Detection of pharmaceutically active compounds in tap water samples by direct injection HPLC/MS-MS: A danger signal in de�ciency in residue management.

The growing and decelerated presence of pharmaceutically active compounds that affect networks of tap water is further studied due to the risks to the health public and the environment. Therefore, a direct injection-HPLC/MS-MS method is developed for the simultaneous determination of 16 active pharmaceutical compounds in tap water samples: amoxicillin, ampicillin, cephalexin, cefotaxime, cefuroxime, cipro�oxacin, clarithromycin, clindamycin, chloramphenicol, cyproterone, erythromycin, �utamide, spironolactone, sulfamethoxazole, tamoxifen, and trimethoprim. The implemented method obtained limits of detection (LOD) of 0.3 – 15 µg/L and quanti�cation limits (LOQ) from 1.5 to 50 µg/L, recoveries percentages in the range of 70 - 125%, time retentions ≤ 2.1 min and allowed the analysis of 22 tap water samples where the highest concentrations corresponding to amoxicillin (147 µg/L) and cipro�oxacin (44 µg/L). The �ndings in this research could be a precedent for establishing safe levels of these compounds and increasing the standards for quality tap water.


Introduction
Water pollution plays a critical role in the maintenance of the health public worldwide.Data from several studies suggest that the high levels of prevalence of emergent pollutants in water are growing continuously [1][2][3].Currently, pharmaceutical compounds are classi ed as priority study emerging pollutants due to a potential risk to environmental and public health reported previously [4].
Traditionally, for the determination of pharmaceutical products in water samples has been implemented diverse techniques such as gas and liquid chromatography [5], which often require pretreatment sample steps for extraction and enrichment of target analytes, being the solid phase extraction more used [6][7][8].However, solvent consumption, higher analysis time and costs, multiple human errors, and poor extraction of high-polar pharmaceutical compounds are disadvantages for the use of this sample pretreatment, and thus have been studied methods by the direct injection of samples for analysis through high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) [9,10].This investigation includes the development of a direct injection-HPLC/MS-MS multianalyte method for the determination of 16 different pharmaceutical compounds in the tap water from 22 sites of the metropolitan area of Guadalajara.The selected pharmaceutical compounds have been considered recalcitrant compounds and previously detected in diverse aqueous compartments of the world [11][12][13][14], being moreover recent interest in the co-presence of antibiotics, hormones, oncologic, and personal care products in water samples study [15] due to a high environmental risk and the evidence of inadequate treatment of wastewater.The ndings of this research work could be a precedent for the use of preliminary methods in the determination of pharmaceutical compounds in water samples; therefore, facilitates the development of framework regulatory and establishes safe levels of these compounds increasing the standards for quality tap water and decreasing the signi cant human health and environmental risks.

HPCL/MS-MS analysis conditions
For the analysis of the tap water samples, an equipment 1200 series high-resolution liquid chromatograph coupled to a model 6430B triple quadrupole mass-mass spectrometer with an Electrospray interface of Agilent Technologies was used.Operated Electrospray Ionization (ESI) source with a capillary voltage of 4000 V, clouding gas temperature of 350 ºC, gas ow of 12 L min -1 , and nebulizer gas pressure of 25 psi.The chromatographic conditions used were a mobile phase acetonitrile 95 % (v/v): water acidi ed with formic acid 0.1 % (v/v), injection volume: 20 µL, mobile phase ow: 0.5 mL/min and a column Zorbax Eclipse XDB-C18 Rapid Resolution 2.1 x 50 mm, 3.5 microns, Agilent, Sample collection and pre-treatment Tap water samples were collected from terminals at 22 different points in the city of Guadalajara (Figure 1).Brie y, the points sample were Centro, Alcalde, Centro Médico, Mezquitán, CUCEI, Olivos, Revolución, and Independencia; also, in different regions inside of the metropolitan zone: Zapopan, Tlaquepaque, Tonalá, Chapala and Tequila.According to Guidelines for drinking-water quality (4th ed.) and NOM-014-SSA1-1993 collected samples were transported to the laboratory in hermetic bottles at 4°C and stored a -18°C until analysis.The tap water samples were just ltered through a 0.45 nm nylon lter due to the small presence of large-size solids or organic matter.
For direct injection by HPLC/MS-MS, tap water samples by spiking 5 mL of water from sampled points with appropriate solutions to obtain 50 mL standard concentrations at 1, 3, and 5 mg/L were prepared.Then, tap water samples were diluted with 5 mL of acetonitrile and puri ed for a week through extraction columns.To assess the applicability and matrix effects of the method, the recovery percentages were calculated, each water sample was spiked with 50 mg/L of standard solutions and calculated by equation preview reported [16]: (C 0 -C f / C 0 ) * 100, where C 0 and C f are the pharmaceutical compounds concentrations before and after the direct analysis procedure.
Therefore, this analytical method requires a short analysis time (< 2.2 min), reduced use of solvents for extraction of target compounds, an easy step pre-treatment water sample procedure, and the capacity for the detection of co-occurrence of antibiotics and endocrine disruptors in tap water samples, two of the most studied pharmaceutical active compounds and recognized as emerging pollutants associated with potentially adverse effects on the aquatic environment [4,14,15].

Method Validation
Table 3 summarizes the obtained analytical parameters using the developed method by direct injection-HPLC/MS-MS in this work.The linearity of the method was calculated by obtaining determination coe cients (r 2 ) higher than 0.978.The limits of detection (LOD) and quanti cation (LOQ) were calculated according to preview reports considering the concentrations at signal-to-noise ratios of 3 and 10, respectively [17].The LODs and LOQs obtained were a range of 0.2 -6.0 µg/L and 0.3 -20.0 µg/L, respectively.The precision of the method (repeatability) was expressed in terms of %RSD (n = 3 injections) ranging from 0.9 to 29.1, which is according to Harmonized Guidelines for Single Laboratory Validation of Methods of Analysis (IUPAC) and Analytical Control Commission and Expansion of Coverage (CCAYAC-P-058) that establish criteria acceptation ≤ 30% for pollutants and residues (as pharmaceutical compounds) in water samples [17,18].Finally, all the obtained percentages recoveries were satisfactory inside the range of 74 to 125 µg/L in agreement with the EPA-821-R-08-002 allowing 70 -130% recoveries [19] without important signal suppression, due to the matrix effect that could be expected when the samples were analyzed by direct injection, Table S1 shows the linearity and retention times results obtained of a calibration curve in the matrix and were consistent with those obtained in the validation.These ndings corroborate that despite the ILIS (Isotope labeled internal standards) not being used the precision, accuracy, and matrix effect of the method was acceptable and, therefore could be applied as a fast alternative for the determination of pharmaceutical compounds in tap water.

Detection of pharmaceutical compounds in tap water samples
The developed and validated method in this work was applied to the analysis of 22 tap water samples.From this analysis by direct injection-HPLC/MS-MS was possible to detect and quanti cation of twelve from sixteen initial pharmaceutical compounds, which could suggest the rapid, low cost, and successful applicability as an alternative for detection of emerging pollutants in sample water with lower organic charge.
The results obtained from the analysis of tap water samples are shown in Table 4, as can be seen, the occurrence of pharmaceutical compounds in tap water samples was from 69 -81% in overall samples, except for cefotaxime, cefuroxime, ampicillin were below the LODs, and trimethoprim was not detected in the samples.These ndings represent an alarm signal for the co-occurrence of pharmaceutical compounds indicating that the management network distribution of tap water has been not adequate for the elimination of this type of pollutants, in addition to the risk assessment that has been previously reported [15,[20][21][22][23].The pharmaceutical compounds detected at higher concentrations were amoxicillin and cipro oxacin in ranges of 2 -147 µg/L and 22 -44 µg/L, respectively.Both compounds are classi ed as antibiotics the priority used in therapy human and veterinary, therefore have been highly detected, reported, and related with antimicrobial resistance in diverse studies worldwide [11,12,[24][25][26][27][28].Currently, cipro oxacin and amoxicillin are included in the European Union watch list of substances for its monitoring in the eld of water policy [26,29].
Another important nding is that higher concentrations of pharmaceutical compounds were detected in samples from zones nearby hospitals (Centro Médico and Independencia) according to previous studies have reported signi cantly higher concentrations of pharmaceutical compounds in water discharge hospital samples [30].Regarding Chapala Lake, the higher concentrations of pharmaceutical compounds could suggest unlawful discharger e uents.The most obvious nding to emerge from the analysis is the need to count on alternative methods (such as direct injection) especially when the analyzed samples come from networks of tap water, which could offer rapid and sensitive detection of these emerging pollutants.Finally, Table (S2) shows the comparative results for detecting antibiotics mixed in water samples, showing the maximum concentrations previously reported in studies offering comparable results according to those obtained in this work.

Conclusions
The results of this work show the importance of the development of methods that represent an alternative reliable, fast, and low cost for the determination of pharmaceutical products in lower complexity water samples.The implemented methodology by HPLC/MS-MS direct injection allowed make evident the occurrence of pharmaceutical compounds in overall tap water samples of 22 diverse sites.
The highest concentrations were antibiotics (amoxicillin and cipro oxacin) which could suggest the need for further legislation.It is necessary to continue conducting studies to understand the risk that the presence of pharmaceutically active compounds implies in human health and ecosystems, mainly in developing countries where the residue management, as well as the construction of infrastructure for its treatment, happens more slowly than disposition and consumption of pharmaceuticals.

Table 2 .
Operating conditions of the mass spectrometer and retention times of HPLC/MS-MS method.

Table 3 .
Analytical parameters of the developed HPLC/MS-MS method for detection of pharmaceutical compounds in tap water samples.
a Low and high level corresponding at high and low concentrations of linear range (n= 3 replicates).

Table S1 .
Linearity and retention times of calibration curves in matrix water sample.
a Retention time of standards solution (50 mg/L) in the matrix (tap water).bThe same points of calibration curve were diluted in tap water.

Table S2 .
Pharmaceutical compounds detected in tap water samples worldwide previously reported.