Indirect spectrophotometric determination of BIDA, DISIDA, DTPA and MDP in labelled compounds

AppL Radiat. Isot. Vol. 40, No. 6, pp. 525-529, 1989 Int. J. Radiat. Appl. Instrum. Part A Printed in Great Britain. All rights reserved

0883-2889/89 $3.00 + 0.00 Copyright © 1989 Pergamon Press plc

Indirect Spectrophotometric Determination of BIDA, DISIDA, DTPA and MDP in Labelled Compounds* T J A A R T N. V A N D E R W A L T , P A U L P. C O E T Z E E a n d P I E T E R J. F O U R I E l Department of Chemistry, Rand Afrikaans University, P.O. Box 524, Johannesburg, 2000 Republic of South Africa and qsotope Production Centre, Atomic Energy Corporation of South Africa, Ltd, P.O. Box 582, 0001 Republic of South Africa (Received 22 June 1988)

N-(4-(n-butyl)-acetanilide)iminodiacetic acid (BIDA), N-(2,6-diisopropylacetanilide)iminodiacetic acid (DISIDA), diethylenetriaminepentaacetic acid (DTPA) and methylene diphosphonic acid (MDP) are used in labelling kits. The contents of BIDA, DISlDA or MDP of the 99mTc-labelledcompounds can be determined (indirectly) spectrophotometrically with copper, eriochrome cyanine R (ECC) and dodecylethyldimethylammonium bromide (DEDA) in a sodium barbital buffered system at pH 8.5. The calibration curves obey Beer's Law from 0 to 40 pg/25 mL for BIDA and DISIDA, 0 to 60/~g/25 mL for DTPA and 0 to 100 #g/10 mL for MDP.

Introduction BIDA (Agha et al., 1986; Hernandez and Rosenthal, 1980; Kapuscinski et al., 1986), DISIDA (Chervu et al., 1982; Hernandez and Rosenthal, 1980; Klingensmith et al., 1981), DTPA (Brookeman and Williams, 1970; Eckelmann and Richards, 1970; Hauser et al., 1970; Klopper et al., 1972) and MDP (Littlefield and Rudd, 1983; Subramanian et al., 1975; Tanabe et al., 1983), labelled with 99rnTc are used in the nuclear medicine field. 99mTc-BIDA and 99mTc-DISIDA are useful for in vivo diagnostic studies of the hepatobiliary functions, 99mTc-DTPA for studies of the kidney functions and 99mTc-MDP is an agent for skeletal imaging. Strict quality control of the labelled compounds is necessary because they are generally considered as radiopharmaceuticals. Sterility, apyrogenicity and chemical purity are relevant characteristics of a pharmaceutical product. Analytical procedures for the determination of the components are seldom included in publications on these labelled compounds. Van der Walt and Fourie recently described a complexometric determination of MDP in labelling kits (van der Walt and Fourie, 1987). Technetium-99m is eluted from the 99mM(y-99mTC generator as the pertechnetate ion, which has to be reduced before labelling of the above mentioned compounds. Stannous chloride is present as reduc*This paper represents part of a M.Sc. Thesis by T. N. van der Walt submitted to the Rand Afrikaans University, Republic of South Africa.

tant in all these labellings kits. The MDP, BIDA, DISIDA and DTPA labelling kits, manufactured at the Isotope Production Centre of the Atomic Energy Corporation of S.A., contain respectively 0.5; 0.5; 0.5 and 0.27 mg of SnC12-2H20 per vial. Calciumtrisodium diethylenetriaminepentaacetic acid (CaNa3DTPA) is used as complexing agent in some of the DTPA labelling kits and 2,5-dihydroxybenzoic acid (DHB) (1.0 mg per vial) is added to the local MDP labelling kits as preservative. Thus, the development of a spectrophotometric method (directly or indirectly) for the determination of BIDA, DISIDA, DTPA and MDP has to deal with possible interferences caused by the presence of the stannous ion, calcium ion or DHB. It was noticed that the presence of BIDA, DISIDA, DTPA or MDP in a solution containing trace amounts of copper interfered seriously with the determination of copper by a spectrophotometric method with ECC and DEDA, recently developed by van der Walt et al. (1989). This effect was used to develop an indirect spectrophotometric method for the determination of microgram amounts of these compounds. The chemical basis of the analytical process is briefly as follows: a specific amount of copper is added to the chelating agent (i.e. MDP, BIDA, DISIDA or DTPA). The chelating agent combines with an equivalent amount of copper and the remaining "free" copper is determined with ECC and DEDA. The absorbance is then less than that it should have been with the added specific amount of copper. The decrease in the absorbance is directly 525

TJAARTN. VANDERWALTet al.

526

proportional to the amount of chelating agent present. A calibration curve is thus prepared by plotting the absorbance against the amount of chelating agent present. A straight line, with a negative slope, is found obeying Beer's Law over a specific concentration range. The use of relatively small aliquots of the sample is due to the radioactive nature of the labelled compounds. This paper describes the indirect spectrophotometric method for the determination of BIDA, DISIDA, DTPA and MDP, using a standard copper solution, ECC and DEDA in a sodium barbital buffered system at pH 8.5.

Experimental Reagents Analytical grade reagents and deionized water were used. The water was deoxygenated by bubbling pure nitrogen through the water for at least 30 min and the water was stored in a sealed container in an inert atmosphere. ECC and DEDA were obtained from Fluka (Switzerland), sodium barbital (SB) and DTPA from E. Merck (South Africa), and MDP from Lancaster Synthesis Ltd (England). BIDA and DISIDA were synthesized according to the method described by Zmbova and Konstantinovska-Djokic (1987).

Stock solutions 1. lO0.Ot~g of Cu/mL. Copper (0.100g) was dissolved in diluted nitric acid. The nitrates were converted to chlorides by repeated evaporation to dryness with concentrated hydrochloric acid. After a final evaporation to incipient dryness, the salts were dissolved in water and the solution diluted with water to IL. 2. 2.01~g of Cu/mL and 5.0~g of Cu/mL. Appropriate aliquots of the 100.0#g of Cu/mL standard solution were diluted with water to 200 mL. 3. 500.Oltg of BIDA/mL. BIDA (0.1000g) was dissolved in 100 mL of water containing 2.0 mL of 0.1 M sodium hydroxide. The solution was diluted with water to 200 mL. 4. lO.O#g of BIDA/mL. An aliquot of stock solution (3) was diluted with water to the appropriate volume in a volumetric flask. 5. 500.O#g of DISIDA/mL and lO.O#g of DIS1DA/mL. The stock solutions were prepared as described above under stock solutions (3) and (4), but using DISIDA instead of BIDA. 6. 500.Ol~g of DTPA/mL. DTPA (0.1000g) was dissolved in 70 mL of 0.01 M sodium hydroxide and diluted with water to 200 mL. 7. lO.Ol~g of DTPA/mL. An aliquot of stock solution (6) was appropriately diluted with water. 8. 1.O00mg of MDP/mL. MDP (0.2500g) was dissolved in and diluted with water to 250 mL. 9. 50.Ot~g of MDP/mL. Appropriate dilution of stock solution (8) was made with water.

10. lO0#g of SnCI2.2H20/mL. SnC12.2H20 (50.0 mg) was dissolved in 10 mL of 5 M hydrochloric acid and diluted with deoxygenated water to 500 mL. 11. lO.Ol~g of SnCI2.2H20/mL. Appropriate dilution of the stock solution (10) was made with deoxygenated 0.1 M hydrochloric acid. 12. lO.Ol~g of DHB. 2,5-dihydroxybenzoic acid (5.0 mg) was dissolved in 5 mL of methanol and diluted with water to 500 mL. 13. lO.Opg of Ca/mL. Calcium carbonate (5.0 rag) was dissolved in 50 mL of 0.01 M hydrochloric acid and diluted with water to 200 mL. 14. l m M ECC. ECC (0.1073 g) was dissolved in and diluted with 0.01 M hydrochloric acid to 200 mL. 15. lOmM DEDA. DEDA (0.6448 g) was dissolved in and diluted with water to 200 mL. 16. O.1M SB. Sodium barbital (2.06g) was dissolved in ca 75 mL of water, neutralized with 0.1 M hydrochloric acid to pH 8.50 and diluted with water to 100 mL. 17. 1% H202. An aliquot of 8.3 mL of perhydrol (30% H202) was diluted to 250mL with water. Apparatus A Varian Super Scan u.v.-vis, spectrophotometer was used for spectrophotometric measurements.

Interference studies Influence of Sn 2+ and Sn 4+ on the determination of DISIDA and BIDA. A series of mixtures, containing 16.0~g of DISIDA (or BIDA)/mL and x/~g of SnClz-2H20/mL (x = 0; 1.6), was prepared in 0.01 M hydrochloric acid. A series of solutions was prepared by measuring out y mL of each mixture (y = 1.00; 2.00) into 25 mL volumetric flasks containing either none or 0.2 mL of 1% hydrogen peroxide. *Two millilitres of water were added and the solutions set aside for ca 15 rain. To each flask the following stock solutions were added in the order: 5.00 mL of 2.0/~g of Cu/mL; 1.0 mL of 1 mM ECC, 3.0 mL of 10 mM DEDA; and 2.5 mL of 0.1 M SB. The solutions were diluted with water to the mark, the air displaced with nitrogen, the flask stoppered and set aside overnight. A reagent blank was prepared similarly, but without copper, tin and DISIDA (or BIDA). The absorbance of each solution was measured against the reagent blank in a h0 cm cell at 567 nm. The results are presented in Table l(a).

Influence of Sn 2+, Sn 4+ and Ca: + on the determination of DTPA. A series of mixtures was prepared containing x/~g of DTPA/mL (x = 10.0; 20.0); y/~g of SnClz.2H20/mL (y = 0; 2.0) and z #g of Ca/mL (z = 0; 1.0) in 0.01 M hydrochloric acid. A series solutions was prepared by measuring out v mL of each mixture (v = 1.00, 2.00) into 25 mL volumetric flasks, containing either none or 2.0 mL of 1% hydrogen peroxide. The same procedure was then followed as from *, as described in the section above, and the results shown in Table l(b).

Spectrophotometric

determination of BIDA, DISIDA,

DTPA

and MDP

527

Table l(a). Interference studies: influence o f Sn ~÷ and Sn 4÷ on the determination o f D I S I D A and B I D A A m o u n t o f component present, # g DISIDA

SnCI:. 2H20

Molar ratio DISIDA/Sn

Tin ion present

Absorbance

-6.4 6.4 -6.4 6.4

-Sn 2+ Sn 4÷ -Sn 2÷ Sn 4÷

0.445 0.439 0.443 0.251 0.241 0.252

Molar ratio BIDA/Sn

Tin ion present

Absorbance

-7.0 7.0 -7.0 7.0

-Sn 2+ Sn 4+ -Sn ~+ Sn 4+

0.444 0.440 0.445 0.234 0.226 0.234

16.0 0 16.0 1.6 16.0 1.6 32.0 0 32.0 3.2 32.0 3.2 Kit:molar ration D I S I D A / S n = 51.5 A m o u n t o f component present, # g BIDA

SnC12 . 2H20

16.0 0 16.0 1.6 16.0 1.6 32.0 0 32.0 3.2 32.0 3.2 Kit: molar ration B I D A / S n = 56.0

Table l(b). Interference studies: influence o f S n ~+, Sn 4+ and Ca 2+ on the determination of D T P A A m o u n t o f component present, # g DTPA

Ca

M o l a r ratio DTPA

DTPA

Ca

Sn

SnCI 2- 2H20

10.0 0 0 -10.0 1.0 2.0 1.0 10.0 1.0 2.0 1.0 20.0 0 0 -20.0 1.0 4.0 2.0 20.0 1.0 4.0 2.0 Kit: Molar ration D T P A / C a = 9.8 and D T P A / S n 0

-2.9 2.9 -2.9 2.9 = 10.6

Tin ion present

Absorbance

Sn z+ Sn 4+ -Sn 2+ Sn 4+

0.537 0.529 0.536 0.425 0.421 0.425

Table l(c). Interference studies: influence o f S n ~+, Sn 4+ and D H B on the determination of MDP A m o u n t of component present, # g MDP

DHB

SnCI 2 • 2H20

Molar ratio MDP DHI ~

MDP Sn

100.0 0 0 --100.0 20 0 4.4 -100.0 0 20 -6.4 100.0 0 20 -6.4 100.0 20 20 4.4 6.4 Kit: M o l a r ration M D P / D H B = 4.4 and M D P / S n = 12.8

Influence o f Sn 2+, Sn 4+ and 2,5-dihydroxybenzoic acid (DHB) on the determination o f MDP. A series of mixtures was prepared containing 50.0#g of MDP/mL and either x/~g of SnCI2. 2HzO/mL (x = 0; 10) or y/~g of DHB/mL (y = 0; 10), or both x #g of SnCI2-2H20 and y g of DHB. A series of solutions was prepared by measuring out 2.0mL of each mixture into 25 mL volumetric flasks. 1% H20 z (0.2 mL) was added to the flasks containing the MDP and both SnCI2.2H20 and DHB. The same procedure was then followed as from *, described in the first section. The results are reported in Table l(c). Calibration curves for the indirect spectrophotometric determination o f BIDA, DISIDA, DTPA and M D P with Cu, ECC and DEDA BIDA. A series of standard solutions was prepared by mixing 5.00mL of the 2.0#g of Cu/mL, in a

Tin ion present

Absorbance

--Sn 2+ Sn 4+ Sn 4+

0.523 0.487 0.079 0.525 0.523

25 mL volumetric flask, with the following stock solutions in the order: x mL of the 10.0#g of BIDA/mL (x = 0; 0.5; 1.0; 1.5; 2.0; 2.5; 3.0; 3.5; 4.0); * 1.0mL of 1 mM ECC; 3.0mL of 10mM DEDA and 2.5 mL of 0.1 M SB. The solutions were diluted with water to the mark and set aside for at least 4 h. The absorbances were measured against a reagent blank (prepared similarly as above, but without copper and BIDA) in a 1.0 cm cell at 567 nm. The calibration curve is shown in Fig. 1. DISIDA and DTPA. Calibration curves were obtained for the determination of DISIDA and DTPA as described under the previous heading, but using either DISIDA or DTPA instead of BIDA. The calibration curves are shown, respectively, in Figs 2 and 3 for DISIDA and DTPA. MDP. A series of standard solutions were prepared by mixing xmL of 50.0/~g of MDP/mL (x = 0.20;

TJAARTN. VAN DER WALT et al.

528

10

O.?'0.6

g 0.5

~

0.4

~ <

03 I 0

0.2

10

_

20

I

I

I

I

30

40

50

60

,u.g D T P A / 2 5 r n [

0.1 0

I

I

10

20

I 50

I 40

Fig. 3. Calibration curve for the indirect spectrophotometric determination of DTPA with copper, ECC and DEDA in a sodium barbital buffered system at pH 8.5.

/zg BIDA/25mt

Fig. 1. Calibration curve for the indirect spectrophotometric determination of BIDA with copper, ECC and DEDA in a sodium barbital buffered system at pH 8.5.

0.50; 1.00; 1.50; 2.00; 2.50); 1.0mL of 10.0pg of SnC12-2H20/mL; 2.00mL of 2 . 0 # g of Cu/mL; 1.0mL of 10.0#g of D H B / m L and 0.1 mL of 1.0% H202, in 10 mL volumetric flasks. The solutions were set aside for 24 h, and the following stock solutions were then added in the order; * 0 . 4 m L of 1 m M ECC; 1.2mL of 1 0 m M D E D A and 1.0mL of 0.1 M SB. The solutions were diluted with water to the mark and left for 4 h. The absorbances of the solutions were measured against a reagent blank (similarly prepared, but without copper, M D P , tin and D H B ) in a micro-cell, with a 1.0 cm pathlength, at 567 nm. The calibration curve is shown in Fig. 4.

umetric flask, containing 1.0 m L of 1% H202. The solution was diluted to volume with 0.05 M hydrochloric acid and left overnight. An aliquot of 1.00 mL was pipetted into a 25 mL volumetric flask containing 10.0PC of Cu and the method followed from * as described under "Calibration c u r v e s - - B I D A " . DTPA. An amount of 25 p L of the 99mTc-DTPA solution were pipetted into a 25 mL volumetric flask, containing 1.0 mL of 0.01 M hydrochloric acid and 0.2 mL 1% H20. The mixture was set aside for 24 h 5.0 mL of 2.0 ~g of Cu were added and the determination carried out following the method from * as described under "Calibration c u r v e s - - B I D A " . MDP. An amount of 2 5 g L of the 99mTc-MDP solution were pipetted into a 10 mL volumetric flask, containing 0 . 2 m L 0.1 M hydrochloric acid and 0.1 mL 1% H202. After 24h 2 . 0 m L o f 2 . 0 # g of Cu were added and the method proceeded from * as described under "Calibration curves M D P " .

Determination o f the DISIDA, DTPA and M D P contents o f solutions containing the 9~Tc-labelled compounds Preparation o f the labelled compounds. Technetium99m was eluted from the 99Mo-99mTc-generator with sterile saline solution (0.9% mass/volume (m/v) sodium chloride). Five mL of this solution were added to the vial containing the labelling kit. The flask was shaken until the freeze-dried material was completely dissolved. DISIDA. An amount of 2 5 # L of the 99mTc D I S I D A solution were pipetted into a 1 0 m L vol0.7

Discussion

The interference studies show that Sn 2+ interfered only slightly with the determination of DTPA, D I S I D A and BIDA, lowering the a b s o r b a n c e - Tables l(a) and (b). The interference was eliminated by the addition of a small amount of hydrogen peroxide in order to oxidize Sn '+ to Sn 4+ which had no influence on the determination. Table I(b) shows that calcium did not interfere with the determination of D T P A (the calciumtrisodium salt of D T P A is used in some labelling kits). In the determination of M D P the relevant amounts of stannous chloride dihydrate

0.6 05

10

8 0.4

o 0.3 <

0.5

0.2 0.1

I

I

I

I

10

20

30

40

#g DISIDA/25mt

Fig. 2. Calibration curve for the indirect spectrophotometric determination of DISIDA with copper, ECC and DEDA in a sodium barbital buffered system at pH 8.5,

__

I

I

[

25

50

75

,__A 100

/zg MDP/IOmt

Fig. 4. Calibration curve for the indirect spectrophotometric determination of MDP with copper, ECC and DEDA in a sodium barbital buffered system at pH 8.5.

Spectrophotometric determination of BIDA, DISIDA, DTPA and MDP Table 2. Determination of the BIDA, DISIDA, DTPA and MDP contents in solutions containing the 9'~Tc labelled compounds Amount of compound Aliquot of (mg per vial) Labelled solution compound take (/~L) Taken Found DISIDA 5 40.0 40.0 + 0.3 BIDA 5 43.0 43.0 + 0.3 DTPA 50 5.0 4.9 ± 0.1 MDP 50 5.1 5.1 + 0.3 *Average of triplicate analyses.

prevented the colour development almost completely, as indicated in Table l(c). This is probably due to the reduction of the Cu 2+ ion, which was not sufficiently stabilized by the M D P , as in the case of BIDA, D I S I D A and D T P A . Although Sn 4+ had no influence on the determination, of M D P , the preservative, 2,5-dihydroxybenzoic acid (DHB) interfered significantly. However, in the presence of Sn 4+ and a small amount of hydrogen peroxide the interference was negligible. F o r this reason it was decided to prepare the calibration curve for the determination of M D P in the presence of the same amounts of stannous chloride and D H B which are used constantly in the labelling kits. The tin(II) chloride contents of the various labelling kits vary widely from one manufacturer to another. Excess amounts of tin(II) chloride (compared with the actual contents of our labelling kits) were used in all the experiments to study the interferences of Sn 2 + and Sn 4+. Table l(a) shows that an eight-fold excess of tin(II) chloride had only a small influence when present as Sn z+, but none when present as Sn 4÷. Tables l(b) and (c) show similar results respectively for a three-fold and a two-fold excess of tin(II) chloride. A ten-fold excess of calcium did not interfere with the determination of DTPA. N o t all commercial M D P kits contain D H B , but the absence of D H B will not cause any problem since the calibration curve for M D P is prepared according to the contents formulation of the kit. The calibration curves (Figs 1-4) show that Beer's Law is obeyed for ( b 4 0 # g / m L for B I D A and D I S I D A ; 0-60 #g/25 m L for D T P A and 0--100 pg/ l0 m L for MDP. The slopes of the calibration curves are negative which is caused by complex formation between copper and these compounds, resulting in a lower absorbance reading due to less "free" copper ions. The results, obtained for the determination of BIDA, D I S I D A , D T P A and M D P contents of solutions containing the relevant 99mTc-labelled compound, show that the method is very sensitive and accurate (Table 2). The relative standard deviation was less than 6%, using aliquots of 5 - 5 0 p L .

529

The proposed method is an excellent means for the determination of microgram amounts of BIDA, D I S I D A , D T P A and M D P in solutions of the 99mTc-labelled compounds.

References Agha N. H., AI-HiUi A. M., Dahir N. D., AI-Hissoni M. S., Jasim M. N., Miran K. M. and Shubber A. H. (1986) Long-term clinical investigation of the hepatobiliary agents: 99mTc-HIDA and ~Tc-p-butyi-IDA. Nuktearmedizin 25, 18 I. Brookeman V. A. and Williams C, M. (1970) Evaluation of 99mTc-DTPA acid as a brain scanning agent. J. Nucl. Med. 11, 733. Chervu L. R., Nunn A. D. and Loberg M. D. (1982) Radiopharmaceuticals for hepatobiliary imaging. Semin. Nucl. Med. 12, 5. Eckelmann W. C. and Richards P. (1970) Instant 99mT~DTPA. J. Nucl. Med. I1, 761. Hauser W., Atkins H. L., Nelson K. G., Richards B. S. and Richards P. (1970) Technetium-99m DTPA: a new radiopharmaceutical for brain and kidney scanning. Radiology 94, 679. Hernandez H. and Rosenthal L. (I980) A cross-over study comparing the kinetics of 99mTc-labelled diisopropyl and p-butyl IDA analogs in patients. Can. Clin. Nucl. Med. 5, 159. Kapuscinski J., Liniecki J., Durski K. and Mikiciuk-Olasik E. (1986) Comparison in rabbits of chole-scintigraphic properties of several ~ T c - I D A derivatives. Nuklearmedizin 25, 188, Klingensmith W. C. III, Fritsberg A. R., Spitzer V. M., Kuni C. C. and Shanahan W. S. M. (1981) Clinical comparison of diisopropropyl-lDA Tc99m and diethylIDA Tc99m for evaluation of the hepatobiliary system. Radiology 140, 791. Klopper J, F., Hauser W., Atkins H. L., Eckelmann W. C. and Richards P. (1972) Evaluation of 99~Tc-DTPA for the measurement of glomerular filtration rate. J. Nucl. Med. 13, 107. Littefield J. L. and Rudd T. G. (1983) Tc-99m hydroxymethylene diphosphonate and Tc-99m methylene diphosphonate: Biological and cliical comparison: Concise communication, J. Nucl. Med. 24, 463. Subramaniam G., McAfee J. G., Blair R. J., Kallfelz F. A. and Thomas F. D. (1975) Technetium-99m-methylene diphosphonate--A superior agent for skeletal imaging: Comparison with other technetium complexes. J. Nucl. Med. 16, 744. Tanabe S., Zodda J. P., Libson K. Deutsch E. and Heineman W. R. (1983) The biological distribution of some technetium-MDP compounds isolated by anion exchange high performance liquid chromatography. Int. J. Appl. Radiat. Isot. 34, 1585. van der Walt T. N. and Fourie P. J. (1987) Complexometric titration of thorium with methylene diphosphonic acid: application to the determination of methylene diphosphonic acid in labelling kits. Appl. Radiat. Isot. 38, 158. van der Walt T. N., Coetzee P. P. and Fourie P. J. (1989) The determination of copper in MIBG solutions before or after labelling with radioiodine. S. Aft. J. Chem. In press. Zmbova B. and Konstantinovska-Djokic D. (1987) Synthesis and quality control of 2,6-diisopropyl IDA and its labelling with technetium-99m. Isotopenpraxis 23, 278.