Development of an Analytical Method for Determination of Carboplatin and Oxaliplatin in Resource Water, Prediction and Environmental Risk Assessment

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 3, Pages: 1168-1175

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal web link: http://www.jett.dormaj.com

Development of an Analytical Method for

Determination of Carboplatin and Oxaliplatin in

Resource Water, Prediction and Environmental

Risk Assessment

M. Alimohammadi ¹^,², M. Asadi-Ghalhari , Y. Ghafuri *

Department of Environmental Health Engineering , School of Public Health , Tehran University of Medical Sciences, Tehran, Iran

Center for Water Quality Research, Institute for Environmental Research (IER), Tehran University of Medical Science, Tehran, Iran

Research Center for Environmental Pollutants, Qom University of Medical Sciences, Qom, Iran

Received: 21/05/2020

Accepted: 16/07/2020

Published: 20/09/2020

Abstract

Carboplatin and Oxaliplatin as cytotoxic drugs have developed in recent years as a group of antineoplastic agents, have the ability

to kill or stop the growth of certain living cells and are used in chemotherapy of cancer. That their function is through the disruption of

cell division. This study aimed to determine carboplatin and Oxaliplatin in wastewater samples by the characterization of the LC-MS/MS

method. The method was carried out on ODS C18 (250 mm, 3.5 µm) in which the mobile phase was A = Water +0.1% formic acid, B=

methanol +0.1% formic acid, (A / B) = 20:80(V/V), flow rate: 0.3 mL/min, and injection volumes: 5 µL. The method was validated for

LOD, LOQ, accuracy and precision. Results of environmental risk assessment showed that RQ (risk quotient) for Carboplatin and

Oxaliplatin were 0.51 and 0.038 respectively that indicates all of platinum compounds had low environmental exposure risk . In this

regard, studies should be carried out to evaluate the toxicity and genotoxicity effects of the metabolites of cytotoxic platinum compounds

and strategies to removal of these compounds.

Keywords: Carboplatin, Oxaliplatin, Risk, Assessment

Introduction¹

especially for platinum ion, in environmental samples, and

high-performance liquid chromatography (HPLC) with UV

detection, to measure the complexity of the platinum drug has

applied. Of course, the above methods are about the relatively

high levels of platinum in environmental samples. HPLC

coupled with triple quadrupole (QqQ) mass spectrometry (MS)

is a commonly used method with high effectiveness for

measurement of pharmaceutical residues in various

environmental samples (7, 8, and 9). Currently, in all oncology

wards of hospitals in Qom Province in the central part of Iran,

platinum complexes containing carboplatin and oxaliplatin are

widely incorporated. This study aimed to develope of an

analytical Method for determination of Carboplatin and

Oxaliplatin in resource water, prediction and environmental

risk assessment.

Very low levels of drug combinations in water and

wastewater environments around the world have been widely

considered (1). Cytotoxic drugs have developed in recent years

as a group of antineoplastic agents, have the ability to kill or

stop the growth of certain living cells and are used in

chemotherapy of cancer. That their function is through the

disruption of cell division (2). Most of the anticancer drugs are

classified as mutations or teratogenic for humans. According to

guidelines and regulations for the safe handling of anticancer

drugs, many studies have shown that the staff and the medical

staff are exposed to these drug (3). Carboplatin and oxaliplatin

are complexes of Pt that utilize anticancer drug and are

effective in the treatment of testicular and ovarian cancer.

Anticancer drugs are also very effective in the treatment of

many malignant tumors, lymphomas, and so on (4). Anticancer

Platinum complex drugs have important antitumor activity

caused particularly by the Cross-linking of DNA and formation

of DNA adducts, subsequently triggering apoptosis and leading

to cell death. Platinum complex drugs are excreted by patients

and released to the urban wastewater system. Health impacts

are considerable with the incomplete removal of

pharmaceutical compounds, especially the platinum complex

2 Materials and Methods

2.1 Chemicals and reagents

Analytical standards of platinum complexes carboplatin

(Car-pt, Cas 41575-94-4) and oxaliplatin (Oxa-pt, Cas 61825-

94-3) were provided by Sigma-Aldrich with the highest

percentage of purity (>99%). All solvents were of HPLC grade,

and all chemicals were of analytical reagent grade. Formic acid

(98–100%), ammonium hydroxide (25%), methanol, and

HPLC-water were purchased from Merck (Darmstadt,

Germany). The selected platinum, according to some

environmental fate and chemical structure, is shown in Table 1.

(

5, 6). Several methods for determining of platinum complex

drugs were provided containing Atomic absorption

Spectrometry (AAS), inductively coupled plasma (ICP),

inductively coupled plasma mass spectrometry (ICP-MS)

Corresponding author: Y. Ghafuri, Research Center for Environmental Pollutants, Qom University of Medical Sciences, Qom, Iran^.

Tel: +9832536602040, E-mail: yadollahghafuri@yahoo.com

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2020, Volume 8, Issue 3, Pages: 1168-1175

Individual solutions of each compound, including Car-pt (4000

µg/mL) and Oxa-pt (2200 µg/mL) were prepared and stored in

the dark at −20ºC.

2.4 Lc-Ms/Ms instruments

Samples were analyzed by an Agilent Technologies

separation module, ZORBAX, SB, C18 column (250 mm, 3.5

µm)in the liquid chromatography/auto-sampler system with a

5mm particle size in which the mobile phase was A= Water

+0.1% formic acid, B= methanol +0.1% formic acid, (A / B) =

20:80 (V/V), flow rate: 0.3 mL/min, and injection volumes: 5

µL. Mass spectrometry was performed using an Agilent G6410

.2 Stability tests

Stability of each analyte in biological matrix should be

confirmed, minimally at low and high short –term storage at

room temperature. The storage time should be based on the

expected duration test that samples are maintained at this

temperature during the intended study (4-24 hr).

Triple Quadrapole Mass spectrometer equipped with

commercial ESI source. For ESI-MS experiments, nitrogen was

used as the sheath gas (100 psi) at the drying gas flow of 6

ml/min. An electro spray voltage of 4.5 kV and a capillary

temperature of 3500 °C with dwell time of 500msand mass

range of 180-1000 amu were used. Multiple reaction

monitoring (MRM) was performed for detection. For

.3 Sample preparation and SPE extraction of carboplatin

and oxaliplatin

For the measurement, monitoring, and extraction of

carboplatin and oxaliplatin in water and wastewater samples,

samples were collected from wastewater treatment plant and

water distribution stations in Qom which serve a population of

carboplatin and oxaliplatin, the [M+H] ions were monitored

at m/z 372.0 and m/z 396.0 as the precursor ion and a fragment

at m/z 355 and 96.0 as the product ion, respectively. Results are

presented in Table 2 (3).

200000. All analytical standard solution samples were

vacuum-ﬁltered through 0.7m-pore size ﬁber glass filters,

followed by Cellulose acetate circles (OE 67), 0.45 µm, ester

membranes (Millipore, Billerica, MA).ENV SPE cartridges

2.5 Calibration standards / quality control

were considered in this study. To improve recovery extraction,

Standard stock solutions of carboplatin (4000 µg/mL) and

oxaliplatin (2200 µg/mL) were prepared in methanol. Standard

stock solutions at the concentration level of 1, 5, 10, 25, and

50ng/mL were prepared for analytical standards and quality

control standards of carboplatin and oxaliplatin in methanol and

then injected. These standard solutions were stored at −20ºCin

a freezer until analyzed.

mL of the sample was acidified to pH 2.5 with hydrochloric

acid, and 1 mL of 5 % (w/v) EDTA was added to obtain an

EDTA concentration of 0.1 % (w/v). The samples were loaded

in an ENV SPE cartridge previously conditioned with 5 mL of

methanol and 5 mL of ultrapure water at the flow rate of 5

mL/min. After conditioning, 5 mL of spiked sample was passed

through the cartridges. The sample flasks were then rinsed with

mL of ultrapure water, and the rinsate was also loaded onto

the cartridges. The samples were eluted of the cartridges with

.5 mL of methanol and acetonitrile (50:50 v/v) and transferred

to HPLC sample vials for analysis.

2.6 Method validation

2.6.1 Linearity, precision, and accuracy

The analytical curves were provided by concentration

levels of carboplatin and oxaliplatin which were previously

described. Correlation coefficients > 0.9998 and 0.999 were

obtained for carboplatin and oxaliplatin, respectively. Precision

and accuracy for the concentration of the calibration point (n =

) of below 15% were considered.

Table 1: Characteristics and some environmental fate of carboplatin and Oxaliplatin extracted by a theoretical model (EPI Suite 4.1)

10)

(

Physico-chemical properties

Compound

Structure

Molecular

mass

Boiling

Point

vapor

pressure

Logkw

Solubility

-1.78

Carboplatin

11.7

6.6

772

780

4.59e -0.19

4.8e-0.19

Oxaliplatin

Compound

397

-1.65

7.9

6.1

Table 2: MRM setting for Carboplatin and Oxaliplatin

Retention Time

Segment

ESI

Product Ion

MS/MS transition

372 > 355.0

[

M+H]⁺

Carboplatin

Oxaliplatin

5.3

ESI+

398 > 96.0

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2020, Volume 8, Issue 3, Pages: 1168-1175

.6.2 Recovery

values of Carbolatin and Oxaliplatin in the wastewater effluent

was considered. For the worst case scenario for PEC

calculation, the highest concentration of Carboplatin and

Oxaliplatin in the measured environmental concentration from

influent of municipal waste water (MEC) were considered as

PEC [3]. The reliability of the PEC value that is a key point for

the assessment of environmental risks for aquatic organisms is

based on the ratio of PEC/MEC estimated with an acceptable

consistency for the ratio 0.2–4 range (13, 14, and 15).

The absolute recovery of carboplatin and oxaliplatin was

determined from the labeled five concentrations standard (1.5,

0.25, and 50.00 ng/mL) of carboplatin and oxaliplatin using

SPE cartridge (ENV ) as the extraction sorbent for analytes

with a wide range of polarity characteristics. The results were

expressed using the equation below:

퐂 spiked−퐂 unspiked

Recovery =

퐂 added

.9 Hazard characterization

In this study, recovery of more than 50% was considered as

the required sensitivity.

For hazard characterization, PNEC (predicted no effect

concentration) of Carbolatin and Oxaliplatin for aquatic

animals were taken from literature data were selected 1.22 µg/l

.6.3 Limit of quantification (LOQ) and detection (LOD)

[

16].

For LOD determination, the minimum amount of

compound analyzed in the lc-ms/ms that product a signal-to-

noise (S/N) ratio of 3, was defined and for LOQ, a minimum

amount of compound that produced an S/N ratio of 10, was

considered. The limits of detection (LOD) and the limits of

quantiﬁcation (LOQ) were calculated using the following

equations:

.10 Risk characterization

The risk quotient (RQ), is used to express the risk

characterization it is calculated by combining the results of

PEC with PNEC (RQ =PEC/PNEC). In this method,

interpretation of data on probable ecological risk effects of

contaminated water contain: RQ <1.0 indicates no signiﬁcant

risk; 1.0 ≤ RQ <10 indicates a small potential for adverse

effects; and 10 ≤ RQ< 100 indicates signiﬁcant potential for

adverse effects (13, 14, and 15).

LOD = 3:3σ/ S

LOQ =10σ/S

Results and Discussion

where σ is the standard error of the intercept and S is the slope

of the standard additions’ calibration curve (11, 12).

.1 LC-MS Results

The analysis of carboplatin and oxaliplatin using the

multiple reactions monitoring (mrm) function was highly

selective with no interfering compound and no internal

standards. Because of the polar, hydrophilic nature of platinum

complexes (oxaliplatin and carboplatin) in the lc-column, we

first investigated the use of reversed–phase C18 for analysis

which showed that the analyte was not retained in the column.

Figures 1 to 8illustrate the chromatograms and mass spectra

obtained from concentrations (10and 50.00 ng/mL) in spiked

samples.

.7 Ecological risk assessment

In this study ecological risk assessment is the process for

evaluating and predict how likely it is that the environment may

be impacted as a result of exposure to Carbolatin and

Oxaliplatin from wastewater effluent that is carried in three

stage approach including determination of PEC, hazard

characterization and risk characterization.

.8 Determination of PEC

Determination of predicted environmental concentration

(

PEC) based on the results of the study to measure residual

Figure 1: Chromatogram of Oxaliplatin at 10 ng/ml

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2020, Volume 8, Issue 3, Pages: 1168-1175

Figure 2: Chromatogram of Carboplatin at 10 ng/ml

Figure 3: Mass spectra for oxaliplatin showing tentative peak assignment at 10 ng/ml

Figure 4: Mass spectra for carboplatin showing tentative peak assignment at 1 0ng/ml

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2020, Volume 8, Issue 3, Pages: 1168-1175

Figure 5: Chromatogram of oxaliplatin at 50 ng/ml

Figure 6: Chromatogram of carboplatin at 50 ng/ml

Figure 7: Mass spectra for carboplatin showing tentative peak assignment at 50 ng/ml

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2020, Volume 8, Issue 3, Pages: 1168-1175

Figure 8: Mass spectra for carboplatin showing tentative peak assignment at 50 ng/ml

In the study by Burns et al. on liquid chromatography–mass

spectrometry for the detection of platinum antineoplastic

complexes for an aqueous mixture of carboplatin, results

indicated that the compound were well separated on the lc

and oxaliplatin, respectively(Table4).In the study by Fleury–

Souverain to provide an overview of analytical methods for the

determination of the most commonly used anticancer drugs, for

carboplatin and oxaliplatin in the pharmaceutical formulation

or biological sample lc-ms/ms have been pointed. In

environmental studies (air, surface, and wastewater),

voltammetry and ICP-MS with a LOD of 0.1ng/mL was

reported. Therefore, in this study, considering the determined

LOD, can be successfully approved for measurement of

carboplatin and oxaliplatin in water and waste water samples.

column and molecular ion (MH )formation had occurred. In the

noted study, ion peaks were observed at m/z 372 for carboplatin

and LOD equaled 35 ng/mL. The results of the noted study

were confirmed in the present work.

.2 Quantification and Method Validation

Linearity, recovery, precision, and accuracy were

considered as the criteria for the validation of the analytical

method. In the present work, calibrations were linear in the

investigated range with the correlation coefficient of greater

than 0.99 for both analytes. As explained in the Materials and

Methods section, instrumental LOD and LOQ were calculated

and, on that basis, LOD and LOQ equaled 0.013ng/L and 0.4

ng/L for carboplatin and 0.09 ng/L and 0.027 ng/L for

oxaliplatin, respectively. Results are presented in Table 3. The

precision and accuracy (analysis with spiking samples for this

method were assessed by QC sample in six replicate at five

concentration (1.5.10.25.50) for carboplatin and oxaliplatin

according to recovery (%) via SPE extraction. A recovery of

more than 50% was considered to obtain the required

sensitivity. This method demonstrated that the recovery (%) of

carboplatin and oxaliplatin equaled 0.78 and 0.74, respectively.

RSD% ranged from 6 to 8.9% and 7.5 to 7.9% for carboplatin

3.3 Estimation of ecological risk assessment

For environmental risk estimation with considering PEC

scenario, PNEC criteria of Carboplatin and Oxaliplatin, RQ

was calculated. The results of the RQ value are represented in

Table (5).

In this study RQ in process of estimation of risk were

determined. The RQ calculated for Carboplatin and Oxaliplatin

(0.51 and 0.038 respectively) showed that these platinum

complex drugs could have no significant risk on aquatic

organism. In the study of Daouk and et al about Dynamics of

active pharmaceutical ingredients loads in a Swiss university

hospital wastewaters and prediction of the related

environmental risk for the aquatic ecosystems, And

determining the values of PEC, confirmed the results of the

current study (16).

Table 3: Calibration curves (n = 6) for Carboplatin and Oxaliplatin

Analyte

Spiking Concentration (ng/ml)

Concentration measured (mean ± SD ng/ml)

Accuracy (%)

102

1.02 ± 0.19

5.14 ± 0.62

9.96 ± 0.17

25.1 ± 0.04

50.4 ± 0.06

1.05 ± 0.08

4.97 ± 0.14

10.22 ± 0.21

25.3 ± 0.06

50.3 ± 03

102.8

100.4

100.8

105

Carboplatin

100.6

102.2

101.2

100.6

Oxaliplatin

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2020, Volume 8, Issue 3, Pages: 1168-1175

Table 4: Precision and quality control parameters (analysis with spiking samples and recovery extraction %) for Carboplatin and

Oxaliplatin

Analyte

Spiking Concentration (ng/ml)

Concentration measured in SPE ( mean ± SD ng/ml)

0.78 ± 0.06

4.09 ± 0.064

7.89 ± 0.078

19.86 ± 0.069

40.4 ± 0.089

0.74 ± 0.075

4.04 ± 0.077

7.81 ±0.099

19.76 ± 0.088

40.3 ± 079

Carboplatin

Oxaliplatin

Table 5: Results of ecological risk assessment of Carboplatin and Oxaliplatin in resource water

Environmental Concentration (µg/L)

Elements

PEC/MEC(mean)

PNEC

µg/L)

Mean

0.38

Typical range

0.63 - < LOQ

0.048 - < LOQ

PEC

0.63

(

Carboplatin

Oxaliplatin

1.65

0.43

1.22

0.51

0.039

0.048

0.038

Results in this study were reasonable to those Cunningham,

et al related to human health risk assessment from the presence

of human pharmaceuticals in the aquatic environment that the

values of drug residues and comparison with PEC

environmental concentration report the risk of exposure to

negligible (17). A survey on anticancer drugs in surface waters

and occurrence and environmental significance of cytotoxic by

Besse et al the PEC values for, platinum drug combinations

including Carboplatin and Oxaliplatin was 0.52 ng/L and 0.72

ng/L respectively, which approved the results of present study

Authors’ contribution

All authors of this study have a complete contribution for

data collection, data analyses and manuscript writing

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