Atomic Absorption Spectrophotometry of Free ... - Clinical Chemistry

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The overall analytical recovery of platinum was 80 ± 2%. .... d 14-mm paper disk Impregnated with strong acid-type catIon-exchange resin. resin was placed in ...
CLIN.CHEM. 24/6,877-880(1978)

Atomic Absorption Spectrophotometry of Free Circulating Platinum Species in Plasma Derived from c/s-Dichlorodiammineplatinum(Il) Steve J. Bannister, Yunik Chang, Larry A. Sternson,’

We describe a method of analysis for free circulating platinum species derived from cis-dichiorodiammineplatinum(ll) in blood plasma. Protein-bound and free platinum species were separated from each other by centrifugal ultrafiltration. Platinum in the ultrafiltrate was converted to a cationic complex by reaction with ethylenediamine, and the product was collected on paper impregnated with cation-exchange resin, where it could be stored indefinitely without loss. The platinum was eluted from the disk with 5 mol/liter hydrochloric acid, and an aliquot of this solution was then analyzed by flameless atomic absorption spectrophotometry. The overall analytical recovery of platinum was 80 ± 2%. The minimum quantity of cis-dichlorodiammineplatinum detectable was 35 tg/liter of plasma at the 99% confidence level. Detector response was linearly related to drug concentration in the range from 80 tg to 290 mg of Pt per liter of plasma. Reaction variables were made optimal, so as to yield maximum sensitivity and reproducibility

(±2%)

consistent

with

minimal

sample

transfers and manipulations. Additional Keyphrases: We recently for monitoring

cancer therapy

trace elements

.

useful analytical method agent, cis-dichiorodiammineplatinum(II) (DDP) in plasma (1). The procedure had advantages over earlier methods (2-5), which could only measure total platinum in samples, in that it distinguishes

between

described a clinically the anti-neoplastic

protein-bound

platinum

and free-circulating

plati-

num-containing species. Ability to do so is particularly important in light of the findings that the protein-bound materials are apparently inactive and 3 h after administration of DDP only about 10% of the dose neither is bound to macromolecules nor has been excreted (1, 6).

HNN

7C1

H3N/

NCI

(1) NH2

and have refined the procedure

fraction,

present

in the ultra-

of Pharmaceutical

Chemistry,

The

Kansas, Lawrence, Kan. 66044. 1 Address correspondence to this author. Received Jan. 27, 1978; accepted Mar. 22, 1978.

University

of

and

by flameless

Materials Crystalline DDP was obtained from the National Cancer Institute. Hydrochloric acid (reagent grade) and ethylenediamine were purchased from J. T. Baker Chemical Co., Phillipsburg, N.J. 08865. For centrifugal ultrafiltration we used Centriflo CF-50A conical filters (Amicon Corp., Lexington, Mass. 02173). Used filters were cleaned for 30 mm in

distilled water in an ultrasonic bath and stored in ethanol! water (10/90 by volume). Paper impregnated with a strongacid type cation-exchange resin (SA-2, 22 X 28cm sheets, from Reeve

Angel/Whatman

Inc., Clifton,

N.J.

07014)

was used

without

pretreatment. Disks about 14 mm in diameter were cut from the sheets with a no. 7 cork borer. The average weight of a disk was 23.3 ± 0.4 mg.

Apparatus Atomic Techtron

absorption measurements were made with a Varian Model 175B atomic absorption spectrophotometer

The platinum

Methods preparation.

Plasma

7 ml, were placed at 1000 X g for 15 mm, and the plasma filtrate was collected. To prepare samples of known DDP concentration, we transferred a 0.9-nil aliquot of plasma ultrafiltrate to a 5-mi scintillation vial and mixed it with 0.1 nil of a solution of DDP in 0.1 mol/liter NaC1.

CF-50A conical

The concentration Department

sensitivity

is monitored

Materials and Methods

Sample

In the earlier procedure (1), protein-bound platinum species were separated from free circulating species by centrifugal

The unbound

by extending

improving reproducibility. Platinum atomic absorption spectrophotometry.

in Centriflo

DDP

ultrafiltration.

filtrate, was converted to a stable cation species by reaction with ethylenediamine (equation 1) and the product collected on a paper disk impregnated with cation-exchange resin by filtration. Platinum was monitored by wavelength dispersive x-ray fluorometry. The minimum detectable quantity of platinum was 240 pg/liter of plasma. The major disadvantages of the method are (a) the relatively high detection limits, (b) the somewhat low and irreproducible recovery of platinum, and (c) the cost of or lack of x-ray fluorescence analytical capabilities at most clinical facilities. We have investigated variables associated with the method

coupled with a CRA-90 carbon rod atomizer. 265.95 nm line was monitored.

/2

+

and A. J. Repta

filters

samples, and

centrifuged

of DDP in the final solution ranged from

100 to 1000 tg/liter. Ethylenediamine was then added to each vial containing sample solution, and the mixture was allowed to stand overnight at room temperature (1). Collection of platinum on paper disks. Alter this overnight standing, a 14-mm paper disk loaded with cation-exchange CLINICAL CHEMISTRY,

Vol. 24, No. 6, 1978

877

Table 1. Percentage of Platinum Remaining In Solution After InteractIon with Cation-Exchange Disk(s) a % PlatInum remaining

Sonlcatlon time, mm

100

41

43

29

45

17

60

15

31 29 29

100

100

35 9

30

9

16

8

15

30 45 60

water

lOmol/l

73

HCI HCI HCI

water water water

5 mol/l 0.83 mol/I 0.1 mol/I

93

HBr

water

48%

b

13

Experiments carried out as descrIbed, with 2-mi samples. Percentageof platinum remaining In solution after agitatIon of the solution

with Ion-exchangedisk(s) an ultrasonic bath for 1 h. Longer ultrasonlcation did not improve recovery. C Valuesrepresentthe averageof three determinations,at each concentration of platinum solutions. d 14-mm paper disk Impregnated with strong acid-type catIon-exchange

resin.

addIng

#{149}Iu.nt d

HCI

Pb(OAc)2 NaCN a Done as

Two diSkS’ 0 15

Conan

Solvent

100

30

aft.r

Eluent’

Saline win.

0 15

b

% of d.poslt.d Pt r.covarsd

In

One diskd

a

Table 2. Elutlon of Platinum from a Single Resin Disk a,b

9%

27