; Yoza, N.; Ohashi, S. J. Chromatogr. 1985,348,27-37.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 13, JULY 1, 1992 31P NMR measwements12J4 of both MFP-I and MFP-I1 solutions (0.1 M, ca. pH 6.5) gave major signals of MFP; a doublet with a chemical shift (6 = +1.3 f 0.1 ppm) and a coupling constant ( 1 J p ~= 870 f 1Hz) and other small signals which appeared at the 6 values of PI, Pz,Ps, and cP3.12J4An unknown NMR signal a t -15 ppm, probably corresponding to the unknown HPLC peak Q in Figure 2b, was observed for MFP-11, but its intensity was too low to evaluate a coupling constant. We have concluded that the HPLC method with the photodiode detector is capable of identifying and quantitating small amounts of MFP, contaminants, and/or reaction products that have been very difficult or impossible to be analyzed by the existing analytical methods.' The NMR
1501
technique was also convenient to give a supporting evidence for the chemical species identified by the HPLC method, because the 3lP NMR signal of MFP (doublet) was clearly resolved from the signals of PI (singlet), Pz (singlet), P3 (doublet and triplet), and cP3 (singlet).l*J4 We hope the techniques will be successfullyapplied not only to the quality control and clinical applications of MFP chemicals but also to the characterization of reactions of MFP with biological substances1 and the fate of MFP in environmental water.10
RECEIVED for review October 25, 1991. Accepted March 19, 1992. Registry No. MFP, 15181-43-8;PI, 14265-44-2;Pz, 1400031-8; P3, 14127-68-5;CP3, 15705-55-2.
