Determination of cisplatin in human blood plasma and urine using liquid chromatography-mass spectrometry for oncological patients with a variety of fatty tissue mass for prediction of toxicity
Aim: The research was aimed to analyze a level of triglycerides in blood serum as a possible new marker of toxicity, particularly in patients with excess body weight, receiving cisplatin. Materials and Methods: Study involved 20 oncological patients with stage III lung cancer, who received palliativ...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2017
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| Цитувати: | Determination of cisplatin in human blood plasma and urine using liquid chromatography-mass spectrometry for oncological patients with a variety of fatty tissue mass for prediction of toxicity / A. Gerina-Berzina, S. Hasnere, A. Kolesovs, S. Umbrashko, R. Muceniece, I. Nakurte // Experimental Oncology. — 2017 — Т. 39, № 2. — С. 124–130. — Бібліогр.: 18 назв. — англ. |
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irk-123456789-1379722018-06-18T03:12:56Z Determination of cisplatin in human blood plasma and urine using liquid chromatography-mass spectrometry for oncological patients with a variety of fatty tissue mass for prediction of toxicity Gerina-Berzina, A. Hasnere, S. Kolesovs, A. Umbrashko, S. Muceniece, R. Nakurte, I. Original contributions Aim: The research was aimed to analyze a level of triglycerides in blood serum as a possible new marker of toxicity, particularly in patients with excess body weight, receiving cisplatin. Materials and Methods: Study involved 20 oncological patients with stage III lung cancer, who received palliative treatment with cisplatin. High-performance liquid chromatography was used for quantitative determination of pure cisplatin in urine and blood samples. Cisplatin concentration of the test samples was determined based on the data obtained from the calibration graph. Results: Quantitative determination of pure cisplatin is quite complicated. The elimination half-time for one of the groups was observed higher almost by half than for other patients. Higher dose of cisplatin showed a significant association with increase in triglyceride levels. We found a close correlation between body mass index and triglyceride changes during chemotherapy (p = 0.001; r = 0.67). The results indicate that a higher body mass index gives higher fluctuations of triglyceride levels in blood serum. Analyses of correlation between level of triglycerides and elimination half-time show that by an increase in the level of triglycerides in the blood serum cisplatin elimination half-time is prolonged (R²Linear = 0.596). Cisplatin concentration in urine is higher and elimination takes longer time at elevated levels of triglycerides, where close correlation between fraction of excreted substance in urine and concentration parameters was seen (p < 0.01). Also good correlation for body mass index with fraction of excreted substance in urine and concentration parameters was observed (p < 0.05). Conclusion: Clearance of cisplatin, which was determined by the chromatographic method, is reduced in individuals with increased adipose tissue mass. Research data suggest that overweight affects cisplatin elimination from the body. The greater body fat mass can contribute to a greater rise of triglyceride level in blood serum. Triglycerides in blood plasma may serve as an additional indicator of higher cisplatin toxicity as a cardiotoxicity marker. 2017 Article Determination of cisplatin in human blood plasma and urine using liquid chromatography-mass spectrometry for oncological patients with a variety of fatty tissue mass for prediction of toxicity / A. Gerina-Berzina, S. Hasnere, A. Kolesovs, S. Umbrashko, R. Muceniece, I. Nakurte // Experimental Oncology. — 2017 — Т. 39, № 2. — С. 124–130. — Бібліогр.: 18 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/137972 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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English |
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Original contributions Original contributions |
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Original contributions Original contributions Gerina-Berzina, A. Hasnere, S. Kolesovs, A. Umbrashko, S. Muceniece, R. Nakurte, I. Determination of cisplatin in human blood plasma and urine using liquid chromatography-mass spectrometry for oncological patients with a variety of fatty tissue mass for prediction of toxicity Experimental Oncology |
| description |
Aim: The research was aimed to analyze a level of triglycerides in blood serum as a possible new marker of toxicity, particularly in patients with excess body weight, receiving cisplatin. Materials and Methods: Study involved 20 oncological patients with stage III lung cancer, who received palliative treatment with cisplatin. High-performance liquid chromatography was used for quantitative determination of pure cisplatin in urine and blood samples. Cisplatin concentration of the test samples was determined based on the data obtained from the calibration graph. Results: Quantitative determination of pure cisplatin is quite complicated. The elimination half-time for one of the groups was observed higher almost by half than for other patients. Higher dose of cisplatin showed a significant association with increase in triglyceride levels. We found a close correlation between body mass index and triglyceride changes during chemotherapy (p = 0.001; r = 0.67). The results indicate that a higher body mass index gives higher fluctuations of triglyceride levels in blood serum. Analyses of correlation between level of triglycerides and elimination half-time show that by an increase in the level of triglycerides in the blood serum cisplatin elimination half-time is prolonged (R²Linear = 0.596). Cisplatin concentration in urine is higher and elimination takes longer time at elevated levels of triglycerides, where close correlation between fraction of excreted substance in urine and concentration parameters was seen (p < 0.01). Also good correlation for body mass index with fraction of excreted substance in urine and concentration parameters was observed (p < 0.05). Conclusion: Clearance of cisplatin, which was determined by the chromatographic method, is reduced in individuals with increased adipose tissue mass. Research data suggest that overweight affects cisplatin elimination from the body. The greater body fat mass can contribute to a greater rise of triglyceride level in blood serum. Triglycerides in blood plasma may serve as an additional indicator of higher cisplatin toxicity as a cardiotoxicity marker. |
| format |
Article |
| author |
Gerina-Berzina, A. Hasnere, S. Kolesovs, A. Umbrashko, S. Muceniece, R. Nakurte, I. |
| author_facet |
Gerina-Berzina, A. Hasnere, S. Kolesovs, A. Umbrashko, S. Muceniece, R. Nakurte, I. |
| author_sort |
Gerina-Berzina, A. |
| title |
Determination of cisplatin in human blood plasma and urine using liquid chromatography-mass spectrometry for oncological patients with a variety of fatty tissue mass for prediction of toxicity |
| title_short |
Determination of cisplatin in human blood plasma and urine using liquid chromatography-mass spectrometry for oncological patients with a variety of fatty tissue mass for prediction of toxicity |
| title_full |
Determination of cisplatin in human blood plasma and urine using liquid chromatography-mass spectrometry for oncological patients with a variety of fatty tissue mass for prediction of toxicity |
| title_fullStr |
Determination of cisplatin in human blood plasma and urine using liquid chromatography-mass spectrometry for oncological patients with a variety of fatty tissue mass for prediction of toxicity |
| title_full_unstemmed |
Determination of cisplatin in human blood plasma and urine using liquid chromatography-mass spectrometry for oncological patients with a variety of fatty tissue mass for prediction of toxicity |
| title_sort |
determination of cisplatin in human blood plasma and urine using liquid chromatography-mass spectrometry for oncological patients with a variety of fatty tissue mass for prediction of toxicity |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2017 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/137972 |
| citation_txt |
Determination of cisplatin in human blood plasma and urine using liquid chromatography-mass spectrometry for oncological patients with a variety of fatty tissue mass for prediction of toxicity / A. Gerina-Berzina, S. Hasnere, A. Kolesovs, S. Umbrashko, R. Muceniece, I. Nakurte // Experimental Oncology. — 2017 — Т. 39, № 2. — С. 124–130. — Бібліогр.: 18 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
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2025-07-10T04:51:18Z |
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2025-07-10T04:51:18Z |
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1837234212183212032 |
| fulltext |
124 Experimental Oncology 39, 124–130, 2017 (June)
DETERMINATION OF cIsplATIN IN HUMAN BlOOD plAsMA
AND URINE UsING lIQUID cHROMATOGRApHY-MAss
spEcTROMETRY FOR ONcOlOGIcAl pATIENTs wITH A vARIETY
OF FATTY TIssUE MAss FOR pREDIcTION OF TOxIcITY
A. Gerina-Berzina1, 2, *, S. Hasnere1, 2, A. Kolesovs3, S. Umbrashko4, R. Muceniece5, I. Nakurte1
1Pauls Stradins Clinical University Hospital, Riga 1002, Latvia
2Latvia University, Riga 1002, Latvia
3Latvia University, Faculty of Psychology, Riga 2015, Latvia
4Riga Stradiņš University, Institute of Anatomy and Anthropology, Riga 2010, Latvia
5Latvia University, Faculty of Chemistry, Riga 2004, Latvia
Aim: The research was aimed to analyze a level of triglycerides in blood serum as a possible new marker of toxicity, particularly
in patients with excess body weight, receiving cisplatin. Materials and Methods: Study involved 20 oncological patients with stage
III lung cancer, who received palliative treatment with cisplatin. High-performance liquid chromatography was used for quantitative
determination of pure cisplatin in urine and blood samples. Cisplatin concentration of the test samples was determined based on the
data obtained from the calibration graph. Results: Quantitative determination of pure cisplatin is quite complicated. The elimination
half-time for one of the groups was observed higher almost by half than for other patients. Higher dose of cisplatin showed a significant
association with increase in triglyceride levels. We found a close correlation between body mass index and triglyceride changes during
chemotherapy (p = 0.001; r = 0.67). The results indicate that a higher body mass index gives higher fluctuations of triglyceride levels
in blood serum. Analyses of correlation between level of triglycerides and elimination half-time show that by an increase in the level
of triglycerides in the blood serum cisplatin elimination half-time is prolonged (R2 Linear = 0.596). Cisplatin concentration in urine
is higher and elimination takes longer time at elevated levels of triglycerides, where close correlation between fraction of excreted sub-
stance in urine and concentration parameters was seen (p < 0.01). Also good correlation for body mass index with fraction of excreted
substance in urine and concentration parameters was observed (p < 0.05). Conclusion: Clearance of cisplatin, which was determined
by the chromatographic method, is reduced in individuals with increased adipose tissue mass. Research data suggest that overweight
affects cisplatin elimination from the body. The greater body fat mass can contribute to a greater rise of triglyceride level in blood serum.
Triglycerides in blood plasma may serve as an additional indicator of higher cisplatin toxicity as a cardiotoxicity marker.
Key Words: cancer, chemotherapy, cisplatin, triglycerides, chromatographic method.
Cancer is a major burden of disease worldwide. Each
year, tens of millions of people are diagnosed with can-
cer around the world, and more than half of the patients
eventually die from it. In many countries, cancer ranks
the second most common cause of death following
cardiovascular diseases [1].
Increase in cancer incidence is due to the aging
of the population (in most cases, the tumors are diag-
nosed in older people), as well as the high prevalence
of risk factors. The risk factors that have a significant
impact on mortality from malignant tumors are: an in-
creased body mass index (BMI), inadequate fruit and
vegetable consumption, low physical activity, tobacco
and alcohol consumption, air pollution, unsafe sex,
various manipulations made in inappropriate environ-
ment and circumstances (preventive and therapeutic
injections, tattooing, etc.). World Health Organiza-
tion (WHO) data show that 35% of cancer deaths are
caused by these risk factors [2].
Survival rates of cancer patients are affected
by various factors, such as tumor localization, clini-
cally morphological risk factors (patient age, tumor
morphological variant, tumor differentiation grade,
stage of disease, reproductive factors, etc.), the pa-
tient’s general health, co-existing conditions, specific
anti-cancer treatment options.
Chemotherapy is one of cancer treatment op-
tions. In chemotherapy, anticancer agents that kill
the cancer cells in the body are used. Even if the
cancer is removed with a surgery, cancer cells may
be located in nearby tissue or elsewhere in the body.
The aim of chemotherapy is to treat cancer in several
ways: either to reduce tumor volume before surgery
or irradiation, or eradicate cancer cells that may
be dispersed in the surrounding tissues or organs
after tumor surgery (i.e. preventive chemotherapy),
or relieve cancer symptoms (such as pain), or to con-
Submitted: March 23, 2017.
*Correspondence: Fax: +37167069918
E-mail: a.gerina@inbox.lv
Abbreviations used: AUC — area under the concentration curve;
BMI — body mass index; C — concentration; CL — clearance;
Cmax — maximum concentration; DDTC — diethyldithiocarbamate;
fe (%) — fraction of excreted substance in urine; HPLC — high-per-
formance liquid chromatography; HPLC-MS — high-performance
liquid chromatography-mass spectrometry; HRMS — high-reso-
lution mass spectra; kel — elimination constant; LC — liquid chro-
matography; LC-MS — liquid chromatography-mass spectrometry;
MRT — mean residence time; MS — mass spectrometry; Pt — plati-
num; t½ — elimination half-time; TOF — time of flight; UHPLC — ul-
tra high performance liquid chromatography; UPLC-TOF — ultra-
performance liquid chromatography — time of flight; WHO — World
Health Organization.
Exp Oncol 2017
39, 2, 124–130
Experimental Oncology 39, 124–130, 2017 (June) 125
trol the tumor growth if the tumor has already spread
(palliative therapy).
Each chemotherapy drug has its own internation-
ally defined dose of administration, which is calculated
by taking into account body square meters. Dose
of drug is calculated individually for each patient
depending on body weight and height. The pa-
tient’s gene ral condition and co-morbidity is also
taken into account. Individuals respond differently
to received chemotherapy and this response may have
significant clinical importance. Calculation of a definite
dose of chemotherapy reduces the possible toxic re-
actions of drugs [3]. There are 5 basic conditions that
determine treatment tactics, i.e., the right medication,
right patient, right dosage, right administration route
and right timing [4]. This helps a physician to make
therapy more rational and avoid therapeutic errors,
as too small doses of medication have no therapeutic
effect and that affects overall survival and disease-free
period, while too high dose increases the toxic effects
in the body for up to a possible death.
High-performance liquid chromatography (HPLC)
is irreplaceable modern analytical method for the
separation and identification of different chemical
compounds in both simple and in highly complex test
samples. Its main advantage is speed and accuracy.
HPLC is currently the most popular method in bio-
chemistry, in particular for pharmacokinetic and phar-
macodynamic studies. When the smaller particle size
of separation column is used (ultra high-performance
liquid chromatography — UHPLC), separation method
goes more effective and more sensitive. UHPLC, with
its shorter analysis time and quicker column equilibra-
tion, is ideally suited to rapid method development.
MATERIAls AND METHODs
Patients. Patient group with 20 cancer patients
was established for determination of cisplatin in bio-
logical liquids — blood and urine. The stage III lung
cancer patients who received palliative treatment with
cisplatin were selected for this study. Based on BMI
patients were divided into two groups. One group were
patients where the BMI < 29.0 kg/m2, another of BMI
> 29.0 kg/m2. Patients received a dose of cisplatin
calculated at 75 mg/m2. The patient blood serum tri-
glyceride levels additional was to evaluated the pharma-
cokinetics of cisplatin relationship with body fat mass.
The serum material for determination of cisplatin
was collected as follows — blood plasma (2 ml, purple
tube), 0 (prior to treatment), 20, 40, 60, 120 and
180 min, 24, 48 and 72 h after the end of the infu-
sion (duration of cisplatin infusion 1.00 h). A sample
of blood allowed clotting for 30 min at the room tem-
perature, then centrifuged for 10 min × 2500 rpm, then
frozen at –20 °C till further analyses.
Cisplatin analysis by high-performance liquid
chromatography-mass spectrometry (HPLC-MS).
HPLC combined with mass spectrometry (MS) was
used to determine the presence of cisplatin in blood
and urine samples. All solvents used were of analytical
grade. Acetonitrile and formic acid were purchased
from Sigma-Aldrich (St. Louis, USA). The used deio-
nized water (18.2 MΩ) was prepared by a Milli-Q water
purification system from Millipore (Billerica, Massa-
chusetts, USA). Chromatographic analyses performed
on a modular UHPLC system, Agilent 1290 Infinity
series (Agilent Technologies). Liquid chromatogra-
phy (LC) separations achieved by using an Extend-
C18 (Agilent) column 2.1 × 15 mm, 1.8 μm. Elution
solvents consist of 0.1% formic acid in acetonitrile
and 0.1% formic acid in water in gradient mode at flow
rate 250 μl•min-1. The injection volume was 1.0 μl. The
high-resolution mass spectra (HRMS) were taken,
respectively, on an Agilent 6230 TOF LC/MS (Agilent
Technologies, Germany) with electrospray ionization.
MS operating conditions were as following: positive
ionization mode, gas temperature of 325 °C, nitro-
gen flow rate of 10 l/min, nebulizer pressure 40 psi,
capillary voltage 3500 V and applied fragmentor was
100 V. Internal reference mass 121.050873 m/z and
922.009798 m/z (G1969-85001 ES-TOF Reference
Mass Solution Kit, Agilent Technologies & Supelco) for
all sample analyses were used. One full mass spectrum
was acquired in profile mode, with mass range from
m/z 50 to 1000.
Direct infusion of the cisplatin showed that there
are no detectable ions found for cisplatin. Peak de-
tection and spectrum extraction were performed with
MassHunter5.00 Software (Agilent).
Sample and standard preparation. Fro-
zen cisplatin-plasma samples were thawed, 5%
diethyldithiocarbamate (DDTC) 0.1N NaOH so-
lution was prepared. Cisplatin derivatives were
prepared as follows — to 500 μl of plasma were
added 100 μl of 5% DDTC solution. The sample
was homogenized by vortexing approximately
15–20 s and incubated for 15 min at 45 °C. After
15 min 1400 μl of 70% acetonitrile were added to the
sample. The sample was homogenized by vortexing
about 15–20 s, and then placed into a centrifuge for
15 min at 10,000 rpm. A 1.0 μl resulting upper layer
aliquot was injected into the liquid chromatography-
mass spectrometry (LC-MS) system. The standard
solutions of cisplatin derivative were prepared in the
following concentrations: 0.5; 1.0; 5.0; 10.0; 25.0;
50.0 and 100.0 μg/ml in acetonitrile. Solutions
of each concentration were injected into LC-MS sys-
tem. All experiments were performed in triplicate.
Calibration curves of standard solutions were con-
structed by plotting the average peak area against
concentration, and a regression equation was com-
puted. A mixture of standard solutions was injected
three times and the corresponding peak areas were
recorded. The relative standard deviation was deter-
mined to be less than 1%. The obtained calibration
curve showed linearity of correlation coefficient (R2)
in the concentration range 0.99994.
Urine sample was collected for 6 h, 12 h, 24 h
(Day 1), 48 h, 72 h (Day 3) and 96 h (Day 5) after re-
ceived cisplatin infusion. Collected urine for ice — 20 °C.
126 Experimental Oncology 39, 124–130, 2017 (June)
Ethical aspects. Positive opinion of the trial
preparation and development given the Latvian Uni-
versity of Research Institute of Cardiology clinical-
physiological study of medicinal and pharmaceutical
products of clinical research ethics committee. Before
the study, the patients were informed about the de-
tails of the study and approved the voluntary consent
to participate in it. The study respondents of main-
tained confidentiality and anonymity.
Statistical analysis. Patient data were analysed
using descriptive statistical methods, making a com-
parative analysis (t-test, chi-square (χ2) test by correla-
tion analysis (Pearson correlation coefficient)) using
Microsoft Excel 2007 and SPSS software.
The aim of statistical analysis of research data
was to evaluate the validity and theoretical probability
distributions of the resulting measurements (distance,
thickness, circumference and weight), as well as follow
up on the statistical hypotheses. Therefore, the com-
mon (popular) descriptive statistical methods were
very widely used in this study.
The variables, measured in relation scale and nor-
mally distributed (by Gaussian normal distribution)
were analysed using parametric statistics methods.
In other cases, non-parametric statistical methods
were used. Hypotheses about the adequacy of the data
to the normal probability distribution were mainly test-
ed by Kolmogorov — Smirnov test. The Student’s t-test
was used to test uniformity of two arithmetic mean
groups, for three or more groups of arithmetic mean
the analysis of variance (ANOVA) was used. For com-
parison of the number of cases different statistical
methods were used, such as χ2-test and Fisher’s exact
test. The correlation and linear regression methods
were used to predict the interaction between different
variables and event analysis.
For statistical data processing first a database was
created in software MS Excel, and then the data were
converted for professional research data statistical
processing program SPSS (Statistical Pacade for So-
cial Sciences) version 16.0 for Windows. All hypothesis
tests used a duplex (2-tailed) statistical hypotheses
and hypothesis was rejected if the probability (signifi-
cance level) was < 5% or p < 0.05.
The statistical software Statistica 6.0 was also used
for processing of the data. In cases where data did not
correspond to the normal distribution, a data transfor-
mation was performed, but after statistical analyses —
opposite transformation. Non-parametric Spearman
rank correlation was done in case of qualitative data.
Differences between groups, correlations and regres-
sions were considered significant, if p < 0.05.
REsUlTs
HPLC-MS is an analytic chemical method, which
combines physical separation of a blend by LC and
mass analysis of a substance by MS. LC-MS analytical
method is characterized by very high sensitivity and
specificity. By using time of flight (TOF) detector,
it is possible to determine compounds of highly com-
plex samples according to the mole mass, avoiding
matrix effect interference.
Quantitative determination of pure cisplatin is quite
complicated. Cisplatin as a chemical compound has
not typical ultraviolet absorption therefore derivatisa-
tion should be performed by sodium DDTC in order
to determine cisplatin by HPLC. This obtained com-
pound absorbs ultraviolet at 254 nm and is determined
using LC.
Direct infusion of the cisplatin showed that there
are no detectable ions found for cisplatin. The direct
infusion into MS system of cisplatin DDTC derivative
generate full scan spectra with a predominant ion
at m/z 492 and m/z 640 correspond to [Pt(DDTC)2]+
and [Pt(DDTC)3]+ ions respectively (Fig. 1). This sug-
gests that after DDTC derivatization, cisplatin is con-
verted to Pt-(DDTC)2 and Pt-(DDTC)3.
x1
04
x1
04
+ESI Scan (rt: 3.18 min) Frag=100.0V
+ESI Scan (rt: 3.18 min) Frag=100.0V
Counts vs. Mass-to-Charge (m/z)
Counts vs. Mass-to-Charge (m/z)
4.5
4
3.5
3
2.5
2
1.5
1
0.5
489.5
490.0112
638.0353
639.0379 640.0385
641.0359 642.0366
643.0369 644.0351645.0355 646.0326
491.0159 492.0171
493.0185
494.0159
495.0201
637 638 639 640 641 642 643 644 645 646637.5 638.5 639.5 640.5 641.5 642.5 643.5 644.5 645.5
490.5 491.5 492.5 493.5 494.5 495.5 496.5 497.5491 492 493 494 495 496 497490
0
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.1
0.2
0
Fig. 1. High-resolution mass spectrum of cisplatin DDTC derivative
from the standard solution, obtained by Agilent 6230 TOF LC/MS
Fig. 1 shows the magnified mass spectrum of a DDTC
derivative of cisplatin from the standard solution, which
is very close to spectrum from study by Yaroshenko
et al. [5]. Other signals in mass spectra correspond to the
signals formed by ions of different Pt isotopes.
In our further experiments cisplatin DDTC derivate
which refers to formation of [Pt(DDTC)3]+ ion was used
because of its higher intensity.
The base peak chromatogram in Fig. 2 shows the
signal of peak from the most intense mass in mass
spectrum of m/z 640, plotted versus time.
x1
06
+BPC(640.0000) Scan
Counts vs. Acquisition Time (min)
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.5 1.5 2.5 3.5 4.51 2 3 5.55 6.56 7.57 9.584
*3.18 Cisplatin
0.2
0.1
0
Fig. 2. Base peak chromatogram at m/z 640 of cisplatin DDTC
derivative Pt-(DDTC)3 in patient’s P01 urine after 12 h
Pharmacokinetic measurements of cispla-
tin in patients’ blood serum. The calibration curve
Experimental Oncology 39, 124–130, 2017 (June) 127
was designed from standard solutions of cisplatin
derivative and cisplatin concentrations in μg/ml were
determined from the standard curve for each sample
of patient serum. Concentrations were converted into
mg/l for calculations of pharmacokinetic data of cis-
platin. From the chromatograms of test samples during
analyses it was concluded that the selected method
can be used in quantitative determination of cisplatin
in human blood serum. Separated peaks were sym-
metrical and did not overlap with other compounds
existing in the matrix.
Changes of cisplatin concentration in blood serum
at different time intervals were identified, where the
key was to find the maximum concentration of cispla-
tin in certain period of time (Cmax). In this study Cmax
of cisplatin was reached 2 h after cisplatin injection.
Small differences were observed in the elimination
half-times (t½) for patients involved in this study. The
t½ for one of the groups (P13; P14; P15 and P16) was
observed higher almost by half than for other patients.
This could be explained by patients being overweight
as the average weight for this group was 97.21 kg.
Data on the t½ for other patients were not significantly
different, they were calculated as average values and
applied to the calculations of following pharmacoki-
netic parameters for all patients (Table 1).
From the equations that were calculated and ob-
tained using Excel 2013 software, individual elimination
constants (kel) were calculated and shown as the de-
gree in equation (see Table 1). They are presented with
a negative (−) sign, because it is a process of elimina-
tion, and kel indicates the percentage (%) of cisplatin
eliminated per hour.
Table 1. Pharmacokinetic data of cisplatin. Calculated individual kel pre-
sented as the degree in equation with a negative (−) sign, because
it is a process of elimination and kel indicates the percentage (%) of cispla-
tin eliminated per hour
Patients Equation C(0),
mg/l
t½
of I phase,
h
AUC,
mg•h/l
CL,
l/h t½ , h
P01 y = 1.17e−0.035x 1.17 16.90 33.43 3.74 19.80
P02 y = 1.40e−0.025x 1.40 22.35 56.00 2.23 27.72
P03 y = 0.73e−0.026x 0.73 11.95 28.08 4.45 26.65
P04 y = 0.85e−0.024x 0.85 18.24 35.42 3.53 28.88
P05 y = 0.91e−0.036x 0.91 12.60 25.28 5.93 19.25
P06 y = 0.74e−0.022x 0.74 16.12 33.64 4.46 31.50
P07 y = 1.58e−0.038x 1.58 18.24 41.58 3.61 20.38
P08 y = 0.76e−0.023x 0.76 15.40 33.04 4.54 30.13
P09 y = 1.72e−0.021x 1.72 15.07 81.90 1.59 33.00
P10 y = 2.22e−0.028x 2.22 16.90 79.29 1.64 24.75
P11 y = 1.92e−0.023x 1.92 27.72 83.48 1.56 30.13
P12 y = 2.24e−0.027x 2.24 23.90 82.96 1.57 25.67
P13 y = 0.45e−0.011x 0.45 8.06 40.91 3.18 63.00
P14 y = 0.69e−0.014x 0.69 9.76 49.29 2.64 49.50
P15 y = 0.62e−0.015x 0.62 9.36 41.33 3.15 46.20
P16 y = 0.74e−0.019x 0.74 9.76 38.95 3.34 36.47
P17 y = 0.98e−0.018x 0.98 38.50 54.44 2.39 38.50
P18 y = 1.07e−0.022x 1.07 31.50 48.64 2.67 43.31
P19 y = 0.97e−0.019x 0.97 36.47 51.05 2.55 46.20
P20 y = 1.17e−0.022x 1.17 31.50 53.18 2.44 38.50
Note: C — concentration; t½ — elimination half-time; AUC — area under the
concentration curve; CL — clearance.
The elimination rate constant kel was expressed
from the established equation and then used to calcu-
late the total area under the concentration curve (AUC)
and the clea rance (discharge of blood plasma volumes
from the amount of substance per unit of time). Within
the framework of our work, the AUC results ranged
from 25.28 to 83.48 mg•h/l (see Table 1). The mean
AUC result for all oncological patients, receiving cis-
platin at a dose of 125–150 mg, was 49.59 mg•h/l.
Clearance estimates were summarized in Table 1.
The results show that the clearance during treatment
ranged 1.56–4.54 l/h. These results were very similar
to the literature data, leading to the conclusion that
results of our pharmacokinetic data are true and can
be used for further research.
One of the pharmacokinetic parameters is the
mean substance resistance time in the body (MRT —
mean residence time). MRT = 1/kel, but since kel =
0.693/t½, then the formula is = 1.44 x t½ (one divided
by 0.693 equals 1.44). The mean t½ in elimination
phase is 33.98 h, the average MRT was 48.93 h.
Determination of cisplatin in urine. Using the
resulting calibration graph, amount of cisplatin in urine
was calculated for all patients. Based on the concen-
tration in the urine, which was determined for 20 pa-
tients, concentration curves were obtained. To show
how visually the curves look for patients depending
on the dose, they were divided into groups of patients,
depending on the received chemotherapy dose. Ac-
cordingly, three groups of patients were acquired.
Pharmacokinetic parameters such as renal clea-
rance, fraction of excreted substance in percentage
and creatinine clearance were defined. It was observed
that the AUC was higher for patients on the first day
compared to the last day. Respectively, for those pa-
tients who received 130 mg or greater dose of cisplatin,
the AUC lowered slowly in last two days of the study
days. There are no data available to explain it. Perhaps
this is related to the significant untapped biochemical
processes, with cisplatin binding to mo lecules that
hold cisplatin and do not let it to be removed gradually,
as well as with cisplatin metabolism, which still has
not been fully understandable. At the cisplatin dose
of 130 mg and above, there was a change in the frac-
tion of excreted substance (%) by increase. Literature
states that cisplatin excretes with urine very long time
after the end of therapy, such as for patients receiving
chemotherapy for a long time and who were followed
for more than 10 years, in the urine were excreted
1.26 ± 0.50 41 μg Pt after 24 h [6]. It would be useful
to determine cisplatin urinary excretion for patients
involved in the study after a long period of time,
in parallel to determination of renal functions. This
would allow the assessment of renal status, if it is not
affected by the long cisplatin elimination after comple-
tion of therapy, or to determine the renal regeneration.
Changes of triglyceride levels in blood serum
in patients receiving cisplatin therapy. Higher dose
of cisplatin showed a significant association with in-
crease in triglyceride levels. Also higher body fat mass
showed a relationship with increase in triglyceride
levels. According to clinical tests, triglyceride levels
in blood serum changed 24 h after received cisplatin
therapy. The highest triglyceride levels were reached
48–72 h after treatment.
128 Experimental Oncology 39, 124–130, 2017 (June)
It was observed that triglycerides have a correlation
with thickness of fatty tissue folds. The closest correla-
tion was with the thickness of fat tissue fold under the
shoulder blade (p < 0.001; r = 0.56). We found a close
correlation between BMI and triglyceride changes
during chemotherapy (p = 0.001; r = 0.67). The results
indicate that a higher BMI gives higher fluctuations
of triglyceride levels in blood serum.
Levels of triglycerides in the blood serum in-
creased more at higher doses of cisplatin (Table 2).
Higher triglyceride changes were observed at higher
doses of cisplatin or greater dose differences. Increase
of triglycerides in blood serum correlated with the
person’s weight, especially the adipose tissue mass,
greater changes were with increased weight. In gene-
ral, gender has not such a big role in the cisplatin dose
application, but fat tissue mass is essential. Amount
of adipose tissue mass contributes to an increase
in serum triglycerides. In conclusion, the greater body
fat mass can contribute to a greater rise of triglyceride
level in blood serum (p < 0.001).
Separately were analysed the data of 20 oncologi-
cal patients, splitting them into two groups depending
on the BMI ≥ 29 kg/m2 (see Table 2).
It was observed by Pearson correlation that the
level of triglycerides in the blood affects the t½ of cis-
platin (p < 0.01). It is likely that cisplatin is eliminated
more slowly from the blood serum at higher levels
of triglycerides. Analyses of correlation between level
of triglycerides and t½ show that by an increase in the
level of triglycerides in the blood serum cisplatin t½
is prolonged (R2 Linear = 0.596) (Fig. 3).
R² Linear = 0,596
2
3
4
5
6
Le
ve
l o
f t
rig
lyc
er
id
es
in
t
he
b
lo
od
1
10 20 30 40 50 60 70
The t½ in blood serum
Fig. 3. Correlation between the levels of triglycerides and the
t½ of cisplatin
Also cisplatin concentration in urine is higher
and elimination takes longer time at elevated levels
of triglycerides (see Table 2), where close correlation
between fe (%) and concentration parameters was
seen (p < 0.01). Also good correlation for BMI with
fe (%) and concentration parameters was observed
(p < 0.05), suggesting that overweight affects cis-
platin elimination from the body. It is concluded that
cisplatin from the serum and urine is excreted more
slowly in patients with obesity and also maintains
a lasting increase of triglycerides in the blood. There
are no research data in the literature at the moment
for comparison.
Table 2. Analysis of triglycerides, pharmacokinetic and anthropometric data of patients receiving cisplatin therapy
BMI,
kg/m2
Dose
of cisplatin
75 mg/m2
Trigly-
cerides
in blood
serum 1
Trigly-
cerides
in blood
serum 2
t½, h CL, l/h C, mg/l fe, %
BMI, kg/m2 Pearson
correlation 1 −0.008 0.060 0.284 0.255 0.227 0.507* 0.502*
Sig. (2-tailed) 0.973 0.802 0.225 0.278 0.337 0.023 0.024
N 20 20 20 20 20 20 20 20
Dose of cisplatin
75 mg/m2
Pearson
correlation −0.008 1 0.105 0.016 −0.275 0.589** 0.381 0.389
Sig. (2-tailed) 0.973 0.659 0.947 0.240 0.006 0.098 0.090
N 20 20 20 20 20 20 20 20
Triglycerides
in blood serum 1
Pearson
correlation 0.060 0.105 1 0.798** 0.731** 0.073 0.077 0.446*
Sig. (2-tailed) 0.802 0.659 0.000 0.000 0.759 0.746 0.049
N 20 20 20 20 20 20 20 20
Triglycerides
in blood serum 2
Pearson
correlation 0.284 0.016 0.798** 1 0.772** 0.093 0.166 0.472*
Sig. (2-tailed) 0.225 0.947 0.000 0.000 0.698 0.483 0.036
N 20 20 20 20 20 20 20 20
t½, h Pearson
correlation 0.255 −0.275 0.731** 0.772** 1 −0.239 −0.059 0.215
Sig. (2-tailed) 0.278 0.240 0.000 0.000 00.311 0.804 0.362
N 20 20 20 20 20 20 20 20
CL, l/h Pearson
correlation 0.227 0.589** 0.073 0.093 −0.239 1 0.777** 0.685**
Sig. (2-tailed) 0.337 0.006 0.759 0.698 0.311 0.000 0.001
N 20 20 20 20 20 20 20 20
C, mg/l Pearson
correlation 0.507* 0.381 0.077 0.166 −0.059 0.777** 1 0.823**
Sig. (2-tailed) 0.023 0.098 0.746 0.483 0.804 0.000 0.000
N 20 20 20 20 20 20 20 20
fe, % Pearson
correlation 0.502* 0.389 0.446* 0.472* 0.215 0.685** 0.823** 1
Sig. (2-tailed) 0.024 0.090 0.049 0.036 0.362 0.001 0.000
N 20 20 20 20 20 20 20 20
Note: *Correlation is significant at the 0.05 level (2-tailed). **Correlation is significant at the 0.01 level (2-tailed). t1/2 — elimination half-time — time in which
the concentration of pharmaceutical agent in the blood (plasma, serum) reduces by half; CL, l/h — the liberation amount of plasma volume from the sub-
stance per unit of time; C — concentration of cisplatin in urine, mg/l; fe, % — fraction of excreted substance in urine, which is usually expressed as percentage.
Experimental Oncology 39, 124–130, 2017 (June) 129
DIscUssION
It is important to determine precisely a dose of che-
motherapy agent as inadequate high dose of medica-
tion can cause serious side effects and even death, but
too low dose reduces the effectiveness of treatment
and overall survival. Accurate determination of the dose
is still debatable both through already mentioned cal-
culation formulas and by taking into account described
toxicity of chemotherapy drugs, when the patient re-
ceives inadequate dose. Described studies on patients
with obesity have trend not to prescribe the calculated
dose, due to fear of side effects, but that is not always
justified. It affirms the need for new and accurate meth-
ods of calculation for doses of chemotherapy drugs.
A LC-MS method was selected to prove that the
body frame has a vital role in chemotherapy dosage,
which would allow better prediction of the therapeutic
toxicity. A new, innovative method was developed for
derivatisation of cisplatin and following determination
by MS with UHPLC-TOF [7], in cooperation with the
Faculty of Chemistry at the University of Latvia.
Differences in the elimination of cisplatin were
observed depending on the content of muscles or fat
tissues in the body mass. It was observed that cisplatin
is eliminated more slowly in patients with overweight
(obesity) than in patients with normal body weight. Com-
paring results of the obtained total AUC with the scientific
literature, we can conclude that they have a slight differ-
ence. In the scientific literature, which studied several
groups of patients with different tumor types, including
lung cancer, the AUC was calculated as the average of all
the groups, which was 23.19 ± 2.052 mg•h/l [8, 9], but
in our study the AUC results ranged 25.28–83.48 mg•h/l.
The mean AUC for all in our study involved oncological
patients was 49.59 mg•h/l. The difference in results
can be explained by the different doses. The peak con-
centration increases with increasing dose of cisplatin
and therefore the total AUC is bigger. In research study,
described in the literature, doses of cisplatin received
ranged from 50 mg to 100 mg [8], but in this work have
been stu died and analysed the patients who received
cisplatin at a doses of 125–150 mg. Comparison of the
cisplatin dose to AUC in both studies, a large difference
between the results was not observed. In the literature
reviewed study clearance calculations were carried out
that during treatment ranged from 2.30 l/h up to 7.98 l/h,
but in our calculations they were between 1.56 l/h and
4.54 l/h. These results were very similar to those in the
described study, which suggests that pharmacokinetic
data of our study are true and can be used for further
research.
HPLC-MS is used for detection of a variety of sub-
stances and medicines in biological solutions. It was
used in our study to determine the concentration of cis-
platin from the constituent Cisplatin in the blood serum
and urine of oncological patients. MS spectra with
a signal at m/z 640, which corresponds to [Pt(DDTC)3]+
ions, were obtained during analysis of samples. Ac-
quired bands were symmetrical and did not overlap
with other existing matrix compounds.
A new, innovative method was developed for derivati-
sation of cisplatin and following determination by MS with
UHPLC-TOF. Pharmacokinetic parameters of cisplatin
were determined for twenty patients with lung cancer
diagnosed in stage III. Cisplatin was measured by stan-
dard curve concentrations in mg/ml. Approximately
at the average dose of 135 mg, the mean Cmax in twenty
patients was 2487.03 mg/ml, which was very close
to the literature data. Cmax for all patients ranged from
795.7 mg/ml up to 6429.1 mg/ml, this could be ex-
plained by a variety of doses administered individually
to each patient. Elderly patients often have impaired
renal function, which does not allow 100% cisplatin dose
application in order to avoid serious adverse reactions.
The t½ was observed almost 50% higher for patient
group (P13, P14, P15, and P16). That could be explained
by the increase in body weight for this group (an average
of 97.21 kg), so that proves the hypothesis that cisplatin
is eliminated more slowly in patients with a higher body
weight (obesity) than in patients with normal body weight.
This confirmed one of our working hypotheses. Clear-
ance during treatment ranged from 1.56 l/h to 4.54 l/h.
These results were very similar to the literature data and
suggest that resulting pharmacokinetic data in our study
are true and can be used for further research.
Despite the fact that there are many studies on phar-
macokinetics of cisplatin, many of these stu dies did not
specify the pharmacokinetic data or often mentioned
that the data are consistent with the norm. It was con-
cluded from the review of literature data on the pharma-
cokinetics of cisplatin that there are no certain limits and
averages, and the mechanism of action of cisplatin is not
clear. There are no whole some data on the pharmaco-
kinetics of cisplatin, which can be influenced by various
factors such as the patient’s overweight, age, gender,
co-morbidities, drug storage and administration, etc.
It was concluded from results of our study that there
are two significant factors that affected the level of tri-
glycerides in the blood. One of them is the higher dose
of cisplatin, the other is the larger body fat mass. Since
patients received multiple courses of chemotherapy,
then a gradual increase in trigly cerides was observed
with each subsequent dose of cisplatin, especially in pa-
tients with obesity. Positive correlation (Pearson) was
found for triglyceride level changes, depending on the
cisplatin dose, between the initial triglyceride levels
and body fat, muscle and bone masses (p < 0.001). In-
creased dose of cisplatin showed a significant associa-
tion with increased levels of triglycerides. Also, a higher
body fat mass showed association with an increased
level of triglycerides. Correlation was found between
level of triglycerides and fat fold thickness. The clo sest
correlation was with fat fold thickness under the shoul-
der blade (p < 0.001; r = 0.56). A close correlation was
found between BMI and triglyceride changes during
chemotherapy (p = 0.001; r = 0.67). Results indicate
that higher BMI gives greater fluctuations of triglyc-
eride levels in blood serum. The adipose tissue mass
became more important for cisplatin dose determina-
tion in women (p < 0.05), although women were not the
130 Experimental Oncology 39, 124–130, 2017 (June)
biggest of statistical groups. Both the adipose tissue
mass (p < 0.001) and muscle mass (p < 0.05) were
essential in dose determination, especially at the dose
differences (p < 0.01). Overall, the gender has not such
a big role in the cisplatin dose application, essential
is the fat tissue mass. Adipose tissue mass contributes
to an increase in serum triglycerides.
Cisplatin is characterized by cumulation (accumu-
lation) of dose, when part of the medication remains
from the previous administration, particularly at doses
higher than 100 mg/m2 [10]. It should further be stud-
ied the relationship between cisplatin dose cumulation
and an increase in triglyceride levels in the blood, and
obesity. Associated between increased triglyceride
levels and cisplatin treatment is unclear. It is important
to mention that cancer patients have abnormal metabolic
processes in the body. Cancer patients with weight loss
have increased triglyceride and fatty acid metabolism
in comparison with patients without weight loss [11].
An increased triglyceride concentration in blood plasma
was observed in weight losing cancer patients compared
to stable weight cancer patients, indicating on enhanced
lipolysis [12]. This makes it difficult to precisely analyse
changes in triglyceride levels in the blood serum.
Results of our study showed a significant rela-
tionship between the dose of cisplatin and the level
of triglycerides in the blood serum. Also the study,
carried out with stem cell tumors, mentioned that the
level of triglycerides in the blood serum tended to in-
crease in the group of cisplatin therapy patients, but
this trend was not statistically significant [13]. However,
in another study by a 5-year follow-up after cisplatin
therapy of testicle tumor, there were no significant dif-
ferences in plasma triglyceride levels among patients
with cisplatin therapy and without it [14]. In the longer
term cisplatin may have no effect on triglycerides in the
blood plasma, as it has been observed in animal stu-
dies that a few days after cisplatin therapy there were
elevated triglyceride levels in blood plasma and also
in proximal renal tubules, thus causing cisplatin toxicity
in the kidneys [15, 16]. This is an important factor for
patients, especially in women with a high-carbohydrate
diet, because additional accumulation of triglycerides
as a result of overproduction of insulin may increase
cisplatin nephrotoxicity [17, 18]. Additional research
study is necessary to carry out on relationship of cis-
platin pharmacokinetics with obesity and levels of tri-
glycerides in the blood. Higher dose of cisplatin and
increased body fat mass are important risk factors
for elevated triglyceride levels in the blood serum.
Determination of the level of triglycerides in blood
plasma may serve as an additional indicator and toxic-
ity marker for higher toxicity of cisplatin.
AcKNOwlEDGEMENTs
AGB would like to thank Silvija Umbrashko, Ilva
Nakurte and Ruta Muciniece, supervisors. Thanks
to all staff of Latvia University, Faculty of Chemistry
and Riga Stradiņš University, Institute of Anatomy and
Anthropology of Latvia.
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