Reagents and materials
Standard NP (CAS 25154-52-3, 99.9% purity) and the internal standard 4–n–nonylphenol (4–n–NP) (CAS 104-40-5, > 99% purity) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). HPLC grade acetone, acetonitrile and ethyl acetate were obtained from Fisher Scientific (Fair Lawn, NJ, USA). β–Glucuronidase and sulfatase were acquired from Sigma–Aldrich (St. Louis, USA). Analytical grade ammonia, ammonium fluoride, acetic acid, sodium acetate hydrochloric acid and other auxiliary reagents were all obtained from the National Pharmaceutical Group Chemical Reagent Co. Ltd. (Shanghai, China). Sample extraction and purification were completed with an Oasis HLB cartridge (Milford, MA, USA). In addition, ultrapure water was used throughout the study.
Preparation of standard solutions
NP and 4–n–NP (1 mg/mL) standard stock solutions were prepared in methanol, and then the stock standard solutions were diluted with methanol (MeOH) to obtain intermediate standard solutions of NP and 4–n–NP (10 μg/mL), which were further diluted and mixed using methanol to prepare 100 ng/mL mixed intermediate standard solutions. A series of mixed standard working solutions were prepared by diluting the mixed intermediate standard solutions with MeOH before use. All solutions described above were stored at 4 °C.
Study site and population
Two communities were chose as study sites. These communities were representative of an urban and a rural area in Wuhan, China. One (the Zongguan communities, S’3) located in the metropolitan downtown center of Wuhan with well-developed industry and commerce in the surrounding areas, industrial and domestic sources are all the sources of NP in there. And another (the Liangzi Island, S’1) located on the periphery of Wuhan, is an isolated island surrounded by Liangzi Lake without any industrial activities, the permanent residents are only 800, and no apparent source of NP presented in there. The corresponding drinking water sources of these two community were Han River water (S’4)and lake water (S’2), respectively. The specific sites of this study are shown in Fig. 1.
A total of 127 participants aged between 18 to 90 years were selected: 58 from the Zongguan communities and 69 from the Liangzi Island. The age range (18-88) was select based on the age distribution of the population in the two areas, besides 3-17 year olds spend most of their day in school where basically not located in study area. Participants were chosen based on the inclusion that they had lived in the area for more than one year and no household water purification facilities had been installed in their home. Informed consent and a brief questionnaire (including data on age, sex, body size, and weight to obtain BMI status) were obtained from all participants before sampling.
Sample collection
Water samples from the drinking water source of the urban and rural areas were collected 100 meters upstream from the drinking water intake. The sampling locations of household tap waters were determined as follows. Because the residential building of Zongguan Community was a seven-floor building, which involved a secondary water supply, we randomly selected three household from the first to third floors and three household from the 4th to 7th floors for sampling. The Liangzi Island residential building was a low-level house, and three house was randomly selected for sampling. Of course, all households were selected from participants who had provided urine samples. Two liters of water sample was collected in a 2.5 L brown glass bottle from each location and stored at 4 °C until pretreatment. This process was repeated four times at an interval of 6 hours each time, and a total of 48 water samplings were collected.
Urine samples in the two areas was completed on the same day in July 2019 with water sample. All participants were asked to provide first-morning urine voids in a 100 ml polypropylene container. And urine samples were stored at -80 °C before analysis. The study was reviewed and approved by the Ethics Committee of Hubei Center for Disease Control and Prevention (Wuhan, China).
Sample preparation
The water samples were prepared in two steps as follows. Filtration: Water samples (500–1000 mL) were weighed accurately and passed through a 0.45 μm glass fiber microporous filter membrane to remove suspended solids. Extraction: Enrichment was performed on an OASIS HLB column (200 mg, 6 ml) previously conditioned with 5 mL of methanol and 5 mL of water. The analytes were eluted from the SPE cartridges with 10 mL of methanol, which were reconstituted in methanol to a final volume of 0.5 mL and passed through a 0.22 μm polytetrafluoroethylene (PTFE) syringe filter before ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) analysis.
The urine samples were prepared briefly as follows: 50 ng of 4–n–NP was added to 2 ml of urine, followed by buffering with 100 µl of sodium acetate (pH 5.5). After the addition of 10 µl of β‐glucuronidase/sulfatase, the mixture was incubated at 37 °C for 3 h. After cooling to room temperature, the dispersed sample solution was centrifuged at 3000 rpm. The supernatant was acidified with hydrochloric acid to a pH of 2–3 and then transferred to OASIS HLB cartridges (60 mg, 3 mL), which were previously conditioned with 5 mL of dichloromethane–methanol (9:1 v/v), 3 mL of methanol, and 3 mL of water (pH 3.0–3.5, adjusted with HCl). Initially, 3 ml of 5% methanol was used for cartridge washing, and subsequently, 5 ml of dichloromethane-methanol (9:1 v/v) was used for elution. Finally, the eluate was evaporated to dryness under a stream of nitrogen and reconstituted in 0.5 ml of methanol for UPLC analysis.
UPLC–MS/MS conditions
UPLC analyses on water samples were carried out using an Agilent 1290 liquid chromatograph (Agilent, CA, USA) with an Agilent Eclipse Plus C18 column (50 mm*2.1 mm, 1.8 μm). Mobile phases A and B were 1 mmol/L ammonium fluoride aqueous solution and 100% methanol, respectively. The system was run with a gradient program: 85% A held for 0.0–1.0 min, 85% A linear reduction to 5% A from 1.0–5.0 min, 5% A held from 5.0–8.0 min, and 5% A linear increase to 85% A from 8.0–8.1 min. The flow rate was 0.3 mL/min, the column temperature was 35 °C, and the injection volume was 5 µl. Mass spectrometry was carried out on a Triple Quad™ 3500 mass spectrometer (AB SCIEX, MA, USA). The selected parameters were as follows: ion source temperature: 550 °C, spray voltage: 5500 V, curtain gas: 35 psi, impact gas: 7 psi, and atomizing gas and auxiliary heating gas: 55 psi.
UPLC analyses on urine samples were carried out using a Waters Alliance Acquity-E2695 high-performance liquid chromatography system (Waters, MA, USA), and the analytical column was a ZORBAX 300SB–C18 (4.6 mm*150 mm, 5 μm). Mobile phases A and B were methanol and water, respectively. The gradient elution program used was as follows: 35% A–90% A (2 min), 90% A–100% A (3 min) and held for 2 min, and 100% A–35% A (0.5 min) and held for 2.5 min. The flow rate was 0.3 ml/min, the column temperature was 20 °C, and the injection volume was 10 µl. Mass spectrometry was carried out with a Waters Xevo TQD Triple quadrupole mass spectrometer (Waters, MA, USA) using electrospray ionization (ESI) in MRM scan mode. The selected parameters were as follows: capillary voltage: 3.0 kV, desolvation temperature: 350 °C, and desolvation gas: 800 L/h.
The optimum cone voltage, collision energy and the characteristic ions for analytes and the internal standards are presented in Table 1.
Urinary creatinine adjustment
To normalize individual variation due to the differing hydration states of each participant at the time of sampling (Wang et al., 2016), NP concentrations were adjusted by creatinine levels. Therefore, urinary NP concentrations were presented in the following two forms: a) as an uncorrected concentration (nanograms per liter, ng/L) and b) as a concentration corrected according to creatinine levels (micrograms per gram creatinine, µg/g creat).
Validation study
The analytical method was validated to illustrate the LOQ (limit of quantitation), accuracy, precision and recovery of the measurements. Standard solutions of NP and 4–n–NP at low, medium and high concentrations were added to the samples, and 6 parallel samples were set at each concentration to calculate the spiked recovery rate and relative standard deviation (RSD). The limits of detection (LODs) and LOQs of the method were determined by three times and ten times the signal–to–noise ratios, respectively. As shown in Table 2, the precision and accuracy of this method met the requirements of detection.
Statistical analysis
Concentrations below the LOD were counted as half the LOD. All statistical analyses were performed with SPSS software, version 16.0 for Windows (SPSS Inc., USA). An independent-samples t test was employed to test the difference in NP concentration in drinking water between the two areas. A nonparametric Mann–Whitney U test was used to analyze the differences in urine NP concentration between two areas. Analysis of variance (one-way ANOVA) was used to compare the baseline characteristics of the participants in two areas. Statistical significance was accepted at P < 0.05 for all comparisons.