Chromatographic quality-control procedures for Tc-99mradiopharmaceuticals are an important part of the totalquality-control program in a nuclear medicine facility. Numerous chromatography procedures for Tc-99m agents havebeen mentioned in the literature—includingWhatman No. 1paper chromatography with 85% methanol (1); Gelmaninstant thin-layer chromatography silica gel (ITLC-SG)with 85% methanol (2) ; and Gelman 1TLC-SG with acetone(3). Other thin-layer and paper-chromatography media andsolvent systems for Tc-99m agents have also been investigated (4,5). In addition, a chromatography system has beenintroduced that not only determines free [‘°Tc]pertechnetate but also determines reduced Tc-99m not bound to theradiopharmaceutical (hydrolyzed reduced Tc-99m) (3—8).Recently, a miniaturized chromatography system has beendescribed for specific Tc-99m radiopharmaceuticals (9).The purpose of the present study was to investigate a rapidminiaturized chromatography system that could be usedeasily for practically all Tc-99m radiopharmaceuticals.

MATERIALS AND METhODS

3 1 ETC chromatography paper, ITLC-SGt chromatography paper, and instant thin-layer chromatography silicic acid(ITLC-SA)t were cut into 1-cm X 6-cm strips. Pencil lines

were drawn across the paper at 1, 3, and 5 cm from the bottorn of the strip and the resultant sections were labeled1 and 2 with a pencil for the 3 1 ET paper and ITLC-SApaper, and 3 and 4 for the 1TLC-SG paper. In order toidentify each strip more readily, colored tape was added tothe top of each type of chromatography paper. In addition,specific marking-pen lines (which move with the solvent andthus identify the solvent front) were drawn on the reversesides of the chromatography strips. The completed strips areshownin Fig. 1.

Various Tc-99m radiopharmaceuticals—including pertechnetate, Tc-99m sulfur colloid, Tc-99m Sn MAA, Tc-99mSn phytate, and Tc-99m stannous chloride—wereeachspotted on the bottom pencil line of 10 strips of 31 ETchromatography. The strips were promptly placed in lO-mlvials, to which approximately 1 ml of acetone was addedand developeduntil the solvent front had migrated to thetop pencil line (approximately 30 sec). The strips were

Received Dec. 14, 1976; revision accepted July 20, 1977.For reprints contact: A. M. Zimmer, Sec. of Nuclear

Medicine, University of Illinois Med Ctr., 840 S. Wood St.,Chicago, IL 60612.

1230 JOURNAL OF NUCLEAR MEDICINE

jIUII/TECHNICAL. NOTE

Rapid Miniatu rized Ch romatog raphic

Quality—Control Procedures for Tc-99m

Radlopharmaceuticais

A. Michael Zimmer and Dan G. Pavel

University of Illinois Medical Ce,uer, Chicago, Illinois

Our laboratory has adopted a complete miniaturized chromatographysystem for Tc-99m radiopharmaceuticals in order to improve upon thecommercial systems currently available. Three distinct, separate, chrometographic procedures are used to determine the labeling efficiencies ofTc-99m-labeled sulfur coiloid, MAA, stannous chloride, phytate, DMSA,DTPA, pyrophosphate, diphosphonate, methylene diphosphonate, polyphosphate, and glucoheptonate. The chromatographic systems include Whatman31 ET paper and acetone,GelmanITLC-SGand 0.9% sodium chloride, andGelman ITLC-SA and acetone. The chromatographic strips are miniaturized (1 x 6 cm), colored-coded, marked, and numbered. AU the chrometographic quality-control procedures are simple, rapid, and can easily beincorporated into the routine quality-control program of any nuclear medicine facility.

JNuclMed 18:1230-1233,1977

RadiopharmaceuticalChromatographyStrip(%of totalactivity)Tc-99m

sulfurTc-99m Tc-99mstannousTc-99mstripand solventsection Pertechnetate colloidSn MAA chlorideSn phytate

TECHNICAL NOTE

dried and cut into eight equal segments: four below thecenter line and four above. Each segment was then countedfor activity.

Tc-99mSn DMSAwas spotted on the bottom pencil lineof ten ITLC-SA paper strips,developedin acetone (approximately 45 sec), sectioned and counted similarly.

A two-strip miniaturized chromatography system was usedfor Tc-99m Sn diphosphonate,Tc-99m Sn pyrophosphate,Tc-99m Sn glucoheptonate,and Tc-99m Sn DTPA. Tendeterminations were performed on each agent. Each determination consisted of spotting the specific radiopharmaceutical on both 31 ET paper strips and ITLC-SG paper stripson the bottom pencil line. The 31 ET strips were immediately placed in a vial containing approximately 1 ml ofacetone and developed until the solvent front migrated tothe top pencilline (about 30 sec). The ITLC-SGstrips weresimultaneously placed in a vial containing approximately1 ml of 0.9% NaCI and developed until the solvent frontreached the top pencil line (about 45 sec). Both strips werethen removed,cut into four segmentsabove and four belowthe center line, and each segmentcounted for activityusinggamma scintillation spectrometry.

For each study, the sum of the net segment counts wasdetermined (ten individual observations for each segment).The total counts for a particular segment were then cxpressed as a percentage of the summed counts of all thesegments.

RESULTS

The results for determiningfree pertechnetateusing the31 Er and acetone chromatography system are given inTable 1. As the data show,free pertechnetatemigratescloseto the acetonesolventfront (Rt = 0.9), whereasthe specificTc-99mradiopharmaceutical(Tc-99m-labeledsulfur colloid,MAA, stannous chloride, or phytate) remains at the origin(Rt = 0.0). From the data, it is quite evident that if thestrips were cut at the center pencil line, separating sections1 and 2, the specific radiopharmaceutical would be locatedin section 1 and free pertechnetate in section 2. A simplifiedprocedure was formulated for these specific radiopharmaceuticals and is listed in Table 2. It is currently used in ourroutine quality-control program.

The results for determining free pertechnetate in Tc-99mSn DMSA preparations using the 1TLC-SA paper and ace

A

B

C

IFIG. 1. Chromatographystrips

Whatman El and ITLC-SG,(C) ITLC-SA.used.(A) Whatman31 ET;(B)

TABLE 1. ACTIVITY DISTRIBUTION (% STRIP COUNTS) FOR SPECIFICTc-99m RADIOPHARMACEUTICALSUSING SINGLE-STRIPMINIATURIZED CHROMATOGRAPHY PROCEDURE

31 ET pap.r andacetone

1 (origin)2

0.60.4

9730.1

97-90.50.20.20.40.40.30.1

99.60.20.00.00.00.10.00.1

9920.20.10.00.00.20.30.0

3 0.4 0.10.10.40.70.50.3

4 0.65 2.4

30.438.0272

678 (solvent

front)

Volume 18, Number 12 1231

11

RadiopharmaceuticalChromatographyStrip(°Ioof totalactivity)Pertech-

Tc-99mstripand solventsectionnetate Sn DMSA

Radiopharmaceutical(%of total activity)Tc-99m

SnTc-99mSnTc-99m Sn methyleneTc-99m SnTc-99mSnChromatographyStripPertech

Tc-99mpyrophos diphos- diphos polyphos.glucostrip and solventsectionnetateSn DTPAphatephonate phonatephateheptonate

ZIMMER ANDPAVEL

the origin. With ITLC-SO paper and 0.9% NaC1, Tc-HRremains at the origin, whereas free pertechnetate and thespecific Tc-99m radiopharmaceutical migrate with the 0.9%NaCl solvent front. Good separation was observed betweenthe various radiochemical components, thus allowing thesectioning of each strip at the center line. The proctocol inuse for these specificradiopharmaceuticalsis listed in Table 5 and is currently used in our daily radiopharmaceuticalquality-control program.

DISCUSSION

We have basically divided the chromatographic evaluationof Tc-99m radiopharmaceuticals into two basic techniques:(a) a procedure involving single chromatography strips,which determine free pertechnetate, and (b) a procedure

TABLE 3. ACTIVITY DISTRIBUTION (% STRIPCOUNTS) FOR Tc-99m Sn DMSA USING

SINGLE-STRIPMINIATURIZED CHROMATOGRAPHYPROCEDURE

Silicic acid 1 (origin) 0.0 71.6instantthin- 2 0.0 20.6layer chromatog- 3 0.0 5.2raphyand 4 0.0 1.0acetone 5 9.2 0.8

6 46.2 0.57 32.8 0.28 (solvent 11.6 0.1

front)

tone chromatography system are shown in Table 3. Technetium-99m Sn DMSA remains at the origin (Rf = 0.0),whereas free pertechnetate migrates close to the solventfront (Rf = 0.75). Separation of the radiochemical components is again achieved by cutting the strip at the centerpencil line. The same simplified procedure, as described inTable 2, is now used in our routine quality-control program.

The results of the chromatographic technique involvingtwo distinct chromatography systemsfor specific Tc-99mradiopharmaceuticals are shown in Table 4. Free pertechnetate migrates close to the acetone solvent front on 31 ETpaper (Rf = 0.9) leaving the specific Tc-99m radiopharmaceutical and hydrolyzed reduced Tc-99m (Tc-HR) at

TABLE 4. ACTIVITY DISTRIBUTION (% STRIP COUNTS) FOR SPECIFICTc-99m RADIOPHARMACEUTICALSUSING TWO-STRIP MINIATURIZED CHROMATOGRAPHY PROCEDURE

31 El paper andacetone

1 (origin)2345678 (solvent

0.60.40.40.62.4

30.438.027.2

97.30.40.00.00.00.40.91.0

77.31.40.10.22.4836.53.4

97.91.90.00.00.00.00.10.0

82.013.44.10.10.10.10.10.1

84.912.12.60.10.10.10.00.1

98.31.30.10.00.10.10.10.0

front)(origin)Silica gel instant

thin-layerchromatographyand 0.9% NaCI

234S678 (solvent

front)

0.00.00.00.00.00.315.783.9

0.60.00.00.00.21.0

26.871.3

0.50.30.20.31.0

11.629.956.1

0.30.20.30.5031.9

10.985.2

0.10.10.10.10.20.41.9

96.9

0.60.10.20.20.41.16.0

91.3

0.20.10.00.00.10.42.5

963

1232 JOURNAL OF NUCLEAR MEDICINE

TABLE 2. DETERMINATION OF FREEPERTECHNETATEIN Tc-99m-LABELEDMAA,

PHYTATE, SULFURCOLLOID, STANNOUSCHLORIDE, AND DMSA

1. Placeapproximately 1 ml acetone into a 10-mI glass vial.2. Spotradiopharmaceuticalon bottompencilline of What

man 31 ET chromatography paper (Gelman ITLC-SA forTc-99m Sn DMSA).

3. Immediately place strip in vial containing acetone, anddevelop until solvent front migrates to top pencil line.

4. Cutstripat centralpencilline,producingsections1 and2.5. Count each section far activity (per unit time) using a

gamma counter, and subtract background.6. % freepertechnetate_I- (netctssect.2) ]<

—[(netctssect.1)+ (netctssect.2)]

TECHNICALNOTE

used for practically all Tc-99m radiopharmaceuticals. Labeling or tagging efficiencies can usually be determinedwithin 3—5mm after spotting. We use these chromatographysystems, as outlined with minimal interference within thenormal routine of the nuclear-medicine department.

Compared with the most commonly used commercialchromatography kit, our chromatographic procedure is morerapid, with a development time of 30—45sec, as opposedto one of about 4 mm for the commercial kit. The shortened development time considerably reduces the time neededto perform the entire chromatographic procedure, and italso reduces the amount of oxidation to free TcO4 thatcan occur in the spotted radiopharmaceutical. The commercml kit also appears to give unusually high values for thehydrolyzed reduced Tc-99m fraction in Tc-99m pyrophosphate (9). The quality-control procedure used in this studycan also be used to evaluate a total of 11 Tc-99m radiopharmaceuticals, whereas the commercial kit can evaluateonly seven.

FOOTNOTES

C Whatman Chromatography Products, Clifton, N.J.

t Gelman Instrument Company, Ann Arbor, Mich.

REFERENCES

1. GUTKOWSKIRF, DWORKIN HJ: A simplified radiochromatographic purity check. I Nuc! Med 12: 513—515,1971

2. Sm@NV, HETZELKR, ICE RD: Radiochemical Purityof Radiopharmaceuticals Using Gelman Seprachrom (JTLC)Chromatography. Ann Arbor, Gelman Instrument Co., 1974

3. BILLINGHURSTMW: Chromatographic quality controlof @‘mTc-labeIedcompounds. I Nuc! Med 14: 793—797,1973

4. ECKELMAN WC, RICHARDS P: Analytical pitfalls with@mTclabeled compounds. I Nuci Med I3: 202—204,19725. ZIMMERAM, HOLMESRA: Chromatographic Quality

Control Procedures for ‘°mTc-LabeledRadiopharmaceuticals.Presented at the 121st Annual Meeting of the AmericanPharmaceutical Association to the Academy of GeneralPractice of Pharmacy, Chicago, 1974

6. ECKELMAN W, MEINKEN 0, RIcHARDS P: Chemicalstate of @SmTcin biomedical products. I Nuci Med 12: 596—600, 1971

7. ECKELMANWC, MEINKEN 0, RICHARDSP: Chemicalstate of @°mTcin biomedical products. 11. The chelation ofreduced technetium with DTPA. I Nuci Med 13 : 577—581,1972

8. ZIMMERAM, HOLMESRA : A precise chromatographysystem for specific Technetium-99m radiopharmaceuticals.NuclearMed (Stuttg)14:192—195,1975

9. COLOMBETTI LO, MOERLIEN S, PATEL GC, et al:Rapid determination of oxidation state of unbound @mTcand labeling yield in @‘Tclabeled radiopharmaceuticals.I Nuci Med 17: 805—809,1976

10. ALBERS JW, JENKINS D, SANDEEN RJ, Ct al: Free(unreacted) pertechnetate in technetium-sulfur colloid preparations. I Nuci Med Tech 2: 14—17,1974

11. ALBERS JW, SANDEEN R, JENKINS D : Quality-controltests for @“Tc-sulfurcolloid. I Nuc! Med 14: 468, 1973

TABLE 5. PROCEDURE2: DETERMINING FREEPERTECHNETATEAND HYDROLYZED REDUCED

Tc-99m IN Tc-99m-LABELEDDTPA,GLUCOHEPTONATE,DIPHOSPHONATE,

METHYLENEDIPHOSPHONATE, PYROPHOSPHATE,AND POLYPHOSPHATE

1. Placeapproximately1 ml of acetoneinto one 10-mIglassvial and 1 ml of 0.9°!. NaCI into an identical vial.

2. Spot radiopharmaceuticalon bottompencil line of Whatman 31 ET paper chromatography and Gelman ITLC-SGchromatography strips.

3. Develop 31 ETpaper strip in acetone solvent,and ITLC-SGstrip in 0.9% NaCI solvent, until solvent front migratesto top pencil line.

4. Cut stripsat center pencil line into sections1, 2, 3, and 4.5. Count all sections for activity (per unit time) using a

gamma counter and subtract backgrounds.

% free pertechnetate

—1 (netctssect.2) 1@L (net cts sect. 2) + (net cts sect. 1) ]

% hydrolyzed reduced Tc-99mF (netctssect.3) 1l lx100.L(netctssect.3)+ (netctssect.4)]

% labeling radiopharmaceutical—100_I %free@ hydrolyzed— [pertechnetatej [reducedTc-99m

involving two chromatography strips, which determines freepertechnetate and Tc-HR. An ideal situation would be thedetermination of Tc-HR for all Tc-99m radiopharmaceuticals, but such a determination is difficult to perform onsome radiopharmaceuticals with paper and/or thin-layerchromatography. Particulate radiopharmaceuticals will remain at the origin, as will Tc-HR, and no differentiation canbe achieved (3). A rapid way to determine unbound Tc-99min particulate radiopharmaceuticals (greater than 0.2 zm) isto filter the suspension through a 0.2-pm millipore filter, inwhich case an indication of Tc-HR is achieved in conjunctionwith the chromatographic determination of free pertechnetate (10,1 1) . An appropriate alternative to determineunbound Tc-99m is centrifugation of particulates with repeat washings.

The chromatography strips are initially marked with pencil lines because these remain stationary with the solventsused. Marking-pen lines, which move with the solvent front,are placed on the back of the strip to ensure that no interaction occurs between the spotted radiopharmaceuticals andthe marker. The strips are also numbered and color-codedto simplify the entire chromatographic procedure. The chromatography strip is immediately placed in the solvent withthe radiopharmaceutical spot still wet to reduce the degreeof radiopharmaceutical oxidation which yields free pertechnetate. Oxidation is further reduced by the rapid solvent development of the miniaturized chromatography strips.

Our miniaturized chromatography procedure is extremelyrapid, easy to perform (see Tables 2 and 5), and can be

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