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Speciation of cisplatin in environmental water samples by Hydrophilic interaction liquid chromatography coupled to inductively coupled plasma mass spectrometry Janja Vidmar, Anže Martinčič, Radmila Milačič, Janez Ščančar www.elsevier.com/locate/talanta
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Received date: 25 October 2014 Revised date: 2 February 2015 Accepted date: 3 February 2015 Cite this article as: Janja Vidmar, Anže Martinčič, Radmila Milačič, Janez Ščančar, Speciation of cisplatin in environmental water samples by Hydrophilic interaction liquid chromatography coupled to inductively coupled plasma mass spectrometry, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.02.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Speciation of cisplatin in environmental water samples by hydrophilic interaction liquid chromatography coupled to inductively coupled plasma mass spectrometry
Janja Vidmara,b, Anže Martinčiča,b, Radmila Milačiča,b, Janez Ščančara,b* a
Department of Environmental Sciences, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana,
Slovenia b
Jožef Stefan International Postgraduate School, Jamova 39, 1000 Ljubljana, Slovenia
*Corresponding author at: Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia. Tel: +386 1 477 3846; fax: +386 1 251 9385. E-mail address: [email protected] (J. Ščančar)
Abstract Cisplatin is still widely used for treatment of numerous types of tumours. Different speciation methods have been applied to study behaviour of the intact drug and its individual biotransformation species in various clinical samples. These methods are mainly based on electrophoresis, size exclusion (SEC) or ion chromatography (IC) techniques coupled to inductively coupled plasma mass spectrometry (ICP-MS). Hydrophilic interaction liquid chromatography (HILIC), which is a common technique for separation of polar substances, was rarely applied for separation of cisplatin and its hydrolysed metabolites. There is also a lack of information available on the occurrence of cisplatin and its hydrolysed complexes in the environmental waters. In the present study the concentrations of Pt were determined in hospital wastewaters by ICP-MS. A procedure for separation of cisplatin and its aqueous hydrolysed complexes by the use of HILIC column was optimized. Quantification of separated Pt species was performed by isotope dilution (ID)−ICP-MS procedure. Low limits of detection (LODs) and quantification (LOQs) were obtained for cisplatin and its hydrolysed
1
complexes ranging from 0.0273 to 0.1726 ng Pt/mL and from 0.0909 to 0.5753 ng Pt/mL, respectively. Good repeatability of the procedure with relative standard deviation (RSD) lower than ± 2.3 % was obtained. The column recoveries, which ranged from 95 to 101 %, indicated that the procedure developed enabled quantitative speciation analysis of aqueous cisplatin complexes. The ZIC-HILIC−ID-ICP-MS procedure was successfully applied in speciation of cisplatin in spiked hospital wastewater samples.
Highlights •
We developed ZIC-HILIC−ICP-MS procedure for speciation of cisplatin.
•
Hydrolysis of cisplatin was studied in water and solutions containing chloride ions.
•
A method developed is repeatable, sensitive and selective.
•
LODs for cisplatin and its hydrolysed complexes ranged from 0.03 to 0.17▒ng▒Pt/mL.
•
The procedure enabled speciation of cisplatin in spiked hospital wastewaters.
Keywords Cisplatin; Speciation analysis; Hydrophilic interaction liquid chromatography; Isotope dilution inductively coupled plasma mass spectrometry; Environmental water samples
1.
Introduction Cancer is a disease characterised by uncontrolled growth, increased division, decreased
cell death and many other abnormalities of cancer cells. For its treatment, various metal-based chemotherapeutics are used [1,2]. Cisplatin (cis-[PtCl2(NH3)2]), the first chemotherapeutic which was introduced into clinical trials in the late 1970s [3], is nowadays still widely used for treatment of numerous types of tumours (lung, bladder, gastric, head and neck, cervical, testicular and ovarian) [4-6]. For better understanding of anticancer therapy with cisplatin the
2
behaviour of the intact drug and its individual biotransformation species in clinical samples has been intensively investigated by applying speciation analysis methods [7,8,3]. To study interaction of cisplatin with serum proteins Szpunar et al. [9] applied size-exclusion chromatography (SEC) coupled to inductively coupled plasma mass spectrometry (ICP-MS). Huang et al. [10] determined cisplatin and its hydrolytic metabolite in human serum by capillary electrophoresis techniques coupled to ICP-MS. Esteban-Fernández et al. [11] performed speciation of Pt in kidney and inner ear tissue of rats treated with Pt-based chemotherapeutics. Pt binding to proteins was followed by two-dimensional (2D) liquid chromatography (SEC plus fast protein liquid chromatography column (FPLC)) coupled to ICP-MS. Later 2D chromatography, comprised of SEC in combination with ion-exchange chromatography, using strong anion-exchange Mono Q FPLC column or monolithic convective interaction media (CIM) weak anion-exchange diethylamino (DEAE) column, was applied. By hyphenation to ICP-MS a study was performed on the kinetics of binding of cisplatin to serum proteins. The same chromatographic set-up was used to investigate the distribution of Pt species in serum of cancer patients receiving cisplatin [12]. Conjoint liquid chromatography (CLC) on short monolithic disks (affinity CIM Protein G disk and anionexchange CIM DEAE) coupled to ICP-MS was then used for simultaneous 2D separation of ionic forms of Pt-based chemotherapeutics from the portions bound to different serum proteins [13]. In this study, the quantification of separated Pt species was performed by postcolumn isotope dilution (ID)-ICP-MS technique. Since the formation of deoxyribonucleic acid (DNA) adducts with cisplatin is crucial pharmacokinetic parameter, which must be optimized in cancer therapy based on Pt drugs, Hann et al. [14] examined the time-resolved interaction of cisplatin with guanosine monophosphate (GMP) by high performance ion chromatography (HPIC)-ICP-sector field (SF)MS. Pt/P ratio was determined by simultaneously measuring 31P and 195Pt.
3
It is well know that in physiological solutions, cisplatin undergoes hydrolysis. The hydrolysed forms of cisplatin react in a different extent with blood components and cell biomolecules [15,3]. The formation of cisplatin hydrolysed complexes is time-dependent and also depends on the concentration of chloride ion [16]. For investigation of cisplatin and its hydrolysed complexes, hydrophilic interaction liquid chromatography (HILIC) coupled to ICP-MS was found to be a promising technique [17,18]. However, HILIC, which is a routine technique for separation of polar drugs [19,20], was rarely used for separation of cisplatin and its hydrolysed complexes [17,18,21]. When HILIC is applied, organic solvents which are used in the separation procedure are not favourable for ICP-MS detection, due to carbon deposition on the sampler and skimmer cones. Hemström et al. [18] reported the choice of possible organic solvents for HILIC separation of cisplatin and highlighted that commonly used acetonitrile (ACN), reacts with cisplatin and upset speciation equilibria. To avoid the latter problems the use of dimethylformamide (DMF) or 20% 1-propanol in 25 mM ammonium formate buffer (pH 6.5) was recommended. Nygren et al. [17] applied HILIC-ICP-MS procedure to study the distribution of Pt species in whole cell lysate from in-vitro grown T289 malignant melanoma cells exposed to cisplatin. Separation of cisplatin and monoaquacisplatin was achieved by using DMF as an eluent. The LODs for cisplatin and monoaquacisplatin were 0.2 ng Pt/mL. Other group of researchers [21] used ACN to separate cisplatin, oxaliplatin and carboplatin in spiked human plasma samples by HILIC-ICP-MS. For cisplatin, the LOD was found to be 9.8 ng Pt/mL. Other
techniques,
like
reversed-phase
ion-pairing
chromatography
were
also
implemented for speciation of cisplatin and enabled separation of neutral complexes from 1+ and 2+ charged Pt species. The LODs obtained for cisplatin were 1 ng Pt/mL [22] and 19.5 ng Pt/mL [23]. ESI-MS-HPLC was an appropriate technique for identification of Pt-based
4
anticancer drugs and their hydrolysis products when Pt concentrations were in µg/mL range [24,25]. Due to the widespread use of Pt-based chemotherapeutics, Pt metabolites that are excreted with urine end-up in hospital or municipal wastewaters. Despite the potential emerging of cisplatin in the environmental waters there is a lack of literature data on occurrence of cisplatin and its hydrolysed complexes in surface waters. Therefore, the aim of our work was first to determine the content of Pt in hospital wastewaters and in wastewaters from inflow and outflow of sewage treatment plants by ICP-MS. The second aim was to optimize the HILIC procedure for separation of cisplatin and its hydrolysed complexes and to perform the speciation analysis of cisplatin in environmental waters by HILIC coupled to ICP-MS, using post-column ID-ICP-MS technique for the quantification of separated cisplatin species.
2. Materials and methods 2.1.Instrumentation Total Pt concentrations were determined by inductively coupled plasma mass spectrometer (ICP-MS), model 7700x, from Agilent Technologies (Tokyo, Japan). HPLC separations were performed on an Agilent (Tokyo, Japan) series 1200 HPLC system with a quaternary pump equipped with a sample injection valve, Rheodyne model 7725i (Cotati, Ca, USA) fitted with a 5 µL injection loop. The column made from poly(etherether ketone) (PEEK) with bonded zwitterionic silica-based HILIC stationary phase (ZIC-HILIC PEEK HPLC column 2.1 ID, Merck, Darmstadt Germany) with a 20 x 2.1 mm guard column was used for separation of Pt species. The outlet of the chromatographic column was directly connected to the Micromist nebuliser and Scott-type spray chamber of the ICP-MS instrument. Quantification of separated Pt species was performed by post-column isotope 5
dilution ICP-MS. ICP-MS operating parameters for determination of total Pt concentrations and Pt species after the chromatographic separation are given in Table 1.
Place Table 1 about here
2.2.Reagents and materials Ultrapure water (18.2 MΩ cm) was obtained from a Direct-Q 5 Ultrapure water system (Millipore Watertown, A, USA). All chemicals were of analytical reagent grade. Ammonium formate, n-propanol, formic acid and sodium chloride were obtained from Merck (Darmstadt, Germany). Eluent A consisted of aqueous solution of 95 % n-propanol + 5 % of 20 mM ammonium formate (pH 4). Eluent B was composed of aqueous solution of 50 % n-propanol + 50 % 20 mM ammonium formate (pH 4), while buffer C was aqueous solution of 100 mM ammonium formate (pH 4). Cisplatin was obtained from Medoc (Hamburg, Germany). Merck stock Pt solution (1000 µg Pt/mL in 8 % HCl) was diluted daily with water for the preparation of fresh calibration standard solutions that were used for the determination of the total concentration of Pt in the samples analysed. Platinum enriched in
194
Pt isotope (Pt metallic
plate, 15 mg) obtained from Oak Ridge National Laboratory (Oak Ridge, TN, USA) was dissolved in 1 mL of aqua regia and diluted to 10 mL with an appropriate amount of HCl, so that the final concentration of HCl was 8 %. The declared composition of the enriched Pt plate was 96.45 ± 0.05 %, 2.46 ± 0.05 %, 0.87 ± 0.02 %, 0.18 ± 0.01 %, 0.03 ± 0.00 % and 0.01 ± 0.00 % for the isotopes 194, 195, 196, 198, 192 and 190, respectively. Sartorius (Goetingen, Germany) 0.45 µm cellulose nitrate membrane filters of 25 mm diameter were used in the filtration procedure.
6
2.3.
Sample preparation Samples of wastewaters were collected in Slovenian and Spanish oncological hospitals
and in inflows and outflows of the sewage treatment plants. After sampling, samples were filtered through 0.45 µm membrane filters. For the determination of total Pt, samples were acidified (0.1 mL of HNO3 per 100 mL of sample), while non-acidified samples were used for speciation analysis. Samples were kept frozen until analysis. It was experimentally found that concentrations of total Pt in filtered and unfiltered samples did not vary more than ± 5 %. So, determination of total Pt concentrations and Pt speciation was performed in filtered samples.
2.4.
Quantification of separated Pt species by post-column isotope dilution ICP-MS Quantification of separated Pt species was performed by the post-column ID-ICP-MS
technique. Isotopically enriched
194
Pt was added continuously by a peristaltic pump via a T-
piece after the separation of Pt species. To calculate the content of the eluted Pt species, the mass flow of Pt was plotted versus time throughout the whole chromatographic run. Calculations were performed using equations derived for species unspecific post-column IDICP-MS analysis [26-28]. If not stated otherwise, all the experiments were performed in duplicate.
3. Results and discussion 3.1 Determination of total Pt concentrations in hospital wastewater samples Total Pt concentrations in Slovenian and Spanish wastewater samples from oncology hospitals and environmental wastewater samples from inflows and outflows of sewage treatment plants were determined by ICP-MS. Results are presented in Table 2.
7
Insert Table 2 about here
As can be seen from data of Table 2, the determined Pt concentrations were the highest in oncological hospital wastewaters, and the lowest in the effluent from sewage treatment plants. These concentrations are for one order of magnitude higher in the Slovenian than in Spanish oncological hospital wastewaters. Nevertheless, the Pt concentrations are low, ranging from 0.013 to 0.35 ng/mL in Slovenian and from 0.006 to 0.014 ng/mL in Spanish wastewaters. ICP-MS technique was sensitive enough for determination of such low Pt concentrations. The limit of detection (LOD) calculated on a 3 s basis (a value of three standard deviation of the blank) for the determination of total Pt concentration was found to be 0.005 ng/mL.
3.2. Speciation of cisplatin by the ZIC-HILIC−ICP-MS procedure: feasibility study 3.2.1. Optimization of the separation procedure In order to investigate the ability of the ZIC-HILIC−ICP-MS procedure for speciation of cisplatin in aqueous samples, the separation procedure was first optimized. Although being commonly applied as eluent for separation of polar substances [19], ACN was not appropriate. It forms complexes with cisplatin and also produces high carbon load on the ICPMS sampler and skimmer cones, even when oxygen is added to the nebulizer gas flow [18]. To preserve the integrity of Pt species in aqueous solutions and to reduce the carbon load in ICP-MS, the use of ACN was omitted in the present work. The best compromise between carbon deposition and separation efficiency was achieved when n-propanol was used as an eluent and the ICP parameters carefully optimized. Moreover, ICP-MS was equipped with the kit that allows organic solvent to be used for separation. Oxygen was introduced into the plasma and the spray chamber chilled to -5 ˚C (Table 1). For avoiding the drawbacks related to the use of ACN, Hemström with co-workers [18] proposed isocratic elution with n-
8
propanol mixed with ammonium formate buffer (20 % n-propanol + 20 % 25 mM ammonium formate) for separation of cisplatin on ZIC-HILIC column. Similar eluents, but using higher concentrations of aqueous solutions of n-propanol buffered with ammonium formate, were applied in the present study. It was experimentally found that separation of cisplatin and its hydrolysed products was the most effective when gradient elution from 100 % of eluent A (aqueous solution of 95 % n-propanol + 5 % of 20 mM ammonium formate, pH 4) to 100 % of eluent B (aqueous solution of 50 % n-propanol + 50 % 20 mM ammonium formate, pH 4) was applied in 20 min, followed by isocratic elution with eluent B for next 4 min. In the following 11 min the column was regenerated with aqueous solution of 100 mM ammonium formate (eluent C) and equilibrated with eluent A. Peaks were well separated when 5 µL of the sample was injected onto the column and the chromatographic procedure performed at a flow rate of 0.3 mL/min. Chromatographic program for the separation of cisplatin and its hydrolysed complexes is presented in Table 3.
Insert Table 3 about here
To obtain repeatable and reproducible chromatographic separations and low LODs for separated cisplatin complexes, efficient regeneration and equilibration of the ZIC-HILIC column was necessary. Therefore, after approximately 20 separations, the column was cleaned at a flow rate of 0.4 mL/min. First, rinsing with MilliQ water was applied for 40 min, followed by elution with 0.5 M NaCl for the next 40 min. The column was again rinsed with MilliQ water for 40 min. After that 5µL of formic acid (100 %) was injected onto the column and the same chromatographic run as for the separation of cisplatin complexes was performed. This step was repeated five times and the column was ready for further use.
9
3.2.2. The behaviour of cisplatin and its hydrolysed complexes on the ZIC-HILIC column In aqueous solutions cisplatin [PtCl2(NH3)2] undergoes fast hydrolysis reactions, yielding monoaquacisplatin [PtCl(OH2)(NH3)2]+ as the prevailing species, which is in equilibrium with minor concentrations of diaquacisplatin [Pt(OH2)2(NH3)2]2+ and monoaquahydroxycisplatin [Pt(OH2)(OH)(NH3)2]+ species. In solutions containing a significant concentration of chloride ion, cisplatin species prevails and is in equilibrium with small concentrations of monoaquacisplatin and monohydroxycisplatin [PtCl(OH)(NH3)2] [15,3]. There are no standard reference materials with hydrolysed forms of cisplatin available. In order to identify cisplatin and its hydrolysed complexes, series of experiments were performed by the dilution of the stock solution of cisplatin (2 µg/mL) to a concentration of 10 ng Pt/mL. Samples were diluted in water or in solutions containing various amounts of chloride ions and analysed in different time intervals over a period of 48 hours by the ZICHILIC−ID-ICP-MS procedure. For obtaining stable solution of cisplatin [16] sample containing 10 ng Pt/mL was prepared from the stock cisplatin solution in 0.9 % NaCl, and injected onto the column immediately after the dilution. To identify monoaquacisplatin, which is the first hydrolysis product of cisplatin, the same solution was also injected 15 min after the dilution. The chromatograms of these separations are presented in Fig. 1.
Insert Fig. 1. about here
As evident from Fig. 1. (left chromatogram), immediately after preparation cisplatin elutes as a single species at retention time 19.7 min. 15 min after the sample preparation (Fig. 1., right chromatogram)
cisplatin,
as
the
prevailing
10
species,
and
its
hydrolysed
form
monoaquacisplatin are present. The latter species, which represents 10 % of the injected cisplatin, is eluted at retention time 16.4 min. To verify the mass balance over the chromatographic step, the column recovery, which is the ratio between the concentration of cisplatin species eluted from the column and the concentration of cisplatin injected, was calculated. The concentrations of Pt species eluted were determined by the post-column ID-ICP-MS. The column recoveries obtained for separation of cisplatin (Fig. 1., left chromatogram) and cisplatin in the presence of monoaquacisplatin (Fig. 1., right chromatogram) were 96 % and 99 % respectively, indicating on quantitative elution of separated Pt species. Once the retention times of cisplatin and monoaquacisplatin were identified, the hydrolysis of cisplatin in water and in 0.3 % HCl was studied 15 min and 48 h after the preparation of samples. Results are presented in Fig. 2.
Insert Fig. 2. about here
Data from Fig. 2. (upper row) indicate rapid hydrolysis of cisplatin in water. After 15 min 52.5 % of cisplatin was hydrolysed in monoaquacisplatin. The column recovery was found to be 101 %. As time elapsed, hydrolysis of cisplatin after 48 hours resulted in appearance of two more hydrolysis products diaquacisplatin (species eluted at retention time of 13.7 min) and monoaquahydroxycispaltin (species eluted at retention time 15.1 min), which represented 4.0 and 4.1 % of cisplatin complexes in solution, respectively. The column recovery for the cisplatin complexes separated 48 hours after the preparation of aqueous solution was 93 %. Data from Fig. 2. (lower row) demonstrate that hydrolysis of cisplatin in 0.3 % HCl is less pronounced than in aqueous solutions, which is in accordance with findings of ElKhateeb et al. [15,16,3]. After 15 min only 16 % of cisplatin is hydrolysed into
11
monoaquacisplatin. The column recovery was 100 %. 48 hours after the sample preparation, as a result of progressive hydrolysis, monohydroxycisplatin species (eluted at retention time of 17.8 min) also appeared, being in equilibrium with cisplatin and monoaquacisplatin. Cisplatin represented 84.8 %, monoaquacisplatin 6.4 % and monohydroxycisplatin 9.1 % of initial cisplatin concentration. The column recovery was found to be 100 %. Data from Fig. 2. additionally revealed that the ZIC-HILIC−ID-ICP-MS procedure enables separation and the quantification of cisplatin and its hydrolysed complexes by IDICP-MS in aqueous solutions and solutions containing chloride ions.
3.2.3. The influence of suspended particulate matter and humic substances on the speciation of cisplatin in water samples Environmental water samples frequently contain suspended particulate matter (SPM) and humic acids (HA). To find out how the presence of SPM and humic acids influence the speciation of cisplatin by the ZIC-HILIC−ICP-MS procedure, environmentally relevant concentrations 35 mg/L of SPM or 5 mg/L of HA were added to the river water sample (Pt concentration below 0.005 ng/mL) and samples shaken on the horizontal shaker for 24 hours. After that, samples were spiked with cisplatin (10 ng Pt/mL), left for 24 hours, filtered and analysed. The results are shown in Fig. 3.
Insert Fig. 3. about here
As it is evident from Fig. 3., 24 hours after the spiking, cisplatin and its hydrolysis product monoaquacisplatin species are present. However, low column recoveries about only 25 % were obtained, indicating that cisplatin complexes are in a great extent adsorbed on SPM and/or HA. Therefore, the speciation procedure enables quantitative determination of
12
dissolved concentrations of cisplatin and its hydrolysed products in environmental water samples. The latter data are in agreement with findings of Lentz et al. [29] who demonstrated that Pt species excreted via urine of oncology patients showed high affinity to suspended solids of hospital wastewaters.
3.2.4. Figures of merits of the ZIC-HILIC−ID-ICP-MS procedure Under optimised chromatographic conditions (Table 3) and optimised ICP-MS operating parameters (Table 1), the limits of detection (LODs) and limits of quantification (LOQs) for the determination of Pt species were calculated as the concentration that provides a signal (peak area) equal to 3s and 10s of the blank sample in the chromatogram, respectively. To calculate the LODs and LOQs, 6 blank samples (MilliQ water) were injected. The LODs and LOQs for separated Pt species are presented in Table 4.
Insert Table 4 about here
As evident, LODs and LOQs were different for individual Pt species. The lowest LOD and LOQ were found for monoaquahydroxycisplatin (0.0273 and 0.0909 ng Pt/mL, respectively), and the highest for cisplatin (0.1726 and 0.5753 ng Pt/mL, respectively). Similar LOD for separation of cisplatin (0.2 Pt/mL) on the ZIC-HILIC column with ICP-MS detection was reported by Nygren et al. [17], while slightly higher LOD for separation of cisplatin (0.5 Pt/mL) was obtained by the use of adsorption chromatography on Hypersil-Keystone Hypercrab column hyphenated to ICP-MS [16]. The linearity of measurement for each particular cisplatin species was obtained from LOQ to 100 ng Pt/mL.
13
In order to evaluate the repeatability of the procedure developed, six consecutive speciation analyses of standard solution of cisplatin (10 ng Pt/mL in 0.3 % HCl), injected 15 min after preparation from stock cisplatin solution, were performed. The chromatograms are presented in Fig. 4.
Insert Fig. 4. about here
As evident from data of Fig. 4., good repeatability of measurement was obtained. For each of individual Pt species eluted (cisplatin and monoaquacisplatin), the relative standard deviation (RSD) between concentrations of Pt species determined by post-column ID-ICP-MS was found to be lower than ± 2.3 %, while the column recoveries ranged from 95 to 101%.
3.2.5. Speciation of Pt in hospital wastewater samples spiked with cisplatin The applicability of the procedure developed was verified by the analysis of hospital wastewater samples. In the Slovenian hospital wastewater sample (sample A) the total Pt concentration was 0.352 ± 0.008 ng Pt/mL, while in Spanish hospital wastewater sample (sample B) 0.0144 ± 0.0016 ng Pt/mL. These Pt concentrations were lower than LOQ for cisplatin, and hence were too low to perform speciation analysis. Therefore, samples were spiked with cisplatin (10 ng Pt/mL) and analysis performed 15 min and 48 hours after the spiking. The results of speciation analysis are shown in Fig. 5.
Insert Fig. 5. about here
From chromatograms of Fig. 5. it can be seen that 15 min after spiking, cisplatin and monoaquacisplatin are present in hospital wastewater samples A and B. 48 hours after the
14
spiking, in sample A cisplatin and monoaquacisplatin are further hydrolysed, resulting in the appearance
of
diaquacisplatin
species,
while
in
sample
B
only
cisplatin
and
monoaquacisplatin are present. The column recoveries for spiked samples A and B ranged between 96 and 104 %. There are only few reports on the determination of cancerostatic Pt compounds in real environmental water samples. Lenz and co-workers reported speciation of cisplatin in wastewaters from Vienna oncologic hospital [29]. Authors used RP-HPLC based on pentafluorophany terminated stationary phase for separation of Pt compounds and ICP-MS for detection of separated Pt species. The concentrations of Pt in samples from the hospital wastewater influent ranged from 3 to 250 ng Pt/mL, and from effluent from 2 to 150 ng Pt/mL, and were much higher than those from the hospital wastewaters analysed in the present study. The data of the present investigation revealed that the ZIC-HILIC−ID-ICP-MS procedure developed can be reliably applied in speciation of soluble concentrations of cisplatin and its hydrolysed products in hospital and environmental water samples, when the total concentration of Pt is higher than the sum of concentrations for LOQs for the individual Pt species.
4. Conclusions The selective and highly sensitive ZIC-HILIC−ICP-MS procedure developed enables simultaneous determination of cisplatin and its hydrolysed products: monoaquacisplatin, monoaquahydroxyciplatin, diaquacisplatin and monohydroxycisplatin in aqueous solutions containing different chloride concentrations at the low ng/mL concentration range. The use of post-column ID-ICP-MS enabled the determination of separated Pt species. LODs for individual cisplatin complexes ranged from 0.03 to 0.2 ng Pt/mL and LOQs from 0.09 to 0.58 ng Pt/mL. The procedure is reliable (RSD lower than ± 2.3 %) and allows quantitative 15
speciation analysis of aqueous cisplatin complexes (column recoveries from 95 to 101 %). It also enables determination of cisplatin and its metabolites in environmental waters at extremely low Pt concentrations, when the total concentration of Pt is higher than the sum of concentrations for LOQs for the individual Pt species. Due to increasing growth of oncology patients, it can be expected that concentrations of Pt-based chemotherapeutics will increase in environmental waters. So, speciation of Pt in environmental waters is becoming an issue of great concern. Low total Pt concentrations determined in Slovenian and Spanish hospital wastewaters (0.35 ng/mL and 0.014 ng/mL, respectively) facilitated speciation analysis only in spiked hospital wastewaters, demonstrating the presence of cisplatin and its hydrolysed complexes monoaquacisplatin and diaquacisplatin. Analysis of environmental water samples, to which the relevant concentrations of SPM or HA were added, and were spiked with cisplatin, revealed that about 75 % of Pt species were adsorbed by SPM or HA.
Acknowledgements This work was supported by the 7th FW EU Project: Fate and effects of cytostatic pharmaceuticals in the environment and identification of biomarkers for an improved risk assessment on environmental exposure (CYTOTHREATH), Grant agreement No. 265264 and Ministry of Education, Science and Sport of the Republic of Slovenia (Programme group P10143).
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[10] Z. Huang, A.R. Timerbaev, B.K. Keppler, T. Hirokawa, J. Chromatogr. A 1106 (2006) 75-79. [11] D. Esteban-Fernández, M.M. Gómez-Gómez, B. Cañas, J.M. Verdaguer, R. Ramirez, M.A. Palacios, Talanta 72 (2007) 768-773. [12] A. Martinčič, R. Milačič, M. Čemažar, G. Serša, J. Ščančar, Anal. Methods 4 (2012) 780-790. [13] A. Martinčič, M. Čemažar, G. Serša, V. Kovač, R. Milačič, J. Ščančar, Talanta 116 (2013) 141-148. [14] S. Hann, A. Zenker, M. Galanski, T.L. Bereuter, G. Stingeder, B.K. Keppler, Fresenius J. Anal. Chem. 370 (2001) 581-586. [15] M. El-Khateeb, T.G. Appleton, L.R. Gahan, B.G. Charles, S.J. Berners-Price, A.-M. Bolton, J. Inorg. Biochem. 77 (1999) 13-21. [16] S. Hann, G. Koellensperger, Zs. Stefánka, G. Stingeder, M. Fürhacker, W. Buchberger, R.M. Mader, J. Anal. At. Spectrom. 18 (2003) 1391-1395.
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[17] Y. Nygren, P. Hemström, C. Åstot, P. Naredi, E. Björn, J. Anal. At. Spectrom. 23 (2008) 948-954. [18] P. Hemström, Y. Nygren, E. Björn, K. Irgum, J. Sep. Sci. 31 (2008) 599-603. [19] P. Jandera, Anal. Chim. Acta 692 (2011) 1-25. [20] M. Staňková, P. Jandera, V. Škeříková, J. Zrban, J. Chromatogr. A 1289 (2013) 47-57. [21] T. Falta, G. Koellensperger, A. Standler, W. Buchberger, R.M. Mader, S. Hann, J. Anal. At. Spectrom. 24 (2009) 1336-1342. [22] Z. Zhao, K. Tepperman, J.G. Dorsey, R.C. Elder, J. Chromatogr. B. 615 (1993) 83-89. [23] D.N. Bell, J.J. Liu, M-D. Tingle, M.J. McKeage, J. J. Chromatogr. B. 837 (2006) 29-34. [24] M. Chui, Z. Mester, Rapid Commun. Mass Spectrom. 17 (2003) 1517-1527. [25] C. Brauckmann, C.A. Wehe, M. Kieshauer, C. Lanvers-Kaminsky, M. Sperling, U. Karst, Anal. Bioanal. Chem. 405 (2013) 1855-1864. [26] K.G. Heumann, S.M. Gallus, G. Rädingler, J. Vogl, Spectrochim. Acta Part B 53 (1998) 273-287. [27] P. Rodrígez-González, J.M. Marchante-Gayón, J.I. García Alonso, A. Sanz-Medel, Spectrochim. Acta Part B 60 (2005) 151-207. [28] United States Environmental Protection Agency (USEPA), Method 6800, Elemental and Speciated Isotope Dilution Mass Spectrometry, Test Methods for Evaluating Solid Waste, Physical/Chemical Methods, SW 846, Update IVA, US Government Printing Office (GPO), Washington DC, 2007. [29] K. Lenz, G. Koellensperger, S. Hann, N. Weissenbacher, S.N. Mahnik, M. Guerhacker, Chemosphere 69 (2007) 1765-1774.
18
Research highlights Captions to Figures
Fig. 1 Separation of solutions of cisplatin (10 ng Pt/mL) on ZIC-HILIC column followed by ICP-MS detection. Sample was prepared from the stock solution by dilution in 0.9 % NaCl and injected onto the column immediately and 15 min after preparation. Pt mass flow is based on measurement of isotope ratios m/z 194 and 195. Fig. 2 Separation of solutions of cisplatin (10 ng Pt/mL) on ZIC-HILIC column followed by ICP-MS detection. Samples were prepared from the stock solution by dilution in water and in 0.3 % HCl and injected onto the column 15 min or 48 h after preparation. Pt mass flow is based on measurement of isotope ratios m/z 194 and 195. Fig. 3 Chromatograms of water samples containing 35 mg/L of SPM or 5 mg/L of HA, spiked with cisplatin (10 ng Pt/mL) on ZIC-HILIC column followed by ICP-MS detection. Samples were injected onto the column 24 h after spiking. Pt mass flow is based on measurement of isotope ratios m/z 194 and 195. Fig. 4 Six consecutive speciation analyses of standard solution of cisplatin (10 ng Pt/mL in 0.3 % HCl) on ZIC-HILIC column followed by ICP-MS detection. Samples were injected onto the column 15 min after their preparation. Pt mass flow is based on the measurement of isotope ratios m/z 194 and 195. Fig. 5 Separation of Pt species in hospital wastewater samples (A: Slovenian Hospital, B: Spanish Hospital) spiked with cisplatin (10 ng Pt/mL) on ZIC-HILIC column followed by ICP-MS detection, 15 min and 48 h after spiking. Pt mass flow is based on the measurement of isotope ratios m/z 194 and 195.
19
Graphical abstract
Mass flow Pt 195 (ng/min)
0,1
monoaquacisplatin
0,08 cisplatin
0,06 0,04 monoaquahydroxycisplatin diaquacisplatin
0,02 0 0
5
10 15 Time (min)
Cisplatin in 0.9 % NaCl immediately after preparation 0,12
Mass flow Pt 195 (ng/min)
cisplatin 0,08
0,04
0 0
5
10 15 Time (min)
20
25
Cisplatin in 0.9 % NaCl 15 min after preparation
0,12
Mass flow Pt 195 (ng/min)
20
cisplatin
0,08
0,04
monoaquacisplatin
0
25
0
Fig. 1.
20
5
10 15 Time (min)
20
25
Cisplatin in water 48 h after the dilution
Cisplatin in water 15 min after the dilution 0,1
monoaquacisplatin cisplatin
0,08
Mass flow Pt 195 (ng/min)
Mass flow Pt 195 (ng/min)
0,1
0,06 0,04 0,02
monoaquacisplatin
0,08 0,06
cisplatin
0,04 monoaquahydroxycisplatin diaquacisplatin
0,02 0
0 0
5
10 15 Time (min)
20
0
25
Cisplatin in 0.3 % HCl 15 min after the dilution
5
0,1
25
20
25
cisplatin
Mass flow Pt 195 (ng/min)
cisplatin
Mass flow Pt 195 (ng/min)
20
Cisplatin in 0.3 % HCl 48 h after the dilution
0,1 0,08 0,06 monoaquacisplatin
0,04
10 15 Time (min)
0,02
0,08 0,06 monohydroxycisplatin
0,04
monoaquacisplatin
0,02 0
0 0
5
10 15 Time (min)
20
0
25
Fig. 2.
21
5
10 15 Time (min)
Spiked water sample containing 35 mg L-1 of SPM
Spiked water sample containing 5 mg L-1 of HA 0,05
Mass flow Pt 195 (ng/min)
Mass flow Pt 195 (ng/min)
0,05 0,04 0,03 monoaquacisplatin
0,02
cisplatin
0,01 0
0,04 0,03 monoaquacisplatin
0,02
cisplatin
0,01 0
0
5
10 15 Time (min)
20
25
0
Fig. 3.
22
5
10 15 Time (min)
20
25
0,04
0,00
0
5
10 15 Time (min)
20
Fig. 4.
23
25
(ng/min)
195
0,08
Mass flow Pt
0,12
Cisplatin in sample A 48 h after spiking
Cisplatin in sample A 15 min after spiking monoaquacisplatin
0,08
0,1 monoaquacisplatin
cisplatin
Mass flow Pt 195 (ng/min)
Mass flow Pt 195 (ng/min)
0,1
0,06 0,04 diaquacisplatin
0,02 0 0
5
10 15 Time (min)
20
0,08
cisplatin
0,06 0,04 diaquacisplatin
0,02 0
25
0
Cisplatin in sample B 15 min after spiking 0,1
cisplatin
0,08
10 15 Time (min)
20
25
Cisplatin in sample B 48 h after spiking
Mass flow Pt195 (ng/min)
Mass flow Pt195 (ng/min)
0,1
5
monoaquacisplatin
0,06 0,04 0,02
monoaquacisplatin cisplatin
0,08 0,06 0,04 0,02 0
0 0
5
10 15 Time (min)
20
0
25
Fig. 5.
24
5
10 15 Time (min)
20
25
Table 1 Optimised ICP-MS operating parameters for the determination of the total Pt concentrations and quantification of Pt species after their separation on ZIC-HILIC column.
ICP-MS parameters Forward power Plasma gas flow (Ar) Carrier gas flow (Ar) Dilution gas flow (Ar) Optional gas flow (20 % v/v O2 in Ar) Nebuliser type Isotopes monitored Integration time Total acquisition time Spray chamber temperature
Total Pt concentrations 1550 W 15.0 L/min 1.10 L/min / / Micromist 193 Ir, 194Pt, 195Pt 0.3 s 17.1 s -5 ˚C
Pt species after chromatographic separation 1600 W 15.0 L/min 0.57 L/min 0.10 L/min1 9% Micromist 194 Pt, 195Pt 0.4 s 2100 s -5 ˚C
Table 2 Total concentrations of Pt in wastewater samples determined by ICP-MS. Results represent average of triplicate sample analysis ± standard deviation of measurements.
Sample
Total Pt concentration (ng/mL)
Slovenian oncological hospital wastewater
0.352 ± 0.008
Slovenian wastewater influent
0.0233 ± 0.0008
Slovenian wastewater effluent
0.0128 ± 0.0011
Spanish oncological hospital wastewater
0.0144 ± 0.0016
Spanish wastewater influent
0.0079 ± 0.0011
Spanish wastewater effluent
0.0059 ± 0.0008
25
Table 3 Chromatographic program for the separation of cisplatin and its hydrolysed complexes on ZIC-HILIC column. 5 µL of sample was injected and chromatographic procedure carried out at a flow rate of 0.3 mL/min.
Time A B (min) (%) (%) 0.00 100 0 20.00 0 100 24.00 0 100 26.00 0 0 28.00 0 100 30.00 100 0 35.00 100 0 A: 95 % n-propanol, 5 % 20 mM ammonium formate
C (%) 0 0 0 100 0 0 0
B: 50 % n-propanol, 50 % 20 mM ammonium formate C: 100 mM ammonium formate
Table 4 LODs and LOQs for separated Pt species determined by ZIC-HILIC−ID-ICP-MS, calculated as the concentration that provides a signal (peak area) equal to 3s and 10s of the blank sample in the chromatogram, respectively.
Pt species
LOD
LOQ
(ng Pt/mL)
(ng Pt/mL)
cisplatin
0.1726
0.5753
monoaquacisplatin
0.1292
0.4307
monohydroxycisplatin
0.0605
0.2015
diaquacisplatin
0.0988
0.3287
monoaquahydroxycisplatin
0.0273
0.0909
26
Speciation of cisplatin in environmental water samples by Hydrophilic interaction liquid chromatography coupled to inductively coupled plasma mass spectrometry Janja Vidmar, Anže Martinčič, Radmila Milačič, Janez Ščančar www.elsevier.com/locate/talanta
PII: DOI: Reference:
S0039-9140(15)00081-8 http://dx.doi.org/10.1016/j.talanta.2015.02.008 TAL15381
To appear in:
Talanta
Received date: 25 October 2014 Revised date: 2 February 2015 Accepted date: 3 February 2015 Cite this article as: Janja Vidmar, Anže Martinčič, Radmila Milačič, Janez Ščančar, Speciation of cisplatin in environmental water samples by Hydrophilic interaction liquid chromatography coupled to inductively coupled plasma mass spectrometry, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.02.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Speciation of cisplatin in environmental water samples by hydrophilic interaction liquid chromatography coupled to inductively coupled plasma mass spectrometry
Janja Vidmara,b, Anže Martinčiča,b, Radmila Milačiča,b, Janez Ščančara,b* a
Department of Environmental Sciences, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana,
Slovenia b
Jožef Stefan International Postgraduate School, Jamova 39, 1000 Ljubljana, Slovenia
*Corresponding author at: Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia. Tel: +386 1 477 3846; fax: +386 1 251 9385. E-mail address: [email protected] (J. Ščančar)
Abstract Cisplatin is still widely used for treatment of numerous types of tumours. Different speciation methods have been applied to study behaviour of the intact drug and its individual biotransformation species in various clinical samples. These methods are mainly based on electrophoresis, size exclusion (SEC) or ion chromatography (IC) techniques coupled to inductively coupled plasma mass spectrometry (ICP-MS). Hydrophilic interaction liquid chromatography (HILIC), which is a common technique for separation of polar substances, was rarely applied for separation of cisplatin and its hydrolysed metabolites. There is also a lack of information available on the occurrence of cisplatin and its hydrolysed complexes in the environmental waters. In the present study the concentrations of Pt were determined in hospital wastewaters by ICP-MS. A procedure for separation of cisplatin and its aqueous hydrolysed complexes by the use of HILIC column was optimized. Quantification of separated Pt species was performed by isotope dilution (ID)−ICP-MS procedure. Low limits of detection (LODs) and quantification (LOQs) were obtained for cisplatin and its hydrolysed
1
complexes ranging from 0.0273 to 0.1726 ng Pt/mL and from 0.0909 to 0.5753 ng Pt/mL, respectively. Good repeatability of the procedure with relative standard deviation (RSD) lower than ± 2.3 % was obtained. The column recoveries, which ranged from 95 to 101 %, indicated that the procedure developed enabled quantitative speciation analysis of aqueous cisplatin complexes. The ZIC-HILIC−ID-ICP-MS procedure was successfully applied in speciation of cisplatin in spiked hospital wastewater samples.
Highlights •
We developed ZIC-HILIC−ICP-MS procedure for speciation of cisplatin.
•
Hydrolysis of cisplatin was studied in water and solutions containing chloride ions.
•
A method developed is repeatable, sensitive and selective.
•
LODs for cisplatin and its hydrolysed complexes ranged from 0.03 to 0.17▒ng▒Pt/mL.
•
The procedure enabled speciation of cisplatin in spiked hospital wastewaters.
Keywords Cisplatin; Speciation analysis; Hydrophilic interaction liquid chromatography; Isotope dilution inductively coupled plasma mass spectrometry; Environmental water samples
1.
Introduction Cancer is a disease characterised by uncontrolled growth, increased division, decreased
cell death and many other abnormalities of cancer cells. For its treatment, various metal-based chemotherapeutics are used [1,2]. Cisplatin (cis-[PtCl2(NH3)2]), the first chemotherapeutic which was introduced into clinical trials in the late 1970s [3], is nowadays still widely used for treatment of numerous types of tumours (lung, bladder, gastric, head and neck, cervical, testicular and ovarian) [4-6]. For better understanding of anticancer therapy with cisplatin the
2
behaviour of the intact drug and its individual biotransformation species in clinical samples has been intensively investigated by applying speciation analysis methods [7,8,3]. To study interaction of cisplatin with serum proteins Szpunar et al. [9] applied size-exclusion chromatography (SEC) coupled to inductively coupled plasma mass spectrometry (ICP-MS). Huang et al. [10] determined cisplatin and its hydrolytic metabolite in human serum by capillary electrophoresis techniques coupled to ICP-MS. Esteban-Fernández et al. [11] performed speciation of Pt in kidney and inner ear tissue of rats treated with Pt-based chemotherapeutics. Pt binding to proteins was followed by two-dimensional (2D) liquid chromatography (SEC plus fast protein liquid chromatography column (FPLC)) coupled to ICP-MS. Later 2D chromatography, comprised of SEC in combination with ion-exchange chromatography, using strong anion-exchange Mono Q FPLC column or monolithic convective interaction media (CIM) weak anion-exchange diethylamino (DEAE) column, was applied. By hyphenation to ICP-MS a study was performed on the kinetics of binding of cisplatin to serum proteins. The same chromatographic set-up was used to investigate the distribution of Pt species in serum of cancer patients receiving cisplatin [12]. Conjoint liquid chromatography (CLC) on short monolithic disks (affinity CIM Protein G disk and anionexchange CIM DEAE) coupled to ICP-MS was then used for simultaneous 2D separation of ionic forms of Pt-based chemotherapeutics from the portions bound to different serum proteins [13]. In this study, the quantification of separated Pt species was performed by postcolumn isotope dilution (ID)-ICP-MS technique. Since the formation of deoxyribonucleic acid (DNA) adducts with cisplatin is crucial pharmacokinetic parameter, which must be optimized in cancer therapy based on Pt drugs, Hann et al. [14] examined the time-resolved interaction of cisplatin with guanosine monophosphate (GMP) by high performance ion chromatography (HPIC)-ICP-sector field (SF)MS. Pt/P ratio was determined by simultaneously measuring 31P and 195Pt.
3
It is well know that in physiological solutions, cisplatin undergoes hydrolysis. The hydrolysed forms of cisplatin react in a different extent with blood components and cell biomolecules [15,3]. The formation of cisplatin hydrolysed complexes is time-dependent and also depends on the concentration of chloride ion [16]. For investigation of cisplatin and its hydrolysed complexes, hydrophilic interaction liquid chromatography (HILIC) coupled to ICP-MS was found to be a promising technique [17,18]. However, HILIC, which is a routine technique for separation of polar drugs [19,20], was rarely used for separation of cisplatin and its hydrolysed complexes [17,18,21]. When HILIC is applied, organic solvents which are used in the separation procedure are not favourable for ICP-MS detection, due to carbon deposition on the sampler and skimmer cones. Hemström et al. [18] reported the choice of possible organic solvents for HILIC separation of cisplatin and highlighted that commonly used acetonitrile (ACN), reacts with cisplatin and upset speciation equilibria. To avoid the latter problems the use of dimethylformamide (DMF) or 20% 1-propanol in 25 mM ammonium formate buffer (pH 6.5) was recommended. Nygren et al. [17] applied HILIC-ICP-MS procedure to study the distribution of Pt species in whole cell lysate from in-vitro grown T289 malignant melanoma cells exposed to cisplatin. Separation of cisplatin and monoaquacisplatin was achieved by using DMF as an eluent. The LODs for cisplatin and monoaquacisplatin were 0.2 ng Pt/mL. Other group of researchers [21] used ACN to separate cisplatin, oxaliplatin and carboplatin in spiked human plasma samples by HILIC-ICP-MS. For cisplatin, the LOD was found to be 9.8 ng Pt/mL. Other
techniques,
like
reversed-phase
ion-pairing
chromatography
were
also
implemented for speciation of cisplatin and enabled separation of neutral complexes from 1+ and 2+ charged Pt species. The LODs obtained for cisplatin were 1 ng Pt/mL [22] and 19.5 ng Pt/mL [23]. ESI-MS-HPLC was an appropriate technique for identification of Pt-based
4
anticancer drugs and their hydrolysis products when Pt concentrations were in µg/mL range [24,25]. Due to the widespread use of Pt-based chemotherapeutics, Pt metabolites that are excreted with urine end-up in hospital or municipal wastewaters. Despite the potential emerging of cisplatin in the environmental waters there is a lack of literature data on occurrence of cisplatin and its hydrolysed complexes in surface waters. Therefore, the aim of our work was first to determine the content of Pt in hospital wastewaters and in wastewaters from inflow and outflow of sewage treatment plants by ICP-MS. The second aim was to optimize the HILIC procedure for separation of cisplatin and its hydrolysed complexes and to perform the speciation analysis of cisplatin in environmental waters by HILIC coupled to ICP-MS, using post-column ID-ICP-MS technique for the quantification of separated cisplatin species.
2. Materials and methods 2.1.Instrumentation Total Pt concentrations were determined by inductively coupled plasma mass spectrometer (ICP-MS), model 7700x, from Agilent Technologies (Tokyo, Japan). HPLC separations were performed on an Agilent (Tokyo, Japan) series 1200 HPLC system with a quaternary pump equipped with a sample injection valve, Rheodyne model 7725i (Cotati, Ca, USA) fitted with a 5 µL injection loop. The column made from poly(etherether ketone) (PEEK) with bonded zwitterionic silica-based HILIC stationary phase (ZIC-HILIC PEEK HPLC column 2.1 ID, Merck, Darmstadt Germany) with a 20 x 2.1 mm guard column was used for separation of Pt species. The outlet of the chromatographic column was directly connected to the Micromist nebuliser and Scott-type spray chamber of the ICP-MS instrument. Quantification of separated Pt species was performed by post-column isotope 5
dilution ICP-MS. ICP-MS operating parameters for determination of total Pt concentrations and Pt species after the chromatographic separation are given in Table 1.
Place Table 1 about here
2.2.Reagents and materials Ultrapure water (18.2 MΩ cm) was obtained from a Direct-Q 5 Ultrapure water system (Millipore Watertown, A, USA). All chemicals were of analytical reagent grade. Ammonium formate, n-propanol, formic acid and sodium chloride were obtained from Merck (Darmstadt, Germany). Eluent A consisted of aqueous solution of 95 % n-propanol + 5 % of 20 mM ammonium formate (pH 4). Eluent B was composed of aqueous solution of 50 % n-propanol + 50 % 20 mM ammonium formate (pH 4), while buffer C was aqueous solution of 100 mM ammonium formate (pH 4). Cisplatin was obtained from Medoc (Hamburg, Germany). Merck stock Pt solution (1000 µg Pt/mL in 8 % HCl) was diluted daily with water for the preparation of fresh calibration standard solutions that were used for the determination of the total concentration of Pt in the samples analysed. Platinum enriched in
194
Pt isotope (Pt metallic
plate, 15 mg) obtained from Oak Ridge National Laboratory (Oak Ridge, TN, USA) was dissolved in 1 mL of aqua regia and diluted to 10 mL with an appropriate amount of HCl, so that the final concentration of HCl was 8 %. The declared composition of the enriched Pt plate was 96.45 ± 0.05 %, 2.46 ± 0.05 %, 0.87 ± 0.02 %, 0.18 ± 0.01 %, 0.03 ± 0.00 % and 0.01 ± 0.00 % for the isotopes 194, 195, 196, 198, 192 and 190, respectively. Sartorius (Goetingen, Germany) 0.45 µm cellulose nitrate membrane filters of 25 mm diameter were used in the filtration procedure.
6
2.3.
Sample preparation Samples of wastewaters were collected in Slovenian and Spanish oncological hospitals
and in inflows and outflows of the sewage treatment plants. After sampling, samples were filtered through 0.45 µm membrane filters. For the determination of total Pt, samples were acidified (0.1 mL of HNO3 per 100 mL of sample), while non-acidified samples were used for speciation analysis. Samples were kept frozen until analysis. It was experimentally found that concentrations of total Pt in filtered and unfiltered samples did not vary more than ± 5 %. So, determination of total Pt concentrations and Pt speciation was performed in filtered samples.
2.4.
Quantification of separated Pt species by post-column isotope dilution ICP-MS Quantification of separated Pt species was performed by the post-column ID-ICP-MS
technique. Isotopically enriched
194
Pt was added continuously by a peristaltic pump via a T-
piece after the separation of Pt species. To calculate the content of the eluted Pt species, the mass flow of Pt was plotted versus time throughout the whole chromatographic run. Calculations were performed using equations derived for species unspecific post-column IDICP-MS analysis [26-28]. If not stated otherwise, all the experiments were performed in duplicate.
3. Results and discussion 3.1 Determination of total Pt concentrations in hospital wastewater samples Total Pt concentrations in Slovenian and Spanish wastewater samples from oncology hospitals and environmental wastewater samples from inflows and outflows of sewage treatment plants were determined by ICP-MS. Results are presented in Table 2.
7
Insert Table 2 about here
As can be seen from data of Table 2, the determined Pt concentrations were the highest in oncological hospital wastewaters, and the lowest in the effluent from sewage treatment plants. These concentrations are for one order of magnitude higher in the Slovenian than in Spanish oncological hospital wastewaters. Nevertheless, the Pt concentrations are low, ranging from 0.013 to 0.35 ng/mL in Slovenian and from 0.006 to 0.014 ng/mL in Spanish wastewaters. ICP-MS technique was sensitive enough for determination of such low Pt concentrations. The limit of detection (LOD) calculated on a 3 s basis (a value of three standard deviation of the blank) for the determination of total Pt concentration was found to be 0.005 ng/mL.
3.2. Speciation of cisplatin by the ZIC-HILIC−ICP-MS procedure: feasibility study 3.2.1. Optimization of the separation procedure In order to investigate the ability of the ZIC-HILIC−ICP-MS procedure for speciation of cisplatin in aqueous samples, the separation procedure was first optimized. Although being commonly applied as eluent for separation of polar substances [19], ACN was not appropriate. It forms complexes with cisplatin and also produces high carbon load on the ICPMS sampler and skimmer cones, even when oxygen is added to the nebulizer gas flow [18]. To preserve the integrity of Pt species in aqueous solutions and to reduce the carbon load in ICP-MS, the use of ACN was omitted in the present work. The best compromise between carbon deposition and separation efficiency was achieved when n-propanol was used as an eluent and the ICP parameters carefully optimized. Moreover, ICP-MS was equipped with the kit that allows organic solvent to be used for separation. Oxygen was introduced into the plasma and the spray chamber chilled to -5 ˚C (Table 1). For avoiding the drawbacks related to the use of ACN, Hemström with co-workers [18] proposed isocratic elution with n-
8
propanol mixed with ammonium formate buffer (20 % n-propanol + 20 % 25 mM ammonium formate) for separation of cisplatin on ZIC-HILIC column. Similar eluents, but using higher concentrations of aqueous solutions of n-propanol buffered with ammonium formate, were applied in the present study. It was experimentally found that separation of cisplatin and its hydrolysed products was the most effective when gradient elution from 100 % of eluent A (aqueous solution of 95 % n-propanol + 5 % of 20 mM ammonium formate, pH 4) to 100 % of eluent B (aqueous solution of 50 % n-propanol + 50 % 20 mM ammonium formate, pH 4) was applied in 20 min, followed by isocratic elution with eluent B for next 4 min. In the following 11 min the column was regenerated with aqueous solution of 100 mM ammonium formate (eluent C) and equilibrated with eluent A. Peaks were well separated when 5 µL of the sample was injected onto the column and the chromatographic procedure performed at a flow rate of 0.3 mL/min. Chromatographic program for the separation of cisplatin and its hydrolysed complexes is presented in Table 3.
Insert Table 3 about here
To obtain repeatable and reproducible chromatographic separations and low LODs for separated cisplatin complexes, efficient regeneration and equilibration of the ZIC-HILIC column was necessary. Therefore, after approximately 20 separations, the column was cleaned at a flow rate of 0.4 mL/min. First, rinsing with MilliQ water was applied for 40 min, followed by elution with 0.5 M NaCl for the next 40 min. The column was again rinsed with MilliQ water for 40 min. After that 5µL of formic acid (100 %) was injected onto the column and the same chromatographic run as for the separation of cisplatin complexes was performed. This step was repeated five times and the column was ready for further use.
9
3.2.2. The behaviour of cisplatin and its hydrolysed complexes on the ZIC-HILIC column In aqueous solutions cisplatin [PtCl2(NH3)2] undergoes fast hydrolysis reactions, yielding monoaquacisplatin [PtCl(OH2)(NH3)2]+ as the prevailing species, which is in equilibrium with minor concentrations of diaquacisplatin [Pt(OH2)2(NH3)2]2+ and monoaquahydroxycisplatin [Pt(OH2)(OH)(NH3)2]+ species. In solutions containing a significant concentration of chloride ion, cisplatin species prevails and is in equilibrium with small concentrations of monoaquacisplatin and monohydroxycisplatin [PtCl(OH)(NH3)2] [15,3]. There are no standard reference materials with hydrolysed forms of cisplatin available. In order to identify cisplatin and its hydrolysed complexes, series of experiments were performed by the dilution of the stock solution of cisplatin (2 µg/mL) to a concentration of 10 ng Pt/mL. Samples were diluted in water or in solutions containing various amounts of chloride ions and analysed in different time intervals over a period of 48 hours by the ZICHILIC−ID-ICP-MS procedure. For obtaining stable solution of cisplatin [16] sample containing 10 ng Pt/mL was prepared from the stock cisplatin solution in 0.9 % NaCl, and injected onto the column immediately after the dilution. To identify monoaquacisplatin, which is the first hydrolysis product of cisplatin, the same solution was also injected 15 min after the dilution. The chromatograms of these separations are presented in Fig. 1.
Insert Fig. 1. about here
As evident from Fig. 1. (left chromatogram), immediately after preparation cisplatin elutes as a single species at retention time 19.7 min. 15 min after the sample preparation (Fig. 1., right chromatogram)
cisplatin,
as
the
prevailing
10
species,
and
its
hydrolysed
form
monoaquacisplatin are present. The latter species, which represents 10 % of the injected cisplatin, is eluted at retention time 16.4 min. To verify the mass balance over the chromatographic step, the column recovery, which is the ratio between the concentration of cisplatin species eluted from the column and the concentration of cisplatin injected, was calculated. The concentrations of Pt species eluted were determined by the post-column ID-ICP-MS. The column recoveries obtained for separation of cisplatin (Fig. 1., left chromatogram) and cisplatin in the presence of monoaquacisplatin (Fig. 1., right chromatogram) were 96 % and 99 % respectively, indicating on quantitative elution of separated Pt species. Once the retention times of cisplatin and monoaquacisplatin were identified, the hydrolysis of cisplatin in water and in 0.3 % HCl was studied 15 min and 48 h after the preparation of samples. Results are presented in Fig. 2.
Insert Fig. 2. about here
Data from Fig. 2. (upper row) indicate rapid hydrolysis of cisplatin in water. After 15 min 52.5 % of cisplatin was hydrolysed in monoaquacisplatin. The column recovery was found to be 101 %. As time elapsed, hydrolysis of cisplatin after 48 hours resulted in appearance of two more hydrolysis products diaquacisplatin (species eluted at retention time of 13.7 min) and monoaquahydroxycispaltin (species eluted at retention time 15.1 min), which represented 4.0 and 4.1 % of cisplatin complexes in solution, respectively. The column recovery for the cisplatin complexes separated 48 hours after the preparation of aqueous solution was 93 %. Data from Fig. 2. (lower row) demonstrate that hydrolysis of cisplatin in 0.3 % HCl is less pronounced than in aqueous solutions, which is in accordance with findings of ElKhateeb et al. [15,16,3]. After 15 min only 16 % of cisplatin is hydrolysed into
11
monoaquacisplatin. The column recovery was 100 %. 48 hours after the sample preparation, as a result of progressive hydrolysis, monohydroxycisplatin species (eluted at retention time of 17.8 min) also appeared, being in equilibrium with cisplatin and monoaquacisplatin. Cisplatin represented 84.8 %, monoaquacisplatin 6.4 % and monohydroxycisplatin 9.1 % of initial cisplatin concentration. The column recovery was found to be 100 %. Data from Fig. 2. additionally revealed that the ZIC-HILIC−ID-ICP-MS procedure enables separation and the quantification of cisplatin and its hydrolysed complexes by IDICP-MS in aqueous solutions and solutions containing chloride ions.
3.2.3. The influence of suspended particulate matter and humic substances on the speciation of cisplatin in water samples Environmental water samples frequently contain suspended particulate matter (SPM) and humic acids (HA). To find out how the presence of SPM and humic acids influence the speciation of cisplatin by the ZIC-HILIC−ICP-MS procedure, environmentally relevant concentrations 35 mg/L of SPM or 5 mg/L of HA were added to the river water sample (Pt concentration below 0.005 ng/mL) and samples shaken on the horizontal shaker for 24 hours. After that, samples were spiked with cisplatin (10 ng Pt/mL), left for 24 hours, filtered and analysed. The results are shown in Fig. 3.
Insert Fig. 3. about here
As it is evident from Fig. 3., 24 hours after the spiking, cisplatin and its hydrolysis product monoaquacisplatin species are present. However, low column recoveries about only 25 % were obtained, indicating that cisplatin complexes are in a great extent adsorbed on SPM and/or HA. Therefore, the speciation procedure enables quantitative determination of
12
dissolved concentrations of cisplatin and its hydrolysed products in environmental water samples. The latter data are in agreement with findings of Lentz et al. [29] who demonstrated that Pt species excreted via urine of oncology patients showed high affinity to suspended solids of hospital wastewaters.
3.2.4. Figures of merits of the ZIC-HILIC−ID-ICP-MS procedure Under optimised chromatographic conditions (Table 3) and optimised ICP-MS operating parameters (Table 1), the limits of detection (LODs) and limits of quantification (LOQs) for the determination of Pt species were calculated as the concentration that provides a signal (peak area) equal to 3s and 10s of the blank sample in the chromatogram, respectively. To calculate the LODs and LOQs, 6 blank samples (MilliQ water) were injected. The LODs and LOQs for separated Pt species are presented in Table 4.
Insert Table 4 about here
As evident, LODs and LOQs were different for individual Pt species. The lowest LOD and LOQ were found for monoaquahydroxycisplatin (0.0273 and 0.0909 ng Pt/mL, respectively), and the highest for cisplatin (0.1726 and 0.5753 ng Pt/mL, respectively). Similar LOD for separation of cisplatin (0.2 Pt/mL) on the ZIC-HILIC column with ICP-MS detection was reported by Nygren et al. [17], while slightly higher LOD for separation of cisplatin (0.5 Pt/mL) was obtained by the use of adsorption chromatography on Hypersil-Keystone Hypercrab column hyphenated to ICP-MS [16]. The linearity of measurement for each particular cisplatin species was obtained from LOQ to 100 ng Pt/mL.
13
In order to evaluate the repeatability of the procedure developed, six consecutive speciation analyses of standard solution of cisplatin (10 ng Pt/mL in 0.3 % HCl), injected 15 min after preparation from stock cisplatin solution, were performed. The chromatograms are presented in Fig. 4.
Insert Fig. 4. about here
As evident from data of Fig. 4., good repeatability of measurement was obtained. For each of individual Pt species eluted (cisplatin and monoaquacisplatin), the relative standard deviation (RSD) between concentrations of Pt species determined by post-column ID-ICP-MS was found to be lower than ± 2.3 %, while the column recoveries ranged from 95 to 101%.
3.2.5. Speciation of Pt in hospital wastewater samples spiked with cisplatin The applicability of the procedure developed was verified by the analysis of hospital wastewater samples. In the Slovenian hospital wastewater sample (sample A) the total Pt concentration was 0.352 ± 0.008 ng Pt/mL, while in Spanish hospital wastewater sample (sample B) 0.0144 ± 0.0016 ng Pt/mL. These Pt concentrations were lower than LOQ for cisplatin, and hence were too low to perform speciation analysis. Therefore, samples were spiked with cisplatin (10 ng Pt/mL) and analysis performed 15 min and 48 hours after the spiking. The results of speciation analysis are shown in Fig. 5.
Insert Fig. 5. about here
From chromatograms of Fig. 5. it can be seen that 15 min after spiking, cisplatin and monoaquacisplatin are present in hospital wastewater samples A and B. 48 hours after the
14
spiking, in sample A cisplatin and monoaquacisplatin are further hydrolysed, resulting in the appearance
of
diaquacisplatin
species,
while
in
sample
B
only
cisplatin
and
monoaquacisplatin are present. The column recoveries for spiked samples A and B ranged between 96 and 104 %. There are only few reports on the determination of cancerostatic Pt compounds in real environmental water samples. Lenz and co-workers reported speciation of cisplatin in wastewaters from Vienna oncologic hospital [29]. Authors used RP-HPLC based on pentafluorophany terminated stationary phase for separation of Pt compounds and ICP-MS for detection of separated Pt species. The concentrations of Pt in samples from the hospital wastewater influent ranged from 3 to 250 ng Pt/mL, and from effluent from 2 to 150 ng Pt/mL, and were much higher than those from the hospital wastewaters analysed in the present study. The data of the present investigation revealed that the ZIC-HILIC−ID-ICP-MS procedure developed can be reliably applied in speciation of soluble concentrations of cisplatin and its hydrolysed products in hospital and environmental water samples, when the total concentration of Pt is higher than the sum of concentrations for LOQs for the individual Pt species.
4. Conclusions The selective and highly sensitive ZIC-HILIC−ICP-MS procedure developed enables simultaneous determination of cisplatin and its hydrolysed products: monoaquacisplatin, monoaquahydroxyciplatin, diaquacisplatin and monohydroxycisplatin in aqueous solutions containing different chloride concentrations at the low ng/mL concentration range. The use of post-column ID-ICP-MS enabled the determination of separated Pt species. LODs for individual cisplatin complexes ranged from 0.03 to 0.2 ng Pt/mL and LOQs from 0.09 to 0.58 ng Pt/mL. The procedure is reliable (RSD lower than ± 2.3 %) and allows quantitative 15
speciation analysis of aqueous cisplatin complexes (column recoveries from 95 to 101 %). It also enables determination of cisplatin and its metabolites in environmental waters at extremely low Pt concentrations, when the total concentration of Pt is higher than the sum of concentrations for LOQs for the individual Pt species. Due to increasing growth of oncology patients, it can be expected that concentrations of Pt-based chemotherapeutics will increase in environmental waters. So, speciation of Pt in environmental waters is becoming an issue of great concern. Low total Pt concentrations determined in Slovenian and Spanish hospital wastewaters (0.35 ng/mL and 0.014 ng/mL, respectively) facilitated speciation analysis only in spiked hospital wastewaters, demonstrating the presence of cisplatin and its hydrolysed complexes monoaquacisplatin and diaquacisplatin. Analysis of environmental water samples, to which the relevant concentrations of SPM or HA were added, and were spiked with cisplatin, revealed that about 75 % of Pt species were adsorbed by SPM or HA.
Acknowledgements This work was supported by the 7th FW EU Project: Fate and effects of cytostatic pharmaceuticals in the environment and identification of biomarkers for an improved risk assessment on environmental exposure (CYTOTHREATH), Grant agreement No. 265264 and Ministry of Education, Science and Sport of the Republic of Slovenia (Programme group P10143).
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D. Esteban-Fernández, E. Moreno-Gordaliza, B. Cañas, M.A. Palacios, M.M. Gómez Gómez, Metallomics 2 (2010) 19-38.
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B. Michalke, J. Trace Elem. Med. Biol. 24 (2010) 69-77.
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J. Szpunar, A. Makorov, T. Pieper, B.K. Keppler, R. Łobinsky, Anal. Chim. Acta 387 (1999) 135-144.
[10] Z. Huang, A.R. Timerbaev, B.K. Keppler, T. Hirokawa, J. Chromatogr. A 1106 (2006) 75-79. [11] D. Esteban-Fernández, M.M. Gómez-Gómez, B. Cañas, J.M. Verdaguer, R. Ramirez, M.A. Palacios, Talanta 72 (2007) 768-773. [12] A. Martinčič, R. Milačič, M. Čemažar, G. Serša, J. Ščančar, Anal. Methods 4 (2012) 780-790. [13] A. Martinčič, M. Čemažar, G. Serša, V. Kovač, R. Milačič, J. Ščančar, Talanta 116 (2013) 141-148. [14] S. Hann, A. Zenker, M. Galanski, T.L. Bereuter, G. Stingeder, B.K. Keppler, Fresenius J. Anal. Chem. 370 (2001) 581-586. [15] M. El-Khateeb, T.G. Appleton, L.R. Gahan, B.G. Charles, S.J. Berners-Price, A.-M. Bolton, J. Inorg. Biochem. 77 (1999) 13-21. [16] S. Hann, G. Koellensperger, Zs. Stefánka, G. Stingeder, M. Fürhacker, W. Buchberger, R.M. Mader, J. Anal. At. Spectrom. 18 (2003) 1391-1395.
17
[17] Y. Nygren, P. Hemström, C. Åstot, P. Naredi, E. Björn, J. Anal. At. Spectrom. 23 (2008) 948-954. [18] P. Hemström, Y. Nygren, E. Björn, K. Irgum, J. Sep. Sci. 31 (2008) 599-603. [19] P. Jandera, Anal. Chim. Acta 692 (2011) 1-25. [20] M. Staňková, P. Jandera, V. Škeříková, J. Zrban, J. Chromatogr. A 1289 (2013) 47-57. [21] T. Falta, G. Koellensperger, A. Standler, W. Buchberger, R.M. Mader, S. Hann, J. Anal. At. Spectrom. 24 (2009) 1336-1342. [22] Z. Zhao, K. Tepperman, J.G. Dorsey, R.C. Elder, J. Chromatogr. B. 615 (1993) 83-89. [23] D.N. Bell, J.J. Liu, M-D. Tingle, M.J. McKeage, J. J. Chromatogr. B. 837 (2006) 29-34. [24] M. Chui, Z. Mester, Rapid Commun. Mass Spectrom. 17 (2003) 1517-1527. [25] C. Brauckmann, C.A. Wehe, M. Kieshauer, C. Lanvers-Kaminsky, M. Sperling, U. Karst, Anal. Bioanal. Chem. 405 (2013) 1855-1864. [26] K.G. Heumann, S.M. Gallus, G. Rädingler, J. Vogl, Spectrochim. Acta Part B 53 (1998) 273-287. [27] P. Rodrígez-González, J.M. Marchante-Gayón, J.I. García Alonso, A. Sanz-Medel, Spectrochim. Acta Part B 60 (2005) 151-207. [28] United States Environmental Protection Agency (USEPA), Method 6800, Elemental and Speciated Isotope Dilution Mass Spectrometry, Test Methods for Evaluating Solid Waste, Physical/Chemical Methods, SW 846, Update IVA, US Government Printing Office (GPO), Washington DC, 2007. [29] K. Lenz, G. Koellensperger, S. Hann, N. Weissenbacher, S.N. Mahnik, M. Guerhacker, Chemosphere 69 (2007) 1765-1774.
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Research highlights Captions to Figures
Fig. 1 Separation of solutions of cisplatin (10 ng Pt/mL) on ZIC-HILIC column followed by ICP-MS detection. Sample was prepared from the stock solution by dilution in 0.9 % NaCl and injected onto the column immediately and 15 min after preparation. Pt mass flow is based on measurement of isotope ratios m/z 194 and 195. Fig. 2 Separation of solutions of cisplatin (10 ng Pt/mL) on ZIC-HILIC column followed by ICP-MS detection. Samples were prepared from the stock solution by dilution in water and in 0.3 % HCl and injected onto the column 15 min or 48 h after preparation. Pt mass flow is based on measurement of isotope ratios m/z 194 and 195. Fig. 3 Chromatograms of water samples containing 35 mg/L of SPM or 5 mg/L of HA, spiked with cisplatin (10 ng Pt/mL) on ZIC-HILIC column followed by ICP-MS detection. Samples were injected onto the column 24 h after spiking. Pt mass flow is based on measurement of isotope ratios m/z 194 and 195. Fig. 4 Six consecutive speciation analyses of standard solution of cisplatin (10 ng Pt/mL in 0.3 % HCl) on ZIC-HILIC column followed by ICP-MS detection. Samples were injected onto the column 15 min after their preparation. Pt mass flow is based on the measurement of isotope ratios m/z 194 and 195. Fig. 5 Separation of Pt species in hospital wastewater samples (A: Slovenian Hospital, B: Spanish Hospital) spiked with cisplatin (10 ng Pt/mL) on ZIC-HILIC column followed by ICP-MS detection, 15 min and 48 h after spiking. Pt mass flow is based on the measurement of isotope ratios m/z 194 and 195.
19
Graphical abstract
Mass flow Pt 195 (ng/min)
0,1
monoaquacisplatin
0,08 cisplatin
0,06 0,04 monoaquahydroxycisplatin diaquacisplatin
0,02 0 0
5
10 15 Time (min)
Cisplatin in 0.9 % NaCl immediately after preparation 0,12
Mass flow Pt 195 (ng/min)
cisplatin 0,08
0,04
0 0
5
10 15 Time (min)
20
25
Cisplatin in 0.9 % NaCl 15 min after preparation
0,12
Mass flow Pt 195 (ng/min)
20
cisplatin
0,08
0,04
monoaquacisplatin
0
25
0
Fig. 1.
20
5
10 15 Time (min)
20
25
Cisplatin in water 48 h after the dilution
Cisplatin in water 15 min after the dilution 0,1
monoaquacisplatin cisplatin
0,08
Mass flow Pt 195 (ng/min)
Mass flow Pt 195 (ng/min)
0,1
0,06 0,04 0,02
monoaquacisplatin
0,08 0,06
cisplatin
0,04 monoaquahydroxycisplatin diaquacisplatin
0,02 0
0 0
5
10 15 Time (min)
20
0
25
Cisplatin in 0.3 % HCl 15 min after the dilution
5
0,1
25
20
25
cisplatin
Mass flow Pt 195 (ng/min)
cisplatin
Mass flow Pt 195 (ng/min)
20
Cisplatin in 0.3 % HCl 48 h after the dilution
0,1 0,08 0,06 monoaquacisplatin
0,04
10 15 Time (min)
0,02
0,08 0,06 monohydroxycisplatin
0,04
monoaquacisplatin
0,02 0
0 0
5
10 15 Time (min)
20
0
25
Fig. 2.
21
5
10 15 Time (min)
Spiked water sample containing 35 mg L-1 of SPM
Spiked water sample containing 5 mg L-1 of HA 0,05
Mass flow Pt 195 (ng/min)
Mass flow Pt 195 (ng/min)
0,05 0,04 0,03 monoaquacisplatin
0,02
cisplatin
0,01 0
0,04 0,03 monoaquacisplatin
0,02
cisplatin
0,01 0
0
5
10 15 Time (min)
20
25
0
Fig. 3.
22
5
10 15 Time (min)
20
25
0,04
0,00
0
5
10 15 Time (min)
20
Fig. 4.
23
25
(ng/min)
195
0,08
Mass flow Pt
0,12
Cisplatin in sample A 48 h after spiking
Cisplatin in sample A 15 min after spiking monoaquacisplatin
0,08
0,1 monoaquacisplatin
cisplatin
Mass flow Pt 195 (ng/min)
Mass flow Pt 195 (ng/min)
0,1
0,06 0,04 diaquacisplatin
0,02 0 0
5
10 15 Time (min)
20
0,08
cisplatin
0,06 0,04 diaquacisplatin
0,02 0
25
0
Cisplatin in sample B 15 min after spiking 0,1
cisplatin
0,08
10 15 Time (min)
20
25
Cisplatin in sample B 48 h after spiking
Mass flow Pt195 (ng/min)
Mass flow Pt195 (ng/min)
0,1
5
monoaquacisplatin
0,06 0,04 0,02
monoaquacisplatin cisplatin
0,08 0,06 0,04 0,02 0
0 0
5
10 15 Time (min)
20
0
25
Fig. 5.
24
5
10 15 Time (min)
20
25
Table 1 Optimised ICP-MS operating parameters for the determination of the total Pt concentrations and quantification of Pt species after their separation on ZIC-HILIC column.
ICP-MS parameters Forward power Plasma gas flow (Ar) Carrier gas flow (Ar) Dilution gas flow (Ar) Optional gas flow (20 % v/v O2 in Ar) Nebuliser type Isotopes monitored Integration time Total acquisition time Spray chamber temperature
Total Pt concentrations 1550 W 15.0 L/min 1.10 L/min / / Micromist 193 Ir, 194Pt, 195Pt 0.3 s 17.1 s -5 ˚C
Pt species after chromatographic separation 1600 W 15.0 L/min 0.57 L/min 0.10 L/min1 9% Micromist 194 Pt, 195Pt 0.4 s 2100 s -5 ˚C
Table 2 Total concentrations of Pt in wastewater samples determined by ICP-MS. Results represent average of triplicate sample analysis ± standard deviation of measurements.
Sample
Total Pt concentration (ng/mL)
Slovenian oncological hospital wastewater
0.352 ± 0.008
Slovenian wastewater influent
0.0233 ± 0.0008
Slovenian wastewater effluent
0.0128 ± 0.0011
Spanish oncological hospital wastewater
0.0144 ± 0.0016
Spanish wastewater influent
0.0079 ± 0.0011
Spanish wastewater effluent
0.0059 ± 0.0008
25
Table 3 Chromatographic program for the separation of cisplatin and its hydrolysed complexes on ZIC-HILIC column. 5 µL of sample was injected and chromatographic procedure carried out at a flow rate of 0.3 mL/min.
Time A B (min) (%) (%) 0.00 100 0 20.00 0 100 24.00 0 100 26.00 0 0 28.00 0 100 30.00 100 0 35.00 100 0 A: 95 % n-propanol, 5 % 20 mM ammonium formate
C (%) 0 0 0 100 0 0 0
B: 50 % n-propanol, 50 % 20 mM ammonium formate C: 100 mM ammonium formate
Table 4 LODs and LOQs for separated Pt species determined by ZIC-HILIC−ID-ICP-MS, calculated as the concentration that provides a signal (peak area) equal to 3s and 10s of the blank sample in the chromatogram, respectively.
Pt species
LOD
LOQ
(ng Pt/mL)
(ng Pt/mL)
cisplatin
0.1726
0.5753
monoaquacisplatin
0.1292
0.4307
monohydroxycisplatin
0.0605
0.2015
diaquacisplatin
0.0988
0.3287
monoaquahydroxycisplatin
0.0273
0.0909
26
