Reaction of β-blockers and β-agonist pharmaceuticals with aqueous chlorine. Investigation of...

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ORIGINAL PAPER Reaction of β-blockers and β-agonist pharmaceuticals with aqueous chlorine. Investigation of kinetics and by-products by liquid chromatography quadrupole time-of-flight mass spectrometry José Benito Quintana & Rosario Rodil & Rafael Cela Received: 10 November 2011 / Revised: 16 December 2011 / Accepted: 29 December 2011 / Published online: 2 February 2012 # Springer-Verlag 2012 Abstract The degradation of two β-blockers (atenolol and propranolol) and one β-receptor agonist (salbutamol) during water chlorination was investigated by liquid chromatographymass spectrometry (LC-MS). An accurate-mass quadrupole time-of-flight system (QTOF) was used to follow the time course of the pharmaceuticals and also used in the identification of the by-products. The degradation kinetics of these drugs was investigated at different concentrations of chlorine, bromide and sample pH by means of a BoxBehnken experimental design. Depending on these factors, dissipation half-lives varied in the ranges 68145 h for atenolol, 1.333 min for salbutamol and 428362 min for propranolol. Normally, an increase in chlorine dosage and pH resulted in faster degradation of these pharmaceuticals. Moreover, the presence of bromide in water samples also resulted in a faster transformation of atenolol at low chlorine doses. The use of an accurate-mass high-resolution LC- QTOF-MS system permitted the identification of a total of 14 by-products. The transformation pathway of β-blockers/ago- nists consisted mainly of halogenations, hydroxylations and dealkylations. Also, many of these by-products are stable, depending on the chlorination operational parameters employed. Keywords Pharmaceuticals . Chlorination . By-products . Liquid chromatographymass spectrometry (LC-MS) . Time-of-flight (TOF) José Benito Quintana was a postdoc at the Technical Univer- sity of Berlin (20042006) and later a research associate (Isidro Parga Pon- dalprogram) at the University of A Coruña (20062008). He was awarded a Ramón y Cajalresearch fellowship in the area of chemistry, and since 2008, he has held this position at the Univer- sity of Santiago de Compostela. His cur- rent research interests are emerging pollutants in water, with particular em- phasis on drugs of abuse; environmental transformation processes, e.g. transfor- mation due to chlorination; LC-MS and GC-MS, including high-resolution analyzers; and the development of novel extraction techniques. Rosario Rodil is currently a researcher of Analytical Chemistry at the University of Santiago de Compostela (Spain) supported by the Ramon y Cajalprogram. She received a Ph.D. degree from Santiago de Com- postela University (Spain) in 2005 and then she had two postdoctoral appoint- ments at the Helmholtz Centre of Envi- ronmental Research-UFZ Leipzig (Germany; 20052006) and at the Uni- versity of A Coruña (Spain; 20062009). Rosario Rodils research interests in- clude the development of analytical methodology for the determination of emerging organic pollutants, the study of the presence and distribution of emerging pollutants in the marine environ- ment and transformation of pollutants during water treatment processes. Published in the special issue Young Investigators in Analytical and Bioanalytical Science with guest editors S. Daunert, J. Bettmer, T. Hasegawa, Q. Wang and Y. Wei. Electronic supplementary material The online version of this article (doi:10.1007/s00216-011-5707-7) contains supplementary material, which is available to authorized users. J. B. Quintana (*) : R. Rodil (*) : R. Cela Department of Analytical Chemistry, Nutrition and Food Science, IIAAInstitute for Food Analysis and Research, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain e-mail: [email protected] e-mail: [email protected] Anal Bioanal Chem (2012) 403:23852395 DOI 10.1007/s00216-011-5707-7

Transcript of Reaction of β-blockers and β-agonist pharmaceuticals with aqueous chlorine. Investigation of...

ORIGINAL PAPER

Reaction of β-blockers and β-agonist pharmaceuticalswith aqueous chlorine. Investigation of kineticsand by-products by liquid chromatography quadrupoletime-of-flight mass spectrometry

José Benito Quintana & Rosario Rodil & Rafael Cela

Received: 10 November 2011 /Revised: 16 December 2011 /Accepted: 29 December 2011 /Published online: 2 February 2012# Springer-Verlag 2012

Abstract The degradation of two β-blockers (atenolol andpropranolol) and one β-receptor agonist (salbutamol) duringwater chlorination was investigated by liquid chromatography–mass spectrometry (LC-MS). An accurate-mass quadrupoletime-of-flight system (QTOF) was used to follow the timecourse of the pharmaceuticals and also used in the identificationof the by-products. The degradation kinetics of these drugs wasinvestigated at different concentrations of chlorine, bromideand sample pH by means of a Box–Behnken experimentaldesign. Depending on these factors, dissipation half-lives variedin the ranges 68–145 h for atenolol, 1.3–33 min for salbutamoland 42–8362 min for propranolol. Normally, an increase inchlorine dosage and pH resulted in faster degradation of thesepharmaceuticals. Moreover, the presence of bromide in watersamples also resulted in a faster transformation of atenolol at lowchlorine doses. The use of an accurate-mass high-resolution LC-QTOF-MS system permitted the identification of a total of 14by-products. The transformation pathway of β-blockers/ago-nists consisted mainly of halogenations, hydroxylations anddealkylations. Also, many of these by-products are stable,depending on the chlorination operational parameters employed.

Keywords Pharmaceuticals . Chlorination . By-products .

Liquid chromatography–mass spectrometry (LC-MS) .

Time-of-flight (TOF)

José Benito Quintanawas a postdoc at the Technical Univer-sity of Berlin (2004–2006) and later aresearch associate (“Isidro Parga Pon-dal” program) at the University of ACoruña (2006–2008). He was awardeda “Ramón y Cajal” research fellowshipin the area of chemistry, and since 2008,he has held this position at the Univer-sity of Santiago de Compostela. His cur-rent research interests are emergingpollutants in water, with particular em-phasis on drugs of abuse; environmentaltransformation processes, e.g. transfor-

mation due to chlorination; LC-MS and GC-MS, including high-resolutionanalyzers; and the development of novel extraction techniques.

Rosario Rodilis currently a researcher of AnalyticalChemistry at the University of Santiagode Compostela (Spain) supported by the“Ramon y Cajal” program. She receiveda Ph.D. degree from Santiago de Com-postela University (Spain) in 2005 andthen she had two postdoctoral appoint-ments at the Helmholtz Centre of Envi-ronmental Research-UFZ Leipzig(Germany; 2005–2006) and at the Uni-versity of A Coruña (Spain; 2006–2009).Rosario Rodil’s research interests in-clude the development of analytical

methodology for the determination of emerging organic pollutants, the studyof the presence and distribution of emerging pollutants in the marine environ-ment and transformation of pollutants during water treatment processes.

Published in the special issue Young Investigators in Analytical andBioanalytical Science with guest editors S. Daunert, J. Bettmer, T.Hasegawa, Q. Wang and Y. Wei.

Electronic supplementary material The online version of this article(doi:10.1007/s00216-011-5707-7) contains supplementary material,which is available to authorized users.

J. B. Quintana (*) : R. Rodil (*) : R. CelaDepartment of Analytical Chemistry, Nutrition and Food Science,IIAA—Institute for Food Analysis and Research,University of Santiago de Compostela,15782 Santiago de Compostela, Spaine-mail: [email protected]: [email protected]

Anal Bioanal Chem (2012) 403:2385–2395DOI 10.1007/s00216-011-5707-7

Introduction

Many pharmaceuticals have been detected in the last 15 yearsas important water contaminants [1–4]. Among the differentpharmaceutical classes, β-adrenergic receptor antagonist (orsimply, β-blockers) and β-adrenergic receptor agonist (inshort, β-agonists) drugs act on the different β-adrenergicreceptors, located at different muscles, such as cardiac, andsmooth muscle (e.g. pulmonary tissue). Hence, β-blockersand β-agonists are used in order to block or stimulate, respec-tively, the response of one or several β-adrenergic receptors.Therefore, their therapeutic action is mostly focused on cardiac(β-blockers) and respiratory (β-agonists) disease treatment [5].

As a result of their common usage to control chronicdiseases, they are frequently detected in municipal waste-water samples in the mid-nanograms per litre to mid-micrograms per litre range [6–10]. Then, ought to the highinfluent concentrations and incomplete removal duringwastewater treatment, they are released to surface water,where they are detected in the nanograms per litre range[6–12], and can finally reach potable water if drinking watertreatment is unsuccessful in pharmaceuticals removal. In theparticular case of β-blockers/agonist, they have beendetected at low concentrations in finished drinking water(up to 20 ng/L) [9, 13]. Although Pinkston and Sedlak haveshown that β-blockers can react with chlorine quite rapidly,they expected them to be mostly transformed to thecorresponding chloramine and N-dealkylated product [14],but this fact has not been confirmed. On the other hand, fieldstudies have shown that pre-chlorination and final chlorina-tion at drinking water treatment plants (DWTP) do not yielda complete elimination [13]. Even so, measured eliminationvalues at DWTP might be misleading, as these pharmaceuti-cals may have been just transformed to new compounds,whose ecotoxicological properties are unknown.

Therefore, bearing in mind that chlorination is one of thecommonest operations during drinking water production,used in ca. 90% DTWPs in Europe [15], it is important tostudy what the actual kinetics of elimination are and howpharmaceuticals and other contaminants are transformed. Inthis sense, several investigations have been focussed in thisfield of research during the last years, where it has beenshown that chlorine reacts with micropollutants to produceundesired by-products, as reviewed elsewhere [16, 17].Thus, some of these by-products have been identified tobe more toxic than the parent compounds, as it is forexample the case of acetaminophen, producing a toxic ben-zoquinone [18]; triclosan, producing chlorinated phenols[19]; or paraben-type bactericides, whose halogenated prod-ucts have a higher toxicity towards aquatic organisms [20,21]. Some more recent studies have shown that severalpharmaceuticals [22–26] and other micropollutants [27,28] react with free chlorine to produce several stable

transformation by-products, some of which are finally en-countered in drinking water [25, 27].

Therefore, the aim of this work was to study the chlorina-tion of two β-blockers (atenolol and propranolol) and a β-receptor agonist (salbutamol). Thus, the effect of differentchlorine dose, pH and bromide concentrations on reactionkinetics was investigated. This was done by means of anexperimental design methodology, which permits the minimi-zation of experimental effort and the integrative considerationof the three variables. Also, several transformation productswere identified and measured at different environmentalconditions by liquid chromatography-mass spectrometry (LC-MS), with an accurate-mass quadrupole time-of-flight (QTOF)system.

Materials and methods

Chemicals and stock solutions

Atenolol, salbutamol and propranolol hydrochloride wereobtained from Sigma-Aldrich (Steinheim, Germany). Stocksolutions containing the compounds (ca. 2 mg/mL, expressedas neutral substance) were prepared in methanol (Romil,Barcelona, Spain) and diluted as necessary. Ultra-pure waterwas obtained in the lab from a Milli-Q water generator (Milli-pore, Billerica, MA, USA). Ammonium acetate was from Fluka(Steinheim, Germany), potassium bromide from Merck (Darm-stadt, Germany), and ascorbic acid, potassium dihydrogenphosphate, dipotassium hydrogen phosphate and sodium hy-pochlorite solution (∼10%) from Sigma-Aldrich. Sodium hy-pochlorite stock solutions at the desired level were prepareddaily by dilution in Milli-Q water, and the chlorine concen-tration was determined using the N,N-diethyl-p-phenylenedi-amine procedure with photometric detection.

Chlorination experiments

Chlorination of pharmaceuticals was performed on 16 mLamber closed vials that were maintained at 20±2 °C. Parallelcontrol samples (without chlorine) were also measured.

Preliminary experiments to determine the stability of drugswere done with 10 mL of Milli-Q water, adjusted to pH 7.1with a phosphate buffer and spiked with the tested drug at the1-μg/mL level and 10 mg/L Cl2. Five aliquots of 1 mL eachwere taken at different reaction times, and the reaction stoppedwith ascorbic acid (0.6 mg/mL). Such experiments, eitherwithout or with bromide added (100 μg/L), were also con-ducted for identification of chlorination by-products.

Further experiments to study chlorination kinetics wereperformed in a similar way, but with lower drug concentrations(50 μg/L) and variable concentrations of Cl2 (1–10 mg/L),bromide (0–100 μg/L) and pH of sample (5.7–8.3) being

2386 J.B. Quintana et al.

considered, within the typical ranges encountered duringdrinking water production. In these experiments, at least fivealiquots were taken at different reaction times and the reactionquenched with ascorbic acid.

These samples were injected in the LC-MS system and thehalf-lives calculated from the pseudo-first-order kinetic fitting.

LC-QTOF-MS

The LC-QTOF-MS instrument was an Agilent 1200 Seriesliquid chromatographic system consisting of a membranedegasser, a binary high-pressure gradient pump, a thermostattedLC column compartment and an autosampler, interfaced to aQTOF-MS instrument (Agilent 6520 Series, Agilent Technol-ogies, Santa Clara, CA, USA) equipped with a dual electro-spray ion source. Nitrogen, used as nebulising and drying,was provided by a nitrogen generator (Erre Due srl, Livorno,Italy).

Separation of analytes was carried out on a 2.1×100-mm,3-μm Nucleosil 100-3C18 HD column (Macherey-Nagel,Düren, Germany) at a flow rate of 0.2 mL/min at 40 °C. EluentA consisted of Milli-Q water and B MeOH, both containing5 mM ammonium acetate. Gradient was as follows: 0 min, 5%B; 10 min, 100% B; 12 min, 100% B; 12.1 min, 5% B; and22 min, 5% B. Injection volume was set to 50 μL.

Nitrogen (99.9995%) used for collision-induced dissocia-tion (for MS/MSmeasurements) was purchased from CarburosMetálicos (A Coruña, Spain). Electrospray ion source and MS/MS parameters were as follows: gas temperature, 275 °C;drying gas, 9 L/min; nebulizer, 42 psi; capillary, 4,000 V;fragmentor, 180 V; skimmer voltage, 65 V; and octapole

RFPeak, 750 V. The instrument was operated in the 2-GHz(extended dynamic range) mode, which provides a FWHMresolution of ca. 4,800 at m/z113 and ca. 12,000 at m/z980.The second sprayer was continuously infused with a referencesolution according to manufacturer specifications during thechromatographic run. Two masses from the components of thisreference solution (positive mode, m/z121.050873 and m/z922.009798; negative mode, m/z112.985587 and m/z980.016375) are automatically employed to continuously re-calibrate the QTOF, maintaining the mass accuracy. Quantifi-cation was carried out in single MS mode, from the accurate-mass extracted chromatogram from the [M+H]+ ion, with amass accuracy window of ±10 ppm. The limits of detection, fora signal-to-noise ratio of 3, achieved were 0.3 ng/mL foratenolol (m/z267.1705), 0.5 ng/mL for salbutamol (m/z240.1595) and 0.2 ng/mL for propranolol (m/z260.1645), whileprecision was kept at relative standard deviation values lowerthan 8% for the three drugs.

By-products were screened in both positive and negativeelectrospray modes, but could only be detected in positive.The by-products were discovered by the function “Find bymolecular feature” of the Mass Hunter software, whichgenerates a list of chemical qualified discrete molecularentities (molecular features) using the mass spectral data[29]. Such function uses an extractor algorithm capable ofremoving background signals, resolving co-eluting interfer-ences and recognizing and grouping isotopic patterns. Mo-lecular features were extracted from a blank reaction sampleand a “time 0” aliquot. Both lists of compounds weresubtracted from that of the samples at different reactiontimes. Then, the identification of the compounds was done

Table 1 Box–Behnken designexperimental plan and measuredhalf-lives (t1/2)

Exp. No. pH Chlorine(mg/L)

Bromide(μg/L)

t1/2

Propranolol (min) Salbutamol (min) Atenolol (h)

1 8.3 1 50 851 2.6 88

2 5.7 10 50 42 1.4 75

3 7 5.5 50 649 1.3 70

4 7 5.5 50 464 1.3 94

5 7 10 100 167 1.3 112

6 5.7 5.5 0 154 12.0 98

7 8.3 5.5 0 945 1.5 91

8 7 1 0 4,265 20.9 145

9 8.3 10 50 630 1.5 91

10 5.7 5.5 100 152 32.5 112

11 8.3 5.5 100 728 1.5 76

12 7 1 100 8,362 5.5 105

13 5.7 1 50 3,122 17.8 132

14 7 10 0 232 1.5 68

15 7 5.5 50 579 1.3 105

Reaction of β-blockers with aqueous chlorine 2387

by generation of candidate formulae with a mass accuracylimit of 5 ppm from the scan (single MS) spectrum. Todetermine the most probable elemental composition, isotopepattern matching was combined to mass accuracy values inthe so-called score value, where 100 represents a perfectmatch of mass accuracy and isotope distribution with theo-retical values. More details on the Mass Hunter algorithmhave been previously reported by Gómez et al. [29]. Subse-quently, product ion MS/MS spectra were acquired at dif-ferent collision energies (10 to 40 V). Thus, formulae for thedifferent fragments were generated. Among the generatedformulae possibilities for each product ion, the best candi-date was selected based on the lowest mass deviation fromtheoretical formulae that was consistent with the precursorformula.

Results and discussion

Chlorination kinetics

A first chlorination test was performed in order to assess thedegradability of the studied pharmaceuticals with chlorine.

Thus, they were treated for 24 h with a 10-mg/L NaClOconcentration at neutral pH (7.1). Under these conditions,atenolol was degraded into a 46% extent, and the otheranalytes were fully removed (mean of two replicates).

Hence, a deeper study on the parameters influencing thechlorination of these pharmaceuticals was carried out by anexperimental design methodology, which has already beensuccessfully applied to study the chlorination of other phar-maceuticals and antioxidants [25–27], or the production oftrihalomethanes from natural organic matter upon chlorina-tion [30]. As mentioned in the “Materials and methods”section, the concentration of the drugs was lower than inthe preliminary experiments (50 μg/L), and the factors consid-ered here were pH (5.7–8.3), chlorine dose (1–10 mg/L) andbromide concentration (0–100 μg/L). This study was done bymeans of a Box–Behnken experimental design, as it providesthe best compromise between number of experiments anddegrees of freedom for three factors [31]. Thus, the finalnumber of experiments was 15 (including three centre points),each experiment being sampled at least at five different reac-tion times: between 0 and 60 min (salbutamol), 0 and 6 h(propranolol), and 0 and 312 h (atenolol). Then, degradationhalf-lives (t1/2) were calculated from the pseudo-first-orderkinetics plots for each experiment and the design analysedfor each pharmaceutical. The correlation coefficients (R)obtained from the logarithmic kinetic plots were usually higherthan 0.9. The factorial experimental plan and calculated t1/2values for each analyte are given in Table 1, where experiments3, 4 and 15 are the three replicates of the central points of theexperimental domain.

The results of the design were analysed with a chemo-metric software (Statgraphics Centurion 16.1, StatpointTechnologies, Warrenton, VA, USA) in order to study theeffect of the considered parameters and their statistical sig-nificance. Table 2 compiles the standardised main effects(SMEs) obtained. It can be observed that chlorine concen-tration was a negative factor, statistically significant forpropranolol and atenolol. This negative value means thatthe t1/2 decreases as the concentration of chlorine increases,i.e. the higher the chlorine concentration, the faster degra-dation proceed, as it was expected.

a b

Chlorine (mg/L)

Bromide=50.0

5.77

8.3pH

14

710

60

80

100

120

140

160

Atenolol Half-life (h) Atenolol Half-life (h)60.070.080.090.0100.0110.0120.0130.0140.0

Hal

f-lif

e (h

)

Chlorine (mg/L)

Hal

f-lif

e (h

)

pH=7.0

14

710 0

25 5075

100

60

80

100

120

140

160

60.070.080.090.0100.0110.0120.0130.0140.0

Bromide (µg/L)

Fig. 1 Response surface plots of atenolol half-lives as a function of pH and chlorine concentration (a), and chlorine and bromide concentrations (b)

Table 2 Standardised main effects (SMEs) obtained from the half-lifeanalysis of the Box–Behnken design

Propranolol Salbutamol Atenolol

A: pH −0.07 −2.78 −2.13

B: Chlorine −3.35 −2.03 −3.80

C: Bromide 0.82 0.24 0.13

AA −1.27 1.21 −0.53

AB 0.87 1.07 2.55

AC −0.06 −1.42 −1.20

BB 1.97 −0.01 1.69

BC −1.26 1.06 3.62

CC 1.19 1.61 1.27

Statistically significant factors are presented in bold. Statistical signif-icance boundary for 95% confidence level: Student t02.57

2388 J.B. Quintana et al.

The pH was also significant and negative for salbutamol.Salbutamol is the only phenolic compound evaluated in thisstudy. Thus, the role of the pH observed is the same as thatfound in the literature for other phenolic compounds [20,28], where this fact is explained due to the higher reactivityof the phenolate ion as compared to neutral phenol towardselectrophilic aromatic substitution, which leads to fasterkinetics at basic pH values.

The case of atenolol was more complex, as beside thestatistically significant effect of chlorine concentration al-ready mentioned, the effect of pH was also close to thesignificance level and negative, and also the interactionbetween the pH and chlorine was very close to statisticallysignificance boundary. This effect is visualised in the re-sponse surface presented in Fig. 1a, where it can be appre-ciated that on one hand, at high chlorine doses, the pH hasno effect on the atenolol disappearance half-lives; on theother hand, at low chlorination doses, the reaction is faster atbasic pH values. However, this fact cannot be explained inthe same way as for salbutamol, since atenolol is not aphenol, and the reaction mechanism is completely different(see next section). Moreover, the interaction term BC (chlo-rine–bromide) is statistically significant for atenolol. This in-teraction is the result of the accelerated kinetics as chlorinedoses increases at low bromide contents, whereas chlorinedose has no effect if the sample contains a high bromideconcentration (Fig. 1b). This can be interpreted as a fasterreaction rate with HBrO, rapidly formed from bromide uponchlorination, but only if the chlorine dose is not much higherthan bromide levels.

Identification of by-products

Chlorination by-products of each drug were investigated at the1-μg mL−1 level, both without and with bromide addition(100 μg L−1) by LC-MS with QTOF instrument in scan andproduct ion scanmodes (see “Materials andmethods” section).

O NH

OH

O NH

OH

X

O NH

OH

OH

O NH

OH

OHOH

Pro

Cl-Pro or Br-Pro

X = Cl or BrOH-Pro (OH)2-Pro

a

b

Fig. 2 Proposed propranolol degradation pattern (a) and by-productformation time profiles (b)

Table 3 LC-QTOF-MS scan data on by-product identification

By-product tR (min) Experimental m/z Proposed formula Calculated m/z Difference(mDa)

Difference(ppm)

DBE Score

Propranolol 13.60 260.1645 C16H21NO2 260.1645 0.02 0.08 7 96.05

Cl-Pro 14.50 294.1257 C16H20NO2Cl 294.1255 −0.17 −0.59 7 99.57

Br-Pro 14.55 338.0750 C16H20NO2Br 338.0750 0.06 0.18 7 80.91

OH-Pro 10.45 and 10.80 276.1597 C16H21NO3 276.1594 −0.25 −0.9 7 92.52

(OH)2-Pro 11.90 292.1547 C16H21NO4 292.1543 −0.36 −1.25 7 65.39

Salbutamol 8.80 240.1595 C13H21NO3 240.1594 −0.05 −0.22 4 99.96

Cl-Sal 10.40 274.1201 C13H20NO3Cl 274.1204 0.39 1.43 4 99.29

Cl-Sal-MeOH 10.75 244.1098 C12H18NO2Cl 244.1099 0.09 0.38 4 99.56

Br,Cl-Sal-MeOH 11.10 322.0201 C12H17NO2ClBr 322.0204 0.24 0.73 4 99.72

Cl2-Sal-MeOH 10.80 278.0709 C12H17NO2Cl2 278.0709 0.01 0.05 4 99.83

Atenolol 9.20 267.1705 C14H22N2O3 267.1703 −0.22 −0.82 5 99.63

OH-At-NH2 7.50 268.1543 C14H21NO4 268.1543 0.03 0.1 5 99.80

OH, Cl-At-NH2 9.70 302.1151 C14H20NO4Cl 302.1154 0.28 0.93 5 93.81

OH, Br-At-NH2 10.10 346.0644 C14H20NO4Br 346.0648 0.39 1.12 5 98.86

OH-At-NH2CHO 10.50 238.1434 C13H19NO3 238.1438 0.34 1.42 5 89.43

At-C3H6 5.50 225.1232 C11H16N2O3 225.1234 0.13 0.56 5 99.17

DBE double bond equivalents

Reaction of β-blockers with aqueous chlorine 2389

The empirical formulae of the products was identified by scanmeasurements in the QTOF instrument, based on the concor-dance between themeasured exact mass and the isotopic profileof the [M+H]+ ions with the theoretical one as expressed in

terms of a “score” (see “Materials and methods” section formore details). A summary of the detected by-products scanidentification data is compiled in Table 3, where it can beappreciated that proposed formulae have deviations lower than

3x10

00.20.40.60.8

11.21.41.61.8

22.22.42.62.8

33.23.43.63.8

44.24.44.64.8

260.1645

56.0501

74.0603

157.0638

116.1067

183.079798.0964

83.0856 244.2051

Counts vs. Mass-to-Charge (m/z)50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260

[M+H]+

C13H11O+

(+0.69 mDa)

-H2O, -C3H9N

C11H9O+

(+0.99 mDa)

-C2H2

C6H14NO+

(+0.32 mDa)

C6H12N+

(+0.07 mDa)

C3H8NO+

(-0.3 mDa)

C3H6N+

(-0.3 mDa)

-H2O

CH2

+

NH

OH

-H2O

-C3H6

3x10

00.10.20.30.40.50.60.70.80.9

11.11.21.31.41.51.61.71.81.9

22.1 58.0656

294.124972.0810

116.1070

98.0966217.0408

182.0719

86.0966252.0775

Counts vs. Mass-to-Charge (m/z)50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290

[M+H]+

-C3H6

C13H15NO2Cl+

(+1.06 mDa)

C13H10OCl+

(+0.46 mDa)

-H2O, -NH3

191.0260

C11H8OCl+

(-0.06 mDa)

-C2H2

C13H10O+

(+1.07 mDa)

-Cl-

C6H14NO+

(+0.21 mDa)C6H12N+

(-0.04 mDa)

C5H12N+

(-0.17 mDa)

C3H8NO+

(-0.5 mDa)74.0605

C4H10N+

(-0.18 mDa)

C3H8N+

(-0.51 mDa)

56.0500

C3H6N+

(-0.51 mDa)

CH2

+

NH

OH

-H2O

-C3H6-H2O

2x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3292.1528

116.1069

103.0540

131.0459

56.049874.0588 237.1104

159.0426

Counts vs. Mass-to-Charge (m/z)50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290

[M+H]+

C13H17O4+

(+1.74 mDa)C10H7O2

+

(+1.45 mDa)

C9H7O+

(+3.25 mDa)

C6H14NO+

(+0.13 mDa)

C8H7+

(+0.19 mDa)

C6H12N+

(+2.84 mDa)98.0936

C3H8NO+

(+1.26 mDa)

72.0814

C4H10N+

(-0.61 mDa)

C3H6N+

(-0.3 mDa)

-C3H5N

C+

OHOH

-C3H6

-H2O-C2H2

-H2O

-CO

-CO

a

b

c

Pro

Cl-Pro

(OH)2-Pro

Fig. 3 QTOF product ion spectra of propranolol and two by-products

2390 J.B. Quintana et al.

±0.5 mDa (less than ±2 ppm) of mass accuracy. Also, the scorevalues were also higher than 98, with a few exceptions due totheir low intensity, which did not permit to obtain a betterisotopic pattern fit. Moreover, the structure of the productswas tentatively elucidated by interpretation of their MS/MSfragmentation pattern in the product ion scan mode.

Propranolol

The degradation of propranolol yielded four peaks, two ofthem corresponding to isomers of the mono-hydroxylatedderivative (OH-Pro) and the other two to the di-hydroxylated ((OH)2-Pro) and chlorinated (Cl-Pro) deriva-tives. If bromide was present in the sample, then also bro-minated propranolol (Br-Pro) is formed (Fig. 2a). Also, aspresented in Fig. 2b, all compounds are formed simulta-neously. The most intense chromatographic peaks were thosecorresponding to the halogenated derivatives, which togetherwith OH-Pro were found at very long reaction time samples(72 h), indicating that these compounds are stable (data notshown).

The study of the MS/MS fragmentation pattern (Fig. 3and Electronic Supplementary Material Figure S1) did notallow knowing the exact position of the introduction of thehalogen or hydroxyl groups. However, the typical losses ofpropranolol, such as H2O, C3H6, NH3 and C2H2, wereobserved in all the spectra (Fig. 3 and Electronic Supple-mentary Material Figure S1). On the other hand, the 1-isopropylamino-2-propanol product ion (C6H14NO

+, m/z116.1067) and its typical fragments at m/z98.0964 (C6H12N

+),m/z74.0603 (C3H8NO

+), m/z72.0810 (C4H10N+), m/z58.0656

(C3H8N+) and m/z56.0501 (C3H6N

+) confirmed that the halo-gen and hydroxyl group are not located in the 1-isopropylamino-2-propanol fragment of the molecules. Hence, as proposed inFig. 2a, it remains clear that this introduction of hydroxyl orhalogen group took place in the naphthalene ring.Moreover, thisfact is strengthened by the presence of ions corresponding to twoCO losses in the spectrum of (OH)2-Pro (Fig. 3c), which wouldnormally be lost as H2O in aliphatic alcohols. Also, hydroxyl-ation of the naphthalene ring has been detected as the main pathof biological degradation of propranolol with the fungus Tra-metes versicolor [32].

The reaction pathway discovered in this work contrastswith the suggestions of Pinkston and Sedlak [14], whoexpected formation of the chloramine followed by N-dealkylation as the main route of β-blockers reaction withchlorine. Hence, in the case of propranolol, neither the chlo-ramine nor the N-dealkylated products were detected. Obvi-ously, the chloramine could have been formed but thentransformed back to propranolol. However, at long reactiontimes, the pharmaceutical completely disappeared, provingthat the chloramine route is not relevant upon typical chlori-nation conditions.

Salbutamol

In the case of salbutamol, two mono-chlorinated derivativesare produced by electrophilic aromatic substitution (Fig. 4a).In one of them, the chlorine atom is introduced in the freeortho-position to the phenolic group (Cl-Sal), and in thesecond one, chlorination occurs in the other ortho-position,thus eliminating methanol during chlorination (Cl-Sal-MeOH). Moreover, these two compounds, Cl-Sal and Cl-Sal-MeOH, finally lead to the dihalogenated compounds:Cl2-Sal-MeOH and Cl,Br-Sal-MeOH, if bromide is presentin the sample (Fig. 4a).

The QTOF MS/MS product ion scan spectra of theseproducts show the typical sequential losses of salbutamol:first, elimination of H2O and C4H8 (Fig. 5 and ElectronicSupplementary Material Figure S2) followed also by lossesof H2O and CHN for the mono-chlorinated derivatives(Fig. 5b and Electronic Supplementary Material FigureS2a). Thus, as in the case of propranolol, the introductionof the halogen atoms is confirmed to place in the aromatic

a

b

OH

OH

OH

NH

OH

OH

OH

NH

Cl

Cl

OH

OH

NH

Y

OH

OH

NH

X

Sal

Cl-SalCl-Sal-MeOH

Br,Cl-Sal-MeOH or Cl2-Sal-MeOH

X, Y = Cl or Br

Fig. 4 Proposed salbutamol degradation pattern (a) and by-productformation time profiles (b)

Reaction of β-blockers with aqueous chlorine 2391

ring and not in the amine side chain. Though the exactposition could not be elucidated by the spectra, it is expectedto occur at ortho-positions to the phenol group, based on theortho-activating character of phenol, as for other phenolic

compounds [20, 25], and the fact that the ortho-hydroxy-methyl group is lost in three of the by-products.

As shown in Fig. 4b, Cl-Sal and Cl-Sal-MeOH are veryrapidly formed and are then transformed into the dihalogenated

4x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4 148.0752

166.0856

222.1484

Counts vs. Mass-to-Charge (m/z)50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240

121.0649

130.064657.0704

91.0539103.0544

79.0544

240.1594

[M+H]+

C13H20NO2+

(+0.44 mDa)

-H2O

C9H12NO2+

(+0.64 mDa)

-C4H8

C9H10NO+

(+0.49 mDa)

-H2O

C8H7NO+

(+0.17 mDa)133.052

-CH3

C8H9O+

(-0.12 mDa)

-CHN

C8H7+

(-0.21 mDa)

-H2O

C7H7+

(+0.33 mDa)

C4H9+

(-0.51 mDa)

-CH2O

C+

CH 2

+

3x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7182.0362

200.0469

256.1092155.0255

91.054357.0706118.0648

Counts vs. Mass-to-Charge (m/z)50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270

274.1196

[M+H]+C13H19NO2Cl+

(+0.64 mDa)

-H2O

C9H11NO2Cl+

(+0.43 mDa)

-C4H8

C9H9NOCl+

(+0.5 mDa)

-H2O

C8H8OCl+

(+0.3 mDa)

-CHN

C9H9NO+

(+0.62 mDa)

147.0672

-Cl

C7H7+

(-0.06 mDa)C4H9

+

(-0.7 mDa) C8H8N+

(+0.29 mDa)

-CHO

-CHO, -Cl

C+

3x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6 247.9466

169.0282

133.051557.0708

Counts vs. Mass-to-Charge (m/z)50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320

322.0203

[M+H]+

C8H8NOClBr+

(+0.61 mDa)

C8H8NOCl+

(+0.68 mDa)

-H2O -C4H8

-Br

212.9768

C8H8NOBr+

(+1.54 mDa)

-Cl

C8H7NO+

(+0.74 mDa)

-HCl

C4H9+

(-0.92 mDa)

C+

a

Sal

b

Cl-Sal

c

Br,Cl-Sal-MeOH

Fig. 5 QTOF product ion spectra of salbutamol and two by-products

2392 J.B. Quintana et al.

compounds. However, at very long reaction time (72 h), noneof these four products remained in the sample (data notshown). This indicates that reaction continues to yield verysimple molecules that could not be detected by LC-QTOF-MS.

Atenolol

Atenolol chlorination undergoes two mechanisms: the first onN-dealkylation to produce At-C3H6, and the second one ahydrolysis of the amide group to yield the carboxylic acid,OH-At-NH2 (Fig. 6a). In this case, the formation of At-C3H6

matches the expected mechanism proposed by Pinkston andSedlak [14], but again, it was a secondary route of degrada-tion. Actually, the second product, OH-At-NH2, was the mostintense product peak at all the investigated reaction times. Inaddition, this last compound has already been encountered asthe major transformation product during biodegradation ofatenolol with activated sludge by Radjenovic et al. [33].

The structures of these two products are also confirmedby the QTOF product ion spectra. Hence, in the spectrum ofAt-C3H6, the loss of C3H6 and the fragment at m/z116.1069(C4H14NO

+), typical of atenolol, are not observed, whereasthere is a fragment at m/z74.0610 (C3H8NO

+; same as m/z116.1069, but without C3H6), confirming that there is noiso-propyl group in the structure (Fig. 7a, b). On the otherhand, the spectrum of OH-At-NH2 is very similar to thatdescribed by Radjenovic et al. [33]. This product ion spec-trum shows the same characteristic fragmentation patternthan atenolol as regards of the iso-propyl-amino-propoxygroup, m/z116.1069 and subsequent products, indicatingthat changes are not produced in this part of the molecule(Fig. 7a, c). Moreover, the atenolol typical loss of CH3NO(corresponding to a fragmentation of the acetamide group) isnot observed, but instead of that, there is a loss of H2O andCO (typical of carboxylic acids; Fig. 7c).

The by-product, OH-At-NH2, further reacts to produce Cl,OH-At-NH2 or Br,OH-At-NH2 (the second one only in thepresence of bromide) by halogenation and to OH-At-NH2CHOby decarboxylation. The spectra of these three products showthe same fragmentation pattern than atenolol and OH-At-NH2

as regards the iso-propyl-amino-propoxy group, again provingthat changes were not produced in this part of the molecule(Fig. 7a and Electronic SupplementaryMaterial Figure S3). Onthe other hand, in the spectra of the halogenated derivatives,there is a loss of H2O and CO (as for OH-At-NH2), typical ofcarboxylic acids, and also a fragment at m/z199.0141 or m/z242.9645 corresponding to C9H8O3X

+ (instead of C9H9O3+ of

OH-At-NH2), indicating than the halogen atom is located inthe benzene ring (Electronic Supplementary Material FigureS3a, b). The spectrum of OH-At-NH2CHO shows the loss ofCO (instead of CH3NO for atenolol or H2O+CO for OH-At-NH2), pointing to an aldehyde group attached to the ben-zene ring (Electronic Supplementary Material Figure S3c).

Obviously, if OH-At-NH2 could already present in environ-mental water samples ought to biotic processes [33], but thenit would be expected to follow the same degradation mecha-nism during chlorination.

All the by-products were still found at long reaction times(144 h), and only a slightly degradation of OH-At-NH2 isobserved in Fig. 6b. However, this compound was still themost intense one at all studied contact times.

Conclusions

The β-blocker/agonist drugs propranolol, salbutamol and ate-nolol are transformed during water chlorination. The opera-tional parameters, pH and chlorine dose, have an importantimpact on reaction kinetics, with half-lives varying from42 min to almost 6 days for propranolol, between 1.5 and33min for salbutamol, and between 68 and 145 h for atenolol.Thus, the increase in chlorine dose or the pH speeds up thedegradation of these drugs. Moreover, the reaction of atenololis also enhanced by the presence of bromide in the sample, butonly at low chlorine concentrations.

The by-products formed were determined with an accurate-mass LC-QTOF-MS instrument. Hence, propranolol yieldsthree hydroxylated and two halogenated derivatives, which arestable under strong and long reaction conditions. Salbutamol

a

b

NH2

O

OH

NH

O

OH

O

OH

NH

O

NH2

O

OH

NH2

O

O

OH

NH

O

OH

O

OH

NH

O

X

At

OH-At-NH2 At-C3H6

OH-At-NH2CHOOH, Cl-At-NH2 or OH, Br-At-NH2

X = Cl or Br

Fig. 6 Proposed atenolol degradation pattern (a) and by-product for-mation time profiles (b)

Reaction of β-blockers with aqueous chlorine 2393

produces four halogenated compounds, which are only stableat low chlorination dosages. Finally, atenolol is transformedby dealkylation of the amine and, as a major route, by oxida-tion of the amide, which can then further react to yield three

other minor products. All atenolol by-products are formedrelatively slowly and are expected to be formed in a significantextent under strong chlorination conditions. Further researchis needed in order to assess the occurrence and possible

3x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6267.1693

74.0603 190.0857145.0643

56.0504116.1069

98.0964208.0966 225.1226

178.0854

133.0644 164.0709

Counts vs. Mass-to-Charge (m/z)50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270

[M+H]+

C11H17N2O3+

(+0.8 mDa)

-C3H6

250.1428

C14H20NO3+

(+0.94 mDa)

-NH3

C11H14NO3+

(+0.17 mDa)

-NH3

-C3H6

C11H12NO2+

(+0.58 mDa)

-H2O

C10H12NO2+

(+0.9 mDa)

-CH2OC9H10NO2

+

(-0.31 mDa)

-C2H4O

180.1007

C10H14NO2+

(+1.24 mDa)-CH3NO162.0903

C10H12NO+

(-0.31 mDa)-H2O

C10H9O+

(+0.53 mDa)

-NH3

-CH3NO

C9H9O+

(+0.42 mDa)

-CH3NO

C6H14NO+

(+0.05 mDa)

C6H12N+

(+0.06 mDa)

-H2O

C3H8NO+

(+0.06 mDa)

-C3H6

72.0811

C4H10N+

(-0.31 mDa)

-C2H4O

C3H6N+

(-0.92 mDa)

-H2O

OH CH

+NH

3x10

00.20.40.60.8

11.21.41.61.8

22.22.42.62.8

33.23.43.63.8

44.24.44.64.8

145.0643191.0695

268.1533

56.050172.0812

98.0964116.1062

226.1067165.0538

Counts vs. Mass-to-Charge (m/z)50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270

[M+H]+

C11H16NO4+

(+0.8 mDa)

-C3H6

-H2O250.1415

C14H20NO3+

(+2.29 mDa)

C11H11O3+

(+0.7 mDa)

-H2O, -NH3

-C3H6, -NH3

C9H9O3+

(+0.86 mDa)

C10H9O+

(+0.48 mDa)

179.0691

C10H11O3+

(+1.15 mDa)

-CH2O, -NH3

-H2O, -CO

C6H14NO+

(+0.87 mDa)

C6H12N+

(+0.03 mDa)

-H2O

C3H8NO+

(-0.44 mDa)

-C3H6

C4H10N+

(-0.37 mDa)

-C2H4O

74.0605

C3H6N+

(-0.92 mDa)

-H2O

OH CH

+NH

OH

OCH 2

+O

2x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8 225.1227

74.0610

145.0648

208.0949190.084056.0492

69.0695 178.0859107.048289.0388 133.0628

83.0855

Counts vs. Mass-to-Charge (m/z)50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230

[M+H]+

C11H14NO3+

(+1.91 mDa)

-NH3

C11H12NO2+

(+2.28 mDa)

-H2O

C10H12NO2+

(+0.32 mDa)

-CH2O

C10H9O+

(-0.04 mDa)

-CO, -NH3

C9H9O+

(+1.95 mDa)

-CO, -NH3

C7H7O+

(+0.95 mDa)

C3H8NO+

(-0.99 mDa)

C3H6N+

(+0.31 mDa)

OH NH+

-H2O

C+

O

a

At

b

At -C3H6

c

OH-At-NH2

Fig. 7 QTOF product ion spectra of atenolol and two by-products

2394 J.B. Quintana et al.

ecotoxicological concern of these by-products and their pre-cursor drugs.

Acknowledgements This work was funded byMinisterio de Cienciae Innovación and FEDER funds, project no.: CTQ2009-08377 andCTQ2010-18927. J.B.Q. and R.R. also acknowledge Ministerio deCiencia e Innovación for their contracts (Ramón y Cajal researchprogram).

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Reaction of β-blockers with aqueous chlorine 2395