1106
Anal. Chem. 1980, 52, 1106-1110
Separation of Technetium Hydroxyethylidene Diphosphonate Complexes by Anion-Exchange High Performance Liquid Chromatography Thomas C. Pinkerton,‘ William R. Heineman,’ and Edward Deutsch” Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 4522 1
An anion-exchange high performance liquid chromatographic separation of technetium hydroxyethylidene diphosphonate (Tc-HEDP) complexes has been developed. Samples were prepared by the reduction of Tc0,- in the presence of HEDP with NaBH,. Samples contained millimolar concentrations of ”Tc carrier and nanomolar concentrations of the y-emitting BB”rc isotope. Three modes of detection were used: (1) single wavelength at 405 nm, (2) UV-visible rapid scanning from 250 to 600 nm, and (3) single channel radiometric scintillation. The chromatograms illustrate that these radiopharmaceutical analogues consist of mixtures of at least seven Tc-containing species of unknown oxidation state, central metal configuration, and coordination environment. The distribution of components in these mixtures is dependent on reaction time and the presence of molecular oxygen.
Radiopharmaceuticals have been used extensively in recent years as organ imaging agents in nuclear medicine (1-6). Noninvasive procedures have been developed for imaging the skeleton, heart, brain, kidney, and lungs (5). These procedures are based on the tendency of the body t o concentrate some chemical form of a radioisotope (primarily 99mTc)a t the organ of interest (6). An image of the organ can then be obtained by a y scan of the appropriate portion of the body. Diphosphonate complexes of 9 9 m Tare ~ widely used as skeletal imaging agents (1-4). A typical skeletal imaging agent is formulated on the day of use by mixing a solution of Tc04(containing *TCOL and its daughter 99’cOJ with a reducing agent, a diphosphonate ligand, and an antioxidant stabilizer such as ascorbic acid. Some reductants and ligands which have been investigated for this purpose are shown in Table I. The chemistry of the formulation involves reduction of Tc0,- t o lower oxidation states [Tc(V), Tc(IV), Tc(III)] with concommitant formation of Tc-diphosphonate complexes (7, 8). Evidence exists for a complicated reaction process which is strongly dependent on reductant, ligand, pH, temperature, presence of oxygen, ligand-to-metal ratio, and reaction time; hydrolysis, air oxidation, disproportionation, and dimerization may also be involved ( 2 , 6-9). T h e resulting radiopharmaceutical solution includes Tc-diphosphonate complexes, reductant-diphosphonate complexes, excess diphosphonate, and stabilizer. Other possible constituents are Tc-stabilizer complexes, mixed metal complexes (e.g., Tc-Sn-diphosphonate), oligomers and polymers of Tc-diphosphonate, unreacted Tc04-, and Tc02. This reaction mixture is injected into a patient without purification, but after being subjected t o quality control procedures (10-15) which distinguish only between insoluble TcOp, unreacted Tc04-, and the mixture of Tc-diphosphonate complexes referred to as the “desired chemical form”. These procedures do not resolve the component complexes of the “desired chemical form”. It has been Present address: Department of Chemistry, Purdue University. West Lafayette, Ind. 47907. 0003-2700/80/0352-1106$01 .OO/O
proposed that these component complexes vary with reaction conditions, time, etc. ( 1 6 ) , and t h a t different component complexes could exhibit different in-vivo distributions-this could account for the nonreproducible clinical performances of these radiopharmaceuticals ( 1 , 2, 17-20). It is clear t h a t in order to understand and further develop Tc-diphosphonate radiopharmaceuticals (21),it will be necessary t o separate these mixtures into their component complexes, to identify and characterize these complexes, and then evaluate the invivo properties of the separated complexes. This study addresses the first step in this sequence, Le., the separation of a particular Tc-diphosphonate mixture into its component complexes. T o our knowledge this is the first report of such a separation.
EXPERIMENTAL HPLC Chromatographic Apparatus. The chromatographic equipment included a Milton Roy model 396 reciprocating pump, a Rheodyne model 70-10 injection valve with a 20-pL loop, CS-3 instrument rack, PG-5 pressure gauge and a PD-1 pulse damper, all obtained from Bioanalytical Systems Inc., West Lafayette, Ind. A precolumn (70 mm X 2 mm) containing pellicular anion-exchange support material (AS Pellionex, Whatman) was installed between the injection valve and the main column. The analytical column consisted of a 250 mm X 4 mm stainless steel column containing microparticulate (av. dia., 13.7 pm), anion-exchange resin (Aminex A-27, Bio-Rad Laboratories). The Aminex A-27 was chosen primarily because of its greater exchange capacity relative to similar pellicular packings (22). The column was received prepacked in the acetate form at pH 8.4. The column was fitted with a 25-cm glass water jacket (Altex Scientific) with temperature control by a Haake FS constant temperature circulator. UV-Visible Single Wavelength Detection. For single wavelength UV-visible spectrophotometric detection, a Varian Aerograph Variscan variable wavelength liquid chromatographic (LC) detector with an &pL flow cell (1-cmpathlength) was utilized. Detection was at 405 nm unless otherwise stated. Rapid Scanning Spectrophotometric Detection. A Rapid Scan Spectrophotometer (RSS) (Harrick Scientific Co., Ossining, N.Y.) was utilized to produce a broad UV-visible detection display from 250 to 600 nm (23). For this particular application, the data collection was accomplished by means of a controlling 8080 microprocessor interfaced with the rapid scanner ( 2 4 ) . A t 18-s intervals, the microprocessor collected 32 scans from the rapid scanner (scanning at a rate of 10 scans/s), signal-averaged the scans, and stored the resulting average on cassette tape. The running time of 13 s for the storage of the data on the cassette tapes limited the data collection time interval. The Variscan LC flow cell detector assembly was centered in the sample compartment of the Harrick Rapid Scanner at the focal point of the beam producing a 1-cm pathlength with minimum stray light. Radiometric Detection. By spiking a sample preparation with y-emitting 4(140 keV) isotope it was 150 mCi of the 9 9 m T ~ 0 possible to employ a single channel analyzer (Nuclear Chicago Model 8725) equipped with a recording ratemeter (Packard Model 380) and a well scintillation detector (Nuclear Chicago DS-202 V) as an LC detector. After passing the Variscan detector, the eluant entered the scintillation detector, which consisted of a low volume stainless steel tube [1/16 in. (1.6 mm) 0.d. x 0.009 in. (0.23 mm) i d . ] extended into the well scintillation region. 1980 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 7, JUNE 1980
1107
Table I. Preparation of Radiopharmaceutical for Skeletal Imaging 99mTcO;
+
reductant
Reductants structure Sn2+ NaBH, ”,OH electrode
H,O,P-0-PO,H, H, 0,P-CH,-PO,H,
+
ligand
+
imaging agent
Ligands name
abbreviation
pyrophosphate methylene diphosphonate
PPi MDP
hydroxyethylidene diphosphonate
HEDP
OH H,O,P-C-PO,H,
I
CH, Eluant. The chromatographic eluant was 0.85 M sodium acetate, pH 8.4 (Fisher Scientific, HPLC grade) prepared with carbon filtered, triply distilled water. After preparation, the acetate solution was filtered through a 0.22-rm filter (Millipore). Tc(NaBH,)-HEDP Sample Preparation. The following describes a typical Tc(NaBH,)-HEDP sample preparation using the T c ; modifications of this basic preparation include chemical reduction under aerobic and anaerobic conditions, and spiking the preparation with %Tc. First, a ligand solution was prepared by diluting 0.797 g of the disodium salt of 1-hydroxyethylidene diphosphonate (97% Na2H2HEDP,Procter and Gamble) to 10 mL yielding an aqueous solution of pH 5. To this solution was added 1mL of 40 mM N H 4 q c 0 4solution prepared by dissolving solid ammonium pertechnetate (99% pure, Oak Ridge National Laboratories) in triply distilled water. Second, in order to initiate the reduction of pertechnetate, 1 mL of a filtered (0.22 pm) 3 M sodium borohydride (99% pure, Alfa) solution in 0.1 M KOH was added dropwise with stirring over a period of about 5 min, allowing the effervescence of hydrogen to subside after each addition. After the NaBH, addition was complete, the reaction mixture was stirred for approximately 30 min, the final pH being ca. 8.4. With a final volume of 12 mL, the concentrations of %Tc,reductant, and ligand were 3.3 mM, 266 mM, and 266 mM, respectively, producing a ligand t o metal ratio of 80 to 1 in the presence of excess reductant. The above general preparation was carried out with the following modifications: (1)Air Oxidized Samples. Samples were prepared as described above in the presence of air. (2) Air Oxidized and Concentrated Samples. Samples were prepared as described above in the presence of air and then allowed to concentrate by evaporation for 3 d. (3) Anaerobic Samples. Samples were prepared as described above except the reaction flask was fitted with a septum seal and the TcO,--HEDP solutions were deoxygenated with an argon stream for 30 min prior to the reaction. The argon stream was scrubbed of trace oxygen and saturated with water vapor by passage through two acid chromous perchlorate towers (0.1 M Cr(C104)2,1.0 M HClO,, Zn(Hg)) and one water tower. The argon bubbling was continued during the redox reaction as well as during the 30-min period after reagent addition. The sodium borohydride solution was deoxygenated in a similar manner. (4)Treated Anaerobic Samples. Several samples prepared under anaerobic conditions were subsequently subjected to oxidation by an oxygen stream at temperatures of 25, 37, and 60 “C for 1 to 2 h. (5) S a m p l e s S p i k e d u i t h 99m T e c h n e t i u m . Samples were prepared for radiometric detection in 1 mL by the addition of approximately 150 mCi of 99mT~04of saline to HEDP solution just prior to dilution to 10 mL. The standard precautions were exercised in handling radioactive materials. HPLC Sample Injection. The Tc-HEDP complex samples were drawn into a 3-mL Stylex syringe and delivered to the 20-pL loop of the Rheodyne injection valve by passage through a 0.22-pm syringe filter (Millipore).
RESULTS A N D D I S C U S S I O N Selection of Radiopharmaceutical Analogue. A radiopharmaceutical analogue prepared by NaBH, reduction of Tc04- in the presence of HEDP was chosen for study. This analogue is referred to as Tc(NaBH,)-HEDP. H E D P was selected as the diphosphonate ligand since Tc-HEDP radio-
pharmaceuticals exhibit higher tumor uptake (tumor/normal bone ratio) compared to other Tc-diphosphonate imaging agents ( 2 5 ) ,thus increasing their clinical importance in the early diagnosis of bone cancer (26). NaBH, was selected as a n innocuous reductant, the use of which would avoid the possibility of mixed metal complexes which may be formed with metal ion reductants. T h e mixture composition was further simplified by omitting antioxidants such as ascorbic acid, removing any particulate “hydrolyzed reduced” technetium (Tc02.2H20) by microfiltration and removing any unreacted Tc04- by retention on a precolumn. When millimolar concentrations of w T ~ 0 are 4 - reduced in the presence of excess H E D P by t h e addition of a NaBH4 solution, the reaction mixture turns from colorless to a clear green and remains green until all of the NaBH, solution is added. If the sample is prepared under argon, the green color persists as long as anaerobic conditions are maintained. If the preparation is performed with exposure to air, the solution turns a yellow-green and continues to increase in yellow hue during the 30-min post reaction period. If t h e sample is exposed to air for several hours after the initial reaction, t h e solution turns completely yellow. If additional NaBH4 is added t o this yellow solution, the yellow-green color returns. Color changes such as these are commonly observed in the chemistry of technetium phosphate complexes. Coulometric reduction of colorless Tc(VI1) t o green Tc(III), with reoxidation to pink Tc(IV), has been reported in neutral orthophosphate media; analogous reduction to blue Tc(II1) has been reported in neutral pyrophosphate media (7). Similar color changes (colorless to green and yellow) have also been observed with the formation of reduced technetium tripolyphosphate complexes (27). T h e visible spectrum of a NaBH, reduced, technetium-HEDP reaction mixture prepared under argon shows one predominant absorption peak a t 405 nm. Exposure of the preparation to air yields absorption peaks a t 405, 525, and a shoulder a t 450 nm. Since all components exhibit absorption in the blue region, 405 n m was chosen as the wavelength for HPLC detection. Selection of H P L C Conditions. T h e selection of chromatographic conditions was complicated by the abstruse nature of the Tc-HEDP complexes t o be separated. Since the complexes were known t o be anionic, anion exchange was selected as the means of chromatographic separation. Aminex A-27, a moderately cross-linked (87’) polystyrene based quaternary ammonium strong base anion-exchange resin, was chosen as the stationary phase. It was anticipated t h a t the hydrophobic nature of the polystyrene base would minimize interaction of the support with the essentially hydrophilic Tc-HEDP complexes. This is of particular concern since interaction of Tc-diphosphonate complexes with Sephadex degrades the complexes (15). The porous resin was also selected for its high exchange capacity in order to accommodate millimolar concentrations for spectrophotometric detection of the separated components and biodistribution studies of individual components in test animals. Alternative techniques
ANALYTICAL CHEMISTRY, VOL. 52, NO. 7, JUNE 1980
1108
B 0
10
20
0 1 AUFS
a:
25Gnn
01 ALFS
01
4 0 5 rrr
30
LO
T I M E (min)
Figure 1. (A) HPLC of 3.8 mM 99Tc04-+ lo-* M 99mT~04in 0.85 M sodium acetate with detection at 250 nm (flow rate, 0.176 mL/min; pressure, 1550 psi). (B) HPLC of same ligand solution with detection at 405 nm which we are presently evaluating are ion-exchange separation using pellicular resins and reversed phase ion-pairing chromatography. Acetate was chosen as the counterion because of its anticipated noncomplexing properties which would minimize the probability of ligand exchange with the technetium complex during the chromatographic process. Reaction of a Tc(NaBH,)-HEDP preparation with 0.1 M EDTA in pH 7.4 tris/HC104 buffer at 80 “ C produces no spectral change in the UV-visible range in 24 h (28). This suggests that at least some Tc-HEDP complexes are substitution inert, even in the presence of a good complexing ligand such as EDTA. Furthermore, studies have shown that preparations of different T c complexes in the presence of acetate do not yield detectable amounts of Teacetate complexes (29). Acetate was also found to have a sufficiently low selectivity coefficient, relative to other possible counterions, to accommodate competition of the Tc-HEDP complexes for the quaternary ammonium exchange groups of the resin matrix. Other considerations leading to the choice of acetate included its availability in HPLC grade, its lack of corrosive effects on stainless steel components, and its physiological compatibility. The last concern is of particular importance in studies which involve the injection of isolated components into test animals for evaluating biodistribution. Since the mode of separation was ion exchange, the pH of the mobile phase was adjusted to effectively ionize the complexes. At pH 5 , the Tc-HEDP complexes appear to be insufficiently ionized to allow separation on Aminex A-27, but a t p H 8.4 (unbuffered sodium acetate) separation could be achieved. T o minimize the effects of pH changes, the Tc(NaBH,)-HEDP preparations were prepared such that the final sample p H was also 8.4. Finally, the ionic strength was adjusted so that a separation could be effected within reasonable time at a given flow rate and temperature. An ionic strength of 0.85 M was found to produce optimum separation under the conditions described. HPLC of Tc04-and HEDP. Since the radiopharmaceutical analogues have excess HEDP and may contain unreacted Tc04-,the HPLC behavior of these species was first examined. By means of single wavelength UV detection at 250 nm, and y ~detection, HPLC separation of a 3.3 mM NH4 with 9 9 m T ”Tc04- spiked with 99mT~04indicated the presence of an unidentified, nongamma emitting, impurity (Figure 1A). The Tc04- impurity does not absorb at 405 nm (Figure 1B). The TcO,- itself was retained on the precolumn as expected. An HPLC analysis of the HEDP ligand (detection at 250 nm) revealed unidentified H E D P components and/or impurities (Figure 2A). A t 405 nm, the HEDP “impurities” absorb less (Figure 2B). HPLC of Tc(NaBH,)-HEDP Samples. The anion-exchange HPLC analysis of Tc(NaBH,)-HEDP reaction mixtures was first performed with detection a t 405 nm. The resulting chromatograms (Figure 3) show that this Tc-HEDP radiopharmaceutical analogue consists of several Tc-HEDP
Figure 2. (A) HPLC of 0.25 M Na,H,HEDP solution with detection at 250 nm (flow rate, 0.174 mL/min; pressure, 1550 psi). (B) HPLC of same ligand solution with detection at 405 nm
- 1’1
:i
A R
.... ‘h3‘0
I
-
ARZOh
3c
20 YE
c
L”
n
Flgure 3. HPLC of Tc(NaBH,)-HEDP reaction mixture (flow rate, 0.195 rnL/min; pressure, 1380 psi; temperature, ambient) ( - - - - - ) 3.2 mM Tc(NaBH,)-HEDP prepared under argon, (-) 7 0 mM Tc(NaBH,)HEDP prepared in air
A
0
C
23 -IME(m
30
43
n,
Figure 4. HPLC of Tc(NaBH,)-HEDP sample prepared in air and allowed to age for two weeks (flow rate, 0.194 mL/min; pressure, 1450 psi; temperature, ambient) complex components. The anaerobic preparation, with a total technetium concentration of 3 mM, yields peaks e and f which are assigned as “reduced” technetium complexes (e.g., containing Tc(II1)). An equivalent aerobic preparation with a total technetium concentration of 7 mM (obtained by evaporation of a 3 mM solution), gives rise to new peaks, particularly g through m, which are assigned as “oxidized” technetium complexes (e.g., containing Tc(1V) or Tc(V)). Peaks a through d appear to consist of both “oxidized” and “reduced” technetium complexes, plus overlapping impurities characteristic of the free ligand (vide supra). It was observed that a sample prepared in air continued to oxidize with time (2 weeks) until peak k was predominant (Figure 4 ) . If additional NaBH, is added to a mixture which has been air
ANALYTICAL CHEMISTRY, VOL. 52, NO. 7, JUNE 1980
1109
n ii n 360
460
wavelength
560 (nrn)
Figure 7. HPLC of Tc(NaBH,)-HEDP oxidized sample using UV-visible rapid scanning detection (flow rate, 0.170 mL/min; pressure, 1600 psi; 10
20
‘0
33
5c
3
TIME lm n l
temperature, ambient)
Figure 5. HPLC of Tc(NaBH,)-HEDP oxidized sample prepared from the “stable”reduced sample (flow rate, 0.126 mL/min; pressure, 1500 psi; temperature, 25 “C) ‘C
1
h
HPLC
of
9DmTc(NoBHAJHEDP
r-
-
1
m E
301
=
20
= I 20 30 40 50 COLUMN T E M P E R A T U R E ,
GO ‘C
Figure 8. Number of theoretical plates N(measured from peak k) vs. column temperature. Plate number N = 5.5 ( t r / W,,,)* Flgure 6. HPLC of 3.3 mM Tc(NaBH,)-HEDP spiked with 9 9 m T and ~ prepared in air (flow rate, 0.156 mL/min; temperature, ambient); (-) y detection at 140 keV (50-keV window), ratemeter time constant (10 s); ( - - - - ) visible detection at 405 nm (0.1 AUFS)
oxidized, peaks g through m disappear and the resultant chromatogram resembles that of the anaerobic preparation. It thus appears that the green “reduced” complexes and the yellow “oxidized” complexes may be interconverted by the use of appropriate redox reagents. These interconversions are, however, complicated by other kinetic phenomena which are not completely understood. For example, a freshly prepared solution of “reduced” complexes is readily oxidized by air a t 25 “C (Figure 3), whereas a solution prepared and aged (2 h) under argon is resistant to oxidation, even by pure oxygen at 37 “C; however, this stable solution can be oxidized within 2 h by using continuous bubbling of pure oxygen a t 60 “C (Figure 5). These observations imply that there are at least two types of “reduced” complexes-one which is formed immediately upon NaBH, reduction and which is readily oxidized, and a second which is formed only slowly and which is resistant to oxidation. *Tc y Detection. By spiking Tc(NaBH,)-HEDP sample preparations with nanomolar amounts of =TcO;, radiometric y detection and visible detection at 405 nm could be performed simultaneously. T h e resulting chromatograms (Figure 6) illustrate that at least seven of the peaks of both the “oxidized’‘ a n d “reduced” forms contain some 9 9 m T activity ~ with an interestingly high technetium concentration among early eluting components that have relatively low molar absorptivities a t 405 nm. The more strongly retained, or “oxidized” components therefore have much larger molar absorptivities at 405 nm. Consistently 97 t o 100% of the *Tc activity was recovered (maximum counting error of *2%). UV-Visible R a p i d S c a n n i n g Detection. A technique suitable for the characterization of chromatographically pure
h
,
01 A I
!! 0
10
20
30
.3
TIME I m n I
Figure 9. HPLC of Tc(NaBH,)-HEDP well oxidized sample (flow rate, 0.145 mL/min: pressure, 1740 psi: temperature, 60 “C)
Tc-HEDP components is rapid scanning spectrophotometric detection (23). An example of the results obtained via this technique (Figure 7) illustrates the unresolved peaks k and 1 of an oxidized Tc(NaBH,)-HEDP sample preparation. I t can be seen that peak k eluting at a retention time of 40.3 min shows absorbances near 400 nm as well as 450 nm, while peak 1 eluting a t 41.5 min shows only the predominant absorbance near 400 nm. Rapid scan spectra of other chromatographic peaks are presently under evaluation. Effect of Column T e m p e r a t u r e o n Separation. In order to evaluate the effect of column temperature on separation, the column temperature was increased in 5 “C increments from 25 to 60 “C. A well-oxidized sample which had been allowed to stabilize over a period of several weeks was used to provide a constant source of the “oxidized” form represented by peak k. T h e plate height efficiency of this predominant peak was found to increase slowly from 25 to 45 “C and then increase sharply from 50 to 60 “C (Figure 8). T h e
1110
Anal. Chem.
1980, 52, 1110-1114
60 "C chromatogram (Figure 9) gave an absorbance response for peak k five times greater than that observed at lower temperatures. T h e selectivity of some peaks changed slightly with increasing temperature; the broad peak f became more strongly retained at a n approximate rate of +0.12 min/"C, a n d peak k became more weakly retained a t an approximate rate of -0.14 min/'C,. Only minor selectivity changes were observed for t h e remaining peaks.
CONCLUSIONS Radiopharmaceutical analogues prepared by NaBH, reduction of T c 0 4 - in the presence of H E D P are complex mixtures with numerous Tc-containing components. T h e mixture composition is highly dependent on mode of preparation and reaction time. These results stimulate interest in determining the composition of clinically-used skeletal imaging agents in order to elucidate the actual species responsible for imaging. T h e injection of a single, highly specific Tc-HEDP complex, rather than a mixture as is the current practice, could be useful in giving clearer images with lower patient exposure t o radiation. Anion-exchange H P L C is a n efficacious technique for separating these radiopharmaceutical mixtures.
ACKNOWLEDGMENT T h e authors thank T. W. Gilbert for many helpful comments a n d discussions. Also, assistance from C. Becker, K. Libson, D. Ferguson, and K. Hajizadeh was very much appreciated.
LITERATURE CITED (1) Subramanian, G., Rhodes, B. A., Cooper, J. F., Sodd. V . J.. Eds., Radiopharmaceuticals"; The Society of Nuclear Medicine: New York. 1975. (2) Rhodes, B. A,; Craft, B. Y . "Basics of Radlopharmacv"; C. V. Mosbv Co.: St. Louis, Mo.. 1978. (3) Callahan, R . J.; Castronovo, F P. A m J . Hosp. Pharm. 1973 30, 614-17 (4) Yano, Y.; McRae, J.; Van Dyke, D. C.; Angen, H. 0. J. Nucl. Med. 1973, 14, 73-78. (5) Keyes, J. W.; Carey, J.; Moses, D.; Beierwakes, W. "Manual of Nuclear Medicine Procedures", 2nd ed.; CRC Press: Cleveland, Ohio, 1973. (6) Heindel, N. D., Burns, H. D., Honda, T., Brady, L. W., Eds., "The Chemistry of Radiopharmaceuticals"; Masson: New York, 1978; Chapter 17.
(7) Rulfs, C. L.; Pacer, R. A.; Hirsch, R. F. J . Inorg, Nucl. Chem. 1967, 2 9 , 681-91. (8) Russell, C. D.; Cash, A. G . J . Nucl. Med. 1979, 20, 532-37. (9) Steigman. J.; Meinken. G.; Richards, P. Int. J . Appl. Radiat. Isot. 1978, 29. 653-60. (10) Rhodes, B. A., Ed., "Quality Control in Nuclear Medicine"; C. V . Mosby: St. Louis, Mo., 1977; Chapter 21. (11) Zimmer, A. M. J . Nucl. Med. 1977, 18, 1230-33. (12) Taukulis, R. A.; Zimmer, A. M.; Pavel, D. G.; Patel. B. A. J . Nucl. Med. Techno/. 1979, 7. 19-22. (13) Owunwanne, A.; Weber, D. A.; O'Mara, R. E. J . Nucl. Med. 1978, 19, 534-37. (14) Colmbette. L. G.; Pinksy, S.;Moerlieu, S. Radiochem. Radioanal. Lett. 1975, 2 0 , 77-85. (15) Van den Grand, J.A.G.M.; Dekker. B. G.; De Ligny, C. L. I n t . J . Appl. Radiat. Isot. 1979, 30, 129-130. (16) Eckelman, W. C.; Levenson. S. M. Int. J. Appl. Radial. Isot. 1977, 28, 67-82. (17) Front, D.; Hardoff, R.; Mashour, N. J , Nucl. Med. 1978, 19, 974-75. (18) Howman-Giles, R . B.; Gilday, D. L.; Ash, J. M.; Brown, R. G. J . Nucl. Med. 1978, 19, 975-77. (19) Byun, H. H.; Rodman, S. G.; Chung, K. E. J . Nucl. Med. 1976, 77, 374-75. (20) Zimrner, A. M.; Isitrnan, A . T.; Holmes, R. A. J . Nucl. Med. 1975, 16, 352-56. (21) Deutsch, E. I n "Radiopharmaceuticals 11". Proceedings 2nd International Symposium on Radiopharmaceuticals, Seattle, Washington, March 1979; Society of Nuclear Medicine: New York, 1979; p 129-46. (22) Majors, R. E. J . Chromatogr. 1977, 15, 334-51. (23) Denton, M. S.;DeAngelis, T. P.; Yacynych, A. M.; Heineman, W. R.; Gilbert, T. W. Anal. Chern. 1976, 48, 20-24. (24) Hurst, R., unpublished work, University of Cincinnati, 1979. (25) Fogelman, I.; Citrin, D. L.; McKillop, J. H.; Turner, J. G.; Bessent, R . G.; Greig, W. R. J . Nucl. Med. 1979, 20, 98-101. (26) Citrin, D. L.; Bessent, R. G.; Tuohy, J. B. B r . J . Radio. 1975, 4 8 , 118-21. (27) Terry, A. A.; Zittel, H. E. Anal. Chem. 1963, 35, 614-18. (28) Deutsch, E.; Libson, K.; Becker. L. B. J . Nuci. Med.. submitted for publication. (29) Russel, C. D.; Majerik, J. Int. J . Appl. Radiat. Isot. 1978, 2 9 , 109-14.
RECEIVED for review December 10,1979. Accepted February 28, 1980. T h e authors gratefully acknowledge financial support provided by the National Institute of Health Grant No. 1 R01 GM27832-01 (W.R.H.) a n d Grant No. HL-21276 (E. A.D.), the Department of Energy (W.R.H.), the Society of Nuclear Medicine (Education and Research Foundation Pilot Research Grant (E.A.D.)),the Procter & Gamble Company, a Laws Fellowship (T.C.P.), and a University of Cincinnati Summer Fellowship (T.C.P.).
Sampling of Formaldehyde in Air with Coated Solid Sorbent and Determination by High Performance Liquid Chromatography Ronald
K. Beasley, Catherine E.
Hoffmann, Melvin L. Rueppel, and Jimmy W. Worley"
Research Department, Monsanto Agricultural Products Company, 800 North Lindbergh Boulevard, St. Louis, Missouri 63 166
A method for the specific determination of formaldehyde in alr Is described. Formaldehyde is sampled with silica gel coated with 2,4-dinitrophenylhydrazlne. The sorbent is extracted with acetonitrile, and the hydrazone is determined by reverse-phase HPLC with UV detection at 340 nm. The method was validated over the range of 2.5-93.3 pg formaldehyde (0.10-3.8 ppm for a 20-L air sample). Average recovery was 94 percent, with a relative standard deviation of 0.04.
Potential occupational exposure to formaldehyde, a major industrial chemical worldwide, has been a serious concern. T h e concern is based on its significant irritant effects (1-3) 0003-2700/80/0352-1110$01 .OO/O
and on its potential to react with hydrochloric acid to form bis(ch1oromethyl) ether, a known carcinogen ( 4 ) . T h e concern has intensified recently with the announcement by the Chemical Industry Institute of Toxicology ( 5 )that preliminary results of a long-term inhalation study indicate formaldehyde is a carcinogen in rats. Many methods for the determination of formaldehyde in air have been reported (2, 6-12), b u t none allows convenient, reliable, and specific measurement of personnel exposure. We now report a new solid sorhent procedure for formaldehyde which overcomes these problems. T h e sorbent is silica gel coated with 2,4-dinitrophenylhydrazine.Analysis of the resulting hydrazone derivative is by H P L C with UV detection, giving the desired specificity. Humidity and storage effects
c 1980 American Chemical Society
