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
