Anal. Chem. 2006, 78, 2413-2421
Application of On-Line Electrochemical Derivatization Coupled with High-Performance Liquid Chromatography Electrospray Ionization Mass Spectrometry for Detection and Quantitation of (p-Chlorophenyl)aniline in Biological Samples Hao Chen,* Yanhua Zhang, Abdul E. Mutlib, and Min Zhong
Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development, Michigan Laboratories, 2800 Plymouth Road, Ann Arbor, Michigan 48105
A rapid, sensitive, and specific assay for detection and quantitation of (p-chlorophenyl)aniline (CPA) in biological samples was developed. The assay was established based on rapid electrochemical oxidation of CPA to a dimerized product (1.0 V vs Pd) with the enhanced detection sensitivity of electrospray mass spectrometer (ES/MS). A ‘‘head-to-tail” dimer ([M + H]+ at m/z 217) was exhibited as the predominant species after electrochemical conversion of CPA. Optimal detection sensitivity and specificity for the dimer of CPA that was present in the biological matrix (e.g., rat urine) were achieved through on-line electrochemistry (EC) coupled with high-performance liquid chromatography tandem mass spectrometry. No matrix-associated ion suppression was observed. The limit of detection (S/N ∼6) was 20 ng/mL, and the limit of quantitation was 50 ng/mL. The calibration curve was exhibited to be quadratic over the range of 50-2000 ng/ mL with r2 > 0.99 in various biological matrixes. The assay was validated and used to study the biotransformation of p-chlorophenyl isocyanate (CPIC) to CPA in rats administered intraperitoneally with CPIC (50 mg/kg). The present LC/EC/MS/MS assay of CPA brings important technical advantages to assist in the risk assessment of new chemical entities, which have the potential to produce anilines via biotransformation. The coupling of liquid chromatography (LC) and mass spectrometry (MS) has been established as one of the most powerful tools in analytical chemistry since it debuted in the mid 1980s. The application of this technology has resulted in important advances especially in biomedical and biochemical research.1,2 In general, the predominantly used interfaces are atmospheric pressure ionization (API) techniques such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). As the ionization typically occurs on the basis of acid/base reactions, ESI and APCI are particularly well suited for the analysis * To whom correspondence should be sent. Tel: (734) 622-4517. Fax: (734) 622-5115. E-mail:
[email protected]. (1) Fenselau, C. Annu. Rev. Pharmacol. Toxicol. 1992, 32, 555-578. (2) Gelpi, E. J. Chromatogr., A 1995, 703, 59-80. 10.1021/ac051949c CCC: $33.50 Published on Web 02/24/2006
© 2006 American Chemical Society
of pharmaceuticals, drug metabolites, peptides, and other polar compounds. Furthermore, high-performance liquid chromatography coupled with ESI tandem mass spectrometry (HPLC/ESIMS/MS) has recently been demonstrated to be a powerful technique for qualitative and quantitative determination of small molecules including drugs and drug metabolites in biological fluids.3-6 It provides a high degree of specificity, thus minimizing the requirement for sample preparation and chromatographic separation. Nevertheless, the successful application of LC/MS/ MS analytical techniques truly relies on the ionization capability of analytes. The analysis of small and polar aromatic amines such as (p-chlorophenyl)aniline (CPA) with ESI- and APCI-MS techniques leads to poor sensitivity, possibly due to its unfavorable ionization mechanism. Aniline is industrially synthesized on a large scale as the parent compound for a variety of different arylamines, including various drugs and dyes. Occupational poisoning by aniline was common in the past,7 and exposure to aniline still occurs today. Both acute toxicity, characterized by methemoglobinemia and hemolytic anemia, and carcinogenic effects due to chronic aniline exposure have been studied in detail.8-10 In addition to occupational exposure, there is concern about the therapeutic exposure to anilines produced from therapeutic agents. For instance, the antitumor agent sulofenur, which has shown promising activity against a wide range of cancers,11-15 caused hemolytic anemia and methemoglobinemia as the dose-limiting (3) Perchalski, R.; Yost, R. A.; Wilder, B. J. Anal. Chem. 1982, 54, 1466-1471. (4) Lee, M. S.; Yost, R. A. Biomed. Environ. Mass Spectrom. 1988, 15, 193204. (5) Naylor, S.; Kajbaf, M.; Lamb, J. H.; Jahanshahi, M.; Gorrod, J. W. Biol. Mass Spectrom. 1993, 22, 388-394. (6) Jackson, P. J.; Brownsill, R. D.; Taylor, A. R.; Walther, B. J. Mass Spectrom. 1995, 30, 446-451. (7) Neumann, H.-G.; Albrecht, W. Arch. Toxicol. 1995, 57, 1-5. (8) Harrison, J. H. J.; Jollow, D. J. Mol. Pharmacol. 1986, 32, 423-431. (9) Harrison, J. H. J.; Jollow, D. J. J. Pharmacol. Exp. Ther. 1987, 238, 10451054. (10) Malejka-Giganti, D.; Ritter, C. L. Environ. Health Perspect. 1994, 102 (Suppl. 6), 75-81. (11) Wienerman, B.; Eisenhauer, E.; Stewart, D.; Mertens, W.; Tannock, I.; Venner, P.; Spaulding, R. Oncology 1992, 3, 83-84. (12) Munshi, N. C.; Seitz, D. E.; Fossella, F.; Lippman, S. M.; Einhorn, L. H. Invest. New Drugs 1993, 11, 87-90. (13) Roe, D. J.; Plezia, P. M.; Grindey, G. B.; Hamilton, M.; Seitz, D. J. Natl. Cancer. Inst. 1992, 84, 1798-1802.
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toxicities in clinical trials.16,17 In animal studies, it was demonstrated that this compound was metabolized to p-chlorophenyl isocyanate (CPIC), which was characterized as a glutathione conjugate excreted in rat bile.18 The presence of CPA, a hydrolytic product of CPIC, in biological samples was not reported perhaps due to the inability of conventional LC/MS to detect it. Although the mechanism(s) of toxicity induced by sulofenur is(are) not well understood, the exposure to CPA produced from the metabolism of sulofenur may have contributed to the toxicities observed in clinics. In the development of biological monitoring strategies for aromatic amines, several analytical methods have been described, e.g., gas chromatography/mass spectrometry (GC/MS) and HPLC with UV detection.19,20 Although these methods allow selective detection and quantitation of anilines, they present limitations. While GC/MS is specific and sensitive, it requires laborious sample preparation and instrument maintenance. HPLC/ UV lacks the specificity and sensitivity often required for the analysis of biological samples. Liquid chromatography coupled with tandem mass spectrometric analysis has become the method of choice for the detection and quantitation of small molecules in biological samples, since it provides the selectivity and superior sensitivity that are often essential for the quantitation of small molecules in complex biological samples. Despite the superior sensitivity of the LC/MS/MS technique, often we are challenged with compounds that are not amenable to LC/MS analyses. For example, the analysis of anilines, produced either as a metabolite or a degradation product of drug molecules, in biological samples has been a challenge for LC/MS. While the high polarity and small size constrain the choice of HPLC method, the poor ionizability of anilines by API limits the detection sensitivity of LC/MS. As a result, the analysis of trace amounts of anilines produced enzymatically or nonenzymatically from drug molecules has been a challenge in the past. In recent years, some new approaches that improved the applicability of ESI and APCI techniques to potentially analyze compounds with poor ionizability have been presented. These include electron capture ionization in an APCI interface,21,22 atmospheric pressure photoionization,23 and electrochemical conversions.24 In particular, the application of electrochemistry (EC) to enhance the ionizability of compounds that are not amenable to ESI or APCI has gained greater attention. (14) Kamthan, A.; Scarffe, J. H.; Walling, J.; Hatty, S.; Peters, B.; Coleman, R.; Smyth, J. F. Anti-Cancer Drugs 1992, 3, 331-335. (15) Talbot, D. C.; Smith, I. E.; Nicolson, M. C.; Powles, T. J.; Button, D.; Walling, J. Cancer Chemother. Pharmacol. 1993, 31, 419-422. (16) Hainsworth, L. D.; Hande, K. R.; Satterlee, W. G.; Kuttesch, J.; Johnson, D. H.; Grindey, G. B.; Jackson, L. E.; Greco, F. A. Cancer Res. 1989, 49, 52175220. (17) Taylor, C. W.; Alberts, D. S.; Ketcham, M. A.; Satterlee, W. G.; Holdsworth, M. T.; Plezia, P. M.; Peng, Y.-M.; McCloskey, T. M.; Roe, D. J.; Hamilton, M.; Salmon, S. E. J. Clin. Oncol. 1989, 7, 1733-1740. (18) Jochheim, C. M.; Davis, M. R.; Ballie, K. M.; Ehlhardt, W. J.; Ballie, T. A. Chem. Res. Toxicol. 2002, 15, 240-248. (19) Cocker, J.; Boobis, A. R.; Davies, D. S. Biomed. Environ. Mass Spectrom. 1988, 17, 161-167. (20) Sepai, O.; Schutze, D.; Heinrich, U.; Hoymann, H. G.; Henschler, D.; Sabbioni, G. Chem.-Biol. Interact. 1995, 97, 185-198. (21) Singh, G.; Gutierrez, A.; Xu, K.; Blair, I. A. Anal. Chem. 2000, 72, 30073013. (22) Evans, C. S.; Sleeman, R.; Luke, J.; Keely, B. J. Rapid Commun. Mass Spectrom. 2002, 16, 1883-1891. (23) Robb, D. B.; Covey, T. R.; Bruins, A. P. Anal. Chem. 2000, 72, 3653-3659. (24) Van Berkel, G. J.; Asano, K. G. Anal. Chem. 1994, 66, 2096-2102.
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Analytical Chemistry, Vol. 78, No. 7, April 1, 2006
Scheme 1. Biotransformation of CPIC to CPA
Cole and co-workers25 coupled EC on-line with ESI-MS for detection of nonpolar polycyclic aromatic hydrocarbons using a self-constructed electrochemical flow cell inside the electrospray probe. Van Berkel26 extensively studied electrochemical reactions in the ESI interface, considering the electrospray interface itself as an electrochemical reactor. Another approach to enhance ionization of compounds is the use of an external electrochemical flow cell that is placed ahead of the ionization interface. On-line EC/ESI-MS has been used for investigating the generation of reaction intermediates and products and for studying biological redox reactions.27-30 Recently, Diehl31 presented an external electrochemical flow cell coupled on-line with LC/MS. A “coulometric” cell was inserted between HPLC and ESI-MS for analysis of ferrocene-labeled alcohols and phenols. With this approach, the effects of electrochemical derivatization to assist ionization with the ESI or APCI could be demonstrated. The present study describes a novel application of the LC/ EC/ESI-MS method that demonstrated great sensitivity and reliability, as well as suitability for detecting and quantitating the simple aromatic amine, CPA, present in complex biological matrixes. Detailed studies have been carried out to elucidate the electrochemical oxidation product of CPA. Subsequently, a simple and specific HPLC/EC/ESI-MS/MS assay was developed and validated. The assay was used to quantify CPA, a hydrolytic product of CPIC (Scheme 1), excreted in urine and bile samples of rats dosed with the agent. EXPERIMENTAL SECTION Materials and Agents. CPIC, CPA, and (p-iodophenyl)aniline (IPA) were obtained from Sigma-Aldrich (Milwaukee, WI) and used without further treatment. Waters Symmetry C18 columns (2.1 × 50 mm, 5 µm) were obtained from Waters Corp. (Milford, MA). All solvents and reagents used for HPLC and LC/MS were of chromatographic grade. Caution: CPIC, CPA, and IPA are highly toxic and care should be taken in handling, analysis, and disposal of these substances. Instrumentations. The electrochemical system purchased from ESA (Chelmsford, MA) consisted of a GuardStat potentiostat and a model 5021 conditioning cell. The cell contains a coulometric working electrode and a Pd reference electrode. All potentials described in this study were given versus the Pd reference (25) Xu, X. M.; Lu, W. Z.; Cole, R. B. Anal. Chem. 1996, 68, 4244-4253. (26) Van Berkel, G. J.; Quirke, J. M. E.; Tigani, R. A.; Dilley, A. S.; Covey, T. R. Anal. Chem. 1998, 70, 1544-1554. (27) Deng, H.; Van Berkel, G. J. Anal. Chem. 1999, 71, 4284-4293. (28) Zhou, F.; Van Berkel, G. J. Anal. Chem. 1995, 67, 3643-3649. (29) Regino, M. C.; Brajter-Toth, A. Anal. Chem. 1997, 69, 5067-5072. (30) Arakawa, R.; Yamaguchi, M.; Hotta, H.; Osakai, T.; Kimoto, T. J. Am. Soc. Mass Spectrom. 2004, 15, 1228-1236. (31) Diehl, G., Liesener, A.; Karst, U. Analyst 2001, 126, 288-290.
Figure 1. Schematic assembly of HPLC/EC/ESI-MS system.
electrode. For coupling of LC/MS on-line with electrochemistry, the electrochemical cell (ECC) was placed between the column and the ESI interface of the existing LC/MS system (Figure 1). The connection between the ECC and the ESI interface was kept as short as possible to minimize band dispersion. The ECC was grounded as shown in Figure 1. The LC/MS system consisted of an Agilent (Agilent Technologies, Palo Alto, CA) 1100 binary pump, an Agilent 1100 autosampler, an Agilent 1100 degasser, and a triple-stage quadrupole mass spectrometer API 4000 (PESciex, Toronto, ON, Canada) with ESI. The LC effluent was introduced into the source using a turbo ion spray interface. The mass spectrometer was interfaced to a computer operating Analyst 1.4 software (PE-Sciex) for data collection, peak integration, and analysis. Electrochemical Oxidation of CPA. To identify and characterize the electrochemical product of CPA, a solution of CPA [100 ng/mL in the mobile phase (see below)] was directly infused through the ECC at a flow rate of 5 µL/min by a syringe pump into the ESI source of mass spectrometer for analysis. The potential applied to the ECC was increased from 0 to 2.0 V at an interval of 200 mV. The mass spectrometer was operated in the positive ion mode with a mass range of 50-500 Da for the full scan. MS/MS analysis of electrochemical product was carried out using nitrogen as the collision gas. The collision energy was kept at 20-30 eV. Furthermore, the effect of mobile phase on the electrochemical oxidation of CPA was investigated. The infusion flow of CPA was mixed with the mobile phase through a threeway union before reaching the ECC (Figure 1). The mobile phase was delivered at a flow rate of 0.25 mL/min through the HPLC pumps. The flow was then introduced into the ESI-MS. The mobile phases tested were (a) acetonitrile in 10 mM ammonium acetate (pH 5) (1:1, v/v), (b) acetonitrile in 10 mM ammonium acetate (pH 8) (1:1, v/v), and (c) acetonitrile in 10 mM ammonium formate (pH 5) (1:1, v/v). Optimization of HPLC/EC/ESI-MRM. To optimize detection sensitivity and specificity of the electrochemical product of CPA, the mass spectrometer was operated in the multiple reaction monitoring (MRM) mode and the same experimental setup was applied. The optimal voltage of ECC was obtained by flow injection analysis (FIA). Aliquots of CPA (10 µL of 100 pg/µL) in the mobile phase or biological matrix were injected. The voltage of ECC was increased from 0 to 2.0 V at an interval of 200 mV/injection. The voltage resulting in the largest peak area was selected as the optimal operating voltage. The chromatographic analysis was achieved on a Waters Symmetry C18 column (2.1 × 50 mm, 5 µm). The mobile phase consisted of (a) acetonitrile and (b) 10 mM ammonium acetate (pH 8.0), which was delivered through a
gradient program. The percentage of acetonitrile was increased from 25 to 65 (v/v) over 4 min. After 4 min, the percentage of acetonitrile was ramped to 80 in 2 min and then back to 25 in 0.1 min to reequilibrate for 4 min before the next injection. The flow rate was set at 0.25 mL/min. The mass spectrometer was operated in the MRM mode under the optimized conditions. Calibration Standards and Quality Control Samples. Duplicate stock solutions of CPA were prepared in dimethyl sulfoxide (DMSO) to give a concentration of 1 mg/mL designated as A and B. The working solutions were obtained by diluting the stock solution in series with a diluting solvent to known concentrations at 400, 1000, 1600, 2000, 4000, 8000, 16 000, 28 000, and 40 000 ng/mL CPA (from solution A) and 1000, 8000, and 40 000 ng/mL (from solution B). The dilution solvent was made up with acetonitrile and 10 mM ammonium acetate (1:1, v/v) containing 30% ammonium hydroxide (final concentration 10%, v/v), and this solvent was used for any diluting purpose during the course of study. A stock solution of IPA (the internal standard, INSD) was prepared in DMSO (1 mg/mL) and diluted with the dilution solvent to give a final concentration of 10 µg/mL. All stock and working solutions were stored in polypropylene tubes at 4 °C in the refrigerator. Calibration curves were constructed in blank rat urine or bile samples. Calibration standards of CPA were prepared daily by spiking individual working solution of CPA (10 µL) in 1.5-mL microcentrifuge tubes that contained blank rat urine or bile (50 µL) and the dilution solvent (40 µL). Subsequently, aliquots (100 µL) of INSD solution were added to each standard sample to achieve concentrations of 20, 50, 80, 100, 200, 400, 800, 1400, and 2000 ng/mL. Quality control (QC) samples were prepared separately following the same procedure, except that the working solutions of CPA prepared from stock solution B were used to provide three concentrations of CPA at 50, 400, and 2000 ng/mL. Method Validation. Assay Specificity.The specificity of the assay was established by analyzing CPA-free rat urine or bile sample for potential interferences. Predose urine and bile samples collected from rats were mixed with the dilution solvent at a ratio of 1:3 (v/v). Matrix Effects. To test for a possible matrix effect in the ESI process, the peak areas obtained from CPA of 50 ng/mL spiked in the matrix (predose bile or urine of rat) were compared (Student’s t test) to the peak areas of the analyte prepared in the dilution solvent at corresponding concentrations. The level of significance was set at P 1 min) between CPA and IPA offered analytical benefits in terms of minimizing the competition of electrochemical oxidation between CPA and IPA and possible cross-reactions of chemical intermediates from these two compounds. Representative MRM chromatograms obtained from predose and spiked urine (with CPA) samples are shown in Figure 5a and c, respectively. Urine from a rat dosed with CPIC (50 mg/kg, ip) was analyzed and shown to contain CPA as demonstrated in Figure 5b. No interfering peaks from endogenous compounds were observed during the chromatographic run (5 min). Also, there was no measurable carryover after injection, even after the highest concentration standard (2000 ng/mL) used in the assay. Evaluation of the effect of matrix including rat urine and bile revealed no differences between the peak area obtained with the CPA at LOQ level (50 ng/mL) in urine or bile and the corresponding standard solution, indicating that the matrix suppression induced by the endogenous materials from the biological samples was minimum under the set experimental conditions. Analytical Assay Characterization and Method Validation. The calibration curves were shown to be quadratic over the concentration range (50-2000 ng/mL), and the nonlinear regression constant (r2) was >0.99 in all cases. A representative
Figure 3. Mass spectrometric hydrodynamic voltammogram of CPA spiked in rat urine obtained through LC/EC/ESI-MRM analyses. 2418 Analytical Chemistry, Vol. 78, No. 7, April 1, 2006
calibration curve is shown in Figure 6. The LOQ was set at 50 ng/mL, since this was the lowest concentration that showed an RSD of 16 h) freeze (-20 °C) for 1 month std soln (4 °C) for 1 month benchtop for 18 h freeze/thaw (3 cycle, >16 h) freeze (-20 °C) for 1 month std soln (4 °C) for 1 month
Table 4. Percentage of Dose Excreted as CPA in Urine and Bile Samples of Rats Dosed with CPIC (50 mg/kg, ip) % of dose excreted as anilinea 0-4 h 4-24 h total
bile urine bile urine
rat 1
rat 2
rat 3
mean ( SD
0.04 0.03 0.13 0.13
0.07 0.10 0.08 0.25
0.08 0.04 0.13 0.24
0.06 ( 0.02 0.06 ( 0.03 0.11 ( 0.03 0.21 ( 0.05
0.33
0.50
0.49
0.44 ( 0.07
a (Concentration × dilution factor × volume of bile or urine)/amount of CPIC given.
in gas phase. CPIC, which is used as an industrial synthetic material or produced as a metabolite of drug molecules, is a wellknown chemically reactive species that can react with biological nucleophiles such as glutathione, DNA molecules, and proteins. Moreover, CPIC undergoes hydrolysis enzymatically or nonenzymatically to produce CPA, a well-established rodent carcinogen. By applying the current assay, the presence and quantities of CPA excreted in urine and bile were clearly demonstrated. The current assay employing an on-line electrochemistry coupled with LC/ ESI-MS provides a simple, rapid, and specific method for the detection and quantitation of CPA in complex biological matrix with a short chromatographic run time. The method shows excellent sensitivity and requires very little sample preparation, allowing analyses of small sample volumes. These are important improvements for the application of aniline detection for environmental monitoring and, more importantly, for monitoring possible exposure to anilines derived from potentially useful therapeutic agents.
CONCLUSIONS A powerful new hyphenated analytical method based on the combination of electrochemical (coulometric) oxidation and ESIMS/MS has been developed for analysis of (p-chlorophenyl)aniline. Simple and commercially available instruments have been used to set up the analytical system that takes advantage of simplicity of on-line electrochemical derivatization with high sensitivity and specificity of MS/MS detection, resulting in a robust analytical method with high sensitivity (LOD ) 20 ng/ mL), large dynamic range (50-2000 ng/mL with r2 > 0.99), and great precision and accuracy (standard derivation
