ANALYTICAL BIOCHEMISTRY 68, 167-174 (1975)
The Determination of the Radiochemical Purity of Phosphorus-32 and Tritium-Labeled Diisopropylphosphorofluoridate (DFP) R . E . CHRISTOPHER AND (IN PART) G .
Quality Control Department, The Radiochemical Centre, Amersham, Buckinghamshire, United Kingdom Received February 7, 1975; accepted April 23, 1975 A method is described for the determination of the radiochemical purity of labeled diisopropylphosphorofluoridate (DFP), based on the irreversible inhibition reaction with the enzyme c~-chymotrypsin. The nature of the impurities in commercially available 32p_ and 3H-labeled D F P is discussed.
Diisopropylphosphorofluoridate (DFP) is a powerful inhibitor of the cholinesterases, to which it becomes irreversibly bound (1). D F P labeled with radioisotopes (phosphorus-32 and, less commonly, tritium) has been widely used as a cholinesterase label in survival studies of blood platelets (l ,2), red cells (1,3), and white cells (1,4). Knowledge of the radiochemical purity of D F P ("RCP"; defined as the proportion of the total radioactivity present in the form of DFP) is important for its successful use. Gas chromatography has previously been used as an analytical technique for DFP. The thermionic detector (5) has been used for 3"P-labeled DFP, and radiogas chromatography (using a proportional counter as the radioactivity detector) for tritiumlabeled DFP. The glc methods, however, have the fundamental disadvantage that impurities of low volatility, and in particular the hydrolysis product diisopropylphosphoric acid, may be completely retained on the column under the conditions suitable for the separation of D F P from its synthetic precursors. Furthermore, the glc methods require specialised equipment. We present here a method for the determination of the RCP of labeled D F P which does not have the disadvantages of the glc methods. The procedure is based on the quantitative reaction of D F P with the enzyme a-chymotrypsin, and is therefore closely related to the use of D F P as a labeling agent for the cholinesterases. RESULTS AND DISCUSSION
The irreversible inhibition of a-chymotrypsin (CHT) by D F P has been studied in detail (6). The phosphorus-fluorine bond of D F P is 167 Copyright© 1975by AcademicPress, Inc. All rightsof reproductionin any formreserved.
CHROMATOGRAPHIC PARAMETERS OF D F P AND RELATED COMPOUNDS tic R~- v a l u e s
DFP DP-CHT a DCP e DHW IlI ~ IIh I~
a. A c e t o n e
b. H e x a n e / a c e t o n e a
Rel. m o b i l i t y HVE b
R e t e n t i o n vol. glc c
0.95 0.00 -0.85 0.80 0.25 0.15
0.45 0.00 -0.20 0.05 0.05 0.00
-0.10 --0.10 1.30 1.00
1.00 -2.78 2.11 2.06 i --i
a4:l. b R e l a t i v e t o b r o m o p h e n o l b l u e as 1.00, in p H 8.6 buffer. c R e l a t i v e t o D F P as 1.00. a Diisopropylphosphoryl-chyrnotrypsin. e D i i s o p r o p y l p h o s p h o r o c h l o r i d a t e , o b s e r v e d as d i i s o p r o p y l p h o s p h o r i c a c i d (I) in tlc and HVE. s Diis o p r o p y l p h o s p h i t e . Di(isopropyl)(2-hydroxy-iso[or n-]propyl)phosphate, see text. h See t e x t . i Completely retained. D i i s o p r o p y l p h o s p h o r i c a c i d , see text.
cleaved, with the liberation of hydrogen fluoride and the incorporation of the diisopropylphosphoryl group into the enzyme (7). A crystalline 1:1 derivative has been isolated (8), and the kinetics of the inhibition reaction have been investigated (9).
Analytical Method for R CP Determination of DFP The analytical method, details of which are in the experimental section, is as follows: A solution of the D F P , prepared in aqueous buffer at pH 7.6, is kept at room temperature for 5 min in order to hydrolyse any diisopropylphosphorochloridate which may be present. An excess of a-chymotrypsin is then added and the solution allowed to stand for 30 min. The radiochemical composition of the reaction mixture is then determined by thin-layer chromatography (tlc) and high voltage electrophoresis (HVE) as follows: tic using a hexane-acetone (4: 1) eluant is used to confirm that the inhibition reaction has gone to completion. The diisopropylphosphoryl derivative (DP-CHT) remains at the origin and all known impurities and reaction precursors run with retention factors ~<0.2 (see Table 1); any unreacted D F P will be clearly observed at R s 0.45. As expected from the high reaction rate for the inhibition (9) (tl/~ -~ 30 sec at the concentrations used here) unreacted D F P is not normally detectable in the reaction mixture.
DFP RADIOCHEMICAL PURITY
Thin-layer chromatography using the more polar eluant acetone separates the nonionic impurities from D P - C H T (see R s values in Table 1) and is used to quantify these impurities. The nature of the nonionic impurities is discussed below. H V E at the isoelectric point of a-chymotrypsin [pH 8.6 (6)] is used to separate and quantify impurities which are anionic at this pH, for example diisopropylphosphoric acid. The distribution of radioactivity across the tlc plates and H V E papers is determined using a computerised 100 channel Radiochromatogram Analyser (10). The radiochemical purity of the labeled D F P is then calculated by summation of the impurities; the retention factors and mobility values listed in Table 1 allow correlation of the tlc and H V E scans, and identification of the impurities. In order to detect possible impurities with low Rs values in tlc and low electrophoretic mobilities (such impurities remain poorly resolved from the D P - C H T peak in both tlc and HVE), a tic plate is run in acetone TABLE 2 PRECISION AND ACCURACY OF R C P DETERMINATION BY CHYMOTRYPSIN BINDINGa
(a) Precision of Chromatographic Method %I b
Fresh sample Reaction 1 0.7 Reaction 2 0.6 Reaction 3 0.7 Reaction 4 0.5 Partly hydrolysed sample Reaction 1 13.5 Reaction 2 13.3 Reaction 3 13.8 Reaction 4 13.4
RCP c (%)
1.5 1.4 1.2 1.3
0.4 0.5 0.4 0.4
97.4 97.5 97.7 97.8
4.1 3.8 4.0 3.4
14.1 15.6 15.0 14.0
68.3 67.3 67.2 69.1
(b) Accuracy of chromatographic method, compared with ultrafiltration
R C P from tlc/HVE
RCP from ultrafiltration (%)
1 2 3a
91,92 92 64.5
91.5,95 88 66,69
F o r ~ P - D F P in propylene glycol. b I, II, and III are impurities as listed in Table 1. c C o u n t i n g ESD values were ca. +-0.2% for the fresh sample, and -+0.7% for the partly
hydrolysed sample. a Partly hydrolysed sample.
C H R I S T O P H E R A N D SHEPPARD
eluant on the D F P solution before the addition of enzyme. D F P runs at high Rf (0.95) in acetone and an examination of peaks near the origin, in conjunction with the correlations in Table 1, will reveal the presence of these impurities. No such impurities have been detected in any of the batches of D F P examined. The results obtained were independent of the pH of the reaction mixture in the range 7-8; the pH 7.6 used routinely corresponds to the maximum activity pH of a-chymotrypsin (8). Variation of the enzyme reaction time (10 min-4 hr) had no effect on the RCP, although the 5 rain hydrolysis time is important (see below). The reproducibility of the method was found to be acceptable; replicate determinations on fresh and partially hydrolysed samples of D F P gave the results in Table 2(a). The accuracy of the tlc/HVE method was assessed by comparison with ultrafiltration measurements of the DFP-cbymotrypsin reaction mixture. Thus, on filtering the reaction solution through a membrane impermeable to species of molecular weight above 10,000 the D P - C H T derivative is retained while the impurities pass through. Liquid scintillation counting of the filtered and unfiltered solutions allows the RCP of the D F P to be calculated. As Table 2(b) shows, the two methods agree quite well for fresh and partially hydrolysed samples of D F P , although ultrafiltration is less reproducible and does not allow the impurities to be identified. Radioactive Impurities in DFP
Most of the batches of phosphorus-32 and tritium-labeled D F P examined in this study were found to contain varying amounts of three impurities. Table 1 lists the tic retention factors, H V E relative mobilities, and glc relative retention times of these impurities, which are denoted I, II, and III. The three impurities all contain at least one isopropyl group attached to phosphorus as they derive from both phosphorus-32 and tritium-labeled DFP. The radioactive impurities may arise from reaction precursors which are carried through the final purification, or from decomposition of the D F P by hydrolysis, reaction with the propylene glycol solvent, or by radiolytic decomposition. 1. The presence of reaction precursors. Radioactive D F P is prepared from 32p. or 3H(methyl)-labeled diisopropylphosphite by chlorination to diisopropylphosphorochloridate, followed by halogen exchange to give D F P ; final purification is by repeated fractional distillation under reduced pressure. Diisopropylphosphite (DHP) was not found as an impurity in any of the batches of D F P examined. In contrast, diisopropylphosphorochloridate (DCP) was found to be a contaminant of several batches of neat 32P-DFP. As D C P rapidly hydrolyses in aqueous solution to diisopropylphosphoric acid It,2 -~ 45 sec in 10-4 M solution (11)]
DFP RADIOCHEMICAL PURITY
the chymotrypsin binding procedure cannot easily distinguish between D C P and diisopropylphosphoric acid impurities. Infrared spectroscopy, however, has clearly identified D C P as the main contaminant of impure batches of freshly prepared neat DFP. It is significant that those batches of neat 32P-DFP which contained appreciable amounts of D C P appeared to be pure by glc. Separate experiments with inactive DCP, and with D F P - D C P mixtures, showed that the recovery of D C P through the glc column was extremely low, presumably because of on-column hydrolysis due to traces of water in the column packing material. The limitations of glc for the analysis of D F P are therefore obvious. Experiments with 32P-labeled D C P showed that the chromatographic parameters of its hydrolysis product were identical to the D F P impurity I (Table 1); compound I can therefore be identified as diisopropylphosphoric acid. Furthermore, it was shown that on diluting 32P-DCP with propylene glycol the D C P partly reacted with the solvent to give a product which is probably the tri-ester di(isopropyl)(2-hydroxy-iso [or n-] propyl)phosphate, and was partly hydrolysed to diisopropylphosphoric acid by water in the solvent. The chromatographic parameters of the tri-ester were identical to those of the D F P impurity III. The presence of impurities I and III in some of the propylene glycol solutions of D F P can therefore be explained in terms of initial contamination of the D F P by unreacted DCP. 2. Hydrolysis of DFP and its reaction with propylene glycol. The hydrolysis of 32P-DFP and 3H-DFP under strongly acidic (6 M HCI) or strongly alkaline (10 M N a O H ) conditions gave 90% of the radioactivity in the form of diisopropylphosphoric acid, regardless of whether propylene glycol was present or not. Under milder conditions, for example in wateror inphosphate bufferat pH 6.5 and7.6, andintheabsenceofpropylene glycol, ~2P-DFP gave products identical in their chromatographic parameters to the D F P impurities 1 (diisopropylphosphoric acid) and II. The nature of II, which has high electrophoretic mobility at pH 8.6, is not known although monoisopropylphosphoric acid or monoisopropylfluorophosphoric acid are possibilities. Cleavage of both alkoxy groups from phosphorus does not occur under the conditions used here as no peaks corresponding to orthophosphoric or phosphorofluoridic acid appear on electrophoresis. Under milder hydrolysis conditions and in the presence of propylene glycol an additional product was formed, which was chromatographically identical to the D F P impurity III. 3. Effect of Diisopropylphosphorochloridate on the decomposition rate of DFP. In addition to itself producing radioactive impurities (by hydrolysis and reaction with propylene glycol solvent) D C P enhances the decomposition of D F P through the catalytic effect of the acids released on reaction of the D C P with water and propylene glycol. This
CHRISTOPHER AND SHEPPARD
was demonstrated by measuring the radiochemical compositions of a series of mixtures of 32P-DFP and inactive DCP (in propylene glycol) at two time intervals; since the reaction products were inactive, these experiments measured only the decomposition of the DFP itself. A small but significant dependence of the D F P decomposition rate on the proportion of DCP initially present was demonstrated; the levels of impurities I and III were found to be linearly related to the amount of DCP initially in the mixture, reflecting the acid catalysed nature of D F P hydrolysis and tri-ester formation. In contrast, the level of impurity II was independent of the DCP concentration. To summarise, the presence of variable amounts of impurities I (diisopropylphosphoric acid), 11, and III (di(isopropyl)(2-hydroxyiso [or n-] propyl)phosphate) in propylene glycol solutions of labeled DFP may arise from the initial presence of DCP and/or the hydrolysis and reaction with solvent of the D F P itself. The initial presence of DCP will result in impurities I and III in the propylene glycol solution; the relative proportions will depend on the water content of the solvent. The hydrolysis of D F P will give I and II, and its reaction with propylene glycol will give III. The formation of I and III will be accelerated if DCP is initially present in the propylene glycol solution. In this work it has been shown, using 32P-labeled compounds, that none of the impurities I, II, and III bind to o~-chymotrypsin. It was further demonstrated that DHP and DCP do not bind to the enzyme under the conditions used in the analytical method. If, however, the DCP was added directly to the chymotrypsin solution, then approximately 20% of the DCP combined with the enzyme, the remainder hydrolysing to diisopropylphosphoric acid; the 5-min period before the addition of the a-chymotrypsin is therefore necessary to ensure accurate results for the radiochemical purity of the DFP. As expected from the reaction rate (1 1) no detectable hydrolysis of the DFP occurs during the analytical procedure. The sterilisation of propylene glycol solutions of 32P-DFP by 6°Co y-irradiation (2.5 MR) was found to have no effect on the radiochemical purity values obtained. EXPERIMENTAL
Reaction of DFP with o~-chymotrypsin. One drop of a propylene glycol solution of DFP, containing about 5 /~g of D F P at a specific activity of 40-100 mCi/mmole for 32P-DFP or ca. 2 Ci/mmole for 3H-DFP, was added to 0.2 ml of pH 7.6 0.01 M phosphate buffer. The solution was allowed to stand at room temperature for 5 min to hydrolyse diisopropylphosphorochloridate; 30 mg of o~-chymotrypsin (Sigma London Chemical Co.) were added, the solution kept at room tempera-
DFP RADIOCHEMICAL PURITY
ture for 30 min, and then examined by thin-layer chromatography and electrophoresis. Neat samples of D F P were diluted with the phosphate buffer and then reacted with chymotrypsin as above. Chromatography and electrophoresis. For thin-layer chromatography, Merck precoated silica gel plates were used, activated for 1 hr at 110°C before use. The eluant was allowed to ascend 15 cm, in preequilibrated tanks. High voltage electrophoresis was done on Whatman 3MM paper, in pH 8.6 barbiturate buffer, at 4000 V (10 mA), using a Shandon instrument. The tlc plates and electrophoresis paper were scanned using a computerised 100 channel Radiochromatogram Analyser constructed at the Radiochemical Centre (10). The counting times were chosen to give counting ESD values o f ± 0 . 2 % for impurities at the 3% level. Ultrafiltration measurements. An Amicon Corp. ultrafiltration cell was used, equipped with a membrane permeable to species of molecular weight below ca. 10,000 (Diaflo PM 10). The DFP-chymotrypsin reaction mixture, prepared as above, was diluted ca. 30-fold with distilled water; part of this solution was passed through the ultrafiltration membrane, and part accurately diluted 10-fold with distilled water. Onemilliliter portions of each solution were added to a scintillant mixture and counted on a Phillips Liquid Scintillation Counter, operating in the integral mode. The radiochemical purity of the D F P was calculated as 100(1--Ni/N2), where N1 = net cpm for the filtered solution, and N2 = net cpm for the unfiltered solution. Gas chromatography. Perkin-Elmer F l l instruments were used; the columns, 10% diethylene glycol adipate on Chromosorb W, were operated at 150°C with nitrogen carrier gas. For 3H-DFP, a PerkinElmer Radiogas-chromatography detector was used (PE 170); for 32P-DFP, a Perkin-Elmer thermionic detector was used. Source of DFP. All batches of tritium and phosphorus-32 labeled D F P used in this study were prepared at The Radiochemical Centre, using the synthetic route outlined above.
ACKNOWLEDGMENTS We thank Dr. W. P. Grove, Managing Director of The Radiochemical Centre Ltd., for permission to publish this work, and Mr. R. Monks, of The Radiochemical Centre, for valuable discussion.
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5. (a) Giuffrida, L. (1964) J. Ass. Offic. Agric. Chem. 47, 293-300. (b) Brazhnikov, V. V., Gurey, M. V., and Sakodynsky (1970) Chromatog. Rev. 12, 1. 6. Desnuelle, P. (1960) in The Enzymes (Boyer, P. D., Lardy, H., and Myrback, K., eds.), 2nd ed., Vol. 4, pp. 93-118, Academic Press, New York. 7. (a)Jansen, E. F., Nutting, M. F., Jang, R., and Balls, A. K. (1950) J. Biol. Chem. 185, 209-220. (b) Jansen, E. F., Nutting, M. F., and Balls, A. K. (1949) J. Biol. Chem. 179, 201-204. 8. Jansen, E. F., Nutting, M. F., Jang, R., and Balls, A. K. (1949) J. Biol. Chem. 179, 189-199. 9. Ooms, A. J. J., and Van Dijk, C. (1966) Biochem. Pharmacol. 15, 1361-1377. 10. Stanford, F. G. (1972) Precision and Accuracy in the Determination of the Radiochemical Purity of Labelled Compounds by Thin layer Chromatography, TRC Report No. 315, 13 pp., The Radiochemical Centre, Amersham, Bucks., U.K. 11. Hudson, R. F., and Greenhalgh, R. (1969) J. Chem. Soc. (B) 325-329.