Anal Bioanal Chem DOI 10.1007/s00216-013-6794-4
ORIGINAL PAPER
Development of a UPLC-MS/MS method for the determination of ten anticancer drugs in hospital and urban wastewaters, and its application for the screening of human metabolites assisted by information-dependent acquisition tool (IDA) in sewage samples L. Ferrando-Climent & S. Rodriguez-Mozaz & D. Barceló
Received: 2 November 2012 / Revised: 12 January 2013 / Accepted: 25 January 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract In the present work, the development, optimization, and validation (including a whole stability study) of a fast, reliable, and comprehensive method for the analysis of ten anticancer drugs in hospital and urban wastewater is described. Extraction of these pharmaceutical compounds was performed using automated off-line solid-phase extraction followed by their determination by ultra-performance liquid chromatography coupled to a triple quadrupole–linear ion trap mass spectrometer. Target compounds include nine cytotoxic agents: cyclophosphamide, ifosfamide, docetaxel, paclitaxel, etoposide, vincristine, tamoxifen, methotrexate, and azathioprine; and the cytotoxic quinolone, ciprofloxacin. Method detection limits (MDL) ranged from 0.8 to 24 ng/L. Levels found of cytostatic agents in the hospital and wastewater influents did not differ significantly, and therefore, hospitals cannot be considered as the primary Published in the special paper collection Liquid Chromatography– Tandem Mass Spectrometry with guest editors Damià Barceló and Mira Petrovic. Electronic supplementary material The online version of this article (doi:10.1007/s00216-013-6794-4) contains supplementary material, which is available to authorized users. L. Ferrando-Climent : S. Rodriguez-Mozaz (*) : D. Barceló ICRA, Catalan Institute for Water Research, Carrer Emili Grahit, 101, Parc Científic i Tecnològic de la Universitat de Girona, 17003 Girona, Spain e-mail:
[email protected] D. Barceló Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDAEA), CSIC, c/Jordi Girona 18-26, 08034 Barcelona, Spain
source of this type of contaminants. All the target compounds were detected in at least one of the influent samples analyzed: Ciprofloxacin, cyclophosphamide, tamoxifen, and azathioprine were found in most of them and achieving maximum levels of 14.725, 0.201, 0.133, and 0.188 μg/L, respectively. The rest of target cancer drugs were less frequently detected and at values ranging between MDL and 0.406 μg/L. Furthermore, a feasible, useful, and advantageous approach based on information acquisition tool (information-dependent acquisition) was used for the screening of human metabolites in hospital effluents, where the hydroxy tamoxifen, endoxifen, and carboxyphosphamide were detected. Keywords Cytotoxic . Anticancer drugs . Metabolites . Hospital effluent . UPLC-QqLit . IDA
Introduction The occurrence of pharmaceutical compounds in aquatic environment due to the human activity can compromise water quality, as indicated in several studies along the last decades [1–4]. Occurrence of pharmaceuticals in our environment, such as antibiotics, analgesic, and psychiatric drugs, among others, is being therefore studied all over the world and is contributing to the demonstration of their environmental importance. Unfortunately, pharmaceutical levels in urban wastewaters are increasing due to the population aging, to the increase in population density [5], and, in some geographical areas, due to water scarcity associated with climate change [6, 7]. On the other hand, the huge rise
L. Ferrando-Climent et al.
of cancer disease in human population has lead to the increase in the use of chemotherapy drugs, and a further augment of their use in the next years can be foreseen [8]. Although chemotherapy drugs are administered in hospitals, 75 % of oncology patients leave the hospital after their administration [9], and therefore, the cytostatic compounds can reach the aquatic environment via hospital or domestic wastewater, and ultimately through wastewater treatment plants (WWTPs). Moreover, it is also important to take into account that the cytostatic compounds are excreted both as parent compound and as metabolites, which might have same mode of action than the parent cytostatic compound or even higher activity. This is the case for example of one metabolite of tamoxifen, hydroxy tamoxifen, which has higher estrogenicity than the parent compound [10]. Anticancer drugs include different chemical families such as antineoplastic, cytotoxic, and other common drugs used for oncologic treatments. The most consumed anticancer drugs are 5-fluorouracil, gemcitabine, ifosfamide, cyclophosphamide, and methotrexate [11, 12], but other drugs such as azathioprine, tamoxifen, etoposide, vincristine, chlorambucil, docetaxel, and paclitaxel are also broadly used in chemotherapy [11, 13]. Cytostatic agents have been shown to have potent cytotoxic, genotoxic, mutagenic, carcinogenic, or teratogenic effects in several organisms [3], since they have been designed to disrupt or prevent cellular proliferation, usually by interfering in DNA synthesis. Previous studies about ecotoxicological and genotoxicological aspects of several cytostatic agents have shown that, in cases such as 5-fluorouracil, the lowest-observed-effect concentration values (in algal and bacterial assays) were close to the concentration found in sewage effluents [14]. Apart from this, there are a limited experimental data on ecotoxicity of anticancer drugs and their metabolites [15]. In addition, unexpected toxicity interactions among drugs at low concentrations ranges are a cause of concern [16]. Another important issue about cytostatic drugs is that they have been reported to have low biodegradability by conventional wastewater treatments and are thus considered recalcitrant compounds [17–21]. For all these reasons, cytostatic drugs and their metabolites should be considered as an important group of emerging contaminants, and it is necessary to know their occurrence and concentration in aquatic environment. Reports on occurrence of cytostatic compounds in the environment are very recent but still scarce [9, 22–27], and the concentration found in wastewater samples are very low compared with other common pharmaceuticals: Levels of cyclophosphamide ranged from 6 till 143 ng/L and from 19 until 4,500 ng/L in urban and hospital wastewaters, respectively, whereas levels between 6 and 20 ng/L were found in wastewater effluents [28, 29]. Scarcity of information about environmental levels of these compounds is partially explained by the lack of analytical methods for environmental
applications [12, 30–32]: Anticancer drugs belong to different chemical families, and developing a multi-residue method for all of them can be an analytical challenge. In order to undertake a proper environmental risk assessment of these compounds, it is important to provide broad and proper information about their occurrence in the environment, namely, to examine the concentration of this group of drugs in aquatic ambience and their potential ecological implications [1, 33, 34]. Therefore, the main objective of this work was to develop a very sensitive method based on ultra-high-performance liquid chromatography coupled to a triple quadrupole–linear ion trap spectrometer (UPLCQqLit) for the detection of a broad number of compounds covering most of the families considered for cancer therapy. Ten anticancer drugs were selected as target analytes due to their importance, consumption, inherent cytotoxic activity, and potential risk to the environment: cyclophosphamide (CY), ifosfamide (IF), docetaxel (DOC), paclitaxel (PAC), etoposide (ETO), vincristine (VIN), tamoxifen (TAM), methotrexate (MTX), and azathioprine (AZA); and one of the most used antibiotics in oncologic treatments, a cytotoxic quinolone, ciprofloxacin (CIP) [35]. With this purpose, two different analytical approaches were considered and applied for the detection of anticancer drugs and their metabolites in hospital effluents and wastewater influents from WWTPs. A fast and reliable method for quantification of ten anticancer drugs using an UPLC-QqLit system was first developed and validated. A comprehensive method optimization as well as a performance study was accomplished and included: (1) determination of a wide and representative number of anticancer drugs, (2) a stability study for selected compounds, 3) a thorough study about analyte identification criteria based on ion area-ratio, (4) a matrix effects study using matrix-matched calibration method in wastewaters, and (5) new lights about the occurrence of anticancer drugs in wastewaters. In addition, an exploratory analysis in sewage samples for the screening of known cancer drug metabolites was performed using the solidphase extraction (SPE)–ultra-high-performance liquid chromatography (UPLC) method developed but assisted by the information-dependent acquisition (IDA) tool available in the QqLit spectrometer. The work performed here offers, therefore, several advantages and improvements in comparison with the currently available methods for cytostatic agents in the environment.
Material and methods Chemicals and reagents Ciprofloxacin HCl, cyclophosphamide, ifosfamide, methotrexate, azathioprine, etoposide, docetaxel, paclitaxel, vincristine
Development of a UPLC-MS/MS method for anticancer drugs
sulfate, and tamoxifen citrate were purchased by European Directorate for the Quality of Medicines and Healthcare (EDQM) Reference Standards (Strasbourg, France). Isotopically labelled compounds, used as internal standards, [2H4]-cyclophosphamide, [13C6]-tamoxifen citrate, [2H3]-etoposide, [2H3]methotrexate, [2H3]-vincristine sulfate, and [13C4]-azathioprine were purchased from Toronto Chemical Research Inc. (Canada) and [2H8]-ciprofloxacin from EDQM Reference Standards (Strasbourg, France). High-performance liquid chromatography (HPLC)-grade Water and HPLC-grade acetonitrile and water (LiChrosolv) were supplied by Merck (Darmstadt, Germany). Reagents like formic acid 98 % (HCOOH) were provided by Sharlab (HPLC-grade) and the NH3 30 % by Panreac. Ammonium formate salt and ammonium acetate salt for HPLC (Chromanorm) were provided by Prolabo, and the ethylenediaminetetraacetic acid disodium salt 0.1 M solution (SV) was provided by Panreac. Cartridges tested for SPE were Oasis HLB (200 mg/6 mL), Oasis MCX (150 mg/6 mL), and Oasis MAX (150 mg/6 mL) from Waters (Milford, MA, USA). Physico-chemical properties of target compounds were obtained by Syrres Database or, for those not available in databases, estimated using the software Marvin Sketch 5.5.0.© (see Table 1). Samples and standards preparation Individual stock standard solutions of each target compound were prepared on a weight basis in methanol at 1 mg/mL and kept frozen at −20 °C. A mixture of all pharmaceutical standards was prepared by appropriate dilution of individual stock solutions. Stock solutions of internal standards were also prepared in methanol and were stored at −20 °C. A mixture of these internal standards was also prepared by diluting the individual stock solution in methanol. Calibration standard solutions were prepared by appropriate dilution in methanol–water (10:90, v/v) of the stock solution of target compounds. Sample collection and pre-treatment Wastewater samples were collected from different hospitals: University Hospital of Coimbra-Portugal (with around 1,456 beds), General University Hospital of Valencia-CGHV-Spain (with around 518 beds), and Dr. Josep Trueta Hospital of Girona-Spain (with around 400 beds). Also, WWTP influents were collected: Municipal WWTP of Girona-Spain and Urban WWTP of Toulouse-France. Girona, Coimbra, Toulouse, and Valencia are towns with approximately 96,236, 145,000, 446,220, and 798,033 inhabitants, respectively. Amber glass bottles pre-rinsed with ultrapure water were used for sample collection. The samples were immediately vacuum-filtered through 1-μm glass fiber filters followed by
0.45 μm nylon membrane filters (from Whatman, Teknokroma, Barcelona, Spain). The samples were kept frozen at −20 °C in amber polyethylene terephthalate (PET) containers, for a period inferior of 1 month, based on the stability studies performed, and described in the section “Stability study” until their analysis. Solid-phase extraction optimization Samples were pre-concentrated using a method based on SPE. For this purpose, an automatic extraction system, GX-271 ASPECTM (Gilson, Villiers le Bel, France), was used. The lipophilic/hydrophilic balance Oasis HLB (200 mg, 6 mL) cartridge was compared with cationic exchange sorbent Oasis MCX (150 mg/6 mL) and anionic exchange sorbent Oasis MAX (150 mg/6 mL), under different conditions. Addition of Na2EDTA onto the samples was tested by adding a suitable volume of a solution of 0.1 M to achieve a final concentration of 0.1 % (gram solute per gram solution) in order to improve the extraction efficiency of target compounds as recommended by other previous studies for pharmaceutical compounds [36–38]. Pharmaceutical compounds, especially antibiotics, can potentially bind with residual metals present in the sample matrix and glassware, which can thus lead to low extraction recoveries. However, by adding the chelating agent, Na2EDTA, soluble metals get bound to it, and therefore, better extraction efficiency for pharmaceuticals can be obtained. Universal organic solvent, methanol, and a strong solvent, ethyl acetate, were tested as elution solvents during SPE optimization. UPLC-QqLit method Chromatographic separation was carried out with an ultraperformance liquid chromatography system (Waters Corp. Milford, MA, USA) equipped with a binary solvent system (Milford, MA, USA) and a sample manager, using an Acquity HSS T3 column ( 50 mm×2,1 mm i.d. 1,7 μm particle size; Waters Corp. Milford, MA, USA) at flow rate of 0.4 mL/min. The column was kept at 25 °C (room temperature), and the sample manager was maintained at 5 °C. The UPLC instrument was coupled to 5500 QqLit, triple quadrupole–linear ion trap mass spectrometer (5500 QTRAP, Applied Biosystems, Foster City, CA, USA) with a Turbo V ion spray source. Analysis was performed in positive ionization mode in multiple reaction monitoring (MRM) mode. Data were acquired and processed using Analyst 2.1 software. Validation study The linearity of the method was studied by analyzing standard solutions in triplicate at eight different concentrations
L. Ferrando-Climent et al. Table 1 Physico-chemical properties of target compounds
Target Compound
Structure
Classification by mode of action
Azathioprine ( AZA)
Cyclophosphamide (CY)
Alkylating agent
Ifosfamide (IF)
Mw (g/mol)
pKa
Log Kow
277.3
7.8
0.10
260.1
pka1=0.02 pka2=12.1
0.63
260.1
12.4
0.63
Methotrexate (MTX)
Anti-metabolite
454.4
4.7
-1.85
Tamoxifen (TAM)
Anti-estrogen
371.5
8.8
6.35
ranging from 0.1 to 100 ng/mL. Satisfactory linearity using weighed least-squares regression was assumed when the correlation quadratic coefficient (r2) was higher than 0.99, based on compound peak areas measurement and the residuals lower than 30 %. Accuracy of the method was expressed by [(mean observed concentration)/(spiked concentration)]×100 and was determined at the low linearity level (0.1 ng/mL) and at the high linearity level (100 ng/mL) of standard mixture by triplicate. The precision of the method was evaluated
from five repeated injections of 20 ng/mL standard mixture during the same day, which is defined as the intra-day precision (repeatability), and during five successive days that corresponds to the inter-day precision (reproducibility). Method detection limits (MDL) and method quantification limits (MQL) were determined as the minimum detectable amount of each analyte with a signal-to-noise of 3 and 10, respectively. MDL and MQLs were calculated from spiked real samples for the two aqueous matrices studied, hospital effluent and wastewater influent.
Development of a UPLC-MS/MS method for anticancer drugs Table 1 (continued)
Target Compound
Structure
Classification by mode of action
Paclitaxel (PAC)
Mw (g/mol)
853.9
pKa
pka1=9.7 pka2=12.0 pka3=13.3 pka4=14.4
Log Kow
3.54
Anti-mitotic Vincristine (VIN)
824.9
Docetaxel (DOC)
Ciprofloxacin (CIP)
Etoposide (ETO)
Recoveries were also evaluated in both, hospital effluent and wastewater influent. To determine them, the samples were spiked in triplicate with a standard mixture containing all target compounds at three spiking levels, which were selected based on the concentration levels of each pharmaceutical
807.8
Cytotoxic quinolones
331.3
Topoisomerase inhibitor
588.6
5.0
pka1=10.6 pka2=12.0 pka3=13.3 pka4=14.0 pka5=14.8
6.1
pka1=9.3 pka2=12.3 pka3=13.9
2.82
2.92
0.28
0.60
compound found in the water matrices under study according to scientific literature [9, 11]—1, 0.5, and 0.1 ng/mL. For the assessment of effects of wastewater complexity on the analysis of target compounds, the so-called “absolute” matrix effect (ME) was calculated using the
L. Ferrando-Climent et al.
Eq. 1, by comparing the slopes of neat standard curves (N) where standards are prepared in a neat mobile phase, with the slopes of matrix match curves (W), where standards are prepared in post-extraction samples, the so-called matrix-matched calibration [32, 39]: MEð%Þ ¼
W N
100
ð1Þ
ME values were calculated using two approaches: with and without internal standard. Finally, the applicability of the method developed to real samples was evaluated. For this purpose, water samples from three different hospitals: University Hospital of Coimbra (Portugal), General University Hospital of Valencia-CGHV (Spain), and Dr. Trueta Hospital of Girona (Spain) and also from two different wastewater influents: samples from a WWTPs of Girona (Spain) and Toulouse (France) were analyzed. Stability study In some occasions, environmental samples (water, sediment, sludge, etc.) cannot be analyzed right after sampling moment, and they need to be stored for certain time until their analysis using different preservation approaches. Therefore, it is necessary to determine how long these samples can be stored under the proposed storage conditions in order to avoid the potential degradation of target compounds. Within the validation procedure, a stability study is needed, and it has to be conducted to determine the appropriate storage conditions and period of time that the samples can be stored before analysis [40]. For this purpose, wastewater samples were fortified with known quantities of studied compounds (initial concentration was 0.5 ng/mL) and stored under the selected conditions. Samples were periodically analyzed at specified intervals: at initial time and after 1 week, 1 month, 3 months, and 6 months. Stability of the compound in each particular case was considered acceptable if the mean concentration obtained at the specified stability time point agreed with the freshly fortified control sample assay results (initial assay results is needed if incurred samples are used) within ±15 % [40]. Different conditions were evaluated in triplicate for the best storage protocol of the samples: (1) wastewater samples spiked at 0.5 ng/mL and kept at −20 °C in PET containers (WS); (2) wastewater samples spiked at 0.5 ng/mL, loaded onto SPE cartridge, which afterward were kept at −20 °C (CART); (3) wastewater samples spiked at 0.5 ng/mL and with 5 % of formaldehyde as preserving substance [41] and kept at −20 °C in PET containers (PRE). Besides, stability at −20 °C of stock standard solutions prepared in methanol were tested at the same time points to check.
Method for the screening of human metabolites assisted by information-dependent acquisition (IDA) tool Extracts of the hospital effluents samples obtained through the extraction method developed in the present work were also analyzed by UPLC-QqLit system using IDA. This application has been previously described by several authors as an advantageous and feasible instrumental methodology for confirmation of trace levels of pharmaceutical compounds and its metabolites in environmental samples [42, 43]. IDA acquisition and processing method consisted in the acquisition of the so-called enhanced MS scan as a survey scan: all the ions with values between 100 and 1,000 counts of m/z, and were triggered to tandem mass spectrometry (MS/MS) experiment in QqLit mode. Three enhanced product ion scans (EPIs) were performed simultaneously using three different collision energy (CE) values (10, 20, and 30 eV) in order to obtain the maximum information about fragmentation pattern of selected ions. In order to confirm the presence/absence of unknown compounds in the samples, mass fragmentation mechanisms were evaluated with the help of Mass Frontier software (Thermo Science), which simulates mass fragmentation pathways and mechanisms for compounds of interest.
Results and discussion MS/MS optimization MS/MS mass spectra and parameters such as declustering potential (DP), entrance potential, CE collision cell entrance, and collision cell exit potential were obtained by direct infusion of 100 ng/mL individual standard solutions of each compound in methanol/water (50:50, v/v) at flow rate of 10 μL/min (Table 2). All the compounds were determined by positive ionization mode which allowed their fast analysis in one single run. Two selected reaction monitoring (SRM) transitions between the precursor ion and the two most abundant fragment ions were monitored for each compound to have a confirmative method, obtaining in this way the minimum number of IPs required for a safe confirmation [44]. In the case of isotopically labelled internal standards, which are not likely to be found in the environmental matrices, only one transition was monitored (Table 2). Extract ion chromatograms of a standard mixture of the target compounds at 100 ng/mL are shown in Fig. 1. For target compounds, the first transition is used for quantification purposes, whereas the second one is to confirm their identity. Only in the cases of etoposide, docetaxel, and paclitaxel could only one SRM transition be recorded due to their poor fragmentation. However, their identification in real samples could be supplemented
Development of a UPLC-MS/MS method for anticancer drugs Table 2 MS/MS parameters for the analysis of target compounds by MRM positive ionization mode Compound
tR (min)
MRM 1
DP/CE
MRM 2
DP/CE
Ion-ratio for spiked samples (n=3)
Ion-ratio for neat standard (n=3)
Ion-ratio comparison (RSD %)
MTX [2H3]-MTX AZA [13C4]-AZA CIP [2H8]-CIP VIN [2H3]-VIN IF CY [2H4]-CY ETO [2H3]-ETO TAM [13C6]-TAM DOC PAC
1.14 1.15 1.27 1.28 1.28 1.28 1.63 1.63 1.82 1.86 1.86 1.98 1.98 2.62 2.64 2.94 3.01
455>308 458>175 278 >142 282>146 332>228 340>296 825>765 828>810 261>154 261>140 266>141 589>185 592>229 372>72 377>72 808>509 854>569
131/27 111/55 61/17 66/17 66/27 61/24 36/53 46/55 76/31 101/31 61/29 56/75 86/21 141/55 16/51 66/17 56/17
455>175 – 278>85 – 332>245 340>249 825>807 – 261>92 261>106 – – – 372>42 – – –
131/57 – 61/31 – 66/33 61/37 36/61 – 76/33 101/25 – – – 141/125 – – –
1.63±0.01 – 3.88±0.54 – 0.67±0.14 – 1.37±0.78 – 0.95±0.01 3.09±0.04 – – – 0.73±0.01 – – –
1.56±0.01 – 3.54±0.26 – 1.77±0.19 – 2.55±0.06 – 0.91±0.15 2.94±0.24 – – – 1.63±0.19 – – –
3.05 – 8.56 – 64.0 – 42.5 – 0.15 3.53 – – – 54.1 – – –
Ion-ratio calculated for spiking level of 0.5 ng/mL
using a matrix-matched standard. This approach is based on the addition of the target compound standard onto the sample at the same concentration as the “suspected” analyte peak appears; this helps to distinguish if the peak corresponds to an interference or to the target compound: By increasing concentration of the standard added, a proportional increment in the signal of the suspected analyte would be expected. If they are different compounds, no proportional increment in the signal or the display of a new peak would be observed. Although this is not an unequivocal test, it can help to confirm or to discard the identity of analytes in those cases where mass fragmentation is poor. On the other hand, as dictated by EU Decision 2002/657/EC, several authors usually confirm a positive result in real sample by comparing the ion-ratios obtained from the sample with the ion-ratios from a reference standard [38, 45]. However, as it is shown in Fig. 2, the ion-ratios calculated from neat standards at 0.5 ng/mL did not match (relative standard deviation>20 %) with those obtained from a real sample spiked at the same concentration in the case of ciprofloxacin, tamoxifen, and vincristine. This can be attributed to matrix effects, which might lead to incorrect results (false-positives or false-negatives) if not taken into account. With this in mind, confirmation of positive findings was carried out by using as reference the ratios of spiked samples instead of those obtained from neat standards. Settings for source-dependent parameters were determined by flow injection analysis, and the optimal ones were: curtain gas (CUR)=30 V; nitrogen collision gas (CAD)=
medium; source temperature (TEM)=700 °C; ion spray voltage was 5,500 V; ion source gases GS1 and GS2 were set 60 and 40, respectively. Chromatography optimization Different aqueous solutions were considered for optimization of chromatographic separation based on those usually recommended for mass spectrometry detector: ammonium acetate 5 mM at pH=7.00, ammonium formate 5 mM at pH=3.00, and formic acid at 0.1 % (pH=2.8). They were tested with acetonitrile as organic solvent. Formic acid at 0.1 % was selected as the better option since it complied with common requirements in terms of peak shape, resolution, and sensitivity. Separation was performed with a binary mobile phase at flow rate of 0.4 mL/min using 0.1 % formic acid (A) and acetonitrile (B) in gradient elution mode, which was 0.0 min, 5 % of B; 0.1–4.0 min, 5–100 % B; 4.0–4.5 min, 100 % B; 4.5–5.0 min, 100–5 % B; 5.0 min, 5 % B during 0.5 min. Sample injection volume was 5 μL. Solid-phase extraction optimization In order to compare the suitability of the different cartridges considered as indicated in the section on “Solid-phase extraction optimization” (HLB, MAX, MCX), recovery efficiencies for the analytes with each of them were evaluated. Milli-Q water samples were spiked with appropriate concentration (0.5 ng/mL) of the standard mixture containing
L. Ferrando-Climent et al. XIC of +MRM (31 pairs): 455.097/308.200 Da from Sample 2 (Std 100 ppb_a) of Data07082012_Metodo Final_IDA samples.wiff (Turbo Spray)
3.8e6 3.6e6
Max. 4.0e6 cps.
XIC of +MRM (31 pairs): 260.960/140.000 Da from Sample 2 (Std 100 ppb_a) of Data07082012_Metodo Final_IDA samples.wiff (Turbo Spray)
1.14
4.0e6
1.14 min
1.86 min
5.0e6
455>308
3.4e6
Max. 5.5e6 cps.
1.86
5.5e6
261>140
4.5e6
3.2e6 3.0e6
4.0e6
2.8e6
Methotrexate
2.6e6
Cyclophosphamide
3.5e6
Intensity, cps
Intensity, cps
2.4e6 2.2e6 2.0e6 1.8e6
3.0e6
2.5e6
1.6e6 2.0e6
1.4e6 1.2e6
1.5e6
1.0e6 8.0e5
1.0e6
6.0e5 5.0e5
4.0e5 2.0e5 0.0 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
0.0 0.0
5.5
0.5
1.0
1.5
2.0
2.5
3.0
XIC of +MRM (31 pairs): 277.951/142.100 Da from Sample 2 (Std 100 ppb_a) of Data07082012_Metodo Final_IDA samples.wiff (Turbo Spray)
2.3e6 2.2e6
4.5
5.0
5.5
Max. 4.9e4 cps.
1.98 4.8e4
278>142
1.27 min
4.6e4
589>185
1.98 min
4.4e4 4.2e4
2.1e6
4.0e4
2.0e6
3.8e4
1.9e6
3.6e4
Azathioprine
1.8e6 1.7e6
Etoposide
3.4e4 3.2e4
1.6e6
3.0e4
1.5e6
2.8e4
Intensity, cps
Intensity, cps
4.0
XIC of +MRM (31 pairs): 588.752/185.000 Da from Sample 2 (Std 100 ppb_a) of Data07082012_Metodo Final_IDA samples.wiff (Turbo Spray)
Max. 2.5e6 cps.
1.27
2.5e6 2.4e6
3.5
Time, min
Time, min
1.4e6 1.3e6 1.2e6
2.6e4 2.4e4 2.2e4
1.1e6
2.0e4
1.0e6
1.8e4 1.6e4
9.0e5
1.4e4
8.0e5
1.2e4
7.0e5
1.0e4
6.0e5 5.0e5
6000.0
4.0e5
4000.0
3.0e5
2000.0
2.0e5
0.0 0.0
1.0e5 4.0
4.5
5.0
5.5 Max. 1.1e6 cps.
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
XIC of +MRM (31 pairs): 372.134/72.000 Da from Sample 2 (Std 100 ppb_a) of Data07082012_Metodo Final_IDA samples.wiff (TurbopS ray)
1.28
1.05e6
9.50e5
0.5
5.5
Time, min
0.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 XIC of +MRM (31 pairs): 332.041/288.200 Da from Sample 2 (Std 100 ppb_a) of Data07082012_Metodo Final_IDA samples.wiff (Turbo Spray) Time, min
1.00e6
5.24
8000.0
2.62
1.03e6 1.00e6
1.28 min
2.62 min
9.50e5 9.00e5
332>288
9.00e5 8.50e5
Max. 1.0e6 cps.
372>72
8.50e5 8.00e5
8.00e5 7.50e5
7.50e5
Ciprofloxacin
7.00e5 6.50e5
6.00e5
5.50e5 5.00e5 4.50e5
4.50e5
3.00e5
3.00e5
2.50e5
2.50e5
2.00e5
2.00e5
1.50e5
1.50e5
4.99
1.00e5
1.00e5
5.00e4
5.00e4 0.00 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 XIC of +MRM (31 pairs): 825.175/765.400 Da from Sample 2 (Std 100 ppb_a) of Data07082012_Metodo Final_IDA samples.wiff (Turbo Spray) Time, min
4.0
4.5
5.0
0.00 0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Time, min XIC of +MRM (31 pairs): 808.000/509.000 Da from Sample 1 (Std 100ppb - 0.1 % Formic) of Data06_08_2012_Citotoxicos Fases Movil es.wiff (Tur...
5.5 Max. 4.6e5 cps.
1.63
4.6e5
4.5
5.0
5.5 Max. 2.0e5 cps.
2.94
2.0e5 1.9e5
4.4e5
1.63 min
825>765
2.94 min
1.8e5 1.7e5
3.8e5
808>509
1.6e5
3.6e5
1.5e5
3.4e5
1.4e5
3.2e5
Vincristine
3.0e5
Docetaxel
1.3e5 1.2e5 Intensity, cps
2.8e5 Intensity, cps
5.00e5
3.50e5
3.50e5
4.0e5
5.50e5
4.00e5
4.00e5
4.2e5
Tamoxifen
6.50e5
6.00e5
Intensity, cps
Intensity, cps
7.00e5
2.6e5 2.4e5 2.2e5
1.1e5 1.0e5 9.0e4
2.0e5
8.0e4
1.8e5
7.0e4
1.6e5
6.0e4
1.4e5
3.25
5.0e4
1.2e5 4.0e4
1.0e5 3.0e4
8.0e4
2.0e4
6.0e4
1.0e4
4.0e4
0.0 0.0
2.0e4 0.0 0.0
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Time,Final_IDA min XIC of +MRM (31 pairs): 260.961/154.100 Da from Sample 2 (Std 100 ppb_a) of Data07082012_Metodo samples.wiff (Turbo Spray)
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
5.5
XIC of +MRM (31 pairs): 854.000/569.000 Da from Sample 2 (Std 100 ppb_a) of Data07082012_Metodo Final_IDA samples.wiff (Turbo Spray)
Max. 4.6e6 cps.
Max. 1.7e5 cps.
3.01
1.82
4.6e6
1.7e5
4.4e6
1.82 min
4.2e6
3.01 min
1.6e5
261>154
4.0e6
1.5e5
3.8e6
854>569
1.4e5
3.6e6
1.3e5
3.4e6
Ifosfamide
3.2e6 3.0e6
Paclitaxel
1.2e5 1.1e5
2.8e6
1.0e5 Intensity, cps
Intensity, cps
0.5
Time, min
0.5
2.6e6 2.4e6 2.2e6 2.0e6
9.0e4 8.0e4 7.0e4
1.8e6 6.0e4
1.6e6 1.4e6
5.0e4
1.2e6
4.0e4
1.0e6
3.0e4
8.0e5 2.0e4
6.0e5 4.0e5
1.0e4
2.0e5 0.0 0.0
0.0 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
0.5
1.0
1.5
5.5
Time, min
Fig. 1 Extract ion chromatograms (XIC) of a standard mixture of target compounds at 100 ng/mL
2.0
2.5
3.0 Time, min
3.5
4.0
4.5
5.0
5.5
Development of a UPLC-MS/MS method for anticancer drugs Fig. 2 Recoveries obtained for selected pharmaceutical compounds with Oasis HLB, MCX, and MAX cartridge in MilliQ-Water. Spiking concentration of target compounds, 0.5 ng/mL
Recovery %
HLB 180.00 160.00 140.00 120.00 100.00 80.00 60.00 40.00 20.00 0.00
MCX MAX
MTX
all target compounds, and afterward they were loaded onto the cartridge under different conditioning steps. As expected, due to the extremely different physico-chemical properties of the target compounds, the cartridges that provided higher recoveries for all the compounds were the universal Oasis HLB (Fig. 2). Acidification of water samples as well as the conditioning water solution was tested because it can influence the recoveries for analytes, in particular, in the case of relative acid compounds, such as CIP or methotrexate, where better recoveries are expected in acid conditions. With this purpose, different pHs were evaluated by adding appropriate amounts of formic acid. Although the best results were achieved obtained at pH 2.8, still low recoveries for MTX, TAM, DOC, and PAC were obtained. Therefore, the addition of the chelating agent Na2EDTA solution at 0.1 M (pH =4–4.5) was also tested to avoid the potential interference of soluble metals with pharmaceutical compounds. The addition of EDTA was selected as the final extraction procedure due to the better recoveries obtained for all compounds (Fig. 3). As regards elution solvents, the use of ethyl acetate, a polar aprotic solvent with intermediate polarity, was compared with methanol, a polar protic solvent. Despite that ethyl acetate was expected to lead a better elution of non140.00
(%) Recovery
120.00 100.00 80.00 60.00 40.00 20.00 0.00 MTX AZA
CY
IF
TAM
Non EDTA
CIP DOC PAC ETO VIN EDTA
Fig. 3 Recoveries obtained for selected pharmaceutical compounds with Oasis HLB cartridge with and without using EDTA as additive in the samples. Spiking concentration of target compounds, 0.5 ng/mL
AZA
CY
IF
TAM
CIP
DOC
PAC
ETO
VIN
polar compounds, better recoveries were obtained for all compounds using methanol, even for the less polar TAM, DOC, and PAC. The optimal extraction method was finally optimized as follows: The 0.1 M EDTA solution was added to each sample (3 % sample volume), and 50 mL of this sample was loaded at 1 mL/min, in an Oasis HLB cartridge (200 mg, 6 mL) previously conditioned using 5 mL of methanol followed by 5 mL of 0.1 % formic acid at 2 mL/min. Afterward, the cartridge was rinsed with 5 mL of HPLC-grade water and subsequently dried under vacuum for 5 min to remove excess of water. Elution was performed with 10 mL (5+5 mL) of methanol at 1 mL/min. The extract was evaporated under gentle nitrogen stream using a ReactiTherm 18824 System (Thermo Scientific) and reconstituted with 500 μL of methanol–water (10:90, v/v). Finally, 5 μL of standard of internal standard mix at 10 ng/μL was added to the extract for internal standard calibration and to compensate possible matrix effect. Method validation Linearity of method was studied in range of 0.1–100 ng/mL for all selected compounds, obtaining satisfactory results. Residuals were always bellow 30 %, and correlation coefficients by linear curves were greater than 0.99 (see Table 3 and Electronic Supplementary Material Table S1). Isotopically labelled standards (IS) were selected as internal standard for the corresponding target compound except in case of DOC and PAC. The signal response of the latest were compared with those of the internal standards available, and [2H3]-MTX showed the most similar response. As it can be observed in Table 3, for each target compound, accuracy was determined by analyzing two concentration levels in quintuplicate: at low concentration (0.1 ng/mL) and at high concentration of linearity range (80 or 100 ng/mL, depending of the analyte). Intra-day and inter-day precision of the analytical procedure were also determined from five repeated injections of standard mixture (5 ng/mL) where the relative standard deviations (RSD, %) were lower than or
82 (±4) 115 (±12) 46 (±11) 93 (±2) 74 (±9) 73 (±12) 64 (±10) 48 (±10) 47 (±18) 52 (±12) 87 (±4) 82 (±17) 58 (±17) 90 (±2) 83 (±7) 88 (±13) 47 (±4) 46 (±9) 79 (±19) 59 (±8) 86 (±7) 95 (±7) 52 (±2) 106 (±8) 90 (±5) 77 (±6) 61 (±3) 78 (±8) 78 (±10) 74 (±5) 82 (±6) 82 (±4) 58 (±5) 86 (±3) 77 (±2) 96 (±5) 73 (±10) 58 (±1) 129 (±16) 115 (±15) 5.9 12.7 8.1 24.5 5.8 3.6 80.0 2.7 20.9 18.4
MQL (ng/L)
8.9 4.7 12.0 15.6 10.3 8.4 17.6 9.0 10.1 12.2 101 104 99 99 101 102 102 99 97 94
3.0 2.4 4.0 2.2 3.7 1.3 10.9 4.2 3.3 1.1
MDL (ng/L) Reprod. (RSD %) (n=5) Repeat (RSD %) (n=5)
82 125 92 81 114 121 117 110 92 91
Linearity range (ng/mL)
0.10–80 0.10 –100 0.10 –100 0.10–80 0.10–80 0.10–80 0.10–80 0.10–80 0.10–80 0.10–80 MTX AZA CIP VIN IF CY ETO TAM DOC PAC
0.03 0.1 0.3 0.4 0.04 0.15 1.2 0.17 0.9 1.0
Accuracy at high conc. (%) Accuracy at low conc. (%)
With the aim of correcting matrix effects, the use of isotopically labelled compounds as IS was recommended, but, because of their high cost, a matrix-matched calibration approach can alternatively be applied, especially in the case of complex matrices, such as sewage waters or biological matrices (plasma, blood) [39]. These two different approaches as well as a combined strategy using both IS and matrixmatched calibration curve were tested to evaluate best protocol to correct the matrix effects. Matrix effects were thus evaluated in two sets of calibration curves (see Table 4 and Table S1) obtained both with and without using an IS (Set L1 and Set L2, respectively). Within each set, calibration curves were built preparing the standards either in the mobile phase solvent (neat curves) or in the post-extraction samples (matrix-matched curves). Evaluation of the matrix effects (ME %) were performed by comparing the Table 4 % Matrix effect (ME) calculated using the Eq. 1, which compares the slopes of neat standards and matrix match curves Target compound
Target compound
ID (pg injected)
around 15 % for the majority compounds, with the exception of ETO which showed a RSD below of 20 %. MDL and MQL were satisfactory: MDLs achieved for all the types of water analyzed ranged from 0.8 to 24 pg/mL, whereas MQLs ranged from 2.7 to 80 pg/mL. The MDLs and MQLs found for hospital and influent wastewaters did not show significant differences, and for that reason, only the limits from hospital wastewater are shown in Table 3. Recoveries performed in both matrices (hospital and urban wastewater) at three spiking levels, 1, 0.5, and 0.1 ng/mL, were all satisfactory (Table 3), and no significant differences were observed between the recoveries of the hospital effluent and wastewater influent. Matrix effects
1.8 3.8 2.4 7.4 1.7 1.1 24.0 0.8 7.5 5.5
Wastewater influent Hospital effluent
% Recovery (RSD %; n=3) spiked at low level
% Recovery (RSD %; n=3) spiked at medium level Hospital effluent % Recovery (RSD %; n=3) spiked at high level Hospital effluent Precision Accuracy
Table 3 Quality parameters of SPE-UPLC-MS/MS method developed: linearity ranges, instrumental detection limit (ID), accuracy (at high and low concentrations of linearity range), precision (repeatability and reproducibility), MDL (method limit detection), MQL (method quantification limit), and recovery obtained in hospital and urban wastewaters at high (1 ng/mL), medium (0.5 ng/ mL), and low (0.1 ng/mL) spiked levels tested for each type of water
L. Ferrando-Climent et al.
MTX AZA CIP VIN IF CY ETO TAM DOC PAC
I.S.
[2H3]-MTX [13C4]-AZA [2H8]-CIP [2H3]-VIN [2H4]-CY [2H4]-CY [2H3]-ETO [13C6]-TAM [2H3]-MTX [2H3]-MTX
ME (%) Set L1 (IS)
Set L2 (non-IS)
100 84 157 25 82 84 194 83 113 81
66 50 778 13 99 99 138 445 72 55
Two approaches were employed: using IS (Set L1) and not using IS (Set L2)
Development of a UPLC-MS/MS method for anticancer drugs
slopes of these curves as indicated in the section on “Validation study.” A ME value higher than 100 % indicates a signal enhancement during ionization in the MS detector, whereas a ME value lower than 100 % indicates ionization suppression. In the case of calibration curves performed without IS (Set L2), most of the target compounds exhibited either ionization enhancement or suppression due to the complexity of effluent sample. On the contrary, the use of IS (Set L1) permitted to obtain ME values around 100 % (within a 20 % of deviation). However, in the case of VIN, CIP, and ETO, the use of IS was not capable to correct the matrix effects, and ME values were 25, 157, and 194, respectively. In the light of these results, the matrix-matched approach was recommended for the analysis of selected cytostatic agents in wastewaters. Stability study Results from the stability study performed using wastewater samples under conditions described in section “Stability study” are shown in Figs. 4, 5, and 6. Samples preserved with formaldehyde and stored at −20 °C (PRE) showed the most limited storage stability; target compounds were stable only for 1 week (Fig. 4). The presence of formaldehyde, instead of helping in preservation, has contributed to their degradation. In the case of the samples stored in the cartridge at −20 °C (CART), all the compounds remained stable till the third month, except for ETO which exhibited an increase of its concentration after first month (Fig. 5). Various compounds as CIP, ETO, and TAM showed very high concentrations after 6 months storage whereas DOC and azathioprine presented very low concentrations at the same sampling time. Diminution of concentration along the storage period can be due to lack of stability of the compounds inside the cartridge or to conformation changes in the sorbent structure. Processes such as the generation of new physical preferential cavities/channels in the packing material of cartridge along the freeze and thaw steps can happen and cause the increase of recoveries of
Fig. 4 Stability results along the time in experiments performed for wastewater samples preserved with formaldehyde and stored at −20 °C, encoded as PRE. Initial spiked concentration, 0.5 ng/mL
the compounds, affecting the final concentration measured. Finally, in the case of the samples stored at −20 °C (WS), the compounds cyclophosphamide, ifosfamide, ETO, and CIP remained completely stable along 6 months, whereas some degradation was observed for DOC and AZA after 3 months of storage (Fig. 6). Additionally, TAM showed an odd behavior, because it exhibited an abrupt decrease between first and third months followed by a further increase till initial concentration at the sixth month. In summary, the storage protocol recommended in cases where the sample analysis cannot be performed right after sampling is either the storage of wastewater samples at −20 °C or the storage of the cartridge at −20 ° C, in both cases, for not longer than 1 month. Since the standard mixtures kept at −20 °C did not show any degradation along 6 months (data not shown), the concentration variations observed in the samples stored through different conditions can only be attributed to the effects of the preservation protocol and conditions applied. Application of the method to environmental water samples To demonstrate the applicability of the analytical procedure developed, wastewater influents from three hospitals and from two WWTPs were analyzed. A summary of the levels found for target compounds in each of the samples analyzed are shown in Table 5. No significant differences in the levels detected of anticancer drugs were identified between the effluents from hospitals and regular urban effluents, and therefore, hospitals cannot be considered as the primary source of this type of contaminants. As pointed out before, most of patients treated with anticancer drugs leave the hospital after their administration [9], and therefore, the cytostatic compounds are equally released via hospital or domestic wastewater. CIP, CY, IF, ETO, MTX, and VIN were found in hospital effluents and influent wastewater in the range of concentrations that other studies have reported till the moment [27–29, 46]. CIP, in particular, was found at very high levels in both hospital and influent wastewater. However, these high values can not only be attributed to
L. Ferrando-Climent et al. Azathioprine CART
1.20
Cyclophosphamide CART
1.00
Ifosfamide CART
C (ng/mL)
Fig. 5 Stability results along the time in experiments performed for wastewater samples loaded in cartridge and stored at −20 °C, encoded as CART. Initial spiked concentration, 0.5 ng/mL
0.80 Ciprofloxacin CART 0.60 Tamoxifen CART 0.40 Etoposide CART 0.20 Docetaxel CART 0.00 Initial
1 week 1 month 3 months 6 months Time
chemotherapy treatments since CIP is also used as antibiotic to treat a number of human infections as well as for veterinary treatments. CY and IF, on the other hand, are at the moment the most studied cytostatic agents, and despite the low levels found in this study, they are still important contaminants because of their known toxicity, persistence in the environment, and ubiquity: They were detected in almost all the samples analyzed. TAM was found in all the samples studied and at higher levels than reported in the rest of existing studies in sewage samples to date [47–49]. Because of those high levels found, they have to be taken very much into consideration in terms of environmental risk assessment. In fact, because of its suspected endocrine disruption effects and high bioaccumulation potential, TAM has been recently included with 13 other substances as priority compounds that cumulate several risk factors for aquatic ecosystems [50]. Some other studies have already reported levels quite high in river waters up to 200 ng/L [51]. Finally, this is the first time to our knowledge that AZA, DOC, and PAC have been analyzed and detected in hospital effluents and wastewater influents. In terms of potential ecotoxicity, EC50 of 0.8 μg/mL (assay: luminescence cellbased dose response HTS to identify compounds cytotoxic to DRD non-viral oncogenic fibroblast) and 18 μg/mL (induced tubulin assembly assay) have been reported for AZA and PAC, respectively, whereas DOC has IC50 of 2.5 ng/mL (human Burkitt lymphoma CA46 cells assay) [52]; web
Screening of metabolites in real samples A new, fast, and feasible approach based on the information acquisition tool (IDA) was used for the screening of human metabolites and transformation products of anticancer drugs in hospital wastewater samples. Several human metabolites were selected for this targeted search approach, based on the information available about anticancer drug metabolites of the compounds considered in the analytical method. Some of them were suspected to be present in the wastewater samples analyzed, since the parent compound had been already found there. The presence of three selected metabolites was thus searched through the chromatograms obtained from the analysis of hospital samples: (1) hydroxy-tamoxifen, the main human metabolite known of TAM [10, 53]; (2) 4,4-dihydroxy desmethyltamoxifen (endoxifen), which belongs to the same chemical and therapeutic family than TAM and which is also a secondary human metabolite of TAM [54], and (3) carboxyphosphamide, a known human metabolite of CY [55, 56]. The presence of these compounds was explored by searching their theoretical molecular ions in the sample chromatograms acquired using the IDA method: ions m/z 389, m/z 390, and m/z 293 corresponding to hydroxy-tamoxifen, 4,4-dihydroxy desmethyltamoxifen, and carboxyphosphamide, respectively.
1.20
C (ng/mL)
Fig. 6 Stability results along the time in experiments performed for wastewater samples stored at −20 °C, encoded as WS. Initial spiked concentration, 0.5 ng/mL
databases consulted were: http://www.bindingdb.org/ and http://pubchem.ncbi.nlm.nih.gov/).
Azathioprine WS
1.00
Cyclophosphamide WS
0.80
Ifosfamide WS Ciprofloxacin WS
0.60
Tamoxifen WS 0.40
Etoposide WS
0.20
Docetaxel WS
0.00 Initial
1 week
1 month 3 months 6 months Time
Development of a UPLC-MS/MS method for anticancer drugs Table 5 Levels of target compounds (nanograms per liter) in hospitals effluents (as A, B, C, and D) and in urban WWTPs influents (E, F, and G) Target compounds (n=3)
Hospital effluent A (ng/L)
Hospital effluent B (ng/L)
Hospital effluent C (ng/L)
Hospital effluent D (ng/L)
Influent wastewater E (ng/L)
Influent wastewater F (ng/L)
Influent wastewater G (ng/L)
CIP AZA CY IF TAM DOC PAC ETO VIN MTX
679.1±21.1 21.6±2.1 43.4±4.5 31.5±7.5 59.5±3.5 n.d. n.d. 97.5±19.1 49.1±7.2
