culty reproducing product yield and purity using bothmethods. This was undoubtedly due to particulars ofthe synthetic apparatus, but indicated a fundamentaldifficulty with the technique. We describe here a new

and automated method and examine the effects ofseveral parameters on the synthesis.

The synthesis uses the published reaction (9—11):

B(n-C4H9)3 —+n-C4H9OH2.H@O

The reaction is exothermic and very rapid. This newmethod incorporates the suggestion of Ido (12) to usea solid support to allow higher flow rates. Rapid washingwith water completes the hydrolysis and washes thebutanol onto a pair of C-l8 cartridges for @Owaterremoval. Butanol (radiochemically 99+% pure) is theneluted with 10% ethanol in saline. Up to 250 mCi ofbutanol can be produced.

MATERIALSAND METHODSTri-n-butylborane (Aldrich or Alfa) was stored under dry

argon. Silica, alumina, and C-18 Sep-Paks (Waters) wereprepared by flushing with dry argon. Silica and alumina wereobtained from Universal Scientific and Aldrich. A HewlettPackard 5890 gas chromatograph with an Ailtech RSL- 150capillary column was used with flame ionization detection.Conditions: 70°C,He flow 5 mi/mm. Retention times: waterand EtOH, 0.69 mm; sec-butanol, 0.97 mm; n-butanol, 1.3mm. A Spectra-physics 4270/8700 HPLC was used with anAlltech Econosil C- 18 column, 15% CH3CN/0.0 1 MNH4OAc, 2 ml/min, with Knauer refractive index and Beckman 170 radioactivity detectors. Retention times of radioactive products were: water, 1.45 mm; sec-butanol, 4.4 mm; andn-butanol, 5.7 mm. Sterility and pyrogen tests were performedaccording to U.S.P. procedures.

Preparationand SynthesisThe alumina cartridge was injected with 75 @l(0.3 mmol)

tri-n-butylborane under dry argon. It was sealed with sleeveseptum stoppers and stored under argon.

Previously used cartridges can be reused up to ten times.They are recycled by washing with ethanol and acetonitrileand drying in vacuum. Old cartridges are eventually discardeddue to mechanical weakness.

Oxygen-l5 was produced by the ‘4N(d,n)'50reaction with8 MeV deuterons on nitrogen gas containing 0.2% oxygen.

The use of labeled butanol for autoradiographic and positron tomographic measurement of cerebral blood flow hasbeen well established using radiocarbon labels. The advantages of the short half-life of oxygen-i 5 (150)in doingsequential flow studies are also recognized. An automatedprocedure has been developed for the routine rapid andsequential synthesis of 150-labeledbutanol in amounts andwith purity suitablefor use in positron tomography. Butanolcan now replace 15O-labeledwater, which is commonlyused for routine applications. The 14N(d,n)15Oreaction isused, with 8 MeV deuterons on a nitrogen target containing0.2% oxygen. Labeled oxygen is reacted with tn-n-butylborane by passing the gas over an alumina support whichholds the reagent. Washing with water through small C18-bonded phase silica cartridges eliminates labeled waterand the majority of boron-containing impurities. Injectablelabeled butanol is collected at 2.5 mm after the end ofbombardment. The yield is 6 mCi per microampere ofsaturated bombardment, measured at the end of synthesis. Injectableproductup to 250 mCican be obtainedat10-mmintervals.

J NucIMed 1990;31:1727—1731

abeled butanol has been shown to be a moreaccurate regional cerebral blood flow tracer than labeledwater (1—8).However, a very short-lived tracer is required for routine clinical positron tomographic (PET)studies. The 2-mm half-life of @Oallows studies at 10-mm intervals and reduces radiation dose. Therefore,sequential studies with improved accuracy would beachievable with ‘5O-butanol.

We previously described (9) a labeling procedurebased on the work ofKabalka (10,11). A 50/50 productmix of ‘5O-butanoland water was obtained in 40%chemical yield. Subsequently, Ido reported (12) the useof C-l8 cartridges for both reagent support and purification with similar results.

While reinstalling a butanol synthesis, we had diffi

ReceivedOct. 23, 1989; revisionaccepted Mar.27, 1990.For reprintscontact: MarcS. Bemdge, PhD,Departmentof Radiology,

case WesternReserveUniversity,Schoolof Medicine,Cleveland,Ohio44106.

1727Oxygen-i 5-Butanol •Berridge et al

A Routine, Automated Synthesis of Oxygen15-Labeled Butanol for Positron Tomography

Marc S. Berridge, Emily H. Cassidy, and Andrew H. Terris

Case- Western Reserve University, Departments ofRadiology and Chemistry, and University Hospitals of Cleveland,Cleveland,Ohio

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ResultsRangeBestTrapped

02activity250—300330ChemiCalyield(%)80—9094RadiOchemiCal

yield(%)16-2528%ACtivityretained onalumina15-187%Activity to waste (H20 +BuOH)10-156%ACtivityretained on C-i810-128%Recovered injectableBuOH55-6584mCi

RecoveredinjectableBuOH50-7084AnalysisRangeBest

. For each parameter measured, the normal range experienced

in over 200 preparations is given as well as the best resultobtained.Ineachcase,the bombardmentusedwas 15 @Afor 10mm Maximumpracticalbeamcurrentis 40 @A.

was prepared within 3 mm. This product containsacceptably low levels of boric acid and carrier butanoland is sterile and pyrogen-free (30 tests). Our maximumroutine beam current (40 @A)gives a maximum yieldof @@-250mCi of injectable butanol.

An alumina bed containing 0.075 ml tri-n-butylborane provided the highest yield and purity of ‘50-butanol. The cartridge could be stored several weeks underargon but only a few days in room air. Silica, C-18bonded silica, glass, wool, and sand were not as effectiveas reagent supports (Fig. 2) Alumina dried at 200°C

COMPARISONOFREAGENTSUPPORTS

FIGURE2Performanceof various reagent supports. For each support(n > 20; except sand, glass, wool, n = 3), the chemicalyieldof injectabiebutanolbasedon starthgoxygen,andthe percentage of water impurity in that product is shown. Thedescribedoptimum procedure (age, load, etc.) was used ineachcasewiththe exceptionof the reagentsupport.*In thecaseof sand,thepuritywas notdetermined.

TABLE 1Results Obtained Using the Optimum Procedur&

The target (0.251)waspressurizedto 6 atm and holds -@-0.l5mmol of oxygen.

The automatedsyntheticsystemis shownin Figure 1.Tworeverse-phase cartridges were joined with a short glass tubeand placed in the helium-purged system. The reagent cartridgewas installed at the end of bombardment, minimizing exposure to air.

Target gas was emptied through the reagent in 30 sec at 31/mm, using an additional pulse of gas to flush the system.Water (I ml) was then injected (Line 1) onto the reagentcartridge. After 3 sec, it was followed by 3 ml of water and 5ml of air, moving butanol from the reagent to the C-l8cartridges. The C-l8 material was then washed (Line 2) with3 x (0.5 ml water + 10 ml air). Oxygen-l5-butanol was theneluted through a sterilizing filter with 5 ml of 10% ETOH/saline.The productwasobtainedwithin2.5 mm afterthe endof bombardment.

All variables in the procedure were investigated in over 400experiments.We report here those experimentswhich represent alterations of the above optimum procedure.

RESULTS

Table 1 summarizes the results obtained using theoptimum procedure in a routine clinical setting. A 10-nun bombardment of 15 microamps typically produced@@@-400mCi of oxygen at end of bombardment (EOB).

From this amount, over 100 mCi of injectable butanol

% Water in product(labeled)0—0.20%Sec-Butanol inproduct4-6NATotal

butanol in product(mg)19—308.5Solidresidue(boroncompounds)(pg)40—2000

Gas Pressure Target Gas

100@

90@

80@

70@

— %YieldProduct

EJ %0—15Water in Product

60C)

0 50

Q@ 40•

30@

Alumina Silica C—la •SandGlass Wool

Reagent Supports

Sterile Syringe and Filter

FIGURE1Butanolsynthesisapparatus.

1728 The Journalof NuclearMedicine•Vol. 31 •No. 10 •October1990

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under vacuum trapped as much as 40% less oxygen andgave a lower conversion to butanol. Water content onthe alumina between 5% and 20% gives optimum performance, with no clear preference within that range.Untreated chromatographic alumina (gamma) fallswithin this range and can be used as delivered. Neutralalpha alumina performed in a similar fashion, but anadditional 25%—30%of the activity was lost as labeledwater waste. Similarly, acidic rny-aluminasent moreactivity to waste, and basic -y-alumina was less efficientat trapping oxygen.

In order to reduce the carrier butanol level, we studied the effects of the oxygen content of the target gas.A maximum yield of 50% of butanol was obtainablefrom 2% oxygen in the target, with the remainder foundas labeled water. One percent oxygen gave up to 90%butanol yields. The minimum concentration tested was0.2%oxygenwhich, like 0.5%and 1%oxygen,gaveexcellent yields. The entire target yield was consistentlytrapped on the reagent even at the highest gas flowsobtainable. This allowed the @0to be collected in aslittle as 20 sec.

Figure 3 shows the effects of the volume of tri-nbutylborane loaded on the alumina, using 0.2% oxygengas. Clearly, additional reagent causes additional activity to remain on the cartridge. Above 75 j@l(a smallexcess over a stoichiometric ratio with carrier oxygen),the yield decreases. Below this amount, the yield dropsrapidly due to a deficiency ofreagent relative to oxygen.

The age of the loaded alumina cartridges (time sinceloading) clearly affected several parameters. Yield, activity trapped at EOB, and conversion oftrapped activity to butanol were all maximized at 24—72hr afterloading the reagent onto the cartridge. Loaded cartndges have remained usable 21 days after loading.Freshly prepared cartridges (aged 15 mm up to 15 hr)do not show any other differences, aside from a significantly lower yield.

A significant amount oflabeled butanol was consistently retained on the reagent cartridge. Only solutionswith a basic pH removed it slightly better than the purewater reference, but the slight gain in yield was insufficient to offset the additional processing requirementsimposed. Use of solutions containing oxidants, acids,or organic solvents and temperature variations from 0to 70°Chad no desirable effects on the results.

The optimum removal ofbutanol from the aluminaoccurs at a total wash volume of 2.0—2.5ml. It wasnecessary to allow several seconds during the initialwash to hydrolyze the intermediate and release butanol.The optimal wash scheme, therefore, is a 1-ml wash,which is loaded onto the cartridge and allowed to standseveral seconds, followed by a 3-ml water wash. The10% of the total activity remaining on the reagentcartridge is high in labeled butanol (95%), but this wasonly removable with an organic solvent.

100

>@

@- 80

U

@a 600

F-

0 40

CC)U@320

0@

0 %Retainedon AluminaCartridge@ S Yield

t:@@ 0

@ 200

@000

diooo 0.100 oioo 0.300 0.400 0.500mL Borone loaded on Alumina

FIGURE3Effect of borane load on the chemical yield (triangles) ofinjectable butanol (based on starting oxygen), and on theamount of radioactivityretainedon the aluminasupport (cirdes). The optimum wash procedure (see text) was used ineachcase. Eachpoint, n = 10, averagesshown.

The function of the C-l8 cartridges is to hold thelabeled butanol so it can be washed free of labeledwater. Two cartridges were necessary to avoid premature removal of the butanol while washing it. Butanolwas retained on a double cartridge while labeled waterwas reduced to <0.4%, and often to zero. Two 0.5-mlportions of water, each followed by an air bolus gavean acceptable level oflabeled water (1.5%) in the product. We preferred to use three portions, which reducedlabeled water to 0%—0.3%at a cost ofan additional 7%of the product being lost to waste. It was necessary toprevent the C-18 cartridge washes from passing throughthe reagent cartridge. This was due to residual water onthat cartridge which slowly elutes, raising product watercontent to a constant 2%.

DISCUSSION

Roleof the AluminaSupportThe use of alumina as a solid support for tri-n

butylborane is a major modification over our previousmethod, raising the chemical yield ofbutanol from 50%to 80%—90%.Therefore, the alumina is acting not onlyas a support but is also taking part in the chemistry.This is supported by the observation that optimumperformance is reached 15 hr after reagent loading.Modifications of borane reactivity by alumina, silica,and florisil supports has been attributed to Lewis acidproperties ofthe supports (13).

The need for an aged reagent cartridge of hydratedgamma alumina requires that alumina is directing thehydrolysis step of the reaction. This is consistent withthe accepted concept (14—16)that a somewhat randomdistribution of ionic sites is present on the surface ofgamma alumina. This distribution allows groups ofanionic acid and cationic clusters to form catalytic sites.Adsorption of water modifies the arrangement of sitesby addition of hydroxyl ions, which explains the need

oioo

1729Oxygen-i 5-Butanol •Berridgeet al

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for a certain amount of water. This also explains theobservation that only gamma alumina is active in directing the butanol synthesis. Alpha alumina, silica, and

the other supports which were tested either do not have

such sites or have them distributed in a more regularpattern which would preclude the formation of catalytically active clusters. While we have not tested this

hypothesis, this alumina model does provide a basis forunderstanding the otherwise peculiar set of observations.

Very little boron is found in the crude product, whichindicates it is not readily washed off of the alumina.

Also, the only boron compound isolated from the system was boric acid, B(OH)3. The BuB(OH)2, which wasobserved in abundance during previous work (9), eitherwas not stable using this method or was not removedfrom the alumina.

Reduction of the oxygen content of the target gas(from 2% to 0.2% in nitrogen) is another significantimprovement over the original method. Although alumina directs the reaction toward butanol, this does notoccur when more than 1% oxygen (1.5 mmol) is usedwith a corresponding amount of borane reagent. Reduction of oxygen also allowed the borane quantity tobe reduced (Fig. 3), and thereby reduced the boron andcarrier butanol in the product.

Clinical Utility of the Method

A clinical method must be fast, easily repeated within10 mm, and give over 100 mCi of butanol. The useof an alumina support, the low oxygen content in thetarget gas, and a rapid purification were very importantin achieving that goal. In applying this procedure toclinical production, it is important to take advantage ofeach of these factors. In our hands, the maximumproduction yield of 250 mCi is then comfortably inexcess of the clinical requirement. The synthesis requires as little as 2 mm, and cleaning and set-up of themanual procedure can be performed within the preferred 10-mm delay between blood flow measurements.The automation of the procedure speeds both of theseaspects ofthe synthesis. All cartridges and solutions canbe prepared in advance, and as noted above, reagentcartridges prepared well in advance give significantlyhigher product yields.

Product CompositionAnalyses by radio-HPLC of the crude product

showed three labeled compounds, water, n-butanol, andsec-butanol (Table 1). Water was removed during theprocedure. Sec-butanol comprised 4%—6%of the totalproduct butanol activity. It originated from sec-butylspresent in the tri-n-butylborane and was not separablefrom the n-butanol without HPLC. Since the utility ofthe radiopharmaceutical as a blood flow tracer is dependent primarily on its lipophilicity (5, 7,1 7), the presence of sec-butanol as a minor impurity would not be

expected to significantly alter the measurements andwas therefore considered to be acceptable.

The yield and the product composition are significantly affected by small changes in the procedure. Theoptimum reagent and wash volumes may vary with thetarget gas, apparatus size, and flow rates. The data givenhere can be used, however, to adjust the conditions foroptimum performance with any particular apparatus.Minimum wash volumes, optimum reverse-phase material load, and the optimum compromise between yieldand purity are easily determined. In this procedure, wehave opted for very high radiochemical purity, allowingnearly 40% of the total butanol to be wasted.

The quantities ofthe nonradioactive impurities present in the product carrier (butanol and boric acid, Table1) are well below any physiologically active dose. Forcomparative purposes, the toxic doses of butanol andofboric acid are in the range ofgrams to tens of grams,and normal serum contains 0. 1—0.2mg of boron perdeciliter (18—22).

AutomationThis synthesis was originally performed, and most of

the data obtained, with a manual system in whichadditions were made by syringe though the two additionlines (Fig. 1). However, it was a simple matter toautomate the system as shown. A regulated gas supplyis used to drive water and the 10% ETOH/saline solution through the apparatus, as well as provide thenecessary bursts of air. Solenoid valves are used todispense air and fluids and direct their flow through theapparatus. Air and fluid volumes are measured bytiming the period that the dispensing valve is open. Theexact timing is determined empirically, and depends onthe gas pressure, and tubing lengths and diameters.Aside from the obvious advantages of automation, thisapproach also speeds the set-up between clinical studiesby eliminating the need to measure the fluid injections.

CONCLUSION

Oxygen-15-labeled butanol can be produced from trin-butylborane in amounts exceeding that necessary forroutine PET use. A 10-mm, 8 MeV deuteron bombardment at 15 vamps can be expected to produce 100 mCiconsistently. The maximum routinely producible quantity using our cyclotron is 250 mCi. The synthesis issufficiently rapid and simple that sequential doses maybe prepared at 8-mm intervals with a manual or automated system. The actual synthesis and purificationrequires up to 3 mm. The purity and toxicity of theproduct are well within prudent limits. This methodrepresents a significant advance in simplicity, speed,and reliability over our previously published approach.

ACKNOWLEDGMENTSThis work was supported by the Ireland Cancer Center of

the University Hospitals of Cleveland.

1730 The Journal of Nuclear Medicine •Vol. 3i •No. i 0 •October i 990

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Med 1986;27:834—839.10. KabalkaGW. Incorporation ofstable and radioactive isotopes

via organoborane chemistry. Accts Chem Res 1984; 17:215—221.

11. Kabalka GW, Lambrecht RM, Sajjad M, et al. Synthesis of‘5-O-labeledbutanol via organoborane chemistry. mt i AppiRadiat Isot 1985;36:853—855.

12.MurakamiM, HagamiE,SasakiH, etal.Radiosynthesisof‘5O-labeledbutanol for clinical use. Proc 6th Intl Svmp RadiopharmChem 1986;81—82.

I 3. Babler JH, Sarussi SJ. Enhanced reducing properties of pyridine-borane adsorbed on solid supports: a convenient methodfor chemoselectivereduction ofaldehydes. J Org Chem 1983;48:4416—4419.

14. Morrison SR. The chemical physics of surfaces New York:Plenum Press; 1977:133—137.

15. Peri JB. Infrared and gravimetric study of the surface hydration of -y-alumina.J Phys Chem 1965;69:211—219.

16. Pen JB. A model for the surface of -y-alumina.J Phys Chem1965;69:220—230.

17. Dischino DD, Welch Mi, Kilboum MR. Raichle ME. Relationship between lipophilicity and brain extraction of C-l 1-labeled radiopharmaceuticals. J Nuc! Med 1983; 24:1030-1038.

18. Lendl L. Untersuchungen uber die Narkosegeschwindigkeithomologer und isomereer einwertiger Alkohole. Arch ExpPathPharmak 1928;129:85.

19. Rost E, Braun A. Zur Pharmakologie der niederen Gliederder einwertigenaliphatischen Alkohole.Arb. Reichsgesundh.Amt. 1926;50:580.

20. Sax. Dangerous properties of industrial materials, Fourthedition. New York: Van Nostrand; 1975.

21. The Merck Index, Tenth Edition. Rahway, NJ: Merck & Co.Inc.: 1983.

22. Rumack BH, ed. Posindef information system. Denver:Micromedix, Inc.; 1988.

1731Oxygen-i5-Butanol•Bemdgeet al

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1990;31:1727-1731.J Nucl Med.   Marc S. Berridge, Emily H. Cassidy and Andrew H. Terris  TomographyA Routine, Automated Synthesis of Oxygen-15-Labeled Butanol for Positron

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