Ultrasensitive Detection of Antiseptic Antibiotics in Aqueous Media

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Ultrasensitive Detection of Antiseptic Antibiotics in Aqueous Media and Human Urine Using Deep UV Resonance Raman Spectroscopy Christian Domes,† Robert Domes,† Jürgen Popp,†,‡,§ Mathias W. Pletz,∥ and Torsten Frosch*,†,‡,§ †

Leibniz Institute of Photonic Technology, Jena 07745, Germany Friedrich Schiller University, Institute for Physical Chemistry, Jena 07743, Germany § Friedrich Schiller University, Abbe Centre of Photonics, Jena 07745, Germany ∥ Center for Infectious Diseases and Infection Control, Jena University Hospital, Jena 07743, Germany ‡

S Supporting Information *

ABSTRACT: Deep UV resonance Raman spectroscopy is introduced as an analytical tool for ultrasensitive analysis of antibiotics used for empirical treatment of patients with sepsis and septic shock, that is, moxifloxacin, meropenem, and piperacillin in aqueous solution and human urine. By employing the resonant excitation wavelengths λexc = 244 nm and λexc = 257 nm, only a small sample volume and short acquisition times are needed. For a better characterization of the matrix urine, the main ingredients were investigated. The capability of detecting the antibiotics in clinically relevant concentrations in aqueous media (LODs: 13.0 ± 1.4 μM for moxifloxacin, 43.6 ± 10.7 μM for meropenem, and 7.1 ± 0.6 μM for piperacillin) and in urine (LODs: 36.6 ± 11.0 μM for moxifloxacin, and 114.8 ± 3.1 μM for piperacillin) points toward the potential of UV Raman spectroscopy as point-of-care method for therapeutic drug monitoring (TDM). This procedure enables physicians to achieve fast adequate dosing of antibiotics to improve the outcome of patients with sepsis.

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the dynamics of the clinical course and the necessity of organ replacement devices such as hemodialysis or extra-corporal membrane oxygenation (ECMO), the pharmacokinetics of antibiotics shows a high and unpredictable inter- and also intraindividual variability. Antibiotics recommended by major guideline for empiric treatment of critically ill patients are broad-spectrum β-lactams (e.g., meropenem and piperacillin) and fluoroquinolones (e.g., moxifloxacin).14 Several studies have confirmed that up to 30−60% of critically ill patients do not attain the requested blood concentrations after administration of licensed dosages.11,15 Correct dosing of antibiotics without therapeutic drug monitoring (i.e., measuring of the plasma concentration and adaption of the dose) is almost impossible in critically ill patients. Currently, routine TDM is only available for aminoglycosides and vancomycin, to avoid nephrotoxicity by overdosing, but not for β-lactams or fluoroquinolones.16 Furthermore, current TDM is based on enzyme-linked immunosorbent assay (ELISA) or high-performance liquid chromatography (HPLC), which are not suited for point-of-care test. The results are therefore not immediately available for the treating physician. Thus, there is a need for a chemically selective technique that monitors antibiotic concentration at the point-of-care in order to guarantee an

epsis is a life-threatening condition that arises when the body’s response to an infection injures its own tissues and organs.1 With an incidence of 314/100 000 cases and a mortality of over 30% in 2013,2 sepsis is the third most common cause of death in Germany in noncardiologic intensive care units.3 The pathological process of sepsis is a dynamic transition from sepsis to severe sepsis and septic shock with organ dysfunction and failure.4 In this context, 64% of patients with sepsis show severe sepsis within 24 h, and 23% of them develop a septic shock within the next 24 days.5 Thus, the mortality depends significantly on the moment of diagnosis and therapy.6,7 A linear increase of 7% per hour of the delayed antibiotic therapy has been shown8 and is described as the “golden hour”. However, the lethality can rise up to more than 60% (in case of septic shock)9 in the case of inappropriate treatment. Reasons for inappropriate treatment comprise not only the failure to cover the underlying pathogen (e.g., due to resistance) but also underdosing resulting in subinhibitory plasma concentrations with consecutive treatment failure.10 Increasing evidence indicates that antibiotic dosing in critically ill patients is inadequate with fixed-dose regimens.11 Dose-finding studies are usually conducted in healthy, normal-body-weight young adults in most instances. However, critically ill patients have a gross physiological derangement, which profoundly impacts the pharmacokinetics of antibiotics (increased volume of distribution, hyperdynamic status leading to increased renal/hepatic clearance, or organ failure leading to decreased clearance).12,13 Due to © XXXX American Chemical Society

Received: June 22, 2017 Accepted: August 5, 2017

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DOI: 10.1021/acs.analchem.7b02422 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry adequate antibiotic therapy and to decrease the still high mortality in septic patients. Fluoroquinolones show a wide pathogenic spectrum against Gram-negative17 and Gram-positive18 bacteria by complexation of the enzymes topoisomerase II or IV, respectively.19 These complexes are more stable than the topo-DNA-complexes of the bacteria, which inhibit their replication20 and lead to bacterial cell death.21 Other antibiotics with a good efficacy against Gram-positive and Gram-negative bacteria inclusive anaerobes are broad spectrum β-lactams.22,23 Their antibacterial activity is characterized by the covalent interaction with the active center of transpeptidase,24 which results in an opening of the peptidoglycan strands and finally leads to bacteriolysis.25 Since fewer new agents were approved in the last 20 years, there is an increase of resistance against standard antibiotics.26 On the one hand, they occur by mutation of the topoisomerases21 or a change in the transport mechanism of the antibiotics,27 and on the other hand, they result in the formation of β-lactamases28,29 rather than β-lactams. These resistances are also induced by a nonadequate antibiotic therapy if the given dose is too low or too high for the present infection. Hence, a method that can show the effective concentration of several antibiotics in biofluids such as urine or blood is indispensable. Biofluids contain several biomarkers which offer information about the human health condition30 including the early diagnosis of diseases.31 Hence, these fluids can serve as a matrix for antibiotics.32,33

Figure 1. Schematic chemical structures and absorption spectra of 0.1 mM aqueous solutions of the antibiotic agents moxifloxacin (A), meropenem (B), and piperacillin (C). The main ingredients of urine (D), namely, creatinine (E), urea (F), and uric acid (G) are shown. In case of urine, the spectra consist mostly of the absorption bands of creatinine and uric acid. The applied laser excitation wavelengths λexc = 244 nm and λexc = 257 nm are depicted as vertical short-dashed lines. These wavelengths can be tuned into the electronic absorption bands of the chromophores where strong resonance Raman enhancements can be achieved for the vibrational modes that are coupled to these electronic transitions.

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MATERIALS AND METHODS Sample Preparation. The fluoroquinolone moxifloxacin (moxifloxacin hydrochloride, 95%) was purchased from Santa Cruz Biotechnology and the β-lactams meropenem (meropenem trihydrate, 99.8%) and piperacillin (piperacillin sodium salt, 98%) as well as the main ingredients of urine, creatinine (98%), urea (95%), and uric acid (99%) were received from Sigma-Aldrich and used without further purification (see Figure 1). Water was obtained from an ultrapure water feed system from SG Water GmbH (κ > 0.06 μS/cm) and the urine samples were collected from nonsmoker volunteers early in the morning, filtered with 0.20 μm syringe filters (Corning Incorporated) and stored at 8 °C before use. First, artificial urine samples (mixture of its main ingredients creatinine (2150 mg/L), urea (23,300 mg/L), and uric acid (670 mg/L)) were analyzed as an intermediated step with controlled matrix. Only small differences were observed between the intensity patterns of the resonance Raman spectra of artificial and real urine (Figure S1) with λexc = 244 nm. The application of λexc = 257 nm caused fluorescence background in the real urine samples, which was not observed in the artificial urine (Figure S1). This background caused a lower sensitivity in the quantification experiments with λexc = 257 nm. In the next step, real urine samples from two nonsmoker volunteers were analyzed, to exclude variations of the urine matrix. The samples showed very similar Raman spectra (Figure S2). For the application of wavelength λexc = 257 nm, a fluorescence background was excited, which caused a stronger deviation in the higher wavenumber region of the Raman spectra (Figure S2). The variations between the Raman spectra of different urine samples were much smaller than the differences in the urine samples with antibiotics. Urine samples from volunteer 1 were used for further investigations, and all samples were measured three times for the quantification.

For FT-Raman experiments, the antibiotics and ingredients were used as solids, 2 mL of the urine was dried on a glass slide under vacuum, and the resulting yellow solid was investigated. For quantification, the antibiotics were dissolved in water and urine, and solutions of various concentrations were prepared. Deep UV Resonance Raman spectroscopy. The resonance Raman spectra were acquired with a LabRAM system (Jobin Yvon HR800) with frequency-doubled Ion laser (Innova 300C Moto-FRED). The excitation wavelengths were λexc = 244 nm (laser power: 3.58 mW (water) and 1.03 mW (urine) at the samples) and λexc = 257 nm (laser power: 2.78 mW (water) and 2.91 mW (urine) at the samples). Any photodegradation of the sample solutions was strictly avoided. The absorption spectra were measured with a Carry 5000 system (Variant). FT-Raman spectra were acquired with a Nd:YAG laser with an excitation wavelength of λexc = 1064 nm and a Ram II spectrometer from Bruker. The resonance Raman spectra of the solutions were normalized to the Raman band of water and urine at approximately 1650 and 680 cm−1, respectively. The signal-to-noise ratios (SNR) were calculated from the height of a Lorenzian fit of the target peaks and the root-mean-square (RMS) values of the baseline. The signals were derived when the SNR was higher than or equal to three. The limits of detection (LOD) were calculated by plotting the SNR against the analytes concentration. From this linear regression, the concentration at a SNR equal to 3 was taken as the LOD. Density Functional Theory Calculations for the Assignment of Raman Marker Bands. For a better understanding of the assignment and an interpretation of Raman marker bands that were used for the quantification of the fluoroquinolone and the β-lactams, the molecular structures were optimized and vibrational modes and Raman scattering activities were calculated B


Article

Analytical Chemistry using density functional theory (DFT) with Gaussian 09.34 Therefore, the hybrid exchange correlation functional with Beckes three-parameter exchange functional (B3) slightly modified by Stephens et al. coupled with correlation part of functional from Lee, Yang, and Parr (B3LYP) and Dunnings triple (cc-pVTZ) correlation consistent basis sets of contracted Gaussian functions with polarized and diffuse functions were applied. Here, different

scaling factors for the higher (1800) wavenumber region, and an intensity correction were estimated.35

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RESULTS AND DISCUSSION In this work, the potential of UV resonance Raman spectroscopy36−38 as a powerful analytical tool for monitoring clinically relevant concentrations of antibiotics in aqueous media and urine should be demonstrated. Established methods such as gas chromatography coupled with mass spectrometer (GC-MS),39 HPLC,40 and bioassays41 require large sample volumes, extended sample preparation and measurement time, and are destructive. Raman spectroscopy is a direct optical technique,42,43 based on the intrinsic molecular vibrations,44−48 and has a high potential for rapid online drug monitoring.49 This nondestructive technique can be applied for simultaneous detection of several analytes.50−52 Since inelastic

Figure 2. Raman spectra of moxifloxacin (1), meropenem (2), and piperacillin (3) in aqueous solution (4) with excitation wavelengths λexc = 244 nm (A) and λexc = 257 nm (B): Raman marker bands that are utilized for the quantification are marked. The concentrations were 0.1 mM for A1 and B1, 0.5 mM for A2 and B2, 0.1 mM for A3, and 0.5 mM for B3. The average spectra and their deviation are depicted in black and gray, respectively. The spectrum of water was used as an internal standard and does not overlap with the signals of the analytes.

Figure 3. FT-Raman spectra of moxifloxacin (A), meropenem (B), piperacillin (C), and urine (D) and its ingredients creatinine (E), urea (F), and uric acid (G). The five strongest bands of the antibiotic agents are marked (including the marker band). For urine, the signals are marked, which are enhanced with UV resonance excitation.

Figure 4. Assignment of the vibrational modes of the Raman marker bands of moxifloxacin (A), meropenem (B), and piperacillin (C) that were used for their quantification (see Figures 5 and 8). Their atomic displacements are shown for the Raman bands at 1373 cm−1 (A), 1390 cm−1 (B), and 1485 cm−1 (C). C


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Analytical Chemistry Raman scattering is a weak process, several enhancement techniques have been developed, such as fiber-enhanced53−56 and resonance Raman spectroscopy.57,58 Here the excitation wavelength can be tuned in the electronic absorption of the molecules and a strong signal enhancement of vibrational modes coupled to these transitions can be achieved, resulting in detection limits in the micromolar range. UV Resonance Raman Spectroscopy of the Antibiotic Agents in Aqueous Media. First, the potential of UV resonance Raman spectroscopy for the detection of micromolar concentrations of the antibiotics moxifloxacin, meropenem, and piperacillin in aqueous media with excitation wavelengths in the deep UV region should be shown. The absorptions of the analyte molecules (Figure 1A−C) show high potential for a strong resonance enhancement of the Raman signals. Here, a serial dilution based on a stock solution of 100 μM (moxifloxacin, lowest concentration: 0.1 μM) and 1000 μM (β-lactams, lowest concentration: 1 μM) was prepared, and resonance Raman spectra were acquired. For every analyte, a strong Raman band was chosen for quantification (Figure 2). For a better understanding of these Raman marker bands, DFT calculations were

compared with the experimentally acquired FT-Raman spectra (Figure 3A−C and S3), and their vibrational modes (Figure 4) were assigned. In detail, the bands at 1373 cm−1 can be assigned to CH2−wagging vibration of the octahydro−pyrrolo−pyridine group for moxifloxacin (Figure 4A), and the Raman signals of the β-lactams at 1390 and 1485 cm−1 can be interpreted as CH3wagging, C−OH-scissoring, and HC−CH-rocking vibrations of the substituted pyrrole unit for meropenem (Figure 4B), and as HC−CH-rocking vibrations of the benzyl group and several NH-out-of-plane bending vibrations of piperacillin (Figure 4C), respectively. When plotting the Raman band intensities against the analyte concentrations, all active agents show a good linearity, which can be used for robust quantification of the antibiotic concentrations (Figure 5). The LODs can be assessed from the concentration with a SNR equal to 3. The excitation with wavelength λexc = 257 nm results in minimal values of 13.0 ± 1.4 μM, 43.6 ± 10.7 μM, and 11.3 ± 0.5 μM and an excitation with wavelength λexc = 244 nm in minimal values of 13.9 ± 1.7 μM, 96.9 ± 4.4 μM, and 7.1 ± 0.6 μM for moxifloxacin, meropenem, and piperacillin, respectively. All values are summarized in Table 1 and are in good agreement with the clinically relevant concentration ranges

Figure 5. Quantification of moxifloxacin (A1, A2), meropenem (B1, B3), and piperacillin (B2, B4) in aqueous solution. The Raman intensities were defined by the peak heights of the Raman bands shown in Figure 4 while using deep UV excitation wavelengths λexc = 244 nm (A1, B1, B2) and λexc = 257 nm (A2, B3, B4). The influence of the excitation wavelength can be seen: Piperacillin shows a higher absorbance at λexc = 244 nm which results in a lower LOD value for this wavelength, while the other antibiotic agents have lower LOD values with excitation wavelength λexc = 257 nm.

Figure 6. Resonance Raman spectra of urine (A) and its ingredients creatinine (B), urea (C), and uric acid (D) with excitation wavelength λexc = 244 nm. Several Raman signals were significantly enhanced. The resonance Raman spectra of relevant mixtures (creatinine (2150 mg/L), urea (23,300 mg/L), and uric acid (670 mg/L)) were measured with excitation wavelengths λexc = 244 nm (E) and λexc = 257 nm (F), which show similar results.

Table 1. Summary of the limits of detection of moxifloxacin, meropenem, and piperacillin with laser excitation wavelengths λexc = 244 nm (Plaser = 3.58 mW (aqueous media), 1.03 mW (urine)) and λexc = 257 nm (Plaser = 2.78 mW (aqueous media), 2.91 mW (urine))a LOD/μM aqueous media

urine

antibiotic agent

244 nm

257 nm

244 nm

257 nm

dose/mg

clinical concentration/μM

moxifloxacin meropenem piperacillin

13.9 ± 1.7 96.9 ± 4.4 7.1 ± 0.6

13.0 ± 1.4 43.6 ± 10.7 11.3 ± 0.5

36.6 ± 11.0 358.5 ± 53.4 114.8 ± 3.1

1372.2 ± 226.7 3802.4 ± 410.1 1039.5 ± 200.7

400 500 2000

193.1 ± 111.8 [o]60 1895.1 ± 783.4 [iv]61 25238 ± 15200 [iv]62

a

The values were normalized to the higher laser powers of the two wavelengths, respectively. For a comparison of the results, literature values of clinically relevant concentrations in urine with their administration form (o: oral, iv: intravenous) are listed below in square brackets. These values strongly depend on the given dose and the pharmacokinetics of the antibiotics as well as the physiological conditions of the patient. D


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Analytical Chemistry

Resonance Raman Spectroscopy of Urine and Its Ingredients. The next step toward clinically relevant conditions was the measurement of the antibiotic agents in the biofluid urine. Therefore, urine and its components were characterized using UV/VIS absorption spectroscopy (Figure 1D−G) and FT-Raman spectroscopy (Figure 3D−G) for an overview of the absorption bands and Raman signals. The absorption bands of urine arise mostly from the signals of creatinine and uric acid (Figure 1D,E,G). The bands in the FT-Raman spectrum of urine are dominated by urea (mainly at 1011 cm−1 in Figure 3D,F). The Raman spectra of urine were acquired with excitation wavelengths λexc = 244 nm and λexc = 257 nm and compared with the spectra of the urine ingredients. Also, a mixture of relevant concentrations of creatinine (2150 mg/L), urea (23,300 mg/L), and uric acid (670 mg/L)59 was prepared and the Raman spectra were measured. No significant difference was discovered while using λexc = 244 nm or λexc = 257 nm (Figure 6E,F) and the resonance Raman spectra of urine originate mainly from the Raman signals of creatinine (Figure 6A,B). This can be explained by the higher absorbance and amount of creatinine compared with urea and uric acid, respectively. Using UV resonant wavelengths, several Raman signals of urine were significantly enhanced compared to the FT data (Figure 3 and 6). In detail, the amide I bands of uric acid and urea at 1649 and 1607 cm−1, a CN- and CC-stretching vibration combined with NH- and OH-bending vibrations of uric acid at 1498 cm−1, a combined CH2- and CH3-wagging vibration of creatinine at 1427 cm−1, a CC-stretching vibration of uric acid at 1348 cm−1, a combination of CN-stretch, CH3-rocking, and CH2-twisting vibration of creatinine at 1245 cm−1, a combined N−CH2-bending and a stretching vibration of the imidazolidine ring of creatinine at 843 cm−1, and a C−NH2−, CO−, and imidazolidine ring stretching vibration of creatinine at 681 cm−1 were enhanced. The Raman signal at 1011 cm−1 (C−NH2-stretching vibration of urea) was increased relative to these vibrations.

Figure 7. Raman spectra of moxifloxacin (1), meropenem (2), and piperacillin (3) in human urine (4) with excitation wavelengths λexc = 244 nm (A) and λexc = 257 nm (B) and the utilized marker bands for their quantification. The concentrations shown were 10 mM for A1, 5 mM for B1, 10 mM for A2 and B2, 1 mM for A3, and 5 mM for B3. The average spectra and their deviation are depicted in black and gray, respectively. Excitation with λexc = 257 nm caused fluorescence background, which overlapped the Raman signals of human urine. The signals of urine do not disturb the Raman signals of the analytes, such that biofluid can be used as matrix for quantification.

(Table 1), which demonstrates the potential for ultrasensitive detection of antibiotics with the applied UV excitation wavelengths.

Figure 8. Quantification of moxifloxacin (A1, A2), meropenem (B1, B3), and piperacillin (B2, B4) in human urine. The Raman intensities were defined by the peak heights of the Raman bands (see Figure 4) while using deep UV excitation wavelengths λexc = 244 nm (A1, B1, B2) and λexc = 257 nm (A2, B3, B4). The regression lines from excitation wavelength λexc = 257 nm show a shallower slope compared with excitation wavelength λexc = 244 nm, which is caused by fluorescence background. E


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Analytical Chemistry Resonance Raman Spectroscopy of the Antibiotic Agents in Urine. After the thorough characterization of the UV resonance Raman spectra of urine, this biofluid was used as a matrix for the detection of the investigated antibiotics. Therefore, urine was spiked with the previously used analytes to a 10 mM stock solution, diluted to a concentration of 0.5 mM and the resonance Raman spectra were acquired by using deep UV excitation wavelengths (Figure 7). The quantification was done in analogy with the measurements in aqueous media by plotting the Raman band intensities against the corresponding drug concentration. An excellent linearity was observed (Figure 8), with LODs in the two- to three-digit micromolar range. The excitation with λexc = 257 nm caused fluorescence of the urine matrix, which decreased the sensitivity by 1 order of magnitude. The LODs with excitation wavelength λexc = 244 nm were 36.6 ± 11.0 μM, 358.5 ± 53.4 μM, and 114.8 ± 3.1 μM for moxifloxacin, meropenem, and piperacillin and are in the range of the clinically relevant concentration (Table 1). Thus, the experiments demonstrate the potential of deep UV resonance Raman as a promising tool for future clinical applications.

M.W.P. was supported by a grant from the German Ministry of Education and Research (01KI1501).

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(1) Czura, C. Mol. Med. 2011, 17 (1−2), 2−3. (2) Fleischmann, C.; Hartmann, M.; Hartog, C.; Welte, T.; Heublein, S.; Thomas-Rueddel, D.; Dennler, U.; Reinhart, K. Intensive Care Medicine Experimental 2015, 3, A50. (3) Hall, M.; Williams, S.; DeFrances, C.; Golosinskiy, A. U.S. Department of Health and Human Services - National Center for Health Statistics 2011, 32, 1−7. (4) Bone, R.; Balk, R.; Cerra, F.; Dellinger, R.; Fein, A.; Knaus, W.; Schein, R.; Sibbald, W. Chest 1992, 101 (6), 1644−1655. (5) Rangel-Frausto, M.; Pittet, D.; Costigan, M.; Hwang, T.; Davis, C.; Wenzel, R. Journal of the American Medical Association 1995, 273 (2), 117−123. (6) Rivers, E.; Nguyen, B.; Havstad, S.; Ressler, J.; Muzzin, A.; Knoblich, B.; Peterson, E.; Tomlanovich, M. N. Engl. J. Med. 2001, 345 (19), 1368−1377. (7) Iregui, M.; Ward, S.; Sherman, G.; Fraser, V.; Kollef, M. Chest 2002, 122 (1), 262−268. (8) Kumar, A.; Roberts, D.; Wood, K.; Light, B.; Parrillo, J.; Sharma, S.; Suppes, R.; Feinstein, D.; Zanotti, S.; Taiberg, L.; et al. Crit. Care Med. 2006, 34 (6), 1589−1596. (9) Kumar, A.; Ellis, P.; Arabi, Y.; Roberts, D.; Light, B.; Parrillo, J.; Dodek, P.; Wood, G.; Kumar, A.; Simon, D.; et al. Chest 2009, 136 (5), 1237−1248. (10) Udy, A. A.; Roberts, J. A.; De Waele, J. J.; Paterson, D. L.; Lipman, J. Int. J. Antimicrob. Agents 2012, 39 (6), 455−7. (11) Roberts, J. A.; Paul, S. K.; Akova, M.; Bassetti, M.; De Waele, J. J.; Dimopoulos, G.; Kaukonen, K. M.; Koulenti, D.; Martin, C.; Montravers, P.; Rello, J.; Rhodes, A.; Starr, T.; Wallis, S. C.; Lipman, J.; Study, D. Clin. Infect. Dis. 2014, 58 (8), 1072−1083. (12) Udy, A. A.; Roberts, J. A.; Lipman, J. Nat. Rev. Nephrol. 2011, 7 (9), 539−543. (13) Roberts, D. M.; Roberts, J. A.; Roberts, M. S.; Liu, X.; Nair, P.; Cole, L.; Lipman, J.; Bellomo, R. Crit. Care Med. 2012, 40 (5), 1523− 1528. (14) Dellinger, R. P.; Levy, M. M.; Rhodes, A.; Annane, D.; Gerlach, H.; Opal, S. M.; Sevransky, J. E.; Sprung, C. L.; Douglas, I. S.; Jaeschke, R.; Osborn, T. M.; Nunnally, M. E.; Townsend, S. R.; Reinhart, K.; Kleinpell, R. M.; Angus, D. C.; Deutschman, C. S.; Machado, F. R.; Rubenfeld, G. D.; Webb, S. A.; Beale, R. J.; Vincent, J. L.; Moreno, R. Crit. Care Med. 2013, 41 (2), 580−637. (15) Pletz, M. W.; Bloos, F.; Burkhardt, O.; Brunkhorst, F. M.; BodeBoger, S. M.; Martens-Lobenhoffer, J.; Greer, M. W.; Stass, H.; Welte, T. Intensive Care Med. 2010, 36 (6), 979−83. (16) Joukhadar, C.; Frossard, M.; Mayer, B.; Brunner, M.; Klein, N.; Siostrzonek, P.; Eichler, H.; Müller, M. Crit. Care Med. 2001, 29 (2), 385−391. (17) Stein, G. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy 1988, 8 (6), 301−314. (18) Ullmann, U.; Kämpfer, K.; Pflug-Rolfes, A. Chemotherapie Journal 1998, 16, 1−9. (19) Goss, W.; Deitz, W.; Cook, T. J. Bacteriol. 1964, 88 (4), 1112− 1118. (20) Hawkey, P. J. Antimicrob. Chemother. 2003, 51, 29−35. (21) Wiedemann, B.; Heisig, P. Chemotherapie Journal 1999, 8 (3), 99−107. (22) Roberts, J.; Lipman, J. Crit. Care Med. 2009, 37 (3), 840−851. (23) Bradley, J.; Garau, J.; Lode, H.; Rolston, K.; Wilson, S.; Quinn, J. Int. J. Antimicrob. Agents 1999, 11 (2), 93−100. (24) Tomasz, A. Annu. Rev. Microbiol. 1979, 33 (1), 113−137. (25) Mutschler, E.; Geisslinger, G.; Kroemer, H.; Schäfer-Korting, M. Arzneimittelwirkungen, Lehrbuch der Pharmakologie und Toxikologie; Wissenschaftliche Verlagsgesellschaft mbH: 2001; Vol. 8. (26) Toloo, S.; Rego, J.; Fitzgerald, G.; Aitken, P.; Ting, J.; Quinn, J.; Enraght-Moony, E. Emergency Health Services: Demand and Service Delivery Models; Queensland University of Technology: 2012.

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CONCLUSIONS This work illustrates the potential of deep UV resonance Raman spectroscopy for ultrasensitive monitoring of clinically relevant concentrations of the antiseptic antibiotics moxifloxacin, meropenem, and piperacillin in aqueous media as well as in human urine. These analytes could be traced down in aqueous solutions to minimal concentrations of 11.3 ± 0.5 μM (λexc = 257 nm) and 7.1 ± 0.6 μM (λexc = 244 nm) for piperacillin. In urine, a minimal concentration of 36.6 ± 11.0 μM (λexc = 244 nm) was achieved for moxifloxacin. Thus, UV Raman spectroscopy allows rapid online monitoring of clinically relevant antibiotic concentrations (Table 1). In the case of urine, wavelength λexc = 244 nm is better suited, because fluorescence arises while using excitation wavelength λexc = 257 nm. The results of this study show high potential for deep UV resonance Raman spectroscopy as a novel analytical tool for monitoring of antibiotics and provide the foundation for future clinical studies.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b02422. Additional information regarding the DFT calculations (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mails for T.F.: [email protected], torsten.frosch@ gmx.de. ORCID

Torsten Frosch: 0000-0003-3358-8878 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Funding from the federal state of Thuringia and European Union (EFRE) is highly acknowledged (2015 FE 9012 and 2015-0021). F


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