The Detection of Cisplatin and Other Platinum Containing Compounds
Using Reverse-Phase High Performance Liquid Chromatography
by
Stephanie A. Chesney
5-3-90
The Detection of Cisplatin and Other Platinum Containing Compounds
Using Reverse-Phase High Performance Liquid Chromatography
INTRODUCTION
-
Cisplatin (cis-diaminedichloroplatinum (II)) is an
antineoplastic cancer drug which has shown favorable results in
reducing different forms of cancer. The results include the
reduction of small and large cell adenocarcinomas of the lungs,
testicular and ovarian cancer, as well as head and neck cancer.
Cisplatin appears to be the most active of the simple platinum
(II) compounds; transplatin, in particular, has little or no
effect in treating cancer. Also, the products formed upon
reaction of cisplatin with water are less active than cisplatin
itself. Therefore, cisplatin is usually administered
intravenously in saline to prevent the formation of these
hydrolysis products.
Problems arise when cisplatin and these
other platinum species enter the kidneys.
It is assumed that
cisplatin and/or the other products are unable to pass through
the kidney membranes mainly because of the size of the
molecules. Toxicity occurs as a result of the accummulation
of these heavy-metal compounds.
Since cisplatin differs so radically in its activity
compared to the other isomers and hydrolysis products, it is
essential to have analytical methods which can quantitate trace
amounts of these compounds directly in a sample of blood or
urine.
Following the work of W.A.J. De Waal, F.J.M.J. Maessen,
and J.C. Kraak, Journal of Chromatography, 407 (1987), we
employed reverse-phase high performance liquid chromatography
(RP-HPLC) to separate the various platinum containing species
and on-line inductively coupled plasma atomic emission
spectrometry (ICP-AES) to detect them. The ICP-AES will
simplify the blood serum and urine chromatographs from the HPLC
by only sho\ving those peaks which contain platinum. The ICP-AES
will also simplify quantitation of the peaks because the
detector will respond with uniform sensitivity to any platinum
species (T.W. Avery, C. Chakrabrarty, J.J. Thompson, submitted
to Analytical Chemistry, 1990). Thus, only one calibration
curve will be needed for all platinum species. Thirdly, the
ICP-AES will improve the detection limit of cisplatin.
De Waal,
Maessen, and Kraak established the detection limit of cisplatin
to be 3.5 ng of platinum by ICP-AES.
The purpose of this research is to devise a separation
scheme which would allow the separation of cisplatin from
transplatin and from various platinum species formed by
reactions with water and other common solvents used in
chromatography. We will then obtain chromatographs of cisplatin
and other plat~inum containing products in the blood and urine.
For this preliminary research, we will use a UV-VIS detector to
optimize the separation; the ICP-AES will be used later for the
reasons cited above.
The ultimate goal of this research is to make RP-HPLC
with ICP-AES helpful in the monitoring of cancer patients who
have been treated with cisplatin. One would be able to inject a
patient intravenously with cisplatin and obtain blood and/or
urine samples from the patient at different time intervals and
send these to a lab to determine the amount of cisplatin and/or
other platinum containing products. One would be able to
observe how much cisplatin was released in the urine, how much
remains in the blood, and whether or not transplatin, any
hydrolysis products, or other inactive, toxic platinum compounds
were present.
EXPERIMENTAL
APPARATUS
-
~
The HPLC used consisted of two ISCO 2350 series pumps with
an ISCO v-4 UV-VIS detector.
Several columns were tested before
the Burdick and Jackson (B & J) 005 Octadecyl, 4.6 x 250 mm., 5
micron, C18 column was selected.
COLUMN TESTING
Three C18 columns were tested. The B & J OD5 Octadecyl,
4.6 x 250 ffilll., 5 micron, C18 column had just been purchased. The
Isco 4.6 x 2:50 mm., 5 micron, C18 column and the Alltech
Econosphere, 4.6 x 250 mm., 5 micron, C18 column were both
approximately two years old.
The first column to be tested was the B & J C18 column.
The conditions were as follows:
flow rate=1.25 ml/min,
pressure=1570 psi (without the backpressure device on the outlet
tubing), mobile phase=65/35% acetonitrile/water, uv
wavelength=254 nm, sample volume=20 ~L. The sensitivity changed
with the known solutions used. Our known concentrated solution
was composed of .005 g of uracil, .700 g of phenol, .050 g of
benzaldehyde, .050 ml of toluene, 4.61 ml of ethyl benzene, 90
ml of acetonitrile, and 10 ml of water. This solution was
diluted by mixing 1 ml of our concentrated solution with 99 ml
of acetonitrile to form the dilute solution. Two tests were run
with our dilute known solution and two tests were run with the
known solution supplied with the column. Our retention times
were compared to the test sheet supplied with the column. (N,Ndiethyl-m-toulamide is listed as toulamide.)
The second column to be tested was the Isco C18 column.
The same conditions were used except that the pressure was 33503360 psi (vlith the backpressure device) and the sensitivity was
.05 aufs. The dilute solution used to test the B & J column, 1
ml of the concentrated solution with 99 ml of acetonitrile, was used
to test this column. The retention times for the four peaks
were also compared to the test sheet.
The last reverse-phase column to be tested was the Alltech
CIS column.
The conditions used for testing the column were the
same as for the Isco, except that the flow rate was 1.10 ml/min
and the pressure was 3910-3920 psi (with the backpressure
device). The retention times were compared to the test sheet.
REAGENTS
Cisplatin and transplatin were obtained from Strem
Chemicals, Inc. (Newburyport, MA). The NaCI and platinum
standard containing tetrachloroplatinate ion was obtained from
the Aldrich Chemical Company (Milwaukee, WI). The HPLC grade 2propanol and the HPLC grade methanol were obtained from Fisher
Chemicals (Fair Lawn, NJ). The HPLC grade acetonitrile was
obtained from Alltech Associates (Deerfield, IL).
In addition,
the sodium octadecyl-sulfonate (SOS), the sodium pentasulfonate
(SPS), the sodium dodecane sulfate (SDS), and the sodium
dodecylsulfonate (SDyS) were obtained from Alltech Associates
(Deerfield, IL). The methionine was obtained from Sigma
Chemicals (St. Louis, MO).
PREPARATION OF REFERENCE SOLUTIONS
-.
Cisplatin (.154g) was placed in 100 mL of saline solution
to create a stock 154 ppm cisplatin solution.
The saline
was composed of 9 g of NaCl in 1 L of water. Two dilute
cisplatin solutions were made. One consisted of 1 mL of the
stock cisplatin solution in 99 mL of saline. The other
consisted of 1 mL of the stock cisplatin solution in 99 mL of
water. A transplatin stock solution was prepared in a similar
manner.
Cisplatin and transplatin were also placed separately in
another stock solution of .0154 g of each in 100 mL of water
instead of saline to observe the hydrolysis products formed.
A methionine stock solution was also prepared by adding
.0075 g of solid to 100 mL of water.
Pig's blood was obtained from The Meat Locker
(Indianapolis, IN) and centrifuged at 2000 rpms for 12 minutes
to separate the serum from the whole blood.
Then, the
micropartition system (MPS-1) was used to further filter the
serum by removing proteins larger than 90,000 daltons.
Concentrated cisplatin (1 mL) in
saline was placed in 1 mL of
0
blood serum and incubated at 37 C for various times to simulate
in-vivo reactions occuring at body temperature.
A human urine sample was obtained and filtered.
1 mL of
cisplatin in saline was placed in 1 mL of urine.
RESULTS AND DISCUSSION
-
RESULTS OF COLUMN TESTING
Three columns were tested in order to see if any were in
good enough condition to separate the platinum compounds.
The B & J C18 column tested as follows:
Substance
1) uracil
2) phenol
3) benzaldehyde
4) toulamide
5) toluene
6) ethyl benzene
Our Dilute Known
1) did not show
2) 2.49min
3) 3.15min
Company Known
1) 1.71min
2) 3.86min
3) 4.44min
4) 6.03min
5) 7.47min
6) 9.94min
Test Sheet
1) 1.75min
2) 2.67min
3) 3.37min
4) 3.87min
5) 5.85min
6) 7.48min
4)
5) 6.02min
6) 7.94min
Our peak efficiency plates were also compared to the test sheet
supplied with the column.
1)
2)
3)
4)
5)
6)
Substance
uracil
phenol
benzaldehyde
toulamide
toluene
ethyl benzene
1)
2)
3)
4)
5)
6)
Test Sheet
15758plates
20582plates
19131plates
18349plates
27997plates
29107plates
ComQany Known
1 ) 3620plates
2 ) 15129plates
3) 16920plates
4 ) 20142plates
5 ) 21100plates
6 ) 22938plates
Our
1)
2)
3)
4)
5)
6)
Dilute Known
did not show
5821plates
8112plates
14763plates
16946plates
The high efficiency values (measured in theoretical plates)
indicate that this column is in excellent condition (Figures I
and II). This is the column that we chose for our research.
The ISCO C18 column tested as follows:
Substance
1) uracil
2) phenol
3) benzaldehyde
4) toulamide
5) toluene
6) ethyl benzene
Test Sheet
1) 1. 75min
2) 2.67min
3) 3.37min
4) 3.87min
5) 5.85min
6) 7.48min
Our Dilute Known
1) did not show
2) 2.11min
3) 2.42min
4)
5) 3.46min
6) 4.05min
The peak efficiency values (theoretical plates) were compared to
the test sheet as follows:
Subst.ance
1 ) uracil
2 ) phenol
3) benzaldehyde
4 ) toulamide
5) toluene
6 ) ethyl benzene
1)
2)
3)
4)
5)
6)
Test Sheet
15758plates
20582plates
19131plates
18349plates
27997plates
29107plates
Our
1)
2)
3)
4)
5)
6)
Dilute Known
did not show
2488plates
2870plates
3256plates
3278plates
This column is beginning to show some degradation and would not
be useful for this research because the peaks are too broad
(Figure III).
The last column, the Alltech C18, tested as follows:
-
Substance
1) uracil
2) phenol
3) benzaldehyde
Test Sheet
1) 1.75min
2) 2.67min
3) 3.37min
Our Dilute Known
1) did not show
2) 2.68min
3) 2.98min
4) 3.87min
5) 5.85min
6) 7.48min
4) toulamide
5) toluene
6) ethyl benzene
4)
5) 3.74min
6) 4.21min
The peak efficiency plates were as follows:
1)
2)
3)
4)
5)
6)
Substance
uracil
phenol
benzaldehyde
toulamide
toluene
ethyl benzene
1)
2)
3)
4)
5)
6)
Test Sheet
15758plates
20582plates
19131plates
18349plates
27997plates
29107plates
Our Dilute Known
1) did not show
2) 731plates
3) 1171plates
4)
5) 1252plates
6) 1372plates
This column had to be retired because the peaks are too broad to
achieve sufficient resolution (Figure IV).
RESULTS OF RESEARCH
.-
Following De Waal, Maessen, and Kraak's findings, the
mobile phase selected was a 0.06-F phosphate (pH 2.6), 1 mM
sodium octadecyl-sulfonate (SOS), and 5% 2-propanol solution.
Tests were also run with a new mobile phase consisting of .02-F
phosphate (pH 2.6), 1 mM SOS, and 5% 2-propanol. With the 0.06F phosphate mobile phase, the chromatograph of the stock cisplatin
solution in saline proved to be reproducible with an average
retention time of 132 (+~.1) sec. (Figure V). A second
injection was made using the dilute cisplatin in saline, but no
peaks were observed in the chromatogram at this low level of
cisplatin.
With the .02-F phosphate mobile phase, the
chromatographs also proved reproducible with an average
retention time of 134 (+~.3) sec. (Figure VI).
In addition,
the chromatograph of the stock transplatin solution in saline
proved to be reproducible with an average retention time of 138
(+~.1) sec. with the .02-F phosphate mobile phase (Figure
VII).
From these data, it can be concluded that transplatin
appears approximately 4-6 sec. later than cisplatin coming off
of the column.
The stock transplatin solution in saline was then combined
with the stock cisplatin solution in saline to observe the
results and try to attempt separation of the two in the column.
The two peaks of cisplatin and transplatin appeared as connected
at the base of the peaks (Figure VIII).
Separation was achieved
by varying the concentrations and trying new combinations of the
phosphate, 50S, pH, and 2-propanol. Changing the phosphate,
SOS, and pH had little if any effect on the two connected peaks.
Even changing the instrumental parameters, such as the flow
rate, had no effect in separating the two connected peaks.
Separation was achieved by lowering the percentage of 2-propanol
from 5% to 0.1% in the mobile phase (Figure IX).
The 2-propanol
was kept in the mobile phase to maintain the symmetry of the
peaks.
Tetrachloroplatinate was then added to the cisplatin and
transplatin solution mixture and used as a standard reference
peak for quantitation. The tetrachloroplatinate eluted close to
the void volume of the column, several seconds before cisplatin
(Figure X).
,-
-
Hydrolysis products for both cisplatin and transplatin
appeared when each was placed in water instead of saline.
Gradient elution was used to elute all four peaks in a
reasonable length of time. The 0.1% 2-propanol mobile phase was
used to separate cisplatin from transplatin at the beginning.
Then, the 0.1% 2-propanol was increased to 5% 2-propanol at the
end to allow the hydrolysis products to come off of the column
fast enough to appear on a 20 min. chromatograph (Figure XI).
Time trials were run with the concentrated cisplatin
solution in water to observe the increase in the concentration
of the hydrolysis product(s) over time, while being heated at
37 0 C (body temperature) (Figures XII-XV). A chromatograph was
recorded every 15 min. The hydrolysis product increased
gradually from 0-45 min. At 15 min. a broad peak began to
appear close to the cisplatin peak. This peak may be
transplatin beginning to appear. Other solvents including 2propanol, met~hanol, and acetonitrile were used in the time
trials.
2-propanol (Figures XVI-XIX) and methanol (Figures XXXXIII) showed the same results as the time trial with water.
The hydrolysis product of cisplatin gradually increased with
time and heating. The assumed transplatin peak also appeared
close to the cisplatin peak at 15 min. (Figures XVI-XXIII). The
acetonitrile time trial showed the same basic peaks, but a new
peak also appeared at 7 min. (Figures XXIV-XXVII). This peak
which grew very large after 45 min. is assumed to be an
acetonitrile adduct of cisplatin (Pt-C=N-CH).
Thus,
acetonitrile is a poor choice of chromatogr~phic solvent for
these separa-tions because it forms new products which would not
be present in the blood or urine.
Following additional findings from De Waal, Maessen, and
Kraak, 2 mL of the stock methionine solution was mixed with 1 mL
of the stock cisplatin solution in water to observe any
platinum-methionine bound complexes. The mixture was heated at
0
37 C for approximately 10 min. Many new peaks were observed,
but further research with the ICP-AES is needed to distinquish
or determine which peaks actually contain platinum (Figure
XXVIII).
The next experiment was to replace the 1 mM SOS with 1 mM
of SPS, SDyS, and SDS to observe how the retention times of the
hydrolysis products of the stock cisplatin and transplatin
solutions in water would change upon the addition of these
ion-pairing reagents. The best results were seen with the SPS
and the SDyS.
Both caused the hydrolysis products to come off
of the column almost immediately after the original cisplatin
and transplatin peaks (Figures XXIX-XXXII). The effect of the
replacement of SOS with SDS could not be determined.
A blank was run of the blood serum and cisplatin in saline
was then added to the serum and a chromatograph was run.
Distinguishing the platinum containing peaks is tricky without
the help of the ICP-AES because there are many other compounds
which absorb in the UV spectrum. Further research with the ICPAES will enable the peaks to be identified which contain
platinum (Figures XXXIII and XXXIV).
Cisplat~in in saline and cisplatin in water were both placed
into urine and a chromatograph was obtained.
It is very hard to
distinguish the different peaks. The ICP-AES is needed here
also (Figures XXXV and XXXVI).
CONCLUSION
First, we have been able to reproduce and extend work
done by other scientists in separating platinum compounds.
Cisplatin, transplatin, hydrolysis products, methionine
complexes, and a tetrachloroplatinate internal standard can now
be separated under a single set of conditions (or under gradient
conditions if the analysis time is to be shortened).
Second,
the hydrolysis products of both cisplatin and transplatin were
0
identified upon heating cisplatin and transplatin at 37 C
regardless of whether the chromatographic solvent was water,
methanol, propanol, and acetonitrile. The acetonitrile produced
another major peak which is assumed to be the acetonitrile
adduct.
In addition, many different assumed methionine-platinum
o
bound complexes appeared after heating (37 C) a mixture (1:2) of
the stock cisplatin solution with the stock methionine solution.
Third, the resulting changes in retention times of the
hydrolysis products by replacing SOS with SPS and SDyS were
determined.
The mobile phase containing SOS (which we used in
most of these separations) appears to be the best choice for
separating these platinum compounds, which confirms what De
Waal, Maessen, and Kraak have reported. Last, chromatograms of
blood serum and urine incubated with cisplatin were obtained,
but identifying the exact platinum peaks was impossible.
The next step will be to find which peaks contain platinum in
the complex chromatograms, by employing the ICP-AES detector.
ACKNOWLEDGEMENTS
The author wishes to acknowledge Dr. Joseph J. Thompson,
the DepartmE~nt of Chemistry, and Ball State University for their
support of this project.
REFERENCES
W.A.J. De Waal, F.J.M.J. Maessen, and J.C. Kraak, Journal of
Chromatography, 407 (1987) 253-272.
T.W. Avery, C. Chakrabrarty, J.J. Thompson, submitted to
Analytical Chemistry, 1990.
]
(Figure I)
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COLUMN TESTING)
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.
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'-"
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rt
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:::l
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;
;
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(.II
;
CSI
CSO
.......
( Figure Xl)
CSI
CI"o
rtCl , cisplatill, Lransplatin, cisplatin hydrolysis
4
product, and transplatin hydrolysis product
(same parameters as Figure IX)
cso
CD
cso
""
=
cso
j:I;:;
...,
z
cso
a;
....
...,
.......
-.0
j:I;:;
...........
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....
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U
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~
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cso
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=
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~
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rtl
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G
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.......
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.1
9
(
2
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4
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6
TIME
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a
9
.19
.1.1
.12
(
r,I
:',
I
(Figure XII)
",-,
~i
i
Cisplatin in water at 0 min. (37°C)
(same parameters as Figure VI)
!
,
\
\\
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..
~-
u
~
'"
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3;-
V
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I
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(Figure XIII)
Cisplatin in water at 15 min .
. .; J
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I
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.
~
i
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.
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e ...... ,.
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------~----~~-------~
(Figure XIV)
i
i
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4
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Cisplatin in water at 30 min.
..
T:I"E
La
< .. J:H>
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r
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-- !~
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a
i
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Cisplatin in 2-propanol at 0 min.
(same parameters as Figure VI)
~
-I
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n
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02
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(Figure XVII)
~
Cisplatin in 2-propanol at 15 min.
u
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a
i
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i
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a
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Cisplatin in 2-propanol at 30 min.
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a
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or
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Cisplatin in 2-propanol at 45 min.
I
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.
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(Figure XX)
~----~L~--------
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-,
Cisplatin in methanol at 0 min.
(same parameters as Figure VI)
,
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TIME
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Cisplatin in methanol at 15 min.
t-
'"
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a
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I
TIME
< ........ >
I
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I
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(Figure XXII)
..
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Cisplatin in methanol at 30 min .
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(Figure XXIV)
Cisplatin in acetonitrile at 0 min.
(same parameters as Figure VI)
,
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8
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,:
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(Figure XXV)
,
a
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Cisplatin in acetonitrile a t
i
8
TIME
.e .... ">
I
i
I
i
.1.2
,
i
14
I
15 min.
r
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a :J
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(Figure XXVI)
~
Cisplatin in acetonitrile at 30 min.
I
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g
I
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lSI
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I
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a
os
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(Figure XXVII)
~-
Cisplatin in acetonitrile at 45 min.
co
g
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a
4
.
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a
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--0
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C7'o
(Figure XXVIIl)
2:1 ME~thionjne and cispJatin in water
(same parameters as F'i <Ju n:) V r )
(SI
CO
(SI
r--
C"':J
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--""' x
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III
I----~---.---~-------.--
(SI
2
9
4
6
8
TIME
(
.12
.19
<M(
->
.14
.16
.18
29
(
(Figure XXIX)
Cisplatin in water with 1 mM SPS
(same parameters as Figure VI)
I
J
YIME
-
!Ii-,
-,
(Figure XXX)
I
<SEC>
Transplatin in water with 1 mM SPS
(same parameters as Figure VI)
030--1
~I
"'-'
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I
~l
..... ~j
~
,..
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.
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0
~
=
~
os
..
T.M&
<SEC>
'"
~
(Fiyure XXXI)
Cisplatin in water with 1 mM SDyS
(same parameters as Figure VI)
:
::
-,
~
fill
~
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~
:;:
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~
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~
.. ..
....
TI ... E
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<"IN>
Transplatin in water with 1 mM SDyS
(same parameters as Figure VI)
(Figure XXXI I)
~
::
.......
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~
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. . "i"'~~'iii"i'ii;ii"iliiiiiiiiiiii'i'iiiiriiiii ",II'i.""."",.,
25M
T:lPlit
308
C SEC>
3~a
488
458
S88
•• """""""'"
5~a
.88
!
Blood serum
(same parameters as Figure VI)
(Figure XXXIII)
~
::
-,
~
=
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g
~
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d
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GIl
'58
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.. ~QIo
eo
ag,a
2!SU
3aa
Y'J"ME
(Figure XXXIV)
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ti
58
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i "
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,"
I' • "
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j
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TIME
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4AQ
'!5~
I'
i t -,
"fT""1""T"T rTIT
!'~A
,fi,CjiIKlIII
<SEC>
Blood serum with cisplatin in saline
(same parameters as Figure VI)
Ii i i i Ii i Ii II
2!58
3~g
3....
<SEC>
iii' "
3'5"
Ii
"i
i
i "
......
i "
"
I' i"
....sa
i
Ii "
i jill. Ii
'5aG
Ii.
Ii"
'5S"
i Ii Ii
iii" ,
6818
(Figure XXXV)
~
I
Urine
(same parameters as Figure VI)
I
I
I
ii
If ,
I:11" !l
!
I
I
1
I
I
I
I
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,)
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a
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I
18
TIM':
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]
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!
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j
12
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....
(Figure XXXVI)
..
--
::: !
~
s'
a
,
"
4
..
,
.loa
y:rM";
~
Urine with cisplatin in saline
(same parameters as Figure VI)
~ ~
u ,..
..
...
~
..
,
< .... N:>
<"'IN"
.loa
.1.4
.1.6
.....