In-target production of [13N]ammonia: Target design, products, and operating parameters

Appl. Radiat.ht. Vol. 44, No. 12,pp. 1433-1441,1993 Printedin Grest Britain. All rights reserved

0969-8043/93 $6.00+ 0.00 Copyright 0 1993Pergamon Press Ltd

In-target Production of [13N]Ammonia: Target Design, Products, and Operating Parameters MARC S. BERRIDGE

and BONNIE J. LANDMEIER

Departments of Radiology and Chemistry, Case Western Reserve University and University Hospitals of Cleveland, 2074 Abington Rd, Cleveland, OH 44106, U.S.A. (Received 16 April 1993) [‘%jAmmonia, commonly used in PET, has been prepared in good yield in the target. Up to 800 mCi of radiopharmaceutical was obtained by in-line processing of the irradiated water. Relatively low hydrogen pressure was used to control the target chemistry. Target designs and the product dependence on beam dose, dose rate and pressure are reported. Ethanol as a target additive was also investigated. Hydrogen and ethanol together were more effective than either alone at high beam dose. Parameters are reported for production needs from single doses to synthetic applications. An incidental method for production of [“Njnitrogen gas is also reported.

Introduction Ammonia labeled with r3N is a standard radiopharmaceutical for routine qualitative determinations of myocardial perfusion. It is used frequently in clinical determinations of coronary flow reserve, myocardial viability and myocardial perfusion patterns (Harper et al., 1972; Schelbert and Schwaiger, 1986). Recently (Chan ef al., 1992; Kuhle ef al., 1992) it has been used for quantitative determinations of myocardial perfusion. Nitrogen-13 is commonly produced by the 160(p,a)*3N reaction in a natural water target with combinations of inert gas pressure and recirculating systems (Krizek et al., 1973; Vaalburg et al., 1975; Parks and Krohn, 1978; Lindner et al., 1978; Tilbury and Dahl, 1979; Patt et al., 1991; Wieland et al., 1991). These targets have long been known to produce ammonia directly at low beam dose (Welch and Straatman, 1973; Tilbury and Dahl, 1979). However, when sufficient beam doses are used to make radiopharmaceutical quantities of ammonia, oxidation occurs to give predominantly nitrate and nitrite (Krizek et al., 1973; Parks and Krohn, 1978; Tilbury and Dahl, 1979; Patt et al., 1991). These have been reduced to ammonia for radiopharmaceutical use using wet chemical procedures that require 3-10 min of processing and give chemical yields of SO-90% (Krizek et al., 1973; Vaalburg et al., 1975). More important for a clinical positron emission tomography (PET) operation, wet chemical methods require a synthetic apparatus and the time to prepare measured reagents, maintain the apparatus, and clean and re-set it for sequential use, occasionally with

associated radiation exposure. Such methods also are subject to a certain rate of failure from mechanical, chemical and human variability. Although the use of Devarda’s alloy and titanium trichloride have been standard practice for many years, it has been known for some time that direct production of useful amounts of ammonia in the cyclotron target was possible. Tilbury and Dahl (1979) showed that the addition of small amounts of various organic radical scavengers, including I-10 mM ethanol and acetate, would allow the production of ammonia at higher beam doses. They produced sufficient quantities in this manner (> 200 mCi ammonia) to allow human PET studies. Unfortunately, the goal of the work was to produce much larger quantities for synthetic use and ammonia for clinical studies was not produced. Tantalizing reports by Mulholland et al. (1989, 1990) of work done incidental to target designs, stated that hydrogen gas under pressure was another effective in-target anti-oxidant. This work also was not extended. Recently, another report (Wieland et al., 1991) has appeared in which the use of ethanol as a target additive was successfully revisited specifically for production of ammonia suitable for PET studies. For the past seven years we have also been interested in the elimination of wet chemical processing for ammonia and in the simplicity and speed of in-target production. We have implemented several varieties of in-target ammonia production and investigated the target behavior, chemistry and production parameters from the perspective of single-dose production in the PET clinic, as well as for large-scale

1433

MARC S. BEIWDGEand BONNIEJ. LAND-

1434

production to supply synthetic, multiple-camera, or remote site applications, This report represents data from over 1000 [13N] ammonia preparations during that time period.

Experimental Materials were obtained commercially and used without further purification, except where noted. Gasses were obtained from Gas Technics and Linde and were high-purity grade. HPLC was performed using Beckman 110B pumps with Rheodyne injectors and a Knauer refractive index detector type 198. A radiation detector was used which consisted of a 1 mL flow-through detection volume in front of a shielded 2 x 2 cm sodium iodide crystal with photomultiplier (Harshaw) and associated electronics (Canberra), including analog rate meter output to the Hewlett Packard chromatography data station. An Alltech 5 pm Adsorbosphere SCX cation exchange column was eluted at 2 mL/min with 2 mM aqueous NH,OAc to which 1 mM acetic acid was added (pH = 5) [RT (min): water 2.3, hydroxylamine 2.3, NO, 1.8, NH, 4.9; water and hydroxylamine were quantified by decay curve analysis until water production was eliminated]. An Alltech 10pm Econosil C-18 column eluted with 15% acetonitrile in 0.01 M aqueous ammonium acetate was used for identification of labeled water [RT (mitt): water 1.5, ammonia 41 in the product mix. Gas chromatography was performed on a Hewlett-Packard 5890 chromatograph with a thermal conductivity detector and Beckman Model 170 flow through radiation detector. An Alltech CTR-I column was used with a He gas flow rate of 25 mL/min for identification of radiolabeled nitrogen gas (RT: N, 0.15 and 1.9 min [two concentric columns in the CTR-I]). An Alltech Superox capillary column with He flow of 2.8 mL/min was used for investigation of intermediates in the target solution (RT: NH, 1.4 min, N,H, 1.9 min, water 6.0 min). Gamma spectroscopy was performed with a 3 x 3” NaI well counter using a Canberra multichannel analyzer and associated electronics. A Scanditronix MC-17 cyclotron, producing nominal 17 MeV protons was used to irradiate a natural abundance water target. Two targets were used. Both were of single-foil design and included a gas head space above the beam strike region. Target 1 was nickel-plated copper in which milling had exposed some copper surface, and target 2 was aluminum. The water chamber volumes and dimensions for targets 1 and 2, respectively, were: 2.5 mL (2.5 cm W x 3.5 cm Hx2mmD),and5mL(4cmWx3.7cmHx3mm D). Both targets had a shape shown schematically in Fig. 1 (solid lines). The dotted lines on the target chamber in Fig. 1 indicate the relative shape of each target before adoption of the head space, or “keyhole,” design (Tewson et al., 1987). The optimal water load volumes of the targets were determined

experimentally to be 2.0 mL (Target 1) and 3.0 mL (Target 2). The targets were installed as shown in Fig. 1. Gas pressure was applied to the target above the water chamber, and was used to deliver the water to the hot cell after irradiation. Target water (Millipore Mini-Q System with activated carbon, cation and anion exchange, and organic cartridges, 18 MR) was introduced from the laboratory through a 6 m, 0.2 mm i.d. Teflon tube, equipped with a valve and optional loading pump. An automatic valve (General Valve 9-568-900, rated to 100 psi, 680 kPa) was placed a short distance from the target on the 0.2 mm i.d. Teflon tube leading to the hot cell and allowed the product to be released to the hot cell after bombardment. The targets were pressurized before the water was loaded into them. This was done because hydrogen is trapped in the tubing between the delivery valve (Fig. 1) and the tee at the base of the target during loading. Pressurization after adding the water would force water into the tubing to compress this gas and partially empty the target. The target was routinely loaded and emptied once before irradiation to ensure that atmospheric gasses were flushed from the system. Following irradiation, the target was emptied into a glass vial inside the hot cell. Gasses from the target, which was tested gastight to the highest operating pressures, were led to a trap containing 8-12 mesh 4A molecular sieves (Aldrich Chemical Co. 20860-4) cooled in liquid nitrogen. The gaseous products were measured immediately with a Capinltec CRC-7 radioisotope calibrator. The liquid fraction was measured and analyzed by HPLC. The gas fraction was analyzed by warming the molecular sieves and withdrawing a sample for gas chromatography. The completeness of radioactive gas release from the molecular sieves was verified by flushing the trap briefly with gas and verifying that all residual radioactivity had been removed. For radiopharmaceutical use, the water was removed from the target under hydrogen pressure and

Fig. 1. Schematic diagram of the target and ammonia production system, showing the target till level, beam strike region and water pathways. Valves are indicated by circles, with solid X for manual, dotted line for normally closed solenoid. The dotted line on the target chamber indicates the initial chamber shape, and the solid line shows the enlarged chamber with open headspace.

In-target ammonia production passed through (Fig. 1) an in-line filter with a 1 mL bed of Bio-Rad AGl-X8 100-200 mesh anion exchange resin in the chloride form. The column was prepared before use by washing with 5 mL portions of water, ethanol and water. The water then passed through a Millipore (vented Millex-GS, 0.22pm, pre-wet with 0.5 mL sterile aq. 9% NaCI) sterile and sterilizing filter, and into a sterile vial or syringe. There was a built-in test of filter integrity due to the fact that the hydrogen pressure would forcibly eject the product from the collection syringe or vial if the filter was not intact. The product was tested for sterility using USP procedures, and for pyrogenicity with the Limulus Amebocyte Lysate test (Whittaker Bioproducts, Inc) according to the manufacturer’s instructions.

Results and Discussion Target design There was no performance difference between the two targets in either total 13N yield or product distributions. A target chamber which has both copper and nickel in contact with the target water might be expected to produce various radionuclides in small proportions. However, raw target water from Target 1 gave a gamma spectrum which was unchanged after 4 h of decay, and was consistent with “N. The target chambers as originally configured (Fig. 1, solid lines) had no head space for gasses. Target 2 was the Scanditronix recirculating water target for 13N production. Target 1 was an early design Scanditronix small volume ‘*O-water target for ‘*F production. The targets in these configurations were limited to low total beam dose because of the full water chamber with a narrow vent tube. Gas production through radiolysis or boiling ejected a significant percentage of the load within S-20 mm at beam currents > 10 p A, as has been reported for F- 18 production (Tewson et al., 1987). This prevented high yields and cast doubt on the ability of the target water to equilibrate with hydrogen. The target modifications as shown in Fig. 1 solved the problem. A relatively thick target foil was important to eliminate “0 contamination in the product after rapid ammonia production with 17 MeV protons. The previous method used a recirculating water target, chemical reduction of nitrogen oxides, and distillation of the product. It removed all traces of “0 by decay during the 5-10min process and by the separation of 90% of the target water from the product by the distillation. However, after rapid delivery of the undiluted target water we observed up to 10% [‘SO]water in the product at EOS. Water was identified by GC and HPLC, by half-life, and by distillation in proper proportion with water solutions. Each analytical system was calibrated with [‘sO]water prepared by our standard method (Berridge et al., 1990). Contamination with 10% water would not be acceptable for routine clinical use. The I50 content at

1435

EOB was reduced to zero by increasing the sum of the Havar target entrance foil thicknesses from 0.05 to 0.10 mm (two foils, 0.05 mm ea.). This reduced the incident particle energy from nominal 17 MeV to below the 16.6 MeV threshold of the ‘Q(p,pn)“O reaction (Landolt-Bomstein, 1973). Additive experiments-general

observations

The total 13Nyields followed the theoretical target yield expression [equation (1); A is produced activity, S is saturation yield, I is beam current, 1 is the decay constant 0.0695 mm-‘, and t is irradiation time]. The average saturation yield was 31.7 + 4 mCi/pA. A = SI(1 -e-l’)

(1)

Only the distribution of radiochemical forms differed as a function of irradiation conditions. The labeled products observed were “NO,, 13N2, 13NH3, “NH,OH, ‘*F- and HiSO. Fluoride was present in very low quantity ( 340 KPa) of hydroxylamine was produced when hydrogen and/or ethanol was used, and has also been observed previously (Patt et al., 1991). It was identified by HPLC vs standard hydroxylamine. It was not separated from ammonia by distillation or by short columns of cation or anion exchange resin, C- 18 bonded silica, alumina or silica. The quantity present did not vary systematically with the bombardment time. For brevity in the following discussions and in the figures, yields are expressed in terms of the radiochemical percentage of ammonia in the product. Except where noted, the remaining radioactivity was nitrogen gas, with no more than a few percent of nitrogen oxides, and the total yield was consistent with equation (1) (S = 31.7). Standard deviations of product percentages were generally between 3 and 10% of their measured values. Full sets of results are shown in Table 1 for a sample of representative bombardment parameters.

MARCS. BERRIDGE and BONNIJZ J. LANDMEIER

1436

Tabk I. Full data for rcprcacntative data pointsfrom the dataset used for thii study H2 F’rea.

DUlUtiOll

WW

0 0

140

140 I40 I40 I40 I40

0 0 IO 10

I40 140

:

z 340 340 E

30 0 0 0 0 IO 0 0 0 0 0

520 520 520 520 520 520 520 520 520

1

2 3 IO

520 680 680 680 680 680 MO* MO* MO* g 340’

:8 0 0 0 0 10 0 IO IO IO IO IO

IO IO 30 30 IO 30 30 IO 30 IO IO 30 30 IO IO IO 30 30 30 30 30 30 30 30 30 IO 30 30 30 30 IO IO 30 30 IO 30

@in)

n

% NH,

% N,

% NO,

2 IS 2 2 6 2 I5 2 20 2 I5 20 2 20 2 I5 30 I5 I5 IS 6 30 6 3 2 I5 30 30 2 2 6 30 2 6

IO a 4 6 3 IO a 2 2 4 1 I 2 3 8 6 6 IO 5 I I 3 4 6 2 I 2 4 2 2 I 3 2 I I 2

71.4 43.9 26.8 II.1 83.2 66.4 27.5 98.9 25.1 86.5 62.9 11.2 37.1 99.5 94.9 66.2 81.6 46.9 42.9 13.9 81.9 100.0 88.5 95.1 39.6 96.5 89.5 72.0 62.5 99.4 1.4 96.9 25.6 0.3 69.6 76.0

22.3 50.4 71.2 81.2 0.0 10.9 62.2 0.0 12.4 II.8 36.0 21.2 61.3 0.0 4.2 32.5 16.8 53.8 56.2 25.0 II.0 0.0 9.9 0.1 58.3 3.0 IO.1 28.0 36.8 0.5 16.8 0.3 38.9 II.4 0.0 19.0

6.4 5.8 2.1 1.7 16.8 22.1 10.3 1.2 1.9 I.7 I.1 1.6 0.9 0.5 0.9 I.3 I.5 0.0 0.9 I.1 I.1 0.0 1.6 4.8 2.2 0.5 0.5 0.0 0.7 0.1 81.8 2.1 35.6 88.3 30.4 5.0

Total (mCi)

mCi NH, 83 IO5 32 62 34 68 88 42 I40 41 156 96 220 245 43 I60 I03 270 321 419 549 604 267 683 I41 58 Ill 419 451 740

II7 233 122 556 41 II8 323 42 543 48 250 124 582 247 2:: 127 601 172 648 625 604 304 721 351 60 124 582 129 144 31 44 306 188 41 214

4: 78 2

Hydrogen pressum, ethanol concentration, run parameters and the number of observations for each data point are given with the pmntage yields of the major products and the absolute total and ammonia yields at EDB. The NO, value includes nitrate, nit& and bydroxylaminc, though valoes >3% reprcaent 90+% nitrate plus nitrite. *These experiments were performed using helium pressure instead of hydrogen; nitrogen gave similar results.

- a- - 10uA.allprcds. Hydrogen -

14OKPx,3OuA

4

4w

600

800

1000

1200

Beam Dose (uAyp*min)

Fig. 2. Radiochemical yields of ammonia from the target under hydrogen pressure without ethanol added, as a function of beam dose rate, hydrogen gas pressure and total beam dose. The longest times shown for 10 and 30 PA beams are both 45 min. Points shown are averages of 2-10 determinations.

1400

In-target ammonia production

1437

10 mM EtOH, Non-Hydrogen

90 80 g

70

-

980KR.

-

S2OKP&3OUA

-

14oKPa.3ouA

1ouA

Pfio f 50 .E m40

B

tPm 20 10 0 0

100

200

300

400

500

700

600

a00

900

BeamDose (uAmp*min) Fig. 3. Radiochemical yields of ammonia from the target under helium and nitrogen gas pressure with 10mM EtOH added to the target water, as a function of beam dose rate and pressure.

Hyakogen pressure Application of relatively low (140 KPa) Hz pressure during 10 PA (l-6 min) irradiations increased the percentage of ammonia in the product from zero to near 90% (Fig. 2). The percentage of ammonia in the product mixture decreased as the irradiation time increased (Fig. 2). Operation of the target with higher hydrogen pressures (up to 680KPa) with a 10 PA beam did not alter the yield curve shown. This implies that the full reductive effect of hydrogen gas was achieved at 140 kPa for a IOPA beam.

At higher beam current, 30pA, oxidation in the target increased, and ammonia production at 140 KPa H2 pressure dropped to near 20%, regardless of the irradiation time. However, at this beam current an increase in Hz pressure was effective for increasing the production of ammonia (Fig. 2). Each pressure increase, to the upper limit of 680 KPa, resulted in higher ammonia yields. At the highest pressure the yield curve for a 30pA beam was similar to that obtained at 10 PA and 140 KPa pressure.

Hydrogen with EtOH (>2mM)

-

34olcPa,1ouA

-

14oKPa.3ouA

-52OlCP&3OUA -68oKP8.3oaA 10 j 0

4

0

100

200

3M

400

500

600

700

800

BeamDose (uAmp*min)

Fig. 4. Radiochemical yields of ammonia from the target under hydrogen pressure with EtOH added to the target water, as a function of pressure. All concentrations of EtOH above 2 mM were averaged to give the data shown.

900

MARCS. BEIU~IDGE and BONNIE J. LANDMEIER

1438 Table

2. Optimum bombardment parameters and minimum hydrogen pressures to achieve appropriate yields for some common purposes Irradiation

Application

Activity (ma)

Pressure @Pa)

pA

min

Single dose clinical study

I5

140 680

0 0

IO 10

I I

90 98

Test-retest second dose

30

140 680

0 0

10 IO

2 2

85 96

Test-retest one batch

13s

520 520

0 0

10 30

14 4

70 80

Synthesis (low)

300

520 520

0 10

30 30

10 6

74 95

Synthesis (high)

700

520

10

30

30

95

Irradiations up to 45 min long produced 60-70% (N 500 mCi) ammonia. Though the additional data is not shown in Fig. 2, a similar trend was observed at intermediate beam currents. At each beam current, a curve similar to the highest yield curves in Fig. 2 could be obtained. As beam dose rate increased, the H, pressure which was required to achieve the highest yields also increased. However, the highest yields obtainable were less than quantitative, and further increases in pressure did not produce additional yield. The limitations of the apparatus prevented the proper investigation of higher hydrogen pressures. However, some attempts to perform irradiations at 850 and 1050 KPa indicated that at 30pA the yield does not improve at these pressures. Our inability to operate routinely at higher pressure has made it impractical to explore the behavior of the system at higher beam current. Ethanol additive

As previously observed (Tilbury and Dahl, 1979; Wieland et al., 1991), addition of ethanol (l-10 mM) to the target water consistently raised ammonia production yields (Table 1, Figs 2 and 3). Less than 3 mM ethanol had a measurable concentrationdependent effect. At concentrations above 3 mM an increase in ethanol concentration did not alter the results. High concentrations of ethanol (> 10 mM) led to the formation of tar at high beam doses. The tar was visible in the raw product as a yellow to brown color which was easily removed by filtration or passage through silica or alumina. In the limit of this process successive irradiations of 100 mM ethanol with a 30 PA beam for 15 min produced sufficient tar to block the target passages and require disassembly and cleaning of the target. Under relatively low pressure (140 KPa He) with a 30 PA beam the yield of ammonia with added ethanol was high at low beam dose, and dropped exponentially to zero at high beam dose (Fig. 3). As the ammonia percentage dropped with higher beam dose, a large (up to 90%) fraction of NO, was observed, indicating that the reductive power of ethanol had been exhausted. This curve can be compared to the

[EtOH]

% NH,

curves in Figs 2 and 4 at the same gas pressure and beam dose rate. Higher helium or nitrogen pressure caused higher ammonia yields. This was a direct effect of pressure on the target chemistry, not a result of suppression of target boiling or radiolysis, because the total “N saturation yield remained constant. The pressure effect is probably responsible for the discrepancy between these results and those of Wieland et al. (1991) which were obtained with substantially higher target pressures. Combined activities

At low beam dose the highest percentage of ammonia was obtained when ethanol was used with or without hydrogen, though the increase compared to hydrogen alone was insignificant for practical purposes. However, at high beam dose the effect of added ethanol was large; a combination of ethanol and hydrogen produced a greater yield than either alone. The ammonia yield from 30 PA, 15 min, irradiations under 140 KPa of H, increased by a factor of 3 when 10 mM ethanol was added (55 to 165 mCi). With 520 KPa of hydrogen the yield was even higher, without using ethanol (266 mCi). Addition of ethanol (10 mM) at this pressure increased the yield by an additional factor of 2 (532mCi). At this point 99.96% of the r3N produced was recovered as ammonia. It is, therefore, clear that a combination of hydrogen and organic additive is necessary to maximize production from high dose irradiations at these pressures. The maximum ammonia yield was achieved with 30 min, 30 PA irradiations of 10 mM EtOH solutions under 500-700 KPa hydrogen, which gave an average of 706 mCi of ammonia at the end of the process, or 97% of the 13N produced in the target. Practical considerations: production parameters

An important benefit of in-target ammonia production is the short “synthesis” time. The lack of post-irradiation decay reduces the beam dose needed to produce clinical doses. Short, low beam dose irradiations (10 PA, l-6 min) were more than sufficient for routine clinical production (Table 2). High

In-target ammonia production hydrogen pressure or addition of ethanol were not required to give high ammonia yields at such a low beam dose. Due to concern over the fate of ethanol in an irradiated target and reports (Ferrieri ef al., 1993) that the 2-carbon of ethanol can be converted to small amounts of formaldehyde, we preferred to avoid organic additives during clinical production when possible. At the high beam doses needed to produce ammonia for use as a synthetic precursor, the addition of ethanol with hydrogen pressure gave a greatly increased production capability compared to either method alone. This rapid production of ammonia in large quantity allows one to consider production for delayed or multiple use and for distribution to relatively remote sites. The method reported here produced labeled ammonia with high radiochemical yield (30mCi/pA sat., EOS) and purity suitable for radiopharmaceutical use. No unlabeled impurities were detected in the product. There was, as discussed above, a small quantity of “N-labeled hydroxylamine in the product. It could not be removed even by distillation; however, the percentage present in the product was not great enough to affect quantification in a PET study and was far below the toxic level (< 3 mnol vs 1.8 mmol/kg LD, i.p. in mice). Implementation of the method was straightforward. Minor modifications in target plumbing allowed the use of hydrogen overpressure. Less hardware was required than for most widely used methods. It was possible to flash-distill the product in seconds from a high temperature vessel containing a convenient base (NaOH, Na,CO,) in the event that the use of ethanol or other concerns prevent in-line processing of the target water. Rapid distillation retained many of the advantages of in-target production, including the high yield, speed and convenience. Though the distillation process took more time and gave a lower yield (18.7 mCi/pA, sat., EOS), it remained an improvement over wet reduction methods (TiCl,, 10.7 mCi/pA, sat., EOS). Target chemistry It is clear that the addition of hydrogen and/or ethanol caused a drastic change in target chemistry, shifting the undesired byproduct from NO, to N, . It is less clear what the mechanism is for the change, though the knowledge could be useful. We have made several observations which may contribute to an understanding of the mechanism. There was a -200 PA . min ‘induction period’ for N2 production which is evident in Fig. 2. Also, because labeled N2 contains two nitrogen atoms, it must be produced by a reaction between two nitrogen-containing species. These facts strongly imply that there was an unlabeled, nitrogen-containing, intermediate which was required for production of labeled nitrogen gas and which was formed by irradiation under H, pressure. Identification of this

1439

intermediate could lead to even higher ammonia production and, if the production of ammonia and of Nr are mechanistically linked, to an understanding of the formation of ammonia. Nitrogen gas did not seem to be a direct nitrogen source in the reaction; sparging the target water with helium and vacuum distilling it under an argon stream immediately before use did not alter the product distribution. Also, a target pressurized with nitrogen did not produce large quantities of labeled nitrogen. Nitrogen gas is, however, the most likely starting material for radiolytic production of another nitrogen-containing intermediate. Target water was very unlikely to contain other nitrogenous materials because it was produced in a high quality Milli-Qo (ion exchange and organic removal) system. Also, the addition of inorganic nitrogen to the target as potassium nitrate and nitrite did not increase N, production. Hydroxylamine was observed in the products of irradiation, and could arise from radiolysis of nitrogen or oxidation of ammonia. However, hydroxylamine also did not seem to be involved in the target chemistry. Addition of 25 mM hydroxylamine during irradiation produced a good yield (80%) of labeled hydroxylamine, but did not increase nitrogen gas production. In all of these experiments, ammonia made up the majority of activity which was not N, (or hydroxylamine). Addition of ammonia (> 10 mM) to the target water before irradiation resulted in high yields of nitrogen gas even from low beam dose experiments, consistent with a previous observation under different conditions (Suzuki and Iwata, 1977). In the case of a need for labeled nitrogen gas, this is an excellent production method capable of generating hundreds of millicuries. Target water with 1 mM or less of ammonia was much less effective for nitrogen gas production. This result validates the already obvious concept that an additional nitrogen source contributes to N2 production. Unfortunately, gas chromatographic experiments failed to detect any carrier ammonia (detection limit 0.3 PM) in the target water from high beam dose runs which had produced large quantities of 13N,. This suggests that carrier ammonia, though effective in high concentration, was also not a factor in the normal target chemistry. We also established through decomposition experiments with added carrier that hydrazine, a potential labeled intermediate (Suzuki and Iwata, 1977; Tilbury and Dahl, 1979), was not present after irradiation. Ethanol and hydrogen appear to operate through different mechanisms. Ethanol almost completely suppressed N, and NO, production at low beam doses but allowed NO, production at high beam dose. Hydrogen was more effective as beam dose increased and was completely effective for suppression of NO, production, but a percentage of the Nt production could not be suppressed by hydrogen alone. Further, the effects of ethanol and hydrogen were additive, which implies that ethanol was able to suppress the portion of N2 production which was

1440

MARCS. BERRIDGEand BONNIEJ. LANDMEIER

unaffected by hydrogen. Hydrogen, in turn, maintained the reducing environment after the ethanol was exhausted and prevented NO, formation. The increase in ethanol effectiveness [Fig. 3, and Wieland ef al. (199111caused by applied He or N, gas pressure is also interesting, and may indicate that a molecular volume increase occurs during a rate-limiting step necessary for labeled NO, production from ammoma. It is not clear at this time what that step might be. These observations are consistent with, but do not necessarily require, a mechanism in which a hotatom, partially protonated, nitrogen may be further reduced to ammonia through hot-atom proton abstraction reactions, or may react with an unlabeled nitrogen-containing species. A similar reaction must occur between protonated 13N atoms and an unlabeled nitrogenous species through the thermal chemistry of aqueous radicals, though the specifics of either of these reactions are purely speculative at this point. The abundance of water in the target is more than sufficient for the required proton abstractions. The direct nitrogen source does not appear to be hydroxylamine, ammonia, nitrogen gas, nitrate or nitrite, and it must be produced from some other source of nitrogen in the target during irradiation. A labeled dinitrogen intermediate formed by hot-atom or thermal processes would be an unstable derivative of hydrazine, which is known to decompose to produce ammonia or nitrogen gas. Naturally, this can only occur when a reducing environment prevents accumulation of oxygenated radicals which would oxidize 13N and any stable nitrogen species to NO,. Hydrogen and ethanol both provide the reducing environment necessary to suppress radiolytic oxidation. Under this scheme, hydrogen under sufficient pressure must also be able to suppress the thermal reaction which gives rise to nitrogen gas, but cannot affect the hot-atom reaction. The mechanism of action for ethanol is clearly not the same as for hydrogen. In this scheme ethanol, or its radiolysis products, must prevent the accumulation of species responsible for oxidation of labeled ammonia, as does hydrogen. In addition, ethanol must either inhibit formation of the unlabeled nitrogen species which is necessary for Nz formation, inhibit the decomposition pathway of the hydrazine derivative which produces N,, or enhance the conversion of hot-atom nitrogen species into ammonia and thereby prevent the formation of a hydrazine derivative. Though hydrazine itself has been observed by others (Suzuki and Iwata, 1977), and a mechanism for its formation from carrier ammonia and decomposition to N, was proposed (Tilbury and Dahl, 1979), it is probable that this pathway is not responsible for our observations. We cannot rule out hydrazine as a transient intermediate, but we did not observe any hydrazine in our products. We were also able to rule out the proposed hydrazine formation mechanism (Tilbury and Dahl, 1979) as a factor in labeled

nitrogen production on the basis that the concentration of ammonia present in the target water is insufficient to cause the observed quantity of nitrogen gas.

Conclusion Radiopharmaceutical ammonia labeled with N-13 has been produced directly in the cyclotron target using only hydrogen overpressure in a single-foil target of “keyhole” design. Very short bombardments are sufficient for all clinical applications. Longer bombardments are possible under elevated hydrogen pressures and with the addition of ethanol to the target. In this way 600-800mCi can be produced for delayed clinical use or for radiosynthesis. There is a complex relationship between the target product composition and beam dose, beam dose rate, hydrogen pressure and ethanol concentration. In addition, pressurization with gases other than hydrogen was noted to increase the effectiveness of ethanol. The simple proton abstraction mechanism for ammonia formation has not been challenged, though several potential mechanisms for the production of labeled nitrogen gas have been eliminated. Acknowledgement-This work was supported National Institutes of Health (HL-43884).

by the

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