positive electrospray tandem mass spectrometry

Journal of Chromatography A, 1248 (2012) 84–92

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Direct quantitation of hydroxyethylvaline in hemoglobin by liquid chromatography/positive electrospray tandem mass spectrometry Fagen Zhang ∗ , Kathy A. Brzak, Lynn H. Pottenger, Michael J. Bartels Toxicology and Environmental Research & Consulting, The Dow Chemical Company, 1803 Building, Midland, MI 48674, USA

a r t i c l e

i n f o

Article history: Received 9 December 2011 Received in revised form 27 April 2012 Accepted 7 May 2012 Available online 1 June 2012 Keywords: Quantitation Hydroxyethylvaline Liquid chromatography–tandem mass spectrometry

a b s t r a c t Hemoglobin adducts are often used as biomarkers of exposure to reactive chemicals in toxicology studies. Therefore rapid, sensitive, accurate, and reproducible methods for quantifying these globin adducts are key to evaluate test material dosimetry. A new, simple, fast, and sensitive LC/ESI-MS/MS methodology has been developed and validated for the quantitation of hydroxyethylvaline (HEVal) in globin samples isolated from rats, both control and exposed to ethylene. Globin samples were first hydrolyzed to amino acids (including HEVal), followed by direct LC/ESI-MS/MS analysis. The lower limit of quantitation was 0.0095 ng/mL (0.026 pmol/mg globin). Typical calibration curves obtained over three days were linear over a concentration range from 0.0095 to 9.524 ng/mL, with correlation coefficient R2 > 0.999. The intra-day assay precision RSD values for all QC samples were ≤11.2%, with accuracy values ranging from 90.6 to 105%. The inter-day assay precision RSD values for all QC samples were ≤8.73%, with accuracy values ranging from 89.3 to 104.5%. The stability of HEVal in three freeze–thaw cycles over 48 h and at room temperature over 24 h was also evaluated, and the measured concentrations of HEVal were compared to the nominal values, with accuracy ranging from 94.8% to 109%. In conclusion, this method provides results comparable to those obtained using the traditional and complex Edman degradation phenylthiohydantoin-related quantitation method, but is much simpler and faster to conduct. © 2012 Elsevier B.V. All rights reserved.

1. Introduction It has been demonstrated that internal exposure to the electrophilic compound ethylene oxide (EO) can be monitored by measuring adducts formed with nucleophilic sites in hemoglobin. In particular, the adduct to the N-terminal valines in hemoglobin, N-(2-hydroxyethyl) valine (HEVal), has been used as a biomarker of exposure in toxicological studies of ethylene or ethylene oxide [1–5]. Therefore, reliable, simple, and accurate methods for quantifying HEVal are key to provision of internal dose information for such studies. Very early methods to quantify these alkylated hemoglobin adducts (such as hydroxyethylated hemoglobin adducts) from animal exposures involved the use of radioactively labeled alkylating agents, which, upon adduct isolation and subsequent radioactivity counting, were quantified by virtue of the radioactive label [6–8]. This traditional procedure is expensive because it requires the synthesis of radiolabeled reactive chemicals (or active chemical metabolites) and it necessitates special precautions for proper radiochemical handling of samples. In addition, exposure via relevant routes, such as inhalation, poses additional technical

∗ Corresponding author. Tel.: +1 989 638 4172; fax: +1 989 638 9305. E-mail address: [email protected] (F. Zhang). 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2012.05.019

challenges with radiolabeled test materials. Therefore, methods using radioactive labels are not routinely used in studies. Alternatively, adducts can be quantified using immunological detection systems [9–11]; however, this approach requires the availability of antibodies capable of selectively recognizing the specific hemoglobin adduct either in cells, in isolated protein, or in hydrolyzed protein. The availability and selectivity of antibodies and onerous handling procedures limit this method’s application. The developments in mass spectrometric (MS) technology, coupled with chromatography [gas chromatography (GC) or liquid chromatography (LC)] have resulted in additional technologies for hemoglobin adduct identification and quantitation. With these increasingly sensitive technologies, the HEVal adducts in hemoglobin have been identified and quantified using combinations of GC–MS, GC–MS/MS (gas chromatography with tandem mass spectrometry), LC–MS/MS [12–20], and GC–HRMS (gas chromatography with high resolution mass spectrometry) [5]. Although LC–HRMS instrumentation was used less frequently than the popular LC–triple quadrupole mass spectrometer (LC–MS/MS) in bioanalytical analysis due to a narrow dynamic range of LC/TOF/MS, LC–HRMS (such as LC–TOF/MS) was still used for hemoglobin adduct quantitation [21]. Most of these published methods rely on the well-known procedure combining Edman degradation with quantitation of phenylthiohydantoin. This method, although employing mild basic derivatization conditions and offering high

F. Zhang et al. / J. Chromatogr. A 1248 (2012) 84–92

sensitivity, typically requires a long process of sample derivatization and extraction. To avoid the drawbacks of an Edman degradation and quantitation of phenylthiohydantoin method, we developed a simple and quick LC/ESI-MS/MS method to directly quantify underivatized HEVal in globin samples. This new method relies on the well-established technique of complete acid hydrolysis of proteins, along with appropriate internal standards, and is sensitive enough to quantify HEVal in globin from control rats and ones exposed to ethylene (ET) or ethylene oxide (EO). This new method provided results that were comparable to ones obtained with the more common and lengthy procedure using the Edman degradation and phenylthiohydantoin quantitation method. 2. Materials and methods 2.1. Caution 2.1.1. Chemical hazards Ethylene oxide was handled in accordance with NIH Guidelines for Laboratory Use of Chemical Carcinogens [22]. 2.2. Reagents, solvents and materials Acetonitrile (HPLC grade), methanol (HPLC grade), water (HPLC grade), and acetic acid (HPLC grade) were obtained from Fisher Scientific (Itasca, IL, USA). D4 -HEVal was obtained from Toronto Research Chemicals, Inc. (North York, Ontario, Canada). Both HEValdipeptide-anilide and D4 -HEVal-dipeptide anilide were obtained from Bachem (Torrance, CA, USA). Myoglobin and other reagents including HEVal were purchased from Sigma–Aldrich (St. Louis, MO, USA), unless otherwise stated. 2.3. Animals In accordance with the U.S. Department of Agriculture animal welfare regulations, 9 CFR, subchapter A, Parts 1–4, the animal care and use activities required for conduct of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). Male Fischer 344/DuCrl rats were obtained from Charles River Laboratories Inc. (Portage, MI, USA; ∼175 g, approximately 42 days of age). Groups of 8-week-old male rats were exposed via whole-body inhalation in 8-m3 stainless-steel and glass inhalation chambers to various levels of ET, monitored by Miran infrared spectrophotometer, for 6 h/day (5 days/week) over 4 weeks. At necropsy of treated and control animals, heparinized blood samples were collected from each animal and globin samples were isolated for analysis of HEVal. Additional globin samples were isolated from control and ethylene-exposed male Fischer 344 rats from Dr. J.A. Swenberg’s laboratory (University of North Carolina) from a previous ET exposure study [16]. 2.4. Synthesis of HEVal-globin and D4 -HEVal-globin Both HEVal-globin and D4 -HEVal-globin standards were prepared according to the published procedure by Tornqvist et al. [23] with an approximate ratio of ethylene oxide (EO)/globin of 100 ␮mol EO/g globin. Briefly, 2 mL of whole blood (containing approximately 0.3 g globin) was added to a 20-mL headspace vial. After sealing with a Teflon cap, 1 mL of EO (408.9 ␮mol) from a gas lecture bottle was injected into the vial through the cap with a 10mL gas-tight syringe. The vial was incubated for 2 h at 37 ◦ C, then taken out of the incubator and left at room temperature overnight (16 h). In the same way, the incubations of D4 -EO with blood and control incubations (without EO) were also conducted.

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2.5. Isolation of globin from incubations The globin was isolated based on the procedure developed by Mowrer et al. [24] with minor modifications. Briefly, approximately 3 mL of pooled blood from each incubation was transferred to a 50-mL plastic centrifuge tube and centrifuged at room temperature for 20 min at 2000 × g. Each red blood cell (RBC) pellet was collected and washed three times with isotonic saline (9 mL each time) by centrifugation (2000 × g). The final washed RBCs were mixed with 9 mL deionized water to form hemolysates and held frozen at −80 ◦ C (2 h). After thawing at room temperature, 18 mL of 50 mM hydrochloric acid in 2-propanol was added to the vial and the mixture was centrifuged at 3500 × g for 10 min. Supernatants were mixed with ethyl acetate (24 mL) and the resulting mixture was kept at 4 ◦ C for at least 2 h, and then centrifuged at 2000 × g for 10 min. After centrifugation, the precipitated globin was washed 2 times with 15 mL ethyl acetate and 15 mL n-hexane. The final washed precipitate was dried under vacuum overnight. The final dried globin (the purity of the isolated globin was assumed as 100% without further purity analysis) was stored at −18 ◦ C until analysis. 2.6. Acid hydrolysis of globin samples The acid hydrolysis of globin and myoglobin was based on published procedures [25–27]. Briefly, weighed aliquots of 5 mg pulverized globin from each isolated globin or myoglobin sample and appropriate amount of internal standard (D4 -HEVal) were added to 10-mL headspace vials. After 4 mL aliquots of 6 M HCl were added to each vial, a headspace cap was put on and sealed with a crimper. The solutions were heated at 120 ◦ C for 18 h. After cooling to room temperature, the hydrolysis solutions were transferred to 4-mL vials and concentrated to dryness with a Speedvac concentrator using a medium temperature (40 ◦ C). The final residue was reconstituted in 2 mL of deionized water and stored at −18 ◦ C until analysis. 2.7. Preparation of HEVal phenylthiohydantoin derivative The preparation of HEVal phenylthiohydantoin derivatives was based on the published procedure by Tornqvist et al. [23]. Briefly, 10-mg aliquots of pulverized control globin were weighed into 1-dram glass vials and fortified with equimolar amounts of HEValglobin. The samples were dissolved in 1.5 mL formamide. Internal standards of D4 -HEVal-globin were added to the corresponding samples. The mixed sample was adjusted to a target pH of 6.8 with 1 M NaOH, using pH paper for measurement. Pentafluorophenyl isothiocyanate (7.5 ␮L; PFPITC) was added to the samples, which were then capped and placed on a rocking panel overnight at ambient temperature. After processing overnight, the samples were placed on a heated vortex-mixer at 45 ◦ C for 1.5 h, agitating gently. The samples were then extracted with diethyl ether (3 × 1.5 mL; vortex-mixed 30–60 s, centrifuged 15 min at 1500 × g), and resulting ethyl ether extracts were combined. The combined ether extracts were taken to dryness under a stream of nitrogen. Residues were reconstituted in 1-mL aliquots of toluene, mixing well. The toluene samples were washed with water (1 × 1.5 mL), 0.1 M freshly prepared Na2 CO3 (1 × 1.5 mL), and again with water (1 × 1.5 mL). The toluene extracts were then taken to dryness under a stream of nitrogen and the residues were reconstituted in 75 ␮L of 1/1 acetonitrile/water, mixing well. The final extracts were transferred to glass, high-recovery conical autosampler vials for analysis. In the same way, some of the globin samples prepared from control rats and ones exposed to ethylene at different levels [16], were also processed for globin adduct quantitation.

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2.8. LC–MS/MS and LC–TOF/MS quantitation methods Three LC–MS/MS or LC–TOF/MS methods were used in this study. 2.8.1. Method A: LC–MS/MS quantitation of phenylalanine To validate the completeness of 6 M HCl hydrolysis of hemoglobin and myoglobin, LC–MS/MS quantitation of phenylalanine released from 6 M HCl hydrolysis of hemoglobin or myoglobin was used according to the published procedure by Lee et al. [28] with minor modifications. Chromatographic separations were performed on a Phenomenex Synergy 4 ␮m Polar-RP 4.6 mm × 150 mm column from Phenomenex (Torrance, CA, USA), using an Agilent HP 1200 HPLC system (Agilent, Palo Alto, CA, USA). Mobile phases consisted of (A) deionized water with 0.1% acetic acid and (B) acetonitrile with 0.1% acetic acid. The gradient profile started with 0% B and linearly increased over 13 min to 60% B. The gradient was then increased to 95% B in 3 min and held for 2 min; finally the gradient was decreased to 0% B in 1 min, and a 5-min equilibration time was incorporated between runs. The flow rate was 0.5 ml/min and the entire LC flow was directed into the mass detector between 2.0 and 20 min using a switching valve from the Valco Instrument Co. (Houston, TX, USA). MS detection was done in the positive ESI mode on a Sciex API 4000 tandem mass spectrometer (Concord, Ontario, Canada) equipped with a Sciex DuoSpray source. Instrument interface parameters were optimized by infusing phenylalanine with a combination of the HPLC mobile phase solvents through the interface and were as follows: declustering potential 55 V, heater temperature 550 ◦ C, ion source voltage 5500 V, curtain gas 10, nebulizer gas (GS 1) 45, and sheath gas (GS 2) 45. Tandem mass spectrometric analysis was performed using nitrogen as the collision gas (CAD setting 9). Selective reaction monitoring (SRM) was used for the detection and quantitation of the phenylalanine with its stable isotope internal standard (phenylalanine-13 C6 ). The SRM transitions of positive charged ions of m/z 166.3 → m/z 120.3 and m/z 172.5 → m/z 126.2 (collision energy, 15 eV) were monitored for phenylalanine and phenylalanine-13 C6 , respectively (Fig. 1). A dwell time of 100 ms was used for each transition. The quadrupoles Q1 and Q3 were set on unit resolution. 2.8.2. Method B: direct LC–MS/MS quantitation of underivatized HEVal in globin This LC–MS/MS quantitation method was developed to analyze the low levels of HEVal released by 6 M HCl hydrolysis of isolated globin from control rats or ones exposed to ethylene at different levels. Chromatographic separations were performed on a Phenomenex Synergy 4 ␮m Hydro-RP 4.6 mm × 250 mm column from Phenomenex (Torrance, CA, USA), using an Agilent HP 1200 HPLC system (Agilent, Palo Alto, CA, USA). Mobile phases consisted of (A) deionized water with 0.1% acetic acid and (B) acetonitrile with 0.1% acetic acid. The gradient profile started with 0% B and a flow rate of 0.5 mL/min, and held for 9 min. The gradient was then increased to 95% B and a flow rate of 1.0 mL/min in 2 min and held for 2 min. Finally the gradient was changed to 0% B in 1 min, and a 2-min equilibration time was incorporated between runs. The entire LC flow was directed into the mass detector between 3.5 and 9.0 min using a switching valve from the Valco Instrument Co. (Houston, TX, USA). MS detection was done in the positive ESI mode on a Sciex API 4000 tandem mass spectrometer (Concord, Ontario, Canada) equipped with a Sciex DuoSpray source. Instrument interface parameters were optimized by infusing the HEVal with a combination of the HPLC mobile phase solvents through the interface and were as follows: declustering potential 60 V, heater temperature 550 ◦ C, ion source voltage 5500 V, curtain gas 10, nebulizer gas (GS 1) 45, and sheath gas (GS 2) 45. Tandem mass spectrometric analysis

Fig. 1. Positive ESI product ion spectra of [M+H]+ ions of phenylalanine and 13 C6 phenylalanine at the collision energy 15 eV: (A) positive ESI product ion spectrum of [M+H]+ ion of phenylalanine and (B) positive ESI product ion spectrum of [M+H]+ ion of 13 C6 -phenylalanine.

was performed using nitrogen as the collision gas (CAD setting 9). Selective reaction monitoring (SRM) was used for the detection and quantitation of the HEVal with its stable isotope internal standard (HEVal-D4 ). The SRM transitions of positive charged ions of m/z 162.2 → m/z 116.3 and m/z 166.2 → m/z 120.3 (collision energy, 19 eV) were monitored for HEVal and HEVal-D4 , respectively (Fig. 2). A dwell time of 100 ms was used for each transition. The quadrupoles Q1 and Q3 were set on unit resolution. 2.8.3. Method C: LC–TOF/MS quantitation of HEVal phenylthiohydantoin derivative The LC–TOF/MS quantitation of HEVal, as the phenylthiohydantoin derivative, was based on the procedure developed by Rusyn et al. [16], with modifications. Briefly, chromatographic separations were performed on YMC ODS-AQ C18 , 5 ␮m, 120A, 2 mm × 50 mm column from Waters (Milford, MA, USA) using an Agilent HP 1100 HPLC system (Agilent, Palo Alto, CA, USA). Mobile phases consisted of (A) deionized water with 0.1% acetic acid and (B) acetonitrile with 0.1% acetic acid. The gradient profile started with 0.5% B. The gradient was then increased to 100% B in 9 min and held for 4 min, finally the gradient was decreased to 0% B in 1 min, and a 5-min equilibration time was incorporated between runs. The flow rate was 0.4 mL/min and the entire LC flow was directed into the mass detector between 2.0 and 15 min using a switching valve. MS detection was done in the negative ion TOF/ESI mode on an Agilent MSD/TOF

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dilution of the 6 M HCl hydrolyzed control globin sample prepared as above with Milli-Q water. Quality control (QC) samples at concentration 0.0095, 0.0238, 0.436, and 9.524 ng/mL were prepared by diluting the corresponding standard working solutions and IS working solutions with a matrix solution, formed by a 5-fold dilution of the 6 M HCl hydrolyzed control globin sample prepared as above with Milli-Q water. QC samples were also stored at 4 ◦ C. 2.10. Method validation

Fig. 2. Positive ESI product ion spectra of [M+H]+ ions of HEVal and D4 -HEVal at the collision energy 10 eV: (A) positive ESI product ion spectrum of [M+H]+ ion of HEVal and (B) positive ESI product ion spectrum of [M+H]+ ion of D4 -HEVal.

mass spectrometer (Agilent, Palo Alto, CA, USA) equipped with an ESI source. Instrument interface parameters were as follows: fragmentor 150 V, skimmer 60 V, OCTRF 250 V, drying gas 50 psi, gas temp. 350 ◦ C, capillary 4000 V. The TOF/MS spectrometric analysis was performed using nitrogen as the collision gas. Data were acquired in the full scan mode (100–1100 amu) and a narrow mass range of m/z 367.04–367.07 was used for quantitation of the HEVal phenylthiohydantoin derivative and m/z 373.08–373.11 was used for quantitation of the stable isotope internal standard (D4 -HEVal) phenylthiohydantoin derivative. 2.9. Preparation of stock and working solutions and calibration standards The stock solutions of the analyte and internal standard (HEVal, D4 -HEVal) were prepared at 1.0 mg/mL in deionized water. A series of standard working solutions of HEVal in the range of 0.0005–0.1 mg/mL were obtained by dilution of the corresponding standard stock solution with de-ionized water. The internal standard (IS) working solution (0.1 mg/mL) of D4 -HEVal was prepared by diluting the internal standard stock solution with deionized water. All solutions were stored at −20 ◦ C. The calibration standards of HEVal (0.0095, 0.0238, 0.0476, 0.0952, 0.476, 0.952, 4.762, 9.524 ng/mL) with a fixed amount of IS (9.524 ng/mL) were prepared by diluting the corresponding standard working solutions and IS working solutions with a matrix solution formed by a 5-fold

The performance characteristics of the direct LC/ESI-MS/MS HEVal quantitation method were established by in-house validation procedures employing assays with standard solutions, sample blanks and QC samples. Linearity, selectivity, precision, detection, and quantitation limits were evaluated. For the quantitation of HEVal from 6 M HCl hydrolysis of globin, peak integration was performed with Analyst software (version 1.4.1) from Applied Biosystems. The linearity of calibration was determined by analyzing the calibration standards. Calibration curves were constructed by plotting the corresponding peak-area ratios of analyte/internal standard vs. the corresponding analyte peptide concentration. Weighted (1/x) linear regression analysis was used to determine the slope, intercept, and correlation coefficient (R2 ). The concentration of HEVal was determined from the peak/area ratios by using the equation of linear regression obtained from the corresponding calibration curve. The limit of quantitation was set on the concentration below which the method could not operate with acceptable precision and accuracy and at which the signal-to-noise ratio was greater than 10. The limit of detection was the lowest concentration that was detectable in all replicates but not necessarily quantifiable, although distinguishable from zero (signal/noise ≥ 3). Intra-day accuracy and precision (each n = 4) for HEVal were evaluated by analysis of QC samples at different times during the same day. Inter-day accuracy and precision were determined by repeated analysis of QC samples over three consecutive days (n = 12). The concentration of HEVal in each sample was determined using calibration standards prepared on the same day. Accuracy of the method was determined by the equation: (mean of determined concentration/actual concentration) × 100. Precision was reported as the relative standard deviation (RSD %) of replicate QC samples. The effect of three freeze–thaw cycles over 48 h and compound stability over 24 h at room temperature was evaluated by repeat analysis (n = 4) of QC samples. The concentrations were determined using freshly prepared calibration curves. Stability was also evaluated by means, precision, and accuracy. 2.11. Assay application The present quantitation method was used to determine the concentrations of HEVal in globin samples of control rats and ones exposed to ethylene.

3. Results and discussion 3.1. Synthesis of HEVal-globin and D4 -HEVal-globin HEVal-globin and D4 -HEVal-globin were synthesized by reaction of the nucleophilic N-terminal amino group of valine in globin from rat blood with the electrophilic ethylene oxide (EO) and ethylene oxide-d4 (D4 -EO), followed by isolation of the alkylated globins. The amounts of HEVal were quantified by the direct LC–MS/MS method (Method B) and D4-HEVal was quantified by

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comparing the peak area of D4 -HEVal vs. the peak area of HEVal standard. 3.2. Development of the LC/ESI-MS/MS conditions for quantifying HEVal in adducted globin To quantify the fraction of HEVal-adduction in globin isolated from rat blood incubated with ethylene oxide, early efforts employed a radiolabeled method involving total acid hydrolysis of globin isolated from blood exposed to 14 C-labeled ethylene oxide, separation by ion-exchange chromatography, collection of HEVal fraction, and counting radioactivity of the HEVal fractions [23,25,29]. In contrast, there have been many recently published LC/ESI-MS/MS methods used for direct analysis of amino acids (including valine), with or without derivatization [28,30–33]. In light of the high sensitivity of LC–MS/MS quantitation methods, and the similarity between HEVal and unalkylated valine, a direct LC/ESI-MS/MS quantitation method for this analyte with D4 -HEVal as an internal standard was developed. The positive ESI product ion spectra of HEVal and D4 -HEVal are shown in Fig. 2. The protonated [M+H]+ ion of HEVal (m/z 162.4) provided a major product ion at m/z 116.1 (Fig. 2A), and protonated [M+H]+ ion of D4 -HEVal (m/z 166.2) provided a major product ion at m/z 120.3 (Fig. 2B). Based on the product ion spectra of HEVal and D4 -HEVal, the transitions m/z 162.4 → m/z 116.1 and m/z 166.2 → m/z 120.3 of HEVal and D4 -HEVal (collision energy, 10 eV; Fig. 1), respectively, were monitored for selective reaction monitoring (SRM) quantitation of HEVal. This approach was applied to quantify HEVal levels in acid-hydrolyzed globin samples isolated from blood of control and ethylene-exposed rats (Method B). Phenylalanine was also quantified in HCl-hydrolyzed globin to evaluate the completeness of this digestion method. The published LC/ESI-MS/MS quantitation method for plasma samples [28] was used with modifications. The positive ESI product ion spectra of phenylalanine and 13 C6 -phenylalanine are shown in Fig. 1. The protonated [M+H]+ ion of phenylalanine at m/z 166.3 provided a major product ion at m/z 120.2 (Fig. 1A), and protonated [M+H]+ ion of 13 C -phenylalanine at m/z 172.2 provided a major product ion at 6 m/z 126.2 (Fig. 1B). Based on the product ion spectra for phenylalanine and 13 C6 phenylalanine, the transitions m/z 166.3 → m/z 120.2 and m/z 172.3 → m/z 126.2 of phenylalanine and 13 C6 -phenylalanine (collision energy, 15 eV; Fig. 2), respectively, were monitored for selective reaction monitoring (SRM) quantitation of phenylalanine (Method A). Sensitivity, resolution, and peak shape were optimized by using different combinations of organic solvents, water, and mobile phase additives. Optimum chromatographic settings were achieved by using mobile phases consisting of aqueous phase A (deionized water with 0.1% acetic acid) and the organic mobile phase B (acetonitrile with 0.1% acetic acid). This mobile phase system was finally used for the optimization of the electrospray source parameter settings. The final combined optimized conditions were used for the HEVal quantitation (Method B). 3.2.1. Matrix selection In consideration of the difficulty to obtain HEVal-free 6 M HCl hydrolyzed control globin (an ideal matrix for making calibration standards and QC samples), a recovery study (a part of cross validation study) was conducted. In this recovery study, a known amount HEVal (ranging from 0.0095 to 0.0476 ng/mL) was spiked into the 6 M HCl hydrolyzed globin which contained a known amount of endogenous HEVal to serve as spiked samples. Those spiked samples were then quantified by using calibration standards prepared in the matrix solution formed by a 5-fold dilution of the 6 M HCl hydrolyzed control globin. After subtraction of the

Fig. 3. Analysis of HEVal in a calibration standard containing HEVal at 0.0476 ng/mL and D4 -HEVal at 9.52 ng/mL by positive LC/ESI-MS/MS in SRM mode: (A) SRM chromatogram HEVal (RT 7.3 min) and (B) SRM chromatogram of D4 -HEVal (RT 7.3 min).

background level of HEVal, 99–105% recovery of HEVal was obtained (data not shown), indicating this matrix solution (formed by a 5-fold dilution of the 6 M HCl hydrolyzed control globin) is a good candidate for the matrix used for making calibration standards and QC samples. 3.2.2. Chromatography and selectivity Representative SRM ion chromatograms (Method B) for HEVal and D4 -HEVal from a calibration standard, from a 6 M HClhydrolyzed control globin, and from a 6 M HCl-hydrolyzed globin sample from ET-exposed rats are shown in Figs. 3–5, respectively. The overall chromatography run times were within 16 min. The retention times of HEVal and D4 -HEVal were 7.3 min (Fig. 3A and B). The control hemoglobin matrix did not show any SRM response for HEVal or D4 -HEVal (Fig. 6). As shown in Figs. 3–6, ion chromatograms for both HEVal and D4 -HEVal were free from interference; peak shapes were sharp and essentially indistinguishable in profile from standards of comparable concentration. 3.2.3. Calibration curve linearity and limit of quantitation (LOQ) A series of calibration standards for HEVal were prepared at various concentrations, with fixed concentrations of the corresponding internal standard. These solutions were analyzed by LC/ESI-MS/MS (see Section 2). The calibration curve obtained for HEVal was linear in a concentration range of 0.0095–9.524 ng/mL with correlation coefficients

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Table 1 Intra- and inter-day precision and accuracy for HEVal quantitation. Concentration (ng/mL)

Intra-assay precision and accuracy

0.0095 0.0238 0.476 9.52

Inter-assay precision and accuracy

Mean (n = 4) (ng/mL)

CV (%)

Mean accuracy (n = 4) (%)

Mean (n = 12) (ng/mL)

CV (%)

Mean accuracy (n = 12) (%)

0.0086 0.0230 0.501 9.26

7.93 3.85 11.2 1.65

90.6 96.8 105 97.2

0.0085 0.0229 0.498 9.17

7.93 4.72 8.73 1.65

89.3 96.3 105 96.3

CV, coefficient of variance.

(R2 ) greater than 0.999 (Fig. 7). The accuracy and precision were determined by analyzing replicates of the calibration standards at different concentrations ranging from 0.00952 to 9.524 ng/mL. The accuracy was between 93.4 and 104.5% and the precision was between 0.2 and 5.2% (data not shown) each day. The limit of quantitation (LOQ) for HEVal was 0.0095 ng/mL (0.026 pmol/mg globin).

3.2.4. Precision and accuracy of quantitation of HEVal The intra-day assay precision RSD values for all QC samples were ≤11.2%, with accuracy values ranging from 90.6 to 105%. The interday assay precision RSD values for all QC samples were ≤8.73%, with accuracy values ranging from 89.3 to 105% (Table 1).

Table 2 Stability studies of HEVal. Nominal concentration (ng/mL) 0.0476

1000

9.17 1.56 96.3

A)

HEVal

1.5e5 1.0e5

7.24

5.0e4

2

4

6

8 10 Time, min

12

14

16 0.0

D4-HEVal

3.0e5

4.9e5

2.5e5

2

4

6

8

10

12

14

16

8

10

12

14

16

Time, min

7.22

B)

D 4 -HEVal

4.0e5

Intensity, cps

Intensity, cps

9.59 6.24 101

2.0e5

HEVal

Intensity, cps

Intensity, cps

2.3e5

2000

0

9.524

3.2.5. Stability studies The freeze/thaw stability of HEVal over 48 h was evaluated. The measured concentrations of HEVal were compared to the nominal values, with accuracy ranging from 97.0 to 105% (Table 2). The stability of the QC samples stored at 25 ◦ C for 24 h was also evaluated

4000 3000

0.476

Stability after three cycles of freeze/thaw (n = 4) 0.0465 0.499 Mean 3.39 0.622 RSD % 97.0 105 Accuracy % Stability after storage at room temperature for 24 h (n = 4) 0.0455 0.0496 Mean RSD % 3.92 3.15 Accuracy % 94.8 103

2.0e5 1.5e5

3.0e5 2.0e5

1.0e5 1.0e5

5.0e4 0.0

0.0

2

4

6

8

Time, min

10

12

14

16

Fig. 4. Analysis of HEVal in a control globin sample containing HEVal at 0.015 ng/mL and D4 -HEVal at 9.52 ng/mL by positive LC/ESI-MS/MS in SRM mode: (A) SRM chromatogram HEVal (RT 7.3 min) and (B) SRM chromatogram of D4 -HEVal (RT 7.3 min).

2

4

6

Time, min

Fig. 5. Analysis of HEVal in a globin sample from ET-exposed rat containing HEVal at 0.774 ng/mL and D4 -HEVal at 9.52 ng/mL by positive LC/ESI-MS/MS in SRM mode: (A) SRM chromatogram HEVal (RT 7.3 min) and (B) SRM chromatogram of D4 -HEVal (RT 7.3 min).

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F. Zhang et al. / J. Chromatogr. A 1248 (2012) 84–92

2.4e4 2.2e4

8.02

A)

8.12

2.0e4

Intensity, cps

1.8e4 1.6e4 1.4e4

HEVal

1.2e4 1.0e4 8000.0 6000.0 4000.0 2000.0 0.0

2.0e4 1.8e4

2

4

6

8

10

12

14

16

8

10

12

14

16

Time, min

3.54

B)

1.6e4

Intensity, cps

1.4e4 1.2e4 1.0e4

D4-HEVal

8000.0 6000.0 4000.0 2000.0 0.0

2

4

6

Time,min

Fig. 6. Analysis of HEVal in a control globin matrix by positive LC/ESI-MS/MS in SRM mode: (A) SRM chromatogram HEVal (RT 7.3 min) and (B) SRM chromatogram of D4 -HEVal (RT 7.3 min).

with the same quantitation procedure as the freeze/thaw stability study. The final accuracy was from 94.8 to 103% (Table 2). These results show that no significant degradation occurs after 3 freeze/thaw cycles or after storage at 25 ◦ C for 24 h, indicating that HEVal in isolated globin is stable under the method conditions described above. 3.3. Hydrolysis of globin

Fig. 7. Representative calibration curve of HEVal from 0.0095 ng/mL to 9.52 ng/mL in SRM (positive mode) by HPLC/ESI-MS/MS.

To quantify the amount of HEVal and D4 -HEVal in synthesized, alkylated globin, the isolated globin was originally hydrolyzed to free amino acids with 6 M HCl according to the classical procedure [25–27]. To make sure that the protein hydrolysis was complete, the amount of phenylalanine released from 6 M HCl hydrolysis of globin was quantified by the newly developed LC/ESI-MS/MS method discussed above. For more accuracy, a commercial protein standard of myoglobin, which has a molecular weight similar to rat hemoglobin, was also hydrolyzed with 6 M HCl, and the resulting free phenylalanine was also quantified by the current method. The quantitative results are shown in Table 3. Good recoveries of

F. Zhang et al. / J. Chromatogr. A 1248 (2012) 84–92

91

Table 3 Final phenylalanine content in globin and myoglobin from 6 M HCl hydrolysis. Sample name

Percentage of phenylalanine in total protein from LC/ESI/MS–MS analysis (Method A)

Theoretical percentage of phenylalanine in total protein

Percentage of recovery of phenylalanine

Myoglobin Globin

5.27 5.29

6.07 6.5

86.2 81.4

Table 4 Comparison of the LC/TOF method (Method C) and the direct LC/ESI/MS–MS method (Method B) for analysis of HEVal in globin samples. Sample name

S1 S2 S3 S4

Ethylene exposure level (ppm) 0 0 0 0

Method B (pmol/mg globin) 0.069 0.060 0.059 0.058

S6 S7 S8 S9 S10

300 300 300 300 300

8.4 6.3 7.8 7.5 8.5

S11 S12 S13 S14 S15

1000 1000 1000 1000 1000

8.3 6.7 9.0 9.5 8.3

S16 S17 S18 S19 S20

3000 3000 3000 3000 3000

7.4 7.2 7.0 11 8.2

S21 S22 S24 S25

10,000 10,000 10,000 10,000

8.4 8.2 15 11

Overall RSD

Mean (RSD)

0.062 7.7%

Mean (RSD)

Method C (pmol/mg globin) ND 0.068 0.065 0.050

Percent difference between Method B and Method C

0.061 15.8%

1%

7.7 11.2%

7.0 7.6 7.1 7.1 6.2

7.0 7.0%

10%

8.3 12.8%

8.4 10.0 10 7.1 7.9

8.8 15.9%

5%

8.1 18.2%

7.4 7.9 7.9 11 7.9

8.4 17.2%

4%

11 28.7%

10 9.7 14 11

11 16.2%

7.0%

15.8%

phenylalanine from both myoglobin and hemoglobin were obtained with similar recovery percentages of 86.8% and 81.4%, respectively. 3.4. Comparison of the direct LC/ESI-MS/MS method (Method B) to the LC/TOF method (Edman degradation phenylthiohydantoin method, Method C) for analysis of HEVal in globin In consideration of a published method using a dipeptide standard with the Edman degradation method for HEVal quantitation [34], use of the dipeptide as a standard was also evaluated in the current study by investigating derivatization efficiency (Edman degradation derivatization). The results demonstrated that the pair

14.4%

HEVal-globin/D4 -HEVal-globin is the best standard set to use for quantitation of HEVal in globin among the various standard sets and methods using phenylthiohydantoin derivatization (Edman degradation derivatization) (data not shown). Thus, HEVal-globin plus D4 -HEVal-globin are the best standards to use for quantitation of HEVal in globin for the methods relying on phenylthiohydantoin derivative formation. Finally, the newly developed, simple, and direct LC/ESI-MS/MS method (Method B), and the LC/TOF method with phenylthiohydantoin derivatization using HEVal-globin and D4 -HEVal-globin as standards (Method C), were applied to the quantitation of HEVal in selected globin samples isolated from control rats. The results are summarized in Table 4. As shown in Table 4, these two

Table 5 Amounts of HEVal in globin of rats exposed to ET (0, 40, and 3000 ppm) for up 20 days.a Quantitation method

HEVal (pmol/mg globin) (average)

STDV

RSD %

0

Current method (Method B) Edman-degradation GC/MS–MS method (Rusyn et al. [16]) Edman-degradation GC/HRMS method (Walker et al. [5])

0.06 (n = 7) 0.44 (n = 21) 0.05 (n = 7)

0.01 0.11 0.01

9.8 25.0 20.0

40

Current method (Method B) Edman-degradation GC/MS–MS method (Rusyn et al. [16]) Edman-degradation GC/HRMS method (Walker et al. [5])

2.06 (n = 7) 1.56 (n = 8) 1.69 (n = 7)

0.22 0.11 0.12

10.5 7.1 7.1

3000

Current method (Method B) Edman-degradation GC/MS–MS method (Rusyn et al. [16]) Edman-degradation GC/HRMS method (Walker et al. [5])

9.66 (n = 8) 7.90 (n = 8) 7.34 (n = 7)

1.42 0.41 0.32

14.7 5.2 4.4

Ethylene exposure level (ppm)

a

Samples were provided by Dr. Swenberg’s lab (University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA).

92

F. Zhang et al. / J. Chromatogr. A 1248 (2012) 84–92

methods provide quite comparable results, with differences across mean concentrations at all dose levels within 10%. The overall RSD across dose levels was also quite comparable (15.8 vs. 14.4%). However, the direct LC/ESI-MS/MS method (method B) is a much simpler method which only requires 6 M HCl hydrolysis and vacuum drying procedures (no complex derivatization and extraction procedures necessary, different from Method C). Although complete acid hydrolysis constitutes strong conditions, it works. 3.5. Comparison of direct LC/ESI-MS/MS quantitation of HEVal in globin samples to HEVal quantitation using derivatization from a previous study The direct LC/ESI-MS/MS quantitation method was also applied to quantify the HEVal content in globin samples provided by Dr. Swenberg’s lab from a previous study in rats exposed to control air or ethylene [16]. As shown in Table 5, the amounts of HEVal quantified in globin from rats exposed to various levels of ethylene using the direct LC/ESI-MS/MS method were comparable to the published results obtained by the complex GC/MS–MS method (or GC–HRMS method) with Edman degradation and HEVal-globin as standards [5,16]. However, the amount of HEVal in globin from control rats via the direct LC/ESI-MS/MS method was much lower than that determined with the GC–MS/MS method [16]; however results for control rat globin analyzed with the direct LC/ESI-MS–MS method were comparable to values obtained from control globin analyzed with the GC–HRMS method [5]. 4. Conclusion A novel, direct and simple, and fast LC/ESI-MS/MS method has been developed for quantitation of HEVal in globin. This method showed satisfactory sensitivity, precision, accuracy, and reproducibility, and also provided comparable HEVal concentrations to results obtained using the complex Edman degradation-related LC/TOF (GC/MS–MS or GC–HRMS) methods via HEVal-globin and D4 -HEVal-globin standards (or dipeptide standards). Although the direct total acid hydrolysis LC/ESI-MS/MS method employs strong acid hydrolysis procedure, clearly the quantitation of HEVal is not affected. This methodology for sample preparation and analysis may be applicable to other globin adducts, following appropriate validation criteria. This method will be useful for mechanistic studies evaluating the relationship between macromolecular protein alkylation and biological response to certain alkylating agents. Acknowledgment We are grateful to Dr. James A. Swenberg and his lab (University of North Carolina at Chapel Hill, Chapel Hill, NC 27599,

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