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A simple neutron activation method is deser~ed for platinum determination in urine and serum of dogs when studying the pharmaeokineties of cisplatin, ...
Journal o f Radioanalytical Chemistry, VoL 75, Nos 1-2 (1982} 71-80

DETERMINATION OF PLATINUM IN URINE AND SERUM A F T E R THE ADMINISTRATION OF CISPLATIN BY NEUTRON ACTIVATION ANALYSIS + J. KUCERA,* J. DROBNIK** *Nuclear Research Institute, 25068 l~e~ near Prague (Czechoslovakia) **Institute o f Macromolecular Chemistry, 16206 Prague 6 (Czechoslovakia)

(Received November 16, 1981) A simple neutron activation method is deser~ed for platinum determination in urine and serum of dogs when studying the pharmaeokineties of cisplatin, an antitumottr drug. The procedure is based on the nuclear reaction a9 s Pt(n, 3', #_)199Au' a radiochemieal separation of gold, and gamma-spectrometry of the radionuelide 199 Au. Gold is separated as metal by coprecipitation with selenium after the addition of aseorbic acid in a highly addle medium. The interference contribution of 199Au originating from stable gold is evaluated, too.

Introduction The need for platinum determination in biological materials has significantly increased after the discovery that platinum coordination compounds of the type [(A)2Pt(II)(X)2] or corresponding complexes of platinum(IV) (where A is ammonium or an aliphatic amine and X is chloride, sulfate or carboxylate anion) feature a bro~.d spectrum of antitumour activity in animals and man. 1-3 Nowadays, the simplest complex (A = NH3, X = CI), named cisplatin (cis-DDP), belongs to the clinically useful antitumour drugs. However, the antitumour activity of cisplatin is accompanied by some undesirable toxic effects, which cause abdominal discomfort, loss of hearing and, most dangerous, kidney failure. Therefore, research has been started on the second generation of platinum complexes which aims at both decreasing nefrotoxicity, other side effects of cisplatin and, simultaneously, extending a spectrum of antitumour activity mainly for leukemia. 4-6 While the activity of cisplatin was discovered by chance, 1,7 the second generation of platinum complexes is being chosen from about 800 compounds which have been prepared on the basis of the present knowledge of biological activity of cisplatin, s'9 +Presented at the Instrumental Activation Analysis Conference-IAA 81, June 1-5, 1981, Klu~enice, Czechoslovakia. J. Radioanal. Chem. 75 (1982)

71

J. KULgERA,J. DROBNfK: DETERMINATIONOF PLATINUM The data on the distribution of platinum in organisms and the excretion kinetics are mandatory parts of the pharmacological evaluation of any platinum-containing drug. From this point of view, cisplatin and analogous compounds represent a rather difficult analytical task. Labelling with radionuclides is an inconvenient procedure in the case of platinum-based drugs, because platinum itself has no suitable radioisotopes. Sometimes, =9 I Pt and ~9 5 rapt are used which are difficult to prepare, are relatively short-lived for this purpose and disadvantageous in other respects. 1~ Labelling with other radionuclides (14C, 3H, etc.), commonly used for organic compounds, is an unreliable procedure in the case of platinum-based drugs as both types of ligands may be exchanged in vivo (X-easily, A-less easily). Therefore, other analytical techniques are to be used which are capable of determining micro-amounts of platinum. Neutron activation analysis (NAA) was one of the first methods used)1-13 while atomic absorption spectrometry (AAS), In'Is X-ray fluorescence analysis, ~6 spectrophotometry and I-IPLC of platinum diethyldithiocarbamate complexes ]7'1s were applied for this purpose later on. However, it was found ~8 that the accuracy of the above methods with the exception of NAA may be subjected to dramatic matrix effects. Thus, NAA plays an important role for both routine analyses and quality control of other analytical techniques. Choice o f neutron activation procedures

The most sensitive nuclear reactions and parameters of radionuclides are summarized in Table 1. These reactions were used for platinum determination in various materials in both nondestructive and radiochemical ways as reviewed by GIJBELS ] 9-21 and SAWANT and HALDAR. 22 When choosing a method for platinum determination in biological materials, which usually requires a radiochemical pro-

Table 1 Nuclear parameters of radionuclides24 Radionuclide

72

Reaction

Half-life

[IO -= 4 cm a I

Relative abundance of the target nuclide

Activation cross-section

197~

96pt (n, -/)

0.74

0.253

18.3 h

199pt

198pt (n, 3,)

3.73

0.072

30.8 m

t 99Au

~ 9aPt (n, % 0_)

3.73

0.072

3.13 d

Main v-rays, keV (absolute intensity)

77.4(21); 191.3(5.7) 317.0(5.6); 542.7(16.5) 158.4(76.8); 208.2(16.6)

J. Radioanal. Chem. 75 (1982)

J. KUC~ERA,J. DROBN~K: DETERMINATION OF PLATINUM Table 2 Activation of radionuelides for platinum determination in biological materials*

radionuclide

Analytical

Activity, Bq 9g-I

Main activity of the matrix, Bq

19~pt

5.0 9 lO s

2.8" 10 ~ (2~Na)

tirr = 20 h tde c = 2 d

199pI

4.7" l0 s

4 . 4 - 106 (asCl)

tirr = 5 m** tde c = 30 m

1 . 6 . 1 0 4 (24Na)

I 99Au

4.2 9 l0 T

9.9 9 103 (24Na )

Conditions

tirr = 4 h tde c = 8 d

*Calculated for a thermal neutron flux o f 1 9 101 a cm -2. s-~ ; activity of the matrix calculated for I ml urine provided that the sodium content a m o u n t s to 3 mg and the chlorine content to 5 mg2 s,2e **Maximum irradiation time for polyethylene rabbits at a pneumatic

facility of the N.R.I.

cedure, not only the sensitivity of the reactions in Table 1 but also the activity produced by a matrix should be taken into consideration. Such information is given in Table 2, where both the obtainable activities of radionuclides for platinum determination and the simultaneously produced main activities in a sample of 1 ml o f urine are shown for suitable irradiation and decay times t~r, and tdee,. respectively. It can be seen from Table 2 that the most sensitive reactions for platinum determination are those that are yielding the radioisotopes 197pt and 199pt. However, high activity of the matrix is to be handled when carrying out a radiochemical separation after decay times given in Table 2. On the other hand, the use o f the daughter radionuclide 199Au leads to the lowering of the matrix activity by about three orders of magnitude while the specific activity of ] 99Au is lowered only by about one order of magnitude compared to 197pt and 199pt (a count rate in gamma-ray spectrometry is even less unfavourable for 199Au when we take into consideration the absolute intensities of gamma-ray emissions of the respective radionuclides ~97pt, 199pt, and 199Au cf. Table 1). Moreover, a detection limit of platinum can be made very low when using 199 Au by prolonging a counting time which is not possible for 199pt with regard to a short half-live of the latter radionuclide (cf. Table 1). Obviously, a procedure based on the measurement o f 199Au is the most suitable one for the determination o f platinum in biological materials by neutron activation with radiochemical separation. I. Radioanal. Chem. 75 {1982)

73

J. KUC~ERA,J. DROBNIK: DETERMINATIONOF PLATINUM From above mentioned reasons, a simple and quick procedure was developed for platinum determination which is based on a radiochemical separation of gold and gamma-spectrometry of 199Au" The method was applied when studying platinum distribution in urine and serum of dogs after the administration of cisplatin. Recently, a similar procedure was described for assay of platinum and gold in human plasma, a~ Experimental

Preparation of samples and standards Dogs were injected with 30 ml of physiological saline containing cisplatin in an amount of 1 mg/1 kg body weight over a 30 min infusion. Urine was collected by means of a metabolic cage while serum was obtained from veinous blood at chosen time intervals. Samples of urine and serum of 1 ml volume were placed into quartz ampoules, evaporated to dryness in a vacuum oven, then the vials were sealed. In the same way, samples of urine and blood were prepared before cisplatin administration (blanks). Standards of platinum (11.2/ag) and gold (10.9 ng) were prepared by pippeting 10/al of respective solutions containing known amounts of elements into quartz vials, which were sealed.

Irradiation Samples were irradiated together with standards in a nuclear reactor WWR-S of the Nuclear Research Institute (N.R.I.) at ~e~, in which the thermal neutron flux amounted to 1 9 101 s n cm -2 s -1 . The irradiation time was 4 hours. Each irradiation batch consisted of 10 samples and 2 standards.

Radiochemical procedure Quartz vies were cleaned on their surface by boiling in aqua regis for 10 rain, washed with water, cooled in liquid nitrogen and opened by crushing after 7 to 10 days of decay time. Destruction of samples and dissolution of standards was carried out in a mixture of 70% perchloric acid and 65% nitric acid (1 + 2) in the presence of 100/~g of gold as a carrier and 5 mg of selenium as a collector for precipitation. Simultaneously, a few mg's of vanadium as catalyst and a few mg's of chromium(Ill) as indicator of the end of destruction were added. 2 a After an orange coloured solution of chromium(VI) was obtained, the solution was diluted with water to obtain 2-4M HC104 and splinters of quartz vials were separated by filtration 74

Z Radio~aaL Chem. 75 (1982)

J. KUCe'ERA,J. DROBNfK: DETERMINATION OF PLATINUM through a sintered glass fdter. Selenium and gold were precipitated as metals by the addition of 100 mg ascorbic acid. The precipitate was Filtered off by means of a membrane ultrafdter with a pore diameter of 0.3 #m (Synpor Nr. 7, Synthezia, Czechoslovakia) and washed on the f'dter with warm, dilute hydrochloric acid (1 + 1) and water. An overall yield of the gold separation exceed 98% as ascertained by tracer experiments with 198Au. The whole radiochemical procedure can be completed within 30 minutes by one person.

Measurements and interferences The Filters with separated gold were measured with a Ge(Li) detector (FWHM 2.5 keV for 1332.4 keV gamma-rays, 4.3% relative efficiency) coupled to a computer-analyzer Plurimat 20 (Intertechnique, France). Measuring times varied from 15 to 60 min, depending on the activity of samples. Activities of both radioisotopes 19SAu and 199Au were measured to permit the necessary correction. The radionuclide 199Au originates not only from the reaction 198pt(n,')t, fl-)199 AB but also from the reaction 197Au(n ' y)l 98Au (n, 7) 199Au. The latter interfering reaction cannot be neglected owing to an extremely high activation cross-section of 19SAu for thermal neutrons (25 100.10 -24 cm2) 24. The interference contribution of the formation of 199AB from stable gold can be calculated using the generalized Bateman-Rubinson equation for growth and decay of radionuclides in an activation and/or a decay chain. The number of atoms of the n-th radionudide after an irradiation time t, Nn(t), is given by

i=n e_Ai t Nn(t ) = A 1 A 2 . . . An_l N0 i=~1 Ci j=i II j=l

where Ci A=X+q~o

A* = ),* + ~o*

-

-

O

J. Radioanal. Chem. 75 (1982)

1 Aj -- A i

(1)

(j ~ i )

modified decay ("disappereance") constant where )~ is the decay constant, (I) is the thermal neutron flux and o is the activation cross-section for thermal neutrons; partial modified decay constant where the asterisks indicate that in either branching decay or branching activation ~,* is the partial decay constant and o* is the partial cross section; number of atoms a t t = O ; 75

J. KUCV'ERA,J. DROBNIK: DETERMINATIONOF PLATINUM For the reaction 198p t

o, ~ 199p~

n, 3r (1)

FaX2 ~ 199Au

3-

h3 ~ 199Hg

3-

(2)

(3)

(2)

(4)

it is valid that A 1 =

A* =

(I)Ol

A2 = 7,2; A~' = F27,2 A3 = 7,3 where Fn is the fraction of disintegration of nuclides (n) which produces nuclides ( n + 1). For the reaction

198Hg 197Au _.or ; 198A u ~ (1)

(2)

(3) 199A u

h, ~ 199Hg

(3)

(4)

it is valid that Al = A~ = (I)ol A2 = 7`2 + ~o2; A* = (I)o2 A3 = X3 Hence, the interference contribution of gold, IAu , in the determination of platinum by means of the radionuclide 199Au, is given by the ratio of the number of 199Au atoms originating from g01d via reaction (3), N3 (Au), and the number 76

J. Radioanal. Chem. 75 [1982}

J. KUC~RA, J. DROBN[K: DETERMINATION OF PLATINUM

of 199Au atoms originating from platinum via reaction (2), N3 (Pt) N3 (Au) IAu --

N3 (Pt)

(4)

Introducing the nuclear parameters for reactions (2) and (3) from Table 1 In2

Tll2 and from Ref) 4 into Eqs (1) and (4), provided that cI, = 1 9 1013 n cm -2 s -1 , t = 4 hours, F2 = 1 [reaction (2)], one obtains 0.8 ng of apparent platinum for 1 ng of gold. The experimental assessment of IAu is rather difficult in the given conditions as the most intensive gamma-line of the radionuclide 199Au having the energy of 158.4 keV is positioned just on the high Compton continuum of the radionuclide ~9 SAu" Results and discussion

The method developed represents very easy, quick and safe operation which is insensitive to small deviations from standard conclitions (for instance, coprecipitation of gold with selenium was found to be quantitative up to 9M HCI04) and it is therefore suitable for routine work. In contradistinction to the procedure by SYKES et al.,s~ no losses of gold were found when using the decomposition mixture described in open flasks even for up to 15 minutes-prolonged heating at 215 ~ after the end of destruction of samples and irrespective of the absence or presence of hydrochloric or hydrobromic acid up to 1M concentration, too. Thus, determination of a yield of gold can be omitted because all steps of its separation (decomposition of samples, coprecipitation with selenium, and membrane ultrafiltration) are performed quantitatively with high reproducibility. The method also provides samples of a very suitable shape for gamma-ray spectrometry. The gamma-ray spectrum of a serum sample after the radiochemical separation of gold is shown in Fig. 1. It can be seen from Fig. 1 that, in addition to gold and selenium, there are also traces of antimony retained in the precipitate. However, the presence of the radionuclides 7SSe, ~22Sb, and 124Sb does not interfere in the gamma-spectrometry of neither ~99Au nor ~98Au and does not significantly increase the detection limit of platinum in the majority of biological matrixes. Z Radioanal. Chem. 75 (1982)

77

J. KU~ERA, J. DROBNiK: DETERMINATIONOF PLATINUM

103

g .c t~

2 ~ ~o~

,

!!.

10 ~':~.~,'.i/~.,. ,

i. . ' ~:.,~'1~1.':.

.~......

9

.

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,,..,..