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
<5
73
23
0
saturated
formic acid
NaOH, 0.1 mol/l
2 mmol/l
described In the text.
Amount of platinum deposited on disks was determined from a material
balance, from knowledge of the initial amount of platinum In solution and the
amount remaining In solution after interaction with the exchanger. Disks containing 200 ng-10 tg of platinum were prepared and tested.
C 0.25 ml of eluent was mixed with the disk and the system agitated in an Ultrasonic bath.
d Recovery represents the amount of platinum species removed from one
14-mm resin-impregnated paper disk after addition of 0.25 ml of eluent and
agitation In an ultrasonic bath for 1 h. Continued agitation did not improve recovery In any of the experiments. Values represent the average of three determinations, at each concentration of platinum on the disk.
mm after the platinum
5 to 60
was introduced.
in recovery of platinum
significant
difference
collection
on ion-exchange
disks)
among
There was no
(based on its
these
samples,
resin was placed in the vial, and the mixture was ultrasonisuggesting that there is no loss of platinum owing to interaccated for 45 mm. The disk was then removed, dried at 100 #{176}Ction with plasma filtrate over this time interval.
for 90 mm, and then stored in another scintillation
mini-vial.
Collection
of charged platinum
on cation-exchange
disks.
Platinum
appears to be analytically
stable indefinitely
on the
The cationic platinum compound was collected on a 14-mm
disk.
We added 0.25 ml
of 5 mol/liter hydrochloric acid to the vials containing the
dried resin disks and ultrasonicated
the mixture for 15 mm.
Elution
of platinum
from paper
The resin disk was then
removed
disks.
from the vial and discard-
ed.
Atomic absorption
analysis.
A 2-il aliquot of the above
eluent was then introduced
into the carbon furnace
of a
flameless
atomic absorption
spectrophotometer.
The lamp
current
was maintained
at 10 mA. We used a three-stage
heating program of 100 #{176}C
for 45s, 700 #{176}C
for 15s, and 2300
#{176}C
for 1 s. The ramp rate of the program was 600 #{176}C/s.
Results
Centrifugal
ultrafiltration
of plasma
of plasma
samples
removed
98%
protein and
molecular
mass greater
loss of DDP in or on the
lating platinum species
other macromolecules
with relative
than 50000, without any observable
filtration membrane (1). Free-circuin the ultrafiltrate were converted to
by reaction with ethylenediamine.
The
the cationic species
yield of the product was greatest when the filtrate was mixed
with about 0.2 volume of ethylenediamine.
Lower concentrations of the amine gave poorer and less reproducible
yields
of product
and reaction
proceeded
more slowly. We saw no
improvement
in reaction parameters with higher concentrations of ethylenediamine.
The reaction followed pseudo
first-order kinetics with a reaction hall-life of <10 mm at room
temperature,
as assessed by ultraviolet absorbance changes
at 301 nm.
complete
Therefore,
the
after the reaction
reaction
mixture
was considered
to be
had stood overnight.
The stability
of platinum
species in the ultrafiltrate
was
investigated
to determine
if the time interval between filtration and addition
of ethylenediamine
was critical. We added
DDP (400 zg/liter) to plasma ultrafiltrate
solutions and made
a single addition
of ethylenediamine
during the period from
878
CLINICAL CHEMISTRY, Vol.24,No. 6, 1978
paper
disk impregnated
with cation-exchange
resin (total
capacity equivalent to more than 6mg of DDP). This
process concentrates
the sample, enhancing
overall sensitivity
of the method and maintaining
the analyte in an indefinitely
exchange
stable form until atomic absorption analysis. The platinum
complexes were collected on the resin-loaded disks by a batch
method,
i.e., the disks were added to the reaction mixtures
contained
in 5-ml scintillation
vials and this mixture was agitated in an ultrasonic
bath, to facilitate the exchange process.
The
collection
process
was optimized
with
respect
to the
amount of resin-impregnated
disk used and the amount of
agitation of the reaction mixture required alter addition of the
exchanger disk. Solutions of DDP (in concentrations
from 100
to 1000 pig/liter) were prepared in both plasma ultrafiltrate
and 0.1 mol/liter
sodium chloride. The amount of platinum
remaining
in solution was then determined
in mixtures containing from one to three resin disks after sonication for various lengths of time (Table 1). When one disk was used to
collect platinum from 2-mi samples, the minimum percentage
of platinum
remaining
in solution after sonication
was about
29% from plasma filtrate and 15% from 0.1 mol/liter NaCl
solution.
Maximum
exchange
was reached
after 45 mm of
sonication. Use of two disks improved collection efficiency,
so that only 15 and 8% of the added platinum remained in the
plasma filtrate and NaCl solution, respectively, after sonication. With two disks, maximum
exchange was reached after
sonication for 30 mm. The same results obtained with two
disks and 2 ml of solution were obtained with 1-ml samples
and one exchange disk. The amount of platinum remaining
in solution
was not decreased
was increased
further
if the number
from one to three, or if duration
of disks
of sonication
was increased.
The platinum remaining in 0.1 mol/liter NaC1 samples was
about half that remaining in plasma-filtrate
samples. This
may be due to unknown species in plasma filtrate competing
for available exchange sites on the resin disk. From these results we concluded
that the best conditions
for collection
of
cationic platinum
in 1-mi samples is to add a single disk and
then sonicate the mixture for 45 mm.
Elution
of platinum
from the resin-impregnated
disk.
Several eluents were tested for removing the platinum from
the exchange-resin-impregnated
paper disks, leaving platinum
form amenable
to introduction
into the
of an atomic absorption spectrophotometer
(Table 2); 0.25 ml of each eluent was added to the disk and the
mixture agitated
in an ultrasonic
bath. Hydrochloric
acid (5
mol/liter)
was the most effective eluent, 93 ± 2% of the platinum could be accounted
for after 15 mm of ultrasonication.
Analytical
recovery was concentration
independent
over the
range 200 g to 1 mg of platinum
per liter. Elution characteristics were identical for platinum
deposited
from NaCl or
plasma solutions.
Higher concentrations
of HC1 were unacceptable because they fragmented
the resin disk and caused
irreproducible
results
in sampling
for atomic
absorption
analysis, due to the positive vapor pressure above the liquid
in the syringe. More dilute HC1 was less efficient, i.e., recovery
of platinum
was low.
An aqueous solution of lead acetate was investigated
as a
possible eluent because Pb(II) ion has a strong affinity for
strong cation-exchange
resins (7); however, use of this eluent
gave poor recovery
of platinum.
Aqueous
sodium cyanide
Table 3. Concentrations
Time after admin.,
mm
in a solubilized
graphite furnace
solution
was also tested
as a prospective
eluent.
Divalent
platinum
compounds
nide ion in aqueous
react rapidly and completely
with cyasolution,
forming the anionic tetracyanoplatinous complex [Pt(CN)4]2- (8, 9). However, 2 mmol/
liter NaCN was not an effective eluent for this system; apparently, the reaction did not occur to any appreciable
extent
in this heterogeneous
environment.
Atomic absorption
analysis. A 2-id aliquot of the 5 mol/liter
HC1 eluent was analyzed for platinum by flarneless atomic
absorption
spectrophotometry.
We analyzed clinical samples
by comparing
the absorbance
values determined
for the unknowns to those of a standard
curve, constructed
over the
concentration
range of DDP from 80 ig/ liter to 290 mg/liter
of plasma. Known amounts of DDP were added to plasma and
the samples immediately
taken through the analysis scheme.
Absorbance
response
was linearly related to DDP concentration
over this range (more concentrated
solutions
were
diluted to bring samples into the linear range for the detector).
Linear regression analysis of the data yielded the line A = (5.5
x 10-s) concn + (2.27 X 10-s) with a correlation coefficient
of 0.995. Total recovery of DDP from plasma was 80±2% over
this wide concentration
range. Some platinum
still is lost at
uncontrolled interfaces, because of the colloidal phase formed
at increased
Clinical
determined
pH values.
samples. The clinical usefulness of the method was
by monitoring
filterable platinum
in the blood of
patients receiving DDP chemotherapy.
A patient was administered DDP (100 mg/rn2 of body surface) as an intravenous single injection. Blood samples were drawn at timed intervals and assayed for filterable platinum. The values we
measured
in the blood
are presented
in Table
3.
Discussion
We could separate
the free circulating
DDP from plasma
protein rapidly and quite reproducibly
by centrifugal
ultrafiltration
without significant
loss of platinum
in the filtration
membrane
(6). The filterable
platinum
was converted
to a
stable cationic species, which was collected on a cation-exchange-impregnated
disk. We could collect platinum from the
bulk plasma
manipulation,
method.
filtrate
thus
onto the disk without
further
improving
the reproducibility
sample
of the
of Filterable
Platinum in
Human Blood after a Single Intravenous Injection
of DDP (100 mg/rn2)
DPP concn.,
mg/liter
1
15
30
2.89
2.28
1.69
45
60
120
1.10
0.83
0.28
Ultrasonication
of the vial containing
the sample solution
and the disk was essential
in order to collect the cationic
platinum
species on the resin disk efficiently.
The yield of
platinum
on the disk by the batch method with ultrasonication of the mixture was about 10% higher than that obtained
by filtration
in which the sample solution was slowly passed
through the resin disk (1).
Without ultrasobication,
the batch deposition
of platinum
on the disk was quite low (about 25%) and irreproducible.
The
increased
recovery
of platinum
on ultrasonication
may be
attributed
to the increased diffusivity of the cationic platinum
species through the hydration
layer surrounding
the cation-
exchange resin of the disk, which would enhance
action between platinum and resin.
the inter-
The platinum
on the exchange disk was analytically
stable
indefmitely
(i.e., more than nine months). When samples were
ready for atomic absorption
analysis, the platinum
had to be
redissolved.
Of the eluents that we investigated,
5 mol/liter
HC1 gave the best results. The presence of 5 mol of chloride
ion per liter in the mixture may facilitate
reversal of the reaction shown in equation 1 to regenerate
neutral DDP, which
does not interact strongly with the resin disk (1). The strong
acid environment
created by the eluent protonates
any ethylenediamine
that may be present,
eliminating
it as a nucleophile,
and also protonates
the sulfonate
groups on the
resin-impregnated
disk, minimizing
its ion-exchange
ability.
Furthermore,
the elastic matrix
of the resin is stretched
to
accommodate
the large platinum
complex. Small hydrogen
ions displace the bulky platinum cations, satisfying the resins’
tendency
to contract
(10) and further facilitating
platinum
displacement.
The optimum
concentration
of HCI for elution of cationic
platinum
species from these disks was 5 mol/liter. This seems
to be related, at least in part, to the thermodynamic
activity
of water molecules
in the system. As the HC1 concentration
increases,
the activity
of water molecules
decreases.
The
matrix of the disk becomes more rigid, impeding
diffusion of
eluent into the resin network
(11) and thus diminishing
displacement
efficiency. Contrariwise,
below this optimum
HC1
concentration,
H and Cl ions will be strongly
hydrated,
decreasing
their reactivity.
In addition,
the concentration
of
HC1 used for elution
(5 mol/liter)
is below the azeotropic
composition
of HC1/H2O mixtures
(12) and therefore
the
vapor above the solution will contain less HC1 than is present
in the solution and less HC1 than would be present in the vapor
above a solution containing
HC1 above its azeotropic
composition. The eluent is thus less corrosive, prolonging the lifetime
of the atomic absorption
components,
and making it easier
to work with. At higher HC1 concentrations
sampling
is also
made more irreproducible
due to the positive vapor pressure
above the liquid in the syringe.
The detection
limit for platinum
by the flameless
atomic
absorption
method is 35 pg/liter of plasma at the 99% confidence level (13), nearly a 10-fold increase in sensitivity
over
CLINICALCHEMISTRY, Vol.24,No. 6, 1978
879
the x-ray fluorescence method (1). Because sample transfers
are few and the procedure is simple, precision was ±2%. In
addition, analyte concentration
and absorbance are linearly
related over the range 35mg to 29 g per liter, after appropriate
dilution of the more concentrated
samples.
We conclude that this method provides a simple means for
monitoring filterable platinum in blood plasma with high
sensitivity and reproducibility,
with instrumentation
that is
available at most medical centers. The method is currently
being used to monitor platinum
in the blood of patients receiving DDP chemotherapy,
and modifications of the procedure are being made to permit analysis of other biological
fluids.
This work was supported
in part by NIH grant CA-09242 from the
National Cancer Institute, USPHS.
References
1. Bannister, S. J., Sternson, L. A., Repta, A. J., and James, G. W.,
Measurement of free-circulating cis-dichlorodiammineplatinum(H)
in plasma. Clin. Chem. 23, 2258 (1977).
2. Pera, M. 1., Jr., and Harder, H. C., Analysis for platinum in biological material by flameless atomic absorption spectrometry. Clin.
Chem. 23, 1245 (1977).
880
CLINICAL CHEMISTRY,
Vol.24,No. 6, 1978
3. Deconti, R. C., Toftness, B. R., Lange, R. C., and Creasey, W. A.,
Clinical and pharmacological
studies with cis-diamminedichloroplatinum(II). Cancer Res. 33, 1310 (1973).
4. Litterst, C. L., Gram, T. E., Dedrick, R. L., et al., Distribution and
disposition of platinum following intravenous administration
of cisdiamminedichloroplatinum(II)
(NSC 119875) to dogs. Cancer Res.
36, 2340 (1976).
5. Miller, R. G., and Doerger, J. U., Determination
of platinum and
palladium in biological samples. At. Absorpt. Newsl. 14, 66 (1975).
(A publication of Perkin-Elmer Corp.)
6. Leh, F. K. V., and Wolf, W., Platinum complexes: A new class of
antineoplastic agents. J. Pharm. Sci. 65, 315 (1976).
7. Karger, B. L., Snyder, L. R., and Horvath, C., An Introduction
to
Separation Science, John Wiley and Sons, New York, N.Y. 1973, pp
337-373.
8. Chang, Y., Sternson, L. A., and Repta, A. J., Development of a
specific analytical method for cis-dichlorodiammineplatinum(II).
To be published.
9. Basolo, F., and Pearson, R. G., Mechanisms
of Inorganic Reactions, 2nd ed., John Wiley and Sons, New York, N.Y., 1967, p 351.
10. Helfferich, F. G., Ion Exchange, McGraw-Hill
Book Co., New
York, N.Y. 1962, p 158.
11. Ibid., pp 95-125.
12. Handbook of Chemistry and Physics, 48th ed., CRC Press,
Cleveland, Ohio, 1967, p D14.
13. Rowe, C. J., and Routh, M. W., Ultimate detection limit barrier
to furnace AAS. Res./Dev. 28, 24 (1977).