Development and Validation of the First Assay Method Coupling

Article pubs.acs.org/crt

Development and Validation of the First Assay Method Coupling Liquid Chromatography with Chemiluminescence for the Simultaneous Determination of Menadione and Its Thioether Conjugates in Rat Plasma Mohamed Saleh Elgawish,†,‡ Chikako Shimomai,† Naoya Kishikawa,† Kaname Ohyama,† Mitsuhiro Wada,† and Naotaka Kuroda*,† †

Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan ‡ Pharmaceutical Chemistry Department, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt S Supporting Information *

ABSTRACT: Menadione (2-methyl-1,4-naphthoquinone, MQ), a component of multivitamin drugs with antihemorrhagic, antineoplastic, and antimalarial activity, is frequently used to investigate quinone-induced cytotoxicity. The formation of MQ conjugates with glutathione (GSH) by Michael addition and subsequent biotransformation to yield N-acetyl-L-cysteine conjugates is believed to be an important detoxification process. However, the resulting conjugates, 2-methyl-3-(glutathione-Syl)-1,4-naphthoquinone (MQ-GS) and 2-methyl-3-(N-acetyl-L-cysteine-S-yl)-1,4-naphthoquinone (MQ-NAC), retain the ability to redox cycle and to arylate cellular nucleophiles. Although the nephrotoxicity and hepatotoxicity of MQ-thiol conjugates have been reported in vitro, methods for their determination in vivo have yet to be published. Herein, a highly sensitive, simple, and selective HPLC-chemiluminescence (HPLC-CL) coupled method is reported, allowing for the first time the simultaneous determination of MQ, MQ-GS, and MQ-NAC in rat plasma after MQ administration. Our method exploits the unique redox characteristics of MQ, MQ-GS, and MQ-NAC to react with dithiothreitol (DTT) to liberate reactive oxygen species (ROS) which are detected by a CL assay using luminol as a CL probe. To verify the proposed mechanism, MQ-GS and MQ-NAC were synthetically prepared. Specimen preparation involved solid-phase extraction on an Oasis HLB cartridge followed by isocratic elution on an ODS column. No interference from endogenous substances was detected. Linearity was observed in the range of 5−120 nM for MQ-GS and MQ-NAC and 10−240 nM for MQ, with detection limits (S/N of 3) of 1.4, 0.8, and 128 fmol for MQ-GS, MQ-NAC, and MQ, respectively. The application of our method reported here is the first to extensively study the stability and reversibility of thiol-quinones.

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drugs.2,3 As an xenobiotic agent, MQ can disrupt cellular functions via two distinct chemical pathways: as a redox cycling quinone that promotes the generation of reactive oxygen species (ROS) or as Michael acceptors, in which MQ covalently modifies cellular nucleophiles, most prominently sulfur nucleophiles, thereby creating potentially damaging arylation adducts.4

INTRODUCTION Quinones are among the oldest organic molecules in the universe.1 The quinone motif is found in many biologically relevant molecules and is well represented in pharmacopeia as clinically validated anticancer agents. However, the in vivo uses of quinones pose a major challenge since they can also cause acute cytotoxicity. Menadione (MQ), a representative quinone compound, has been widely used as an anti-inflammatory, anticancer, antimalarial, and a therapeutic agent for hypothrombinemia, in addition to being a component of multivitamin © XXXX American Chemical Society

Received: July 10, 2013

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dx.doi.org/10.1021/tx400253k | Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

Chemical Research in Toxicology

Article

Figure 1. Chemical structures of menadione and its thiol conjugates.

NAC-trapped quinoid-thioether reactive metabolites of endogenous substances, and exogenous xenobiotics such as estradiol, adrenaline, acetaminophen, diclofenac, and bromobenzene. These methods were developed to screen and structurally characterize quinoid-thiol adducts formed in vitro after microsomal incubation.8,12−15 The major limitation of these methods is that an independent analytical method is required to unequivocally identify the metabolites. Currently, no analytical methods exist for quantifying MQ and its thiol conjugates in biological fluids, so the design of an effective quantification method would provide valuable benefits, not only in the areas of analytical and toxicological chemistry but also in drug discovery. The application of chemiluminescence (CL) for the determination of trace and ultratrace concentrations of organic and inorganic species has significantly increased in the last two decades due to the high sensitivity and simplicity of this technique.16,17 However, the direct determination of quinones by CL has been limited because quinones cannot produce a CL signal directly. Nonetheless, many quinones, including MQ, have been detected in our laboratory by CL following specific modifications.18−20 One of these modifications allows liberated ROS to be measured with a CL probe in a medium containing a reductant.20 Because the conjugation reaction between GSH or NAC and MQ does not disturb the quinone nucleus, the resulting conjugates remain redox-active. The aim of this study was to exploit this redox capability to investigate a validated, very simple, and sensitive analytical method for the simultaneous determination of MQ and its thiol conjugates. The principle behind our method is that the reaction of a quinone with a dithiol compound such as dithiothreitol (DTT) liberates ROS that can be measured by a luminol-CL assay. To this end, we developed an HPLC-CL methodology and applied it to the quantification of MQ, MQ-GS, and MQ-NAC in rat plasma after the administration of MQ. Although MQ thiol conjugates have been postulated to be involved in MQ toxicity, they have never been detected in vivo and have only been identified using in vitro methods.21−24 To the best of our knowledge, the study presented here is the first to simultaneously quantify MQ and its thiol conjugates in vivo, even though it has long been known that thiol conjugates are metabolites of MQ.25,26 Our novel method introduces a valuable tool for the identification and quantification of

Glutathione (GSH) is the most abundant nonprotein thiol in cells and acts as a multifunctional intracellular antioxidant. Additionally, it plays a substantial role in the detoxification of electrophilic drugs.5 Although protein thiols are usually more abundant than GSH in a cellular pool, their reactivity is lower than that of GSH, making GSH the most important cellular thiol. GSH thus provides a protective mechanism against the depletion of protein sulfhydryl groups.6 MQ, like other quinones such as benzoquinone and naphthoquinone, can cause the depletion of GSH either through oxidation or conjugation. The resulting conjugate (Figure 1) has similarities to benzoquinol-GSH conjugates but differs in one important respect: the MQ conjugate (2-methyl-3-(glutathion-S-yl)-1,4naphthoquinone; MQ-GS) remains in the quinone form.7 GSH adducts are either directly excreted into bile or undergo a series of biotransformation steps mediated consecutively by γglutamylcysteine transpeptidase, dipeptidase, and N-acetyl transferase to give rise to N-acetyl-L-cysteine (NAC) conjugates (2-methyl-3-(N-acetyl-L-cysteine-S-yl)-1,4-naphthoquinone; MQ-NAC) that are commonly excreted into urine.8 The redox potential of quinones is strongly influenced by the substituent effect. The addition of an electron donating group to MQ, such as GSH or NAC, can decrease the redox potential of MQ.9 The lower the redox potential of a quinone, the higher is the rate constant for the reaction of a semiquinone with dioxygen to form a superoxide anion and the more readily the hydroquinone auto-oxidizes. In this respect, the initial nucleophilic addition of GSH and NAC to MQ makes the conjugate a better redox reagent than MQ itself.7 Besides their redox capability, MQ conjugates can arylate protein thiols. A mechanism for this reaction is provided for the first time in this work. Our results show that the conjugation of MQ with GSH and NAC cannot be considered as a true detoxification reaction, although conjugation can affect both bioavailability and the intracellular distribution of MQ. GSH and NAC conjugates of MQ have been reported to cause severe cytotoxicity;9−11 therefore, the quantification of these conjugates in different matrices is important in order to define these physiological and toxicological processes. HPLC coupled with tandem mass spectrometric (LC-MS/MS) detection is the method of choice for the identification of drugs and metabolites in biological fluids. Many LC-MS/MS methods have been employed for the identification of GSH, B



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Figure 2. Schematic diagram of the HPLC-CL system. buffer pH. Mass spectral data were obtained on a JEOL JMS-700N spectrometer (Tokyo, Japan). Elemental analyses were performed on a Perkin-Elmer 2400II (Norwalk, CT, USA). Melting points were measured with a Yanagimoto MP-53 melting point apparatus (Tokyo, Japan). Animal Treatment. Wistar rats (male, 8 weeks, 270−300 g, Tagawa Experimental Animals, Nagasaki, Japan) were used for the experiments. Animals were housed separately in a metabolic cage and kept on a 12 hour (h) light/dark cycle for 3 days before the experiments. Food and water were available ad libitum. All animal procedures and care were approved by the Nagasaki University Animal Care and Use Committee. MQ was dissolved in corn oil with gentle heat until dissolution. This MQ solution was administered intraperitoneally (i.p.) at a dose of 50 mg/kg in a volume of 10 mL/kg. Blood was withdrawn from the arteria femoralis through indwelling arterial catheters under ethyl carbamate (1.5 g/kg, i.p.) induced anesthesia. Blood samples were transferred to EDTA tubes and centrifuged for 10 min at 4 °C, 5000g; then, the separated plasma was stored at −80 °C in the dark until analysis. Determination of Quinones in Rat Plasma. One hundred microliters of rat plasma was transferred into a 1 mL polypropylene tube, diluted with purified water to approximately 200 μL, and vortexmixed for 30 s. Oasis HLB 1 cm3/30 mg cartridges were used to isolate MQ, MQ-GS, and MQ-NAC from each plasma sample. The cartridges were conditioned with 0.5 mL of methanol and equilibrated with 0.5 mL of purified water. The samples were passed through individual cartridges, after which the cartridges were washed two times with 250 μL of purified water. The target analytes were eluted with 150 μL of 40% ACN, followed by 150 μL of neat ACN. Each mixture was vortexmixed, and 20 μL was then injected into the HPLC-CL system. Method Validation. The method was validated by evaluation of the following parameters: specificity and selectivity were assessed by comparing chromatograms of blank rat plasma, plasma spiked with target analytes, and plasma samples obtained from rats injected with MQ. Sensitivity was termed by limit of detection (LOD) and limit of quantitation (LOQ), which are defined as the concentration with a signal-to-noise (S/N) ratio of at least 3 and 10, respectively. Linearity, expressed by the correlation coefficient (r), was evaluated with calculation of a least-squares regression line. The linearity of each analyte was determined with at least 6 concentration levels, not including the blank on 3 separate days. In order to assess the intra- and interday precision and accuracy, three quality control (QC) samples at low (10 nM), middle (60 nM), and high (100 nM) concentrations of MQ-GS and MQ-NAC and 20, 120, and 200 nM of MQ were prepared as described above. The intraday precision was assessed by calculating the % RSD for the analysis of the QC samples in triplicates, and interday precision was determined by the analysis of the QC samples on three separate days. Accuracy was calculated by comparing the averaged measurements to the nominal values and was expressed in percentage. The recovery for MQ, MQ-GS, and MQ-NAC were determined by comparing the peak height ratios of the analytes in rat plasma at the QC concentrations to those in purified water at equivalent concentrations and expressed in percentage.

quinones, both alone and as conjugates with macromolecules, in different biological matrices. As a result, medical researchers will be able to better understand the health consequences of internal and external quinone exposure.

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EXPERIMENTAL PROCEDURES

Chemicals and Reagents. All chemicals and solvents were of extra pure grade. MQ, acetonitrile (ACN), and methanol (HPLC) grade were supplied by Kanto Chemical Company (Tokyo, Japan). DTT, tetra-n-butylammonium bromide (TBAB), nitric acid, ethanol (99.5%), and diethylether were from Nacalai Tesque (Kyoto, Japan). Luminol, 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione (L-012), L-ascorbic acid, and phosphate buffer saline(PBS) powder (0.01 mol/L) were from Wako Pure Chemical Industry (Osaka, Japan). NAC, cysteine, isoluminol, lucigenine, and 2methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA) were from TCI (Tokyo, Japan). Bovine erythrocytes superoxide dismutase (SOD) (4470 U mg−1) and mannitol were from Sigma Aldrich (St. Louis, MO, USA). Beef liver catalase (20 mg/ mL) was purchased from Boehringer (Mannheim GmbH, Germany). Sodium hydroxide was purchased from Merck (Darmstadt, Germany). Tris (hydroxymethyl) aminomethane (Tris) was obtained from INC biomedical Co. (Eschwege, Germany). GSH was from KOHJIN Co. Ltd. (Tokyo, Japan). SPE cartridges, Oasis hydrophilic lipophilic balance (HLB) 1 cm3/30 mg were from Waters (Milford, MA, USA). MQ-GS and MQ-NAC were synthesized in our laboratory based on the method of Nickerson et al.27 with a small modification and the structures confirmed by MS and elemental analysis (complete data shown in Supporting Information). Milli-Q water was purified by Simpli Lab-UV (Millipore, Bedford, MA, USA), an ultrapure water system. Standard stock solutions of MQ-GS and MQ-NAC prepared in PBS and of MQ in ACN were kept at −30 °C until used. Working solutions were prepared each day from stock solution by appropriate dilution with purified water. All other chemicals were prepared in purified water unless otherwise indicated. Instrumentation. The HPLC system (Figure 2) consisted of three LC-10AS liquid chromatographic pumps (Shimadzu, Kyoto), a Rheodyne 7125 injector (Cotati, CA, USA) with a 20-μL sample loop, a CLD-10A chemiluminescence detector (Shimadzu), and an SIC chromatorecorder (Tokyo, Japan). PTFE tubing (15 m × 0.5 mm i.d., GL Sciences, Tokyo) was used as the reaction coil. Chromatographic separation was performed on Discovery HS C18 (250 × 4.6 mm, i.d., 5 μm, Sigma Aldrich) that used as stationary phase. Isocratic elution with a mixture of 5 mM Tris-HNO3 buffer (pH 8) and ACN (60:40, v/v %) containing 5 mM TBAB was used as mobile phase. The eluent from the column was simultaneously mixed with 1 mM of DTT in ACN and 1 mM of luminol in 20 mM NaOH aqueous solution. The flow rates of the mobile phase, DTT, and luminol solutions were set at 0.50, 0.25, and 0.25 mL/min, respectively. For the comparison study, a JASCO 875-UV detector (Japan) was used at a wavelength of 254 nm. Lumat LB-9507 luminometer (Berthold) was applied for time profile CL measurement, and a HORIBA F22 pH meter was used to adjust C



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Temperature and Time-Dependent Stability of MQ and Thiol Conjugates. The concentration of MQ, MQ-GS, and MQ-NAC after each storage period was related to the initial concentration at zero cycle (samples that were freshly prepared and processed immediately). Analyte stability in rat plasma was evaluated with three replicates at each QC concentration under three conditions: 1, 4, and 12 h at room temperature (RT), three freeze−thaw cycles at −30 °C, and QC samples being stored at −30 °C for 21 days. Reversibility of MQ Thiol Conjugates. The synthesized MQ-GS and MQ-NAC (10 μM) were incubated at 37 °C with10 μM, respectively, NAC and GSH in 0.01 mol/L PBS (pH 7.4). At different time intervals, aliquots of 10 μL were diluted and analyzed by the HPLC-CL system.

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(Figure S-2, Supporting Information) showed that MQ-GS, MQ-NAC, and MQ eluted at 9.5, 14, and 36 min, respectively. Many factors can affect CL response, the most important being the CL probe. Since luminol, isoluminol, L-012, lucigenin, and MCLA are extensively used in the biological detection of ROS,29 the effect of these reagents on the current methodology was tested. Luminol provided the highest CL intensity and high S/N ratio (Figure S-3, Supporting Information). This is important because luminol is a general CL probe exhibiting high CL sensitivity for superoxide anion radicals and other species. The influence of luminol concentration on CL intensity and S/N ratio was also examined. It was found that CL intensity increased linearly as the concentration of luminol increased; however, the background noise also increased. The best S/N ratio was obtained at 1 mM luminol (Figure 3). In

RESULTS AND DISSCUSION

Confirmation of Redox Capability and Liberation of ROS. In a preliminary test using luminol and a luminometer, no CL response was obtained with MQ, MQ conjugates, or reductant alone. In contrast, the combination of quinone with a dithiol reductant such as DTT generated ROS, which reacted with luminol to provide a high CL signal that increased or decreased depending on the quinone concentration. This mechanism was reported previously to explain sulfhydryl oxidation by phenanthraquinone, a component of diesel exhaust particles, using thiol compounds and protein preparations.28 Under aerobic conditions, the oxidation capability of phenanthraquinone was accelerated due to the reaction of semiquinone with dioxygen, leading to the overproduction of ROS. MQ-thiol conjugates provided higher CL signals than MQ (Figure S-1, Supporting Information) because the addition of an electron donating group such as GSH or NAC decreases the redox potential and consequently accelerates the reaction rate between the semiquinone and molecular oxygen to generate ROS.7 Although this result confirmed the liberation of ROS, we also studied the quenching effect of various ROS scavengers such as SOD, catalase, ascorbic acid, and mannitol. The quenching effect of SOD on target analytes showed that the superoxide anion plays an important role in the generation of CL. Method Development. To date, no analytical methods for the simultaneous determination of MQ, MQ-GS, and MQNAC in vivo have been developed, although thiol conjugates have been confirmed as metabolites of MQ.25,26 We therefore developed and validated an analytical method for their quantification in rat plasma. The conditions required to obtain high sensitivity and selectivity were optimized as follows. Optimization of Separation Conditions. We first used an HPLC-UV method to achieve good separation between MQ, MQ-GS, and MQ-NAC. This was challenging due to the different natures of the analytes: both MQ-GS and MQ-NAC are hydrophilic, while MQ is hydrophobic. Many columns and mobile phases comprising different organic modifiers, as well as buffers at different pH values, were studied. Optimum separation was achieved using a C18 column, isocratic elution, and TBAB as an ion-pair reagent. The mobile phase was a mixture of 5 mM Tris-HNO3 buffer (pH 8) and ACN (60:40, v/v %) containing 5 mM TBAB at a flow rate 0.5 mL/min. TABA was used as an ion-pair reagent, not only for its separation properties, but also for its neutral effect on CL response, verified using a luminometer (data not shown). Optimization of CL Conditions. Eluent from the C18 column containing MQ, MQ-GS, and MQ-NAC was mixed with DTT and luminol and allowed to react in the reaction coil before CL detection (Figure 2). A typical chromatogram

Figure 3. Effects of luminol concentration on the S/N ratio of the studied quinones. Conditions: 1 mM DTT, 20 mM NaOH, and 15 m reaction coil.

addition to the type and concentration of the CL probe, the luminol solvent also significantly affected CL intensity. The highest CL response was achieved when NaOH was used, compared to carbonate or borate buffer solutions (data not shown). The influence of NaOH concentration was also studied. High intensity and high S/N ratio were obtained at 20 mM NaOH (Figure S-4, Supporting Information). In a preliminary study using a luminometer, we explored the effect of different reductants; adequate CL response was only achieved with the dithiol compound, DTT. Consequently, DTT was used for further investigation by HPLC. Study of the influence of DTT on CL intensity and S/N ratio showed that the best CL response and S/N ratio were obtained at 1 mM DTT (Figure S-5, Supporting Information). The flow rate of both luminol and DTT also influenced the S/N ratio: increasing the flow rate of each reagent above 0.25 mL/min caused a decrease in S/N ratio. As grow-type CL was observed from MQ-GS, MQ-NAC, and MQ, the mixed solution was passed through a PTEF reaction coil before CL detection. The effect of coil lengths ranging from 5 to 25 m on CL intensity and S/N ratio was examined. CL intensity and S/N ratio increased with increased coil length up to 25 m; however, a 15 m coil was selected as peak broadening was observed with coils longer than 15 m (data not shown). Method Validation. This method was validated according to the criteria described in the Experimental Procedures section and based on the U.S. Guidance of Industry on Bioanalytical Method Validation.30 Under optimum experimental conditions, a linear relationship was observed by plotting relative CL intensity versus target analyte concentration. The calibration D



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NAC, and MQ was studied under various laboratory conditions such as short-term stability at RT for 1, 4, and 12 h, stability to three thaw−freeze cycles, and long-term stability by storing at −30 °C for three weeks. The three analytes showed very low stability when stored at room temperature for 12 h and at −30 °C for three weeks. Both MQ and MQ-GS showed very poor stability either at room temperature for 1 and 4 h or following three thaw-freeze cycles. Conversely, MQ-NAC showed moderate stability under the same conditions, as observed in Table 2. The poor stability of MQ in rat plasma was attributed to its electrophilic nature, making it prone to attack by nucleophilic macromolecules, especially thiol-containing proteins such as albumin, the most abundant protein in plasma that provides the largest pool of thiols in circulation.34 The ability of MQ to arylate protein tyrosine phosphatases (PTPs) has been reported and is believed to be the basis for the anticancer activity of MQ.35 The stability of MQ and its thiol conjugates in plasma could be improved by the rapid plasma pretreatment processes and careful storage of sample at −80 °C. MQ and its thiol conjugates were stable in −80 °C for at least 6 months. Also, the thawed sample should be processed rapidly. Reversibility of MQ-GS and MQ-NAC. The reversibility of the MQ-thiol conjugates was studied by incubation of MQ-GS with NAC and MQ-NAC with GSH in PBS at 37 °C. Additionally, the ability of MQ-GS to conjugate protein thiols was confirmed by incubation of MQ-GS with human serum albumin (HSA) in PBS at 37 °C. It was found that the incubation of MQ-GS with NAC led to a rapid decrease in the concentration of MQ-GS and an increase in MQ-NAC formation; in contrast, the interconversion of MQ-NAC and GSH occurred slowly (Figure S-6, Supporting Information). Also, the incubation of MQ-GS with HSA led to a loss of MQGS with concomitant formation of MQ-HSA adducts (to be published as a separate study). The proposed method demonstrated the relative reactivity of MQ-GS and MQNAC, and that in biological systems, significant covalent binding of quinone-thiol conjugates to tissue protein sulfhydryl groups can be expected, such as the incubation of MQ-GS with HSA to give the MQ-HSA adducts (Scheme 1). The ability of quinone-thioethers to form protein adducts was confirmed previously for quinones containing a free position for adduct formation, such as 2, 2, 5 and 2, 3, 5-mono, di, and triglutathionyl-benzoquinone.36 However, this is the first study in which the GSH group of the MQ-GS adduct was replaced by a thiol protein or nonprotein thiol. The transient nature of MQ-HSA in the presence of nonthiol proteins such as GSH was expected. The transient nature of the glyceraldehyde3-phosphate dehydrogenase (GAPDH)-1,2-naphthoquinone adduct in the presence of GSH, with the release of free GAPDH, was confirmed recently.37 The MQ-NAC conjugate

curve prepared using quinone standard solutions was linear in the range 5−120 nM for MQ-GS and MQ-NAC, and 10−240 nM for MQ, with excellent correlation coefficients (r) > 0.997. The detection limits (S/N of 3) obtained were 1.4, 0.8, and 128 (fmol/injection) for MQ-GS, MQ-NAC, and MQ, respectively (Table S-1, Supporting Information). Our results show that, for quantification in biological fluids, this method is 50, 10, and 8 times more sensitive for MQ than differential pulse polarography, HPLC-UV, and HPLC-FL, respectively.31−33 Additionally, application of our microplate reader CL-based method to the determination of MQ in human serum showed that the proposed method is twice as sensitive.19 Since we believe this is the first method that can simultaneously determine MQ, MQGS, and MQ-NAC, we compared this new method to an HPLC-UV method also developed in our laboratory. The HPLC-CL method was shown to be 1000 times more sensitive than the HPLC-UV method. Method accuracy and precision, both within day and between days, was assessed at low, middle, and high concentrations of MQ-GS, MQ-NAC, and MQ, as shown in Table 1. The accuracy ((mean observed concenTable 1. Accuracy and Precision of the Proposed Method in Rat Plasma within-day (n = 5) quinone

between-day (n = 5)

concn (nM)

accuracy (%)

precision (RSD %)

accuracy (%)

precision (RSD %)

10 60 100

98.82 97.22 92.36

4.68 4.94 5.32

87.07 86.20 89.37

6.26 5.12 8.56

10 60 100

94.78 94.23 102.2

8.33 5.72 5.04

92.68 102.45 97.60

7.24 3.81 7.90

20 120 200

87.12 93.13 85.18

3.76 3.64 4.34

99.11 85.49 85.06

6.07 3.52 5.01

MQ-GS

MQ-NAC

MQ

tration/spiked concentration)·100) of the proposed method was 86.2−98.8%, 92.7−102.5%, and 85.1−99.1% for MQ-GS, MQ-NAC, and MQ, respectively. Our method also showed good reproducibility, calculated by relative standard deviation (RSD %), as shown in Table 1. The rapid processing of the tested samples ensured the desired accuracy and precision. Stability of MQ and Its Thiol Conjugates in Rat Plasma. Our proposed method was successfully used to investigate the stability and reversibility of MQ and its thiol conjugates in the presence of plasma components. The stability of MQ-GS, MQTable 2. Stability of Studied Quinones in Rat Plasma

% remaining (n = 3) MQ-GS (nM)

a

MQ-NAC (nM)

MQ (nM)

condition

10

60

100

10

60

100

20

120

200

room temperature (1 h) room temperature (4 h) room temperature (12 h) three freeze−thaw cycles (−30 °C) freeze at (−30 °C) for 3 weeks

19.56 N/Aa N/A 30.43 N/A

60.65 N/A N/A 38.79 24.59

78.64 17.25 3.73 60.49 29.35

62.68 61.19 25.37 71.64 16.38

97.88 64.29 30.15 97.88 38.09

97.81 88.36 33.63 97.45 42.54

63.15 13.57 N/A N/A N/A

77.88 11.57 N/A 21.05 N/A

77.51 5.42 N/A 26.35 N/A

Not detected. E



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Scheme 1. Reactivity and Reversibility of MQ and Its Thiol Conjugates

Figure 4. Chromatograms of (A) blank rat plasma, (B) standard solution of 100 nM MQ-GS and MQ-NAC, and 200 nM MQ spiked in rat plasma and detected by the proposed HPLC-CL method.

Procedures section. Our method showed high selectivity and specificity as observed in typical chromatograms (Figures 4 and 5). Figure 4 shows blank rat plasma and plasma spiked with MQ, MQ-GS, and MQ-NAC and processed using our method. Figure 5 shows a plasma sample obtained from rats injected with MQ and processed as described above. No interference peaks were observed in any rat plasma samples evaluated, indicating that the proposed method is highly specific and selective for the determination of MQ, MQ-GS, and MQ-NAC. Many elution systems were studied to obtain high recovery. The best system is 40% ACN followed by 100% ACN. The more hydrophilic compounds, MQ-GS and MQ-NAC, eluted first in 40% ACN, then the more hydrophobic MQ eluted later in 100% ACN; elution with 40% ACN or 100% ACN alone provided poor recovery. Using these optimized conditions, recovery was excellent: 89−96%, 88−100%, and 74−87% for MQ-GS, MQ-NAC, and MQ, respectively, at three different concentrations. Thus, SPE provided the high recovery required for the detection of low analyte concentrations in rat plasma

showed the same characteristics but with low reactivity compared to that of MQ-GS. This can be explained by the fact that GS− is a better leaving group than NAC−. The leaving group capability is related to the pKa of the protonated form: the lower the pKa, the better is the leaving capability. The pKa values of GSH and NAC are 8.6 and 9.5, respectively.38 Determination of MQ and Its Thiol Conjugates in Rat Plasma. Both protein precipitation and solid phase extraction (SPE) were applied to remove interfering endogenous substances from the rat plasma samples. Protein precipitation provided poor selectivity and low recovery, especially for MQGS and MQ-NAC, which are hydrophilic. Consequently, SPE was evaluated for the extraction and purification of MQ, MQGS, and MQ-NAC from the plasma samples. Oasis HLB sorbent (polydivinylbenzene-co-N-vinylpyrrolidine) was selected because it delivers highly reproducible recoveries and binds both hydrophilic and hydrophobic compounds. An HLB cartridge was washed and equilibrated, and the sample was loaded, washed, and eluted as described in the Experimental F



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selected as model quinone because it was frequently used to investigate quinone-induced cytotoxicity. Rat plasma was selected as a real sample because there are restrictions related to MQ administration to human beings in Japan and also in the USA. The inevitable exposure to quinones and their inherent reactivity have triggered substantial research into the chemistry and toxicology of these compounds. Quinones are oxidants and electrophiles; and both chemical characteristics confer reactivity in biological systems. The addition of GSH or NAC to MQ generates simple conjugates that remain in the oxidized, quinone form. In general, thiol conjugates are considered products of a detoxification reaction. More recently, however, interest in the structure of thiol conjugates has been heightened by the finding that some classes of conjugates are themselves toxic as a consequence of certain mechanisms. The results of the present study clearly reveal that both MQ-GS and MQNAC show high redox reactivity and more ability to liberate ROS than the parent, MQ. Besides reactivity, we also confirmed for the first time the poor stability and reversibility of these analytes in plasma components and also in the presence of other sulfhydryl-containing compounds. Such quinone-thiol conjugates can be regarded as transporting agents for electrophiles through the body, and initial detoxification can ultimately result in the release of the reactive compound at some other site. Because of the reversibility of MQ-thiol conjugates, such compounds might provide the basis for strategies designed to improve the efficacy of certain chemotherapeutic agents. The proposed method succeeded in detecting MQ-GS and MQ-NAC in rat plasma after MQ administration, even though the reversibility of these conjugates hampered their detection. Our method may be the method of choice for selective determination of quinones and quinone-thiol conjugates regarded as biomarkers of quinone exposure (quinones of polycyclic aromatic hydrocarbons) or quinones formed under pathological conditions (oxidative metabolites of estrogens and catecholamines). Besides thiol conjugates, the proposed method might be extended to determine quinones adducts of DNA and proteins (future research under investigation).

Figure 5. Chromatograms of rat plasma collected at (A) zero time, (B) 30 min, and (C) 240 min following a single-dose administration of 50 mg/kg MQ and analyzed by the proposed HPLC-CL method.

following a single dose of MQ (50 mg/kg) in corn oil administered i.p. Typical chromatograms (Figure 5) of rat plasma prior to injection and 30 and 240 min after dose administration show that the proposed method provides high sensitivity and selectivity for the detection of low concentrations of MQ-GS, MQ-NAC, and MQ. The concentration of MQ-GS and MQ decreased gradually following administration, while MQ-NAC remained at a constant low concentration throughout the measurement period (0−240 min). The high concentration of MQ-GS compared to other adducts formed after MQ administration is due to the physiological concentration of GSH being higher than that of other thiols, which was shown to shift the balance in favor of glutathionyl adduct formation. The low concentration of MQ-NAC throughout the measurement period is attributed to the reversibility or high reactivity of its precursor, MQ-GS. Our method can also detect other MQ adducts in biological samples, as shown in the chromatogram in Figure 5B. Three to four new peaks eluting between 23 and 33 min were evident. Although cysteine is present in high concentration in rat and human plasma, the cysteine adduct of MQ cannot be detected by our method because the MQ-cysteine adduct is labile and might form the thiazine ring owing to cyclization.9,11 The resulting compound lacks quinone structure; therefore, it is difficult to be detected by our method. Therefore, the proposed HPLC-CL method should be valuable for investigating the pharmacokinetic parameters of quinones and quinone conjugates. Under the same conditions, the HPLC-UV method lacked the sensitivity to detect MQ, MQ-GS, or MQ-NAC in rat plasma following MQ administration. In this study, MQ was

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CONCLUSIONS The ubiquitous nature of quinones and the high intracellular concentration of thiols ensure that cells will be exposed to the side effects of the resulting quinone-thioethers. Studies on the formation and biological and toxicological activities of quinonethioethers will be a challenging area for future research. Utilization of the redox ability of MQ and its thiol conjugates was key in designing a highly sensitive and very fast method for their quantification in rat plasma. The high selectivity of the proposed method should allow the pharmacokinetic profiles of MQ, its thiols, and other quinone conjugates, to be studied in unprecedented detail. The proposed method, the first of its kind, verifies the high reactivity of thiol conjugates compared to the parent quinone. The proposed method provides valuable tools for studying the analytical and toxicological chemistry of quinones and thioether-quinones and will be useful for determining their function in transport, storage, and perhaps the toxicity of oxidized quinones. This assay can identify humans exposed to quinones and at risk of developing toxicity. Additionally, this study may provide a means for the dose monitoring of anticancer quinones. G



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ASSOCIATED CONTENT

* Supporting Information S

Details of the synthetic pathway of thiol conjugates, chemical confirmation data, figures of method preparation and optimization, and table of analytical regression. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Fax: +81958192444. E-mail: [email protected]. Funding

This work was supported in part by Grant-in-Aid for Scientific Research (B) (no. 22390116) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. M.S.E. expresses his sincere gratitude to the Japanese Government for a scholarship. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS ACN, acetonitrile; CL, chemiluminescence; FL, fluorescence; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HLB, hydrophilic lipophilic balance; HSA, human serum albumin; L-012, 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione; MCLA, 2-methyl-6-(4-methoxyphenyl)-3,7dihydroimidazo[1,2-a]pyrazin-3-one; MQ, menadione; MQGS, 2-methyl-3-(glutathione-S-yl)-1,4-naphthoquinone; MQNAC, 2-methyl-3-(N-acetyl-L-cysteine-S-yl)-1,4-naphthoquinone; NAC, N-acetyl-L-cysteine; PTP, protein tyrosine phosphatises; QC, quality control; TBAB, tetra-n-butylammonium bromide

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Development and Validation of the First Assay Method Coupling

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