use of model compounds to study removal of pharmaceuticals using

1949-8241/10 $90.00 + .00 DOI: 10.3727/194982410X12741230805380 E-ISSN 1949-825X www.cognizantcommunication.com

Technology and Innovation, Vol. 12, pp. 71–77, 2010 Printed in the USA. All rights reserved. Copyright  2010 Cognizant Comm. Corp.

USE OF MODEL COMPOUNDS TO STUDY REMOVAL OF PHARMACEUTICALS USING OCTOLIG Wen-Shan Chang, Dean F. Martin, and Meagan Small Department of Chemistry, University of South Florida, Tampa, FL, USA The possibility of removing certain pharmaceuticals from wastewater was tested using Octolig, a commercially available material with polyethyldiamine moieties covalently attached to high-surface area silica gel. Selected drugs and drug models were subjected to column chromatography for removal by means of ion encapsulation, the effectiveness of which would depend upon having appropriate anionic functional groups. Removal of methylene blue with quaternary ammonium groups was (statistically) unsuccessful. In contrast, complete success was attained for removal of each of three xanthenylbenzenes (rose bengal, eosin Y, erythrosine) that have both phenolic and carboxylic acid groups, as is the case with two of the top five prescribed drugs in the US. Key words: Pharmaceuticals; Xanthenylbenzenes; Octolig; Encapsulation

INTRODUCTION

sponse in humans, animals, plants, bacteria, or other organisms (11). Ultimately, the impact of pharmaceuticals on the environment becomes a matter of concern because many individual pharmaceuticals and their metabolites are found in the environment, having passed through sewage treatment plants (3,11). Investigations showed that pharmaceutically active compounds cannot be totally eliminated during wastewater treatment, and also can be only partially biodegradable in the environment (3,9). The occurrence of pharmaceuticals has been investigated in many countries, including the US, and throughout the EU. As a consequence, it is known that more than 80 pharmaceutically active compounds are detected up to µg/L range in the aquatic environment (9). Pharmaceuticals can enter the environment through several routes. Most pharmaceuticals enter the environment through sewage treatment plants, agriculture run-off, landfill leaching, or direct application, as for example, some pharmaceuticals used in aquaculture. Pharmaceutical pathways to the environment were summarized by Ku¨mmerer (12), who also described similar pathways for anti-

With the rapid developing of the human population, the demand for pharmaceuticals also has increased, and in 2000, 16,200 tons of antibiotics were produced in the US, of which 70% was used for livestock (11,21). In 1999, some 13,288 tons of antibiotics were reportedly used in the European Union (EU) and Switzerland, of which 65% was for human medicine and 29% was used in the veterinary field (4,11). Wise estimated the total antibiotic market consumption was between 100,000 and 200,000 tons world-wide (23). Pharmaceutical drugs are defined as those organic or inorganic compounds used in the diagnosis, cure, mitigation, treatment, or prevention of disease (2). Pharmaceuticals have been given to humans, of course, but the definition of pharmaceuticals can be extended to veterinary uses, to plant pharmaceuticals, or even illicit drugs (11). What is more, pharmaceuticals have a range of applications in farming and medicine (11). The impact is especially wide because pharmaceutical compounds are made to cause a physiological re-

Address correspondence to Dean F. Martin, Institute for Environmental Studies, Department of Chemistry, University of South Florida, 4202 East Fowler, Tampa, Fl 33620-5250, USA. Tel: (813) 974-2374; Fax: (813) 974-3203; E-mail: [email protected]

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CHANG, MARTIN, AND SMALL

biotics (11). Not only the direct disposal of unused pharmaceuticals will enter our environment, but also those medications that are only partially biodegradable will go into the environment. And large amounts of active pharmaceutical ingredients (APIs) enter our environment by going down to the drains directly. Drugs enter the environment through several pathways and affect organisms in the environment. Some drugs can be excreted through human or animal metabolism, and some pharmaceuticals simply cannot be completely eliminated by sewage treatment systems (3,25). In addition to considering the effects of active pharmaceuticals, it is necessary to remember that pharmaceuticals that are changed in the digestion process by organisms and those additional molecules that are formed by transformation processes can cause pharmaceutical contamination. Drug structures may be altered when they go through the bodies of humans and animals (5). Some structures of drugs are largely changed by the microorganisms in the guts or by the enzymes during human metabolism before they are excreted. Therefore, their pharmaceuticals properties are much different from their parent drugs. Although the discharge of pharmaceuticals could be in low concentration, it may still cause significant effects. Three major concerns have been mentioned previously (3). One concern is that antimicrobials may not be sufficiently destroyed in sewage treatment plants. A second concern is that antimicrobials may affect, qualitatively or quantitatively, the resident microbial community in sediments (18). A third concern is that resistant bacteria and, perhaps, multiple-resistant bacteria, may be involved in sewage, soil, or other environmental components. Such bacteria have been detected in wastewater and in sewage treatment plants (7,24). The magnitude of the problem is uncertain. Drug production plants may make a significant contribution to the total pharmaceutical concentration in the environment (14). Pharmaceutical manufacturing processes involve many series of steps, which can be taken in many different sites. Therefore, there is a risk that pharmaceuticals may enter the environment from many places during the production of APIs. However, according to Williams and Cook (22), no studies have documented whether drug

manufacturers could be main sources for pharmaceuticals in the environment. Hospitals, on the other hand, could also be a concentrated source of waste pharmaceuticals, either through disposal of expired drugs or through the metabolism of patients. Ciprofloxcin was found in a German hospital effluent (0.7–125 µg/L) (8). Ampicillin was found in another German hospital effluent (920–980 µg/L) (10). We may well wonder about the situation in Florida, the fourth most populated state of the US that had 203 hospitals in 2006, and around 2,373 thousand patients being served per day (1). There is an obvious need to develop methods to effect removal of these agents. The present study examines the use of model pharmaceuticals to evaluate whether Octolig might be suitable for their removal from aqueous solutions. A previous study (19) indicated that Octolig, a polyethylenediamine covalently attached to a high-surface-area silica gel, is capable of removing such anions as phosphate, sulfate, nitrate, and nitrite, presumably through a process of encapsulation by the polyethylenediamine rings suitably protonated (19). The working hypothesis is that certain pharmaceuticals might have appropriate functional groups to enable appropriate encapsulation (Fig. 1), and that this possibility might be tested with suitable dyes (e.g., xanthenylbenzenes). MATERIALS AND METHODS Source of Reagents and Materials Octolig (CAS Registry Number 404899-06-5) was a gift from Metre-General, Inc. (Frederick,

Figure 1. Possible structure of encapsulation of anions (An−) by Octolig showing a one-arm model [after Stull and Martin (19 )].

REMOVAL OF PHARMACEUTICALS USING OCTOLIG

CO). Methylene blue was obtained from EMD Chemicals Inc. Rose bengal was obtained from J. T. Baker Chemical Co. Eosin Y was obtained from Sigma Chemical Co. Erythrosine was acquired from J. Preston Ltd. Well water samples were obtained from a private well at 3402 Valencia Road in Original Carrollwood, Tampa, FL. Prior to use, the water was filtered through a 3-µm Millipore membrane filter using an all-glass apparatus. Analyses Measurements of total dissolved solid (TDS) of aqueous samples were done by a Fisher Scientific digital conductivity meter, and the pH values were obtained by an Orion model 290A pH/ISE meter connected with an Orion pH triode electrode model 9107BN. Concentrations of dye solutions were acquired from the absorbance measurements using a Shimadzu UV-2401 PC UV-Vis recording spectrophotometer. Spectra were saved to a disk using Origin Pro 8.0 program for further use. Chromatography Experiments The chromatography process was similar to that used previously (16,19). The Octolig as received was subjected to a pretreatment by suspending the solid in DI water and then decanting the water to remove the fines. A CHEMGLASS column, 2 cm (i.d.) equipped with a glass frit and a Teflon stopcock, was packed with Octolig and washed with about 1 L of solvent [i.e., deionized (DI) water, tap water, or well water, which were used as different matrices]. Aqueous samples of dyes were chromatographed using a rate of 10 ml/min using a Spectra/chronTM peristaltic pump. A series of 50ml fractions was collected, and measurements were made of conductivity, pH, and visible spectra. The concentrations of the effluents (fractions 4 on, typically) were compared with the concentration of the input solutions, and the percent removal was calculated and recorded (Table 1). A “batch check” was made, and the results with rose bengal were compared for two different samples of Octolig. In addition, a matrix effect was checked by using deionized water as well as water from a shallow aquifer. As a precaution, all stock solutions of the dyes were kept covered with aluminum foil.

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Molar Extinction Coefficient Measurements Serial dilutions for each dye were prepared from a known stock solution concentration. Absorbance values were recorded for a wavelength near the λmax for each dilution using a Shimadzu UV-2401 PC spectrophotometer. Concentrations were prepared to ensure that absorbance values did not exceed OD = 1.5. Molar extinction coefficients were determined using the Beer-Lambert law, in which the slope of the absorbance versus concentration plot is equal to the extinction coefficient times the path length, in which the path length was 1.0 cm. Values are reported in Table 2. RESULTS AND DISCUSSION Compound Selection Pharmaceuticals can be characterized in order of number of prescriptions. The top five in the US in 2008 (20) (with uses in parentheses) were hydrocodone (pain), lisinopril (hypertension), simvastatin (high cholesterol), levothyroxine (hypothyroidism), and amoxicillin (bacterial infection). Pharmaceuticals may also be characterized in terms of structural features, the simplest being by functional groups. Accordingly, the second, fourth, and fifth ranked drugs have either carboxyl (second, fourth, fifth) or phenolic groups (fourth, fifth) or both (fourth and fifth). Amino groups could also be of interest, and these are found in the second, fourth, and fifth ranked pharmaceuticals. Accordingly, a series of model compounds was selected for testing their potential for encapsulation. Methylene blue (Fig. 2), for example, has a pair of tertiary amino groups. It is a guanylate cyclase inhibitor used to treat vasoplegia, which is a frequent complication after cardiopulmonary bypass (15). Fluorescein and the halogenated derivatives are substituted xanthenylbenzenes that have both phenolic and carboxylic acid groups. The series includes rose bengal, eosin Y, erythrosine, and sodium fluorescein (Fig. 2). All are sensitizers for the production of singlet oxygen, the best being rose bengal, which has the largest quantum yield for production of singlet oxygen (6). Rose bengal has other interesting uses: as a cosmetic and in the analysis of hepatic function, which entailed swallowing the dye (17).

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Table 1. Passage of Aqueous Fluorscein Dye Solutions Over a 2.2-cm i.d. Chromatographic Column Packed With 22 cm of Octolig (Batch 1) at a Flow Rate of 10 ml/min (50-ml Aliquots Were Collected) Dye, Matrix Methylene blue DI water

Rose Bengal DI water

Well water Eosin Y DI water

Well water Erythrosine DI water

Tap water Well water

Fraction

TDS (ppm)

pH

Concentration (106 M)

Stock 4–15 Stock 4–9 Stock 4–10

2 1.9 ± 0.2 3 4.0 ± 0 7 5.2 ± 0.4

7.34 6.09 ± 0.24 7.61 6.33 ± 0.22 5.56 5.82 ± 0.26

3.321 3.318 ± 0.11 0.876 0.844 ± 0.02 2.429 2.550 ± 0.06

Stock 4–9 Stock 4–9 Stock 4–10 Stock 4–10 Stock 6–10

3 6.5 ± 0.55 4 5.0 ± 0 4 5.0 ± 0 4 5.0 ± 0 159 220.3 ± 3.87

6.09 5.63 ± 0.35 6.58 5.78 ± 0.10 7.05 6.65 ± 0.24 7.18 6.34 ± 0.35 7.93 6.74 ± 0.07

4.170 0.949 ± 0.266 1.042 0.008 ± 0.071 9.600 0.045 ± 0.022 9.647 0.070 ± 0.010 21.921 0.012 ± 0.011

— 77.30 ± 6.4 — 99.20 ± 0.7 — 99.50 ± 0.2 — 99.30 ± 0.1 — 99.90 ± 0.0


9 14.0 ± 0.0 10 20.0 ± 0.0 10 13.0 ± 0.5 10 15.2 ± 0.3 164 205.3 ± 1.3

6.27 6.24 ± 0.08 6.68 5.99 ± 0.08 6.72 5.98 ± 0.08 6.83 5.88 ± 0.04 7.97 6.70 ± 0.09

55.185 0.713 ± 0.009 56.213 0.002 ± 0.005 86.290 0.035 ± 0.031 90.044 0.927 ± 0.065 85.167 0.054 ± 0.037

— 87.10 ± 0.2 — 100.00 ± 0.2 — 100.00 ± 0.0 — 99.00 ± 0.1 — 99.90 ± 0.0


30 31.1 ± 0.8 32 34.9 ± 0.3 28 31.9 ± 0.3 293 352.7 ± 7.5 203 199.9 ± 1.2

7.48 6.71 ± 0.06 9.54 6.22 ± 0.13 8.66 6.13 ± 0.10 7.76 7.45 ± 0.08 8.33 8.42 ± 0.06

59.662 0.642 ± 0.027 73.14 0.094 ± 0.020 78.753 0.046 ± 0.027 94.897 0.122 ± 0.019 105.566 1.504 ± 0.070

— 98.90 ± 0.0 — 99.90 ± 0.0 — 99.90 ± 0.0 — 99.90 ± 0.0 — 98.60 ± 0.1

The data in Table 1 show that methylene blue (Fig. 2) was not removed by passage over an Octolig column, and given the model of encapsulation (19), as represented (Fig. 1), one would not expect neutral species or cations to be removed. Thus, the results with methylene blue, though negative, are supportive of the working hypothesis. Results with xanthenylbenzenes provided positive support of the hypothesis. All three dyes tested

% Removed

— 1.92 ± 2.57 — 3.72 ± 2.71 — 0.00 ± 0.00

(rose bengal, erythrosine, and eosin Y) (Fig. 3) were completely removed, at least within experimental error, as shown in Table 1. And the results show good precision (as good as 0.04% relative standard deviation). The results (Table 1) also indicate that the success applied to all three related xanthenylbenzenes. Not surprisingly, a pH effect was observed (Table 1). Rose bengal when unadjusted solution (pH


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Table 2. Spectral Properties of Selected Dyes Using Shimadzu UV-2401 PC UV-Vis Recording Spectrophotometer Extinction Coefficient Sample Methylene blue Rose Bengal Eosin Y Erythrosine

λmax (nm)

M−1

cm−1

662 544 516 526

73,000 57,900 72,000 71,200

73,004 57,886 71,976 71,235

6.09) was used and only 77% removal was observed. Adjusting the pH of the stock solution closer to neutrality of higher (pH 6.9–7.9) resulted in removals of 99% or greater. The pH of the eluants was lower than the stock solutions (Table 1), which is consistent with proton release from all three xanthenylbenzenes. One would not expect a great difference of the effectiveness of batch-to-batch differences for a commercial product backed by a good QA/QC program, but this was checked anyway using rose bengal and no discernable difference was observed. A matrix effect was tested by comparing results with standard choice of solvent (i.e., DI water or well water) (Table 1), and no notable differences were observed for rose bengal or eosin Y, nor were notable differences observed in comparing results for erythrosine for DI water, tap water, or well water. The results described here are consistent with our working hypothesis and suggest an approach worth considering for further exploration. It is true, for example, that aminopenicillins such as amoxicillin have a short half-life in the aquatic environ-

Figure 3. Substituted xanthenylbenzenes used in this study. Rose bengal (R1 = I, R2 = Cl), erythrosine (R1 = I, R2 = H), and eosin Y (R1 = Br, R2 = CH).

ment as noted in a recent study (13). The authors noted a concern that hypersensitivity-inducing drugs, such as penicillins and their degradation products, may elicit allergic reactions in human consumers of water and food of animal origin (13). While the scope of the present study did not involve investigation of amoxicillin or its degradation products, the two major products have the appropriate groups (phenolic and carboxylic) that might imply successful removal, based on the results described here. ACKNOWLEDGMENTS: We gratefully acknowledge samples of Octolig provided by Metre-General by President Robert L. Alldredge (dec) and by Mark Alldredge. We are grateful to Dr. Randy Larsen for the access to the Shimadzu UV-240 IPC UV-Vis recording spectrophotometer in his laboratory. We thank Dr. Kirpal Bisht for his encouragement. Statement of Dean Martin: I have never received a salary or a stipend from Metre-General, Inc., but when the current owner was grateful for helpful advice and wished to reward me, I suggested that he make a donation to one of our chemistry funds, and he did. ABOUT THE AUTHORS

Figure 2. Structure of methylene blue.

Wen-Shan Chang received a B.S. degree in Chemistry from the Tunghai University in 2007. Currently, she is a graduate

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student in chemistry at the University of South Florida where she is finishing her M.S. degree in Chemistry with Dr. Dean F. Martin. Her research now focuses on Environmental Analytical Chemistry and pharmaceutical compounds. She is also a Teaching Assistant in General Chemistry.

Dean F. Martin, Ph.D., is Distinguished University Professor Emeritus in the Department of Chemistry at the University of South Florida. He was educated at Grinnell College (B.A., 1955), The Pennsylvania State University (Ph.D. 1958), and University College, London (NSF Postdoctoral fellow, 1958– 1959). He was a faculty member at University of IllinoisChampaign/Urbana (1959–1964), before joining USF, where he became a full professor in 1969, a Distinguished University Professor in 1992, and was granted emeritus status in 2006. From 1969 through 1974, he held a PHS Career Development Award, and was a Visiting Professor of Physiology and Pharmacology at Duke University Medical Center (1970–1971). He is the author or coauthor of over 300 publications and several books, including Laboratory Chemistry and Marine Chemistry.

Meagan Small received a B.S. in Chemistry and Biology from the University of South Florida in 2007, in which her research focused primarily on microbial diversity via phylogenetic analysis using 16S rRNA. Currently, she is finishing an M.S. in

Chemistry with Dr. Randy W. Larsen at the University of South Florida. Her current research involves the use of photoacoustic calorimetry and spectroscopic methods to better understand ligand dynamics in myoglobin and heme-copper oxidase model systems.

REFERENCES 1. Bureau of the Census. Statistical abstract of the United States, 128th ed. Community hospitals—states: 2000 and 2006 (Table 165). Washington, DC: United States Department of Commerce; 2009. 2. Buser, H. R.; Poiger, T.; Muller, M. D. Occurrence and environmental behavior of the chiral pharmaceutical drug Ibuprofen in surface water and wastewater. Environ. Sci. Technol. 33:2529–2535; 1999. 3. Daughton, C. G.; Ternes, T. A. Pharmaceuticals and personal products in the environment: Agents of subtle change? Environ. Health Persp. 107(Suppl. 6):907–938; 1999. 4. European Federation of Animal Health (FEDESA) press release. Antibiotic use in farm animals does not threaten human health. July 13, 2001. 5. Golan, D.; Tashjian, A. H.; Armstrong, E. J.; Armstrong, A. W. Principles of pharmacology the pathophysiologic basis of drug therapy. Philadelphia, PA: Williams & Wilkins; 2007. 6. Gollnick, K.; Schenk, G. O. Mechanism and selectivity of the photosensitized oxygen transfer reactions. Pure Appl. Chem. 9:507–525; 1964. 7. Guardabassi, L.; Petersen, A.; Olsen, J. E.; Dalsgaard, A. Antibiotic resistance in Acinetobacter spp. isolated from sewers receiving waste effluent from a hospital and a pharmaceutical plant. Appl. Environment. Microbiol. 64: 3499–3502; 1998. 8. Hartmann, A.; Golet, E. M.; Gartiser, S.; Alder, A. C.; Koller, T.; Widmer, R. M. Primary DNA damage but not mutagenicity correlated with ciprofloxacin concentrates in German hospital waste waters. Arch. Environ Contam. Toxicol. 36:115–117; 1999. 9. Heberer, T. Occurrence, fate, and removal of pharmaceuti-


10.

11. 12.

13.

14.

15.

16.

cal residues in the aquatic environment: A review of recent research data. Toxicol. Lett. 131:5–17; 2002. Ku¨mmerer, K. Drugs in the environment: Emission of drugs, diagnostic aids and disinfectants into wastewater by hospitals in relation to other sources—a review. Chemisphere 45:957–969; 2001. Ku¨mmerer, K. Significance of antibiotics in the environment. J. Antimicrob. Chemother. 52:5–7; 2003. Ku¨mmerer, K., ed. Pharmaceuticals in the environment. Sources, fate, effects and risk, 3rd ed. Berlin/Heidelberg: Springer; 2008. Lamm, A.; Gozlan, I.; Rotstein, A.; Avisar, D. Detection of amoxicillin-diketepiperazine-2′,5′ in wastewater samples. J. Environ. Sci. Health Part A 44:1512–1517; 2009. Larsson, D. G. J.; dePedro, C.; Paxeus, N. Effluent from drug manufactures contains extremely high levels of pharmaceuticals. J. Hazard. Mat. 148(3):751–755; 2007. Leyh, R. G.; Kofidis, T.; Stru¨ber, M.; Fischer, S.; Knobloch, K.; Hagl, C.; Sirnon, A. R.; Haverich, A. Methylene blue: The drug of choice for catecholamine-refractory vasoplegia following cardiopulmonary bypass? J. Thorac. Cardiovasc. Surg. 6:1426–1431; 2003. Martin, D. F.; Aguinaldo, J. S.; Kondis, N. P.; Stull, F. W.; O’Donnell, L. F.; Martin, B. B.; Alldredge, R. L. Comparison of effectiveness of removal of nuisance anions by metalloligs, metal derivatives of Octolig. J. Environ Sci. Health Part A 43:1296–1302; 2007.

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17. Neckers, D. C. The Indian happiness wart in the development of photodynamic action. J. Chem. Educ. 84:649; 1987. 18. Nygaard, K.; Lunestad, B. T.; Hektoern, H.; Bergea, J. A.; Hormazabal, V. Resistance to oxytetracycline, oxolinic acid and furazolidine in bacteria from marine sediments. Aquaculture 104:31–36; 1992. 19. Stull, F. W.; Martin, D. F. Comparative ease of separation of mixtures of selected nuisance anions (nitrate, nitrite, sulfate, phosphate) using Octolig. J. Environ. Sci. Health Part A 44:1545–1550; 2009. 20. Towner, B. The fifty most prescribed drugs. AARP Bulletin October, 39; 2009. 21. Union of Concerned Scientists press release. 70 percent of all antibiotics given to healthy livestock. January 9, 2001. 22. Williams, R. T.; Cook, J. C. Exposure to pharmaceuticals present in the environment. Drug Inf. J. 41:133–141; 2007. 23. Wise, R. Antimicrobial resistance: Priorities for action. J. Antimicrob. Chemother. 49:585–586; 2002. 24. Witte, W. Medical consequences of antibiotic use in agriculture. Science 279:996–997; 1998. 25. Zwiener, C.; Frimmel, F. H. Oxidative treatment of pharmaceuticals in water. Water Res. 34:1881–1885; 2000.