Separation-Oriented Derivatization of Native Fluorescent Compounds

May 21, 2009 - ... felbinac, flurbiprofen, and etodolac) were used as model native fluorescent compounds. Experimental results indicate that the fluor...
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Anal. Chem. 2009, 81, 5039–5045

Separation-Oriented Derivatization of Native Fluorescent Compounds through Fluorous Labeling Followed by Liquid Chromatography with Fluorous-Phase Yohei Sakaguchi, Hideyuki Yoshida, Kenichiro Todoroki, Hitoshi Nohta, and Masatoshi Yamaguchi* Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Johnan, Fukuoka 814-0180, Japan We have developed a new and simple method based on “fluorous derivatization” for LC of native fluorescent compounds. This method involves the use of a column with a fluorous stationary phase. Native fluorescent analytes with target functional groups are precolumn derivatized with a nonfluorescent fluorous tag, and the fluorouslabeled analytes are retained in the column, whereas underivatized substances are not. Only the retained fluorescent analytes are detected fluorometrically at appropriate retention times, and retained substrates without fluorophores are not detected. In this study, biologically important carboxylic acids (homovanillic acid, vanillylmandelic acid, and 5-hydroxyindoleacetic acid) and drugs (naproxen, felbinac, flurbiprofen, and etodolac) were used as model native fluorescent compounds. Experimental results indicate that the fluorous-phase column can selectively retain fluorous compounds including fluorouslabeled analytes on the basis of fluorous separation. We believe that separation-oriented derivatization presented here is the first step toward the introduction of fluorous derivatization in quantitative LC analysis. As reviewed by Przybyciel1 and Zhang,2 fluorinated compounds have been used as stationary phases in liquid chromatography (LC) since the 1980s.3-5 Although the examinations for development of novel stationary phase with fluorinated compounds have been currently in progress,6,7 some LC columns with fluorocarbon stationary phases are commercially available. The stationary phase of these columns is either a pentafluorophenyl-type phase or a perfluoroalkyl-type phase.1,2 The perfluoroalkyl-containing compounds have unique properties such as high electronegativity, low polarizability, strong lipophobicity and hydrophobicity, and good * Corresponding author. E-mail: [email protected]. Fax: +81-92-8630389. (1) Przybyciel, M. LCGC Eur. 2006, 19, 19–27. (2) Zhang, W. J. Fluorine Chem. 2008, 129, 910–919. (3) Pirkle, W. H.; House, D. W.; Finn, J. M. J. Chromatogr. 1980, 192, 143– 158. (4) Berendsen, G. E.; Pikaart, K. A.; de Galan, L.; Olieman, C. Anal. Chem. 1980, 52, 1990–1993. (5) Xindu, G.; Carr, P. W. J. Chromatogr. 1983, 269, 96–102. (6) Shearer, J. W.; Ding, L.; Olesik, S. V. J. Chromatogr., A 2007, 1141, 73– 80. (7) Daley, A. B.; Oleschuk, R. D. J. Chromatogr., A 2009, 1216, 772–780. 10.1021/ac9005952 CCC: $40.75  2009 American Chemical Society Published on Web 05/21/2009

thermal and chemical stability.8,9 However, the most interesting property of these compounds is their high fluorophilicity between perfluoroalkyl compound (structure) and perfluoroalkyl compound (structure), so-called “fluorous”.1,2,8-10 Therefore, a branch of organic chemistry is dedicated to research on fluorous chemistry. Furthermore, a technique involving fluorous solid-phase extraction (F-SPE) based on fluorous chemistry has been developed for the collection or removal of fluorous compounds.10-15 There are major functional differences between a nonfluorous stationary phase, which is commonly known as a reversed-phase (RP), and a fluorous (perfluoroalkyl) stationary phase. A fluorousphase column has the following features: (1) It can retain only fluorous compounds strongly but not hydrophilic compounds. (2) It is suitable for resolving a mixture of fluorinated compounds according to their fluorine content. (3) It is chemically and thermally stable, and therefore, it has high reproducibility and a long lifetime comparable to that of RP-LC columns. Many studies on the separation mechanism in LC with a fluorocarbon stationary phase have revealed that “secondary effects”, e.g., absorption to silanol residue on the particulate, and fluorophilicity affect the retention of analytes.16-21 Therefore, LC columns with fluorocarbon stationary phases have been used to (8) Gladysz, J. A., Curran, D. P., Horva´th, I. T., Eds. Handbook of Fluorous Chemistry; Wiley-VCH: Weinheim, Germany, 2004. (9) Uneyama, K. Organofluorine Chemistry; Blackwell Publishing: Oxford, U.K., 2006. (10) Luo, Z.; Zhang, Q.; Oderaotoahi, Y.; Curran, D. P. Science 2001, 291, 1766– 1769. (11) Curran, D. P.; Hadida, S.; He, M. J. Org. Chem. 1997, 62, 6714–6715. (12) Curran, D. P.; Hadida, S.; Kim, S.-Y.; Luo, Z. J. Am. Chem. Soc. 1999, 121, 6607–6615. (13) Dandapani, S. QSAR Comb. Sci. 2006, 25, 681–688. (14) Zhang, W.; Curran, D. P. Tetrahedron 2006, 62, 11837–11865. (15) Horhant, D.; Lamer, A.-C. L.; Boustie, J.; Uriac, P.; Gouault, N. Tetrahedron Lett. 2007, 48, 6031–6033. (16) Jinno, K.; Nakamura, H. Chromatographia 1994, 39, 285–293. (17) Turowski, M.; Morimoto, T.; Kimata, K.; Monde, H.; Ikegami, T.; Hosoya, K.; Tanaka, N. J. Chromatogr., A 2001, 911, 177–190. (18) Neue, U. D.; Tran, K. V.; Iraneta, P. C.; Alden, B. A. J. Sep. Sci. 2003, 26, 174–186. (19) Euerby, M. R.; McKeown, A. P.; Petersson, P. J. Sep. Sci. 2003, 26, 295– 306. (20) Marchand, D. H.; Croes, K.; Dolan, J. W.; Snyder, L. R.; Henry, R. A.; Kallury, K. M. R.; Waite, S.; Carr, P. W. J. Chromatogr., A 2005, 1062, 65–78. (21) Poole, C. F.; Ahmed, H.; Kiridena, W.; DeKay, C.; Koziol, W. W. Chromatographia 2007, 65, 127–139.

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separate highly hydrophobic compounds22-25 and structural isomers16,17,26-28 that are difficult to separate with conventional RP columns. Furthermore, drug analogues containing some trifluoromethyl structure, a C1 fluorous unit, have been successfully determined by LC separation using a fluorous-phase column.27,29 Because the fluorous stationary phase can retain fluorous compounds effectively, many examinations based on fluorous interaction have been conducted to collect or remove fluorous compounds10,15,30-32 and to investigate the specific separation mechanisms.16-21,33 However, to the best of our knowledge, there are no reports on quantitative analysis of fluorous-labeled compounds using a fluorous-phase LC column. Some native fluorescent compounds are useful for clinical investigations, food analysis, etc. However, because biological, food, and environmental samples contain different types and amounts of native fluorescent compounds, it is very difficult to detect the specific analytes in such samples by a simple procedure. Moreover, the analytes sometimes form complexes with other chemicals in the samples. Therefore, LC analyses are generally performed by using not only pretreatment techniques such as liquid-liquid extraction (LLE), SPE, and ultrafiltration but also sophisticated separation techniques such as gradient elution and column switching. Recently, we developed a simple method for LC analysis of biological native fluorescent compounds based on derivatization in which fluorescent resonance energy transfer (FRET) occurs between analytes and a labeled fluorophore.34,35 This method has sufficient selectivity and is suitable for analyzing biosamples without pretreatment. However, the combination of analytes and fluorescent derivatization reagents used in this method must be optimum in order to achieve effective FRET. Therefore, this method has not been used extensively in practice. In this article, we report a new and simple method for LC of native fluorescent compounds. This method is based on “fluorous derivatization”, and its principle is described as follows: Native fluorescent analytes with target functional groups are precolumn derivatized with a nonfluorescent fluorous tag. The fluorouslabeled analytes are retained in the fluorous-phase column, whereas underivatized substances are not. Only the retained fluorescent analytes are detected fluorometrically at appropriate retention times, and retained substrates without fluorophores are (22) Aboul-Enein, H. Y.; Serignese, V. Anal. Chim. Acta 1996, 319, 187–190. (23) Kamiusuki, T.; Monde, T.; Nemoto, F.; Konakahara, T.; Takahashi, Y. J. Chromatogr., A 1999, 852, 475–485. (24) Lanina, S. A.; Toledo, P.; Sampels, S.; Kamal-Eldin, A.; Jastrebova, J. A. J. Chromatogr., A 2007, 1157, 159–170. (25) Sundstro ¨m, I.; Andre´n, P. E.; Westerlund, D. J. Chromatogr., A 2008, 1189, 503–513. (26) Monde, T.; Kamiusuki, T.; Kuroda, T.; Mikumo, K.; Ohkawa, T.; Fukube, H. J. Chromatogr., A 1996, 722, 273–280. (27) Obayashi, M.; Kosugi, T.; Yamazaki, J.; Matsumoto, Y.; Fukuoka, M.; Matsumoto, M. J. Chromatogr., B 1999, 726, 219–223. (28) Wood, N.; Gibbs, D. D.; Jackman, A. L.; Henley, A.; Workman, P.; Raynaud, F. J. Chromatogr., B 2005, 824, 181–188. (29) Xu, Y.; Willson, K. J.; Musson, D. G. J. Chromatogr., B 2008, 863, 64–73. (30) Curran, D. P.; Luo, Z. J. Am. Chem. Soc. 1999, 121, 9069–9072. (31) Zhang, W.; Luo, Z.; Chen, C. H.-T.; Curran, D. P. J. Am. Chem. Soc. 2002, 124, 10443–10450. (32) Werner, S.; Curran, D. P. Org. Lett. 2003, 5, 3293–3296. (33) Kamiusuki, T.; Monde, T.; Yano, K.; Yoko, T.; Konakahara, T. Chromatographia 1999, 49, 649–656. (34) Yoshitake, M.; Nohta, H.; Yoshida, H.; Yoshitake, T.; Todoroki, K.; Yamaguchi, M. Anal. Chem. 2006, 78, 920–927. (35) Yoshitake, M.; Nohta, H.; Ogata, S.; Todoroki, K.; Yoshida, H.; Yoshitake, T.; Yamaguchi, M. J. Chromatogr., B 2007, 858, 307–312.

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not detected. We believe that separation-oriented derivatization presented here is the first step toward the introduction of fluorous derivatization in quantitative LC analysis, although fluorous labeling and F-SPE have been used in some recent analytical studies.36,37 For the first examination based on this method, we selected carboxylic acids, whose derivatization analysis is more difficult than that of amines and thiols,38,39 as the analyte group and used biologically important carboxylic acids [homovanillic acid (HVA), vanillylmandelic acid (VMA), and 5-hydroxyindoleacetic acid (5-HIAA)] and drugs (naproxen, felbinac, flurbiprofen, and etodolac) as model native fluorescent compounds. HVA, VMA, and 5-HIAA are metabolites of major neurotransmitters in mammals; their concentration in urine is an indicator of a neuroendocrine tumor.40-42 The drugs used in this study are four representative nonsteroidal anti-inflammatory drugs (NSAIDs). These acids are commonly determined by LC with fluorescence or electrochemical detection42-44 and by LC with mass spectrometry (LC-MS).42,45 However, the former needs tedious pretreatment such as LLE and SPE, and the apparatus used for the latter is expensive and large. The fluorous derivatization method has been developed to overcome these drawbacks, and it is a simple and inexpensive method for LC analysis. In this study, 4,4,5,5,6,6,7,7, 8,8,9,9,10,10,11,11,11-heptadecafluoro-n-undecylamine (HFUA) was used as a derivatization reagent for carboxylic acids with a fluorous structure, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) was used as an effective reagent for condensation reaction between HFUA and the analytes.46-49 Fluorous derivatization was carried out for the specific determination of native fluorescent carboxylic acids (Scheme 1). Furthermore, this method has been successfully applied to the analysis of human plasma spiked with NSAIDs. To the best of our knowledge, the present study is the first report on the determination of native fluorescent compounds by separation-oriented derivatization based on fluorous labeling and fluorous-phase LC. (36) Brittain, S. M.; Ficarro, S. B.; Brock, A.; Peters, E. C. Nat. Biotechnol. 2005, 23, 463–468. (37) Go, E. P.; Uritboonthai, W.; Apon, J. V.; Trauger, S. A.; Nordstrom, A.; O’Maille, G.; Brittain, S. M.; Peters, E. C.; Siuzdak, G. J. Proteome Res. 2007, 6, 1492–1499. (38) Lunn, G.; Hellwig, L. C. Handbook of Derivatization Reactions for HPLC; John Wiley & Sons: New York, 1998. (39) Fukushima, T.; Usui, N.; Santa, T.; Imai, K. J. Pharm. Biomed. Anal. 2003, 30, 1655–1687. (40) Russo, S.; Nielen, M. M. A.; Boon, J. C.; Kema, I. P.; Willemse, P. H. B.; de Vries, E. G. E.; Korf, J.; den Boer, J. A. Psychopharmacology 2003, 168, 324–328. (41) Monsaingeon, M.; Perel, Y.; Simonnet, G.; Corcuff, J.-B. Eur. J. Pediatr. 2003, 162, 397–402. (42) Lionetto, L.; Lostia, A. M.; Stigliano, A.; Cardelli, P.; Simmaco, M. Clin. Chim. Acta 2008, 398, 53–56. (43) Kazemifard, A. G.; Moore, D. E. J. Chromatogr. 1990, 533, 125–132. (44) Suh, H.; Jun, H. W.; Lu, G. W. J. Liq. Chromatogr. Relat. Technol. 1995, 18, 3105–3115. (45) Sultan, M.; Stecher, G.; Sto ¨ggl, W. M.; Bakry, R.; Zaborski, P.; Huck, C. W.; El Kousy, N. M.; Bonn, G. K. Curr. Med. Chem. 2005, 12, 573–588. (46) Kunishima, M.; Kawachi, C.; Morita, J.; Terao, K.; Iwasaki, F.; Tani, S. Tetrahedron 1999, 55, 13159–13170. (47) Kunishima, M.; Kawachi, C.; Hioki, K.; Terao, K.; Tani, S. Tetrahedron 2001, 57, 1551–1558. (48) Sekiya, S.; Wada, Y.; Tanaka, K. Anal. Chem. 2005, 77, 4962–4968. (49) Wheeler, S. F.; Domann, P.; Harvey, D. J. Rapid Commun. Mass Spectrom. 2009, 23, 303–312.

Scheme 1. Fluorous Derivatization of Native Fluorescent Carboxylic Acids in the Presence of 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4methylmorpholinium Chloride

Scheme 2. Chemical Structures of Native Fluorescent Carboxylic Acids Examined in This Study

EXPERIMENTAL SECTION Reagents and Solutions. Unless stated otherwise, all chemicals mentioned below were of the highest purity available and were used as received. HVA, VMA, 5-HIAA, naproxen, etodolac, and DMT-MM were purchased from Wako Pure Chemicals (Osaka, Japan). Felbinac, flurbiprofen, and HFUA were obtained from Alfa Aesar (Ward Hill, MA), Sigma-Aldrich (St. Louis, MO), and Fluka (Buchs, Switzerland), respectively. The structures of the analytes are presented in Scheme 2. All organic solvents were of LC grade. It should be noted that these reagents and solvents are toxic to the eyes, lungs, and skin and should be used carefully according to guidelines specified in the latest material safety data sheets. Ultrapure water, purified using a Milli-Q gradient system (Millipore, Billerica, MA), was used to produce all aqueous solutions.

Stock solutions (1.0 mM) of analytes were prepared in water or acetonitrile and stored at room temperature. They were stable for at least 1 week and diluted further with tetrahydrofuran (THF) to the required concentrations before use. A solution of 20 mM HFUA in THF was usable for at least 1 week when stored at room temperature. A solution of 10 mM DMT-MM in 90% (v/v) THF was stored at room temperature and used within 1 day. Derivatization Procedure. The sample solution (200 µL) was placed in a 1.5 mL vial, and 100 µL of 20 mM HFUA and 200 µL of 10 mM DMT-MM were successively added to it. The vial was tightly sealed and left at room temperature for 5 min. After derivatization, 500 µL of water was added to the vial, and the entire reaction solution was placed in the autosampler of an LC system. A reagent blank was prepared by the same procedure using a 200 µL aliquot of 90% (v/v) THF. Plasma Sample and Pretreatment. A plasma sample was obtained from whole blood (8.5 mL) of a healthy male volunteer (23 years old, 57 kg), using a vacuum blood collection tube (SSTII, Becton, Dickinson and Company, Franklin Lakes, NJ). The volunteer understood the purpose and significance of this experiment and donated blood after signing an agreement. The blood was immediately centrifuged at 9 000g for 10 min at 4 °C, and the obtained supernatant were transferred to a screw-capped tube and used as blank plasma. To 40 µL of plasma placed in a 1.5 mL polypropylene tube, 20 µL of a drug solution (0 or 2.0 µM) and 340 µL of THF were added. After vortex mixing for a few seconds, the mixture was immediately centrifuged at 9 000g for 5 min at 4 °C, and the supernatant was passed through a disposable filter (0.20 µm, i.d. 13 mm, polytetrafluoroethylene; Advantec Toyo, Tokyo, Japan). Then, a part (200 µL) of the filtrate was subjected to derivatization, and the remaining part (20 µL) was diluted 5-fold with 60% (v/v) acetonitrile and injected into an LC system with an ODS column without derivatization. LC System and Conditions. Apparatus. We used an isocratic LC system consisting of an LC-10AD liquid chromatography pump, an SIL-10A autoinjector, a DGU-12A online degasser, a CTO-10A column oven, a separation column, an RF-10AXL spectrofluorometer equipped with a 12 µL flow cell, and a CBM-20A controller. With the exception of the separation column, all components of the LC system were manufactured by Shimadzu (Kyoto, Japan). Injection of each 20 µL sample into the system was carried out automatically. The flow rate of the mobile phase was set at 1.0 mL/min, and the column temperature was 40 °C. Both the monochromators in the fluorescence detector had a slit width of 15 nm. Examination of Fluorous Separation. Detailed LC conditions are described in the Supporting Information (Figure S-1). Analysis of Biologically Important Carboxylic Acids by FluorousPhase Separation. For comparison, two fluorous-phase columns: Wakopak Fluofix-II 120E (250 mm × 4.6 mm i.d., 5 µm; Wako Pure Chemicals) and FluoroFlash (50 mm × 4.6 mm i.d., 5 µm; Fluorous Technologies, Pittsburgh, PA) were used as analytical columns. Mixtures of methanol, water, and phosphoric acid were used as the optimum mobile phases in the Fluofix column (mixing ratio ) 78:22:0.1, v/v) and FluoroFlash column (mixing ratio ) 70:30:0.1, v/v). The fluorescence detector was operated at excitation and emission wavelengths of 280 and 320 nm, respectively. Analytical Chemistry, Vol. 81, No. 12, June 15, 2009

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Table 1. Calibration Curves, Repeatabilities, and Detection Limits of the Fluorous Derivatization Method calibration curve (n ) 3)

repeatability (RSD)a (n ) 5)

analytes

range (µM)

R

intraday (%)

interday (%)

detection limit (fmol)b

HVA VMA 5-HIAA naproxen felbinac flurbiprofen etodolac

0.5-10 0.5-10 0.01-10 0.01-25 0.01-25 0.01-25 0.01-25

0.9996 0.9998 0.9999 0.9996 0.9998 0.9992 0.9996

1.2 2.4 0.5 0.3 0.8 0.5 1.8

2.5 6.4 2.3 2.1 2.3 2.1 3.1

500 1600 36 11 6 10 16

a Relative standard deviation of peak height for 4 pmol per 20 µL injection volume. b Defined as the amount per 20 µL injection volume yielding a signal-to-noise ratio of 3.

Analysis of Drugs by Fluorous-Phase Separation. A Wakopak Fluofix-II 120E column (150 mm × 4.6 mm i.d., 5 µm) was used as an analytical column, and a mixture of methanol, water, and acetonitrile (70:20:10, v/v) was used as a mobile phase. The fluorescence detector was operated at excitation/emission wavelengths (nm) of 270/350 (0-22.8 min), 260/316 (22.8-32.0 min), and 280/350 (>32.0 min). Analysis of Drugs Using ODS Column. Detailed LC conditions are described in the Supporting Information (Figure S-2). Fluorescence Spectral Characterization. Fluorescence spectral measurements were performed using a Hitachi (Tokyo, Japan) F-2500 spectrofluorometer having quartz cells with dimensions of 10 mm × 10 mm; the spectral bandwidth of both the excitation and emission monochromators was 5 nm. The fluorescence properties (excitation and emission maxima and relative fluorescence intensity) of the derivatized and underivatized carboxylic acids were measured after dilution to the same concentration (4 nmol per mL in the measurement solution) with aqueous 70% (v/v) methanol. Method Validation. In order to obtain the validation parameters, peak heights that were estimated by a Shimadzu software (LC solution, version 1.23) with baseline-to-baseline method were used for the quantification of all carboxylic acids. For the quantitative analysis, calibration curves, whose ranges are listed in Table 1, were obtained by diluting the stock solutions (n ) 3). The equations of the calibration curves were determined by least-

squares linear prediction. Precisions (intraday and interday) of the present method were estimated during the analytical procedures (sample dilution, derivatization, and LC separation) using the standard solutions (1.0 µM). The intraday and interday precisions were assessed by performing an analysis five times on the same day and by analysis on five different days in a month, respectively. The detection limits were determined as the lowest concentrations at which the signal-to-noise ratio was 3. RESULTS AND DISCUSSION Fluorous Separation. In the preliminary experiments, HVA and 5-HIAA were used as model native fluorescent carboxylic acids. First, the retention mechanism of fluorous derivatives in the fluorous-phase column was compared with that of nonfluorous derivatives under the same separation conditions. Both unlabeled carboxylic acids and their derivatives with n-undecylamine (whose carbon number was the same as that of HFUA) were examined as nonfluorous derivatives. Only HFUA-labeled carboxylic acids were retained in the fluorous-phase column, and alkylaminelabeled acids and carboxylic acids were eluted within the solvent front (see Figure S-1 in the Supporting Information), as in the case with F-SPE11-14,36,37 and LC.30-32 This result indicates that the fluorous-phase column can selectively retain fluorous compounds including fluorous-labeled analytes on the basis of fluorous separation and not hydrophobicity. Some silica-gel-based LC columns with a fluorous stationary phase are commercially available.1,2,17-19 Among them, we investigated Wakopak Fluofix-II 120E (250 mm × 4.6 mm i.d.) and FluoroFlash (50 mm × 4.6 mm i.d.)50 as the model fluorous-phase columns with C6F13-branched type and C8F17-straight type, respectively. As shown in Figure 1, although both columns could retain fluorous-labeled HVA, VMA, and 5-HIAA, the number of theoretical plates of FluoroFlash (50 mm) was less than that of Fluofix (250 mm). In addition, FluoroFlash could not separate fluorous-labeled 5-HIAA and labeled VMA under the separation conditions used in this study. In subsequent examinations, the Fluofix-II column was used for the separation of HFUA-labeled carboxylic acids. In contrast, the Fluofix-II 120E column (150 mm × 4.6 mm i.d.) effectively separated the HFUA derivatives of naproxen, felbinac, flurbiprofen, and etodolac within 40 min. For the separation, a mixture of methanol, water, and acetonitrile (70:20:

Figure 1. Chromatograms obtained with fluorous-labeled HVA, VMA, and 5-HIAA (4 pmol each on column) using (A) FluoroFlash column and (B) Fluofix column. Peaks: 1, HFUA-labeled 5-HIAA; 2, HFUA-labeled VMA; 3, HFUA-labeled HVA. LC conditions are described in the Experimental Section. 5042

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Figure 2. Chromatogram obtained with fluorous-labeled naproxen, felbinac, flurbiprofen, and etodolac (400 fmol each on column) using the Fluofix column. Peaks: 1, HFUA-labeled naproxen; 2, HFUAlabeled felbinac; 3, HFUA-labeled flurbiprofen; 4, HFUA-labeled etodolac. LC conditions are described in the Experimental Section. Table 2. Effect of Derivatization on Fluorescence Properties [Excitation (λEx) and Emission (λEm) Maxima and Relative Fluorescence Intensity (RFI)]a of Derivatized and Underivatized Carboxylic Acids underivatized carboxylic acid

fluorous derivative with HFUA

λEx (nm) λEm (nm) RFIb λEx (nm) λEm (nm) RFIb HVA VMA 5-HIAA naproxen felbinac flurbiprofen etodolac

280 280 280 270 260 255 280

315 310 310 350 315 310 345

7 14 24 55 100 73 33

280 280 280 270 260 255 280

310 310 335 350 315 310 345

2 2 29 62 84 78 57

a All fluorescence properties were measured after dilution to the same carboxylic acid concentration (4 nmol/mL) with aqueous 70% (v/v) methanol. b Fluorescence intensity obtained with underivatized felbinac was 100.

10, v/v) was used as the mobile phase. Figure 2 shows a typical chromatogram obtained with fluorous-labeled naproxen, felbinac, flurbiprofen, and etodolac. It was found that fluorous derivatives that have the same perfluoroalkyl moiety could be separated on this column because of secondary effects26,32,51 such as differences in absorption to silanol residue on particulates and solubility of the mobile phase. Fluorescence from Fluorous-Labeled Derivatives. The fluorescence properties (excitation and emission maxima and relative fluorescence intensity) of seven derivatized and underivatized fluorescent carboxylic acids are presented in Table 2. The fluorescence excitation and emission wavelengths of the fluorouslabeled carboxylic acids are almost the same as those of the unlabeled acids; however, the intensities of the derivatized acids are 0.2-1.8 times those of the underivatized acids. This result suggests that fluorous derivatization does not significantly affect the fluorescence wavelengths. Hence, the proposed method could be used for LC of many types of native fluorescent compounds (50) We used a 50 mm length column in this study, but FluoroFlash columns with longer lengths are also available. (51) Bell, D. S.; Jones, A. D. J. Chromatogr., A 2005, 1073, 99–109.

without considering the combination of analytes and reagents, which is an essential precondition for derivatization involving FRET.34,35 Derivatization Conditions. Optimization studies on derivatization were conducted to maximize the fluorescence peak height. They involved fluorous-phase separation of the HFUA derivatives of HVA, VMA, and 5-HIAA. DMT-MM, of which introduction to the derivatization between carboxylic acids and amines is the first attempt for quantitative analysis as far as we know,48,49 was used as the condensation reagent for native fluorescent carboxylic acids with HFUA (amine) (Scheme 1). It is known that amidation reaction using DMT-MM is faster and milder than that using other condensation reagents such as EDC, HOBt, and diphenylphosphorylazide.38,39 In fact, under this reaction conditions (room temperature, 5 min), the peak intensities of labeled-carboxylic acids obtained with EDC and HOBt were about 1/20 of those with DMTMM. Because HFUA did not dissolve in water easily, the derivatization reaction proceeded in the presence of organic solvents (methanol, acetonitrile, THF, or their mixtures). The best results were obtained when fluorophilic THF was used for the preparation of HFUA solution. The derivatization reaction did not occur when the concentration of water in the reaction solution was over 40% (Figure 3). Therefore, a solution of DMT-MM, which is highly soluble in water, was prepared by using aqueous 90% (v/v) THF and used for the derivatization reaction. When the concentration of HFUA in the reagent solution was more than 1 mM, the peak heights of all the carboxylic acids were maxima. Therefore, the optimum HFUA concentration for the analysis of biological samples was selected as 20 mM. Furthermore, a 10 mM DMT-MM solution was used for the derivatization, because maximum peak heights were obtained when the concentration of DMT-MM in the reagent solution was 0.2-10 mM. Higher concentrations of HFUA and DMT-MM could be used for the analysis of real samples containing many types and high amount of carboxylic acids, because both reagents do not fluoresce. The derivatization reaction using DMT-MM was mild and rapid.46-49 More specifically, it was found that a temperature of 4-60 °C and a time duration of 1-60 min were sufficient for reaction completion, and the fluorescence intensities of fluorouslabeled compounds were stable among the conditions. Therefore, the optimal derivatization conditions were selected at room temperature for 5 min for the convenient conditions. Not only fluorous-labeled HVA, VMA, and 5-HIAA but also labeled naproxen, felbinac, flurbiprofen, and etodolac in the final reaction mixture were stable. Their fluorescence intensities were constant even after the mixture was left to stand for at least a week in the dark at room temperature. Calibration Graph, Precision, and Detection Limits. The validation data are presented in Table 1. The relationships between the amounts of native fluorescent carboxylic acids examined and the peak heights were linear over the concentration range of at least 0.5-10 µM in the standard solution (2.0-40 pmol per 20 µL injection volume). The values of the linear correlation coefficients of all the carboxylic acids exceeded 0.999 (n ) 3). The intraday and interday precisions were estimated during the entire process by repeated determinations (n ) 5 in each case) using mixtures of standard compounds (1.0 µM each in a sample solution, 4.0 pmol each per 20 µL injection volume); the relative standard Analytical Chemistry, Vol. 81, No. 12, June 15, 2009

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Figure 3. Effects of water concentration in the reaction solution on the fluorescence intensity. Graphs: (A) HVA, (B) VMA, (C) 5-HIAA.

Figure 4. Chromatograms obtained with plasma samples by fluorous derivatization and fluorous-phase separation. Chromatograms: A, drugfree plasma; B, plasma spiked with native fluorescent drugs (1 nmol each per mL of plasma). Peaks: 1, HFUA-labeled naproxen; 2, HFUAlabeled felbinac; 3, HFUA-labeled flurbiprofen; 4, HFUA-labeled etodolac; others, endogenous native fluorescent compounds. LC conditions are described in the Experimental Section.

deviations were within 2.4% and 6.4%, respectively. The detection limits (signal-to-noise ratio ) 3) that were dependent on the fluorescence intensities of the analytes were less than 1.6 pmol per 20 µL injection volume (Table 1). In order to increase the detection sensitivity, F-SPE before LC injection is very useful and simple to concentrate the fluorous-labeled compounds such as the derivatives obtained by the present method.11-15,36,37,52 Determination of Native Fluorescent NSAIDs in Human Plasma. To investigate the applicability of the fluorous derivatization method to biomedical analysis, it was used to determine native fluorescent NSAIDs (naproxen, felbinac, flurbiprofen, and etodolac; Scheme 2) in human plasma. Plasmas that were drug free and spiked with NSAIDs were diluted 10-fold with THF to deproteinize and optimize the reaction solvent. Figure 4 shows typical chromatograms obtained with derivatized plasma samples. No peaks of the plasma components were observed at the retention times around and after the objective peaks, because the native fluorescent compounds without carboxyl groups in the plasma were not derivatized and retained in the column. Furthermore, nonfluorescent carboxylic acids were not detected, even though they were retained. The components of peaks 1, 2, 3, and (52) Todoroki, K.; Etoh, H.; Yoshida, H.; Nohta, H.; Yamaguchi, M. Anal. Bioanal. Chem. 2009, 394, 321–327.

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4 in Figure 4B were identified as HFUA-labeled naproxen, felbinac, flurbiprofen, and etodolac, respectively, by a comparison of their retention times with those in Figure 2 and by cochromatography using various mobile phases. When HFUA solution was replaced with only a solvent, it was found that all compounds were eluted in the solvent front. As mentioned above, the biogenic fluorescent compounds did not affect the determination of the NSAIDs. These results support the fact that peaks 1, 2, 3, and 4 in Figure 4 have single components, namely, HFUA-labeled naproxen, felbinac, flurbiprofen, and etodolac, respectively. The peak components observed at retention times of 15-20 min might be attributed to the HFUA derivatives of endogenous native fluorescent carboxylic acids. A comparison between the fluorous-phase column and the ODS column, which is most widely used for RP-LC, indicated that the proposed method is advantageous to biomedical analysis. Figure S-2 in the Supporting Information shows chromatograms obtained with the same plasma samples as those used to obtain the results shown in Figure 4 without fluorous derivatization. The separation conditions used in this study were optimized in advance by using standards of naproxen, felbinac, flurbiprofen, and etodolac. It was difficult to determine the acids because many disturbance peaks appeared at the retention times of the four drugs. In addition, the

appearance of many fluorescence peaks after the objective peaks indicates that a long analysis time, excellent gradient elution condition, or column switching might be required. The combination of fluorous derivatization and LC separation with a fluorous stationary phase results in a simple and effective method for determining native fluorescent carboxylic acids in a short time.

be useful for biomedical and clinical investigations of not only native fluorescent carboxylic acids but also other functional-groupcontaining compounds. Such investigations are currently in progress. In addition, it is expected that a fluorous-phase column packed with sub 2 µm particulates will be developed in order to enable the use of fluorous derivatization for ultrafast LC.

CONCLUSION With the use of fluorous derivatization and fluorous-phase separation, native fluorescent carboxylic acids could be converted easily into perfluoroalkyl-labeled derivatives, are retained specifically, and detected selectively. The condensation reaction between native fluorescent carboxylic acids and perfluoroalkylamine in the presence of DMT-MM was sufficiently rapid and mild for biological analysis. Because perfluoroalkyl-labeled derivatives have properties of fluorous compounds, only analytes can be retained intensely and specifically to fluorous-phase column with fluorous separation. Furthermore, simple chromatograms could be obtained, and the analysis could be completed in a short time, because few disturbance peaks originate from biological components or the environment. Separation-oriented derivatization might

ACKNOWLEDGMENT This study was partly supported by the Grants-in-Aid for Encouragement of Young Scientists (B) (Grant Numbers 18790373, 20750066, and 20790417) from the Japan Society for the Promotion of Science, and by funds (Grant No. 0825002) from the Central Research Institute of Fukuoka University. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review March 23, 2009. Accepted May 1, 2009. AC9005952

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