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Simultaneous Detection of 3‑Nitrotyrosine and 3‑Nitro-4hydroxyphenylacetic Acid in Human Urine by Online SPE LC-MS/MS and Their Association with Oxidative and Methylated DNA Lesions Mu-Rong Chao,†,⊥ Yu-Wen Hsu,†,§,⊥ Hung-Hsin Liu,† Jia-Hong Lin,‡ and Chiung-Wen Hu*,‡,∥ †

Department of Occupational Safety and Health, and ‡Department of Public Health, Chung Shan Medical University, Taichung 402, Taiwan § Department of Optometry, Da-Yeh University, Changhua 515, Taiwan ∥ Department of Family and Community Medicine, Chung Shan Medical University Hospital, Taichung 402, Taiwan S Supporting Information *

ABSTRACT: Reactive nitrogen species (RNS) can modify proteins at tyrosine and tryptophan residues, and they are involved in the pathogenesis of various human diseases. In this study, we present the first liquid chromatography−tandem mass spectrometry (LC-MS/MS)-based method that enables the simultaneous measurement of urinary 3-nitrotyrosine (3NTYR) and its metabolite 3-nitro-4-hydroxyphenylacetic acid (NHPA). After the addition of stable isotope-labeled internal standards, urine samples were purified and enriched using manual solid-phase extraction (SPE) and HPLC fractionation followed by online SPE LC-MS/MS analysis. The limits of quantification in urine were 3.1 and 2.5 pg/mL for 3-NTYR and NHPA, respectively. Inter- and intraday imprecision was 99%. Participants and Urine Samples. This study was approved by the Institutional Review Board of Chung Shan Medical University Hospital. Written informed consent was obtained from each participant before urine sample collection. Spot urine samples were obtained from 65 apparently healthy subjects. Urine samples were collected directly in centrifuge tubes and were stored at −20 °C until analysis. Data on the subject’s age, body mass index (BMI), and smoking status were collected using a questionnaire. Simultaneous Quantification of Urinary 3-NTYR and NHPA Using Online SPE LC-MS/MS. Standard Solution and Calibration Curve. The 3-NTYR and NHPA stock solutions were prepared by individually dissolving 3-NTYR or NHPA in a 10% aqueous methanol solution to a final concentration of 50 μg/mL; they were then diluted with deionized water to obtain the desired concentrations and were stored at −20 °C before use. The calibration curve was prepared by mixing the two working standard solutions of 3-NTYR (100 ng/mL) and NHPA (100 ng/mL) (1:1, v/v) and then performing the appropriate serial dilution with deionized water (1:1, v/v) to yield aqueous standard solutions. Two linear ranges were obtained for 3NTYR and NHPA, ranging from 0.03 to 0.5 ng (low range) and from 0.5 to 8 ng (high range); each calibration standard also contained 5 ng each of d3-3-NTYR and d3-NHPA. Pretreatment of Urine Samples. Urine samples were enriched and purified using manual SPE and preparative HPLC prior to online SPE 998

DOI: 10.1021/acs.chemrestox.5b00031 Chem. Res. Toxicol. 2015, 28, 997−1006

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Chemical Research in Toxicology LC-MS/MS analysis. The urine samples were thawed, vortexed, and then heated to 37 °C for 20 min to release any possible 3-NTYR and NHPA from the precipitate. To 3 mL of urine was added 450 μL of 1 M ammonium acetate (AA, pH 5.25) and 100 μL of the internal standard solution containing 5 ng each of d3-3-NTYR and d3-NHPA. The mixture was then applied to a Sep-Pak Vac C18 cartridge (500 mg, 3 mL, Waters, Milford, MA) that was preconditioned with 3 mL of methanol and 3 mL of deionized water containing 1% (v/v) formic acid (FA). The cartridge was washed with 3 mL of deionized water containing 1% FA and was then eluted with 3 mL of 40% methanol containing 1% FA. The eluate was dried under vacuum and redissolved in 110 μL of deionized water, followed by preparative HPLC purification. Preparative HPLC purification of 3-NTYR and NHPA was performed on a Shimadzu (Tokyo, Japan) HPLC system, which was equipped with a system controller (SCL-10 AVP), two pumps (LC-10 ADVP), an autosampler (SIL-10 ADVP), a UV−visible detector (SPD-10 AVVP) set at 270 nm, and a fraction collector (FRC-10A). Chromatographic separation was performed on a Hypersil BDS C18 column (250 mm × 4.6 mm i.d., 5.0 μm) at room temperature with a flow rate of 1 mL/min. The detailed gradient program is provided in Supporting Information, Table S1. The HPLC fractions containing 3NTYR (eluted from 19.6 to 21.6 min) and NHPA (eluted from 39.0 to 41.0 min) were collected by the fraction collector. The collected fractions were then dried under vacuum and redissolved in 100 μL of 5% methanol containing 0.1% FA prior to analysis by online SPE LCMS/MS. Automated Online SPE. Online cleanup/enrichment was performed using a column-switching system19 that consisted of a switching valve (Valco Instruments, Inc.) and a C18 trap column (75 × 2.1 mm, 5 μm, ODS-3, Inertsil) and was controlled by PESCIEX control software (Analyst, Applied Biosystems). A timetable of the online SPE and LC gradients and the switching valve position programming used during the online cleanup and analytical processes are provided in Table 1. With the valve in position A, 25 μL of the prepared urine sample was loaded onto the trap column using an Agilent 1100 series autosampler and a binary pump (Agilent Technology), which was fractionated for 6.5 min using solvent Ia [5% (v/v) methanol/0.1% (v/v) FA] at a flow rate of 200 μL/min. Subsequently, the valve was switched to position B, and the retained analyte (enriched 3-NTYR) was transferred to the LC system. At 8.0 min after injection, the valve was switched back to loading position A, and the trap column was washed for 7 min using an eluent composed of 60% solvent Ia and 40% solvent Ib [75% (v/v) methanol/0.1% (v/ v) FA]. The valve was then switched to position B again to transfer the sample (enriched NHPA) into the LC system. At the 18.0 min time point, the valve was switched back to position A, and the trap column was washed and reconditioned. Liquid Chromatography. After online sample cleanup/enrichment (see Table 1 at the 6.5 min time point), separation of the analytes was achieved using a C18 column (250 × 2.1 mm, 5 μm, ODS-3, Inertsil). A gradient elution of 3-NTYR from the trap column to the analytical column was initiated by running 100% solvent IIa [20% (v/v) methanol containing 1 mM AA for 1.5 min at a flow rate of 200 μL/ min. At the 15 min time point, the gradient elution was set to 50% solvent IIa and 50% solvent IIb [75% (v/v) methanol containing 1 mM AA to begin the NHPA elution]. The column was then washed and prepared for the next analysis. The total run time was 30 min. Electrospray Ionization MS/MS. An API 3000 triple-quadrupole mass spectrometer (Applied Biosystems) equipped with a TurboIonSpray source operating in the negative mode was used for MS/MS detection. Nitrogen was used as the nebulizing gas. Analyst software (ver. 1.4) was used for instrument control, data acquisition, and quantitative analysis. The multiple reaction monitoring (MRM) mode was used, with two major transitions (quantifier and qualifier) for both of the analytes. Only one transition was selected for each of the stable isotope-labeled internal standards, d3-3-NTYR and d3-NHPA. The MRM transitions, along with their respective declustering potentials (DP), focusing potentials (FP), and collision energies (CE), are provided in Table 2. The dwell time per channel was set at 100 ms for

Table 2. Tandem Mass Spectrometry Parameters for 3NTYR and NHPA compd 3-NTYR d3-3NTYR NHPA d3NHPA a d

Q1 mass (amu)

Q3 mass (amu)

Dwell time (ms)

DPa (V)

FPb (V)

CEc (V)

225 225 228

163d 136 166d

100 100 100

−35 −35 −50

−130 −130 −190

−20 −25 −20

196 196 199

122d 135 125d

100 100 100

−30 −30 −40

−120 −120 −190

−15 −15 −15

Declustering potential. Quantifier transition.

b

Focusing potential.

c

Collision energy.

each MRM transition. The optimized ESI-MS/MS parameters in the negative ion mode were as follows: needle voltage, −4500 V; nebulizer gas flow, 10; curtain gas flow, 10; turbo gas flow, 8 heated at 450 °C; and collisionally activated dissociation gas flow, 4. Artifactual Nitration Test during Sample Pretreatment. To investigate the potential artifactual formation of 3-NTYR and NHPA during sample pretreatments in our method (i.e., manual SPE), an aqueous solution was prepared that consisted of sodium nitrite (10 μM), nitrate (1000 μM), and L-tyrosine and PHPA (each at 10 μg/ mL). After adding isotope-labeled internal standards, 3-NTYR and NHPA were measured before and after manual SPE purification using online SPE LC-MS/MS. The manual SPE process used was the same as that described above. The results of the artifact test are presented in Supporting Information, Table S2. Similar levels of 3-NTYR and NHPA were observed before and after the manual SPE pretreatment, indicating that no apparent artifactual nitration occurred during our pretreatment. Urinary Analysis of 8-OxoGua, 8-OxodGuo, N7-MeG, and N3-MeA Using Online SPE LC-MS/MS. The urinary concentrations of 8-oxoGua and 8-oxodGuo were determined using a validated online SPE LC-MS/MS method as described by Hu et al.19 The urinary concentrations of N3-MeA and N7-MeG were also measured using two different online SPE LC-MS/MS methods.20,21 Representative online SPE LC-MS/MS chromatograms of 8-oxoGua, 8-oxodGuo, N7MeG, and N3-MeA in urine are shown in Supporting Information, Figures S1−S3. All of the urine samples were also analyzed for creatinine using a validated HPLC-UV assay.22 Urinary Analysis of NDMA Using LC-MS/MS. The urinary concentration of NDMA was measured using an isotope dilution LCMS/MS method previously reported by Ripollés et al.23 with several modifications. Briefly, 12 mL of urine was added to a solution containing d6-NDMA as an internal standard. The urine mixture was added to a Supelclean coconut charcoal SPE tube (2 g/6 mL, SigmaAldrich) preconditioned with dichloromethane, methanol, and deionized water. The analyte was then eluted with 12 mL of dichloromethane. The eluate was concentrated under gentle nitrogen flow to 100 μL and was analyzed by LC-MS/MS. A representative LCMS/MS chromatogram of NDMA in urine is provided in Supporting Information, Figure S4. Statistical Methods. Data were analyzed using the SPSS statistical package software (SPSS, version 16.0). The Spearman correlation coefficient was used to estimate the relationship among the urinary biomarkers measured in this study. A multiple linear regression model was used to examine the relationship among the urinary biomarkers after adjusting for other variables (i.e., age, BMI, and smoking status).

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RESULTS Chromatography and Mass Spectra of 3-NTYR and NHPA. The product ion spectra of 3-NTYR, NHPA and their corresponding stable isotopically labeled internal standards are shown in Figure 1. The spectra were recorded by selecting the deprotonated precursor ion ([M−H]−) in the first quadrupole 999


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Chemical Research in Toxicology

Figure 1. Product ion spectra (negative mode) of 3-NTYR (A), d3-3-NTYR (B), NHPA (C), and d3-NHPA (D). cps, counts per second.

chromatogram from a calibration standard is shown in Supporting Information, Figure S6. The retention times for 3-NTYR and NHPA were 13.5 and 26.6 min, respectively. Method Validation. The limit of quantification (LOQ) was estimated using the quantification transition as the concentration that provided a signal-to-noise (S/N) ratio of 10 from the sample chromatograms at the lowest validation level tested. Because 3-NTYR and NHPA are typically present in body fluids, there was no blank matrix available. In the present study, LOQ values were estimated from quantified levels present in nonspiked samples.26 Using the present method, the LOQs in urine were determined as 3.1 and 2.5 pg/mL (9.2 pg and 7.5 pg in 3 mL urine) for 3-NTYR and NHPA, respectively. The method’s limits of detection (LODs), defined as the lowest concentration in neat solution that gave an S/N ratio of at least 3, were 0.67 and 0.33 pg/mL (2.0 and 1.0 pg in 3 mL neat solution) for 3-NTYR and NHPA, respectively. Two linear calibration curves covering the low concentration range (0.03−0.5 ng) and the high concentration range (0.5−8 ng) were obtained by serial dilution of the calibration standards with deionized water. Each calibration standard contained 5 ng

(Q1). After collisional activation of the selected ions in the collision cell, the product ion spectra were recorded by scanning the last quadrupole (Q3). For 3-NTYR, the fragmentation pattern showed that the [M−H]− precursor ion (m/z 225) produced two major product ions [M−H− CH2O3]− (m/z 163, used as the quantifier ion) and [M−H− C2H3NO3]− (m/z 136, used as the qualifier ion), as shown in Figure 1A. The internal standard d3-3-NTYR showed the same fragmentation pattern as that of 3-NTYR (Figure 1B). For NHPA, the [M−H]− precursor ion of NHPA was at m/z 196, which was fragmented to m/z 122 (quantifier ion) and m/z 135 (qualifier ion), resulting from the loss of CNO3 and CHO3, respectively (Figure 1C). A precursor ion at m/z 199 and a product ion at m/z 125 characterized the d3-NHPA (Figure 1D). The transitions obtained for the ESI/MS-MS analysis of 3NTYR and NHPA were in agreement with those previously reported.5,24,25 The proposed fragmentation patterns of the precursors 3-NTYR and NHPA are illustrated in Supporting Information, Figure S5. A representative online SPE LC-MS/ MS chromatogram for the urine analysis of a healthy subject is shown in Figure 2, while an online SPE LC-MS/MS 1000


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Figure 2. Chromatograms of 3-NTYR and NHPA in human urine, as measured by LC-MS/MS coupled with online SPE. Multiple reaction monitoring transitions of m/z 225 → 163 (A) and m/z 225 → 136 (B) for 3-NTYR and m/z 228 → 166 (C) for d3-3-NTYR; m/z 196 → 122 (D) and m/z 196 → 135 (E) for NHPA; and m/z 199 → 125 (F) for d3-NHPA.

Table 3. Method Precision in Human Urine for 3-NTYR and NHPA precisiona mean ± SD (pg/mL) intraday urine 1

urine 2

urine 3

urine 1

urine 2

urine 3

3-NTYR

16.1 ± 0.55 (3.4)b 31.4 ± 1.31 (4.2)

48.3 ± 1.13 (2.3) 48.8 ± 0.76 (1.6)

98.4 ± 2.41 (2.5) 169.5 ± 4.54 (2.7)

16.9 ± 1.42 (8.4) 35.9 ± 0.61 (1.7)

50.4 ± 2.34 (4.7) 51.0 ± 1.95 (3.8)

100.8 ± 3.97 (3.9) 168.1 ± 3.24 (1.9)

NHPA a

interday

compound

Each urine sample was analyzed 6 times in the intraday and interday tests. bCV, %.

each of d3-3-NTYR and d3-NHPA. Linear regressions were calculated without weighting and without forcing the regression through zero, and the following linear equations were obtained: y = 0.0431x + 0.0003 (r2 = 0.9998, low range) and y = 0.0527x − 0.0027 (r2 = 0.9967, high range) for 3-NTYR; y = 0.0644x + 0.0009 (r2 = 0.9991, low range) and y = 0.0749x − 0.0037 (r2 = 0.9945, high range) for NHPA. The correlation coefficients (r2) obtained were >0.99 in all of the cases. For all of the calibration standards, the relative error of the back-calculated concentrations from the nominal concentrations ranged from −12.8% to 3.3% for 3-NTYR and from −1.7% to 8.9% for NHPA, with an imprecision (CV) < 10%. For each analyte in urine, the peak identity was also confirmed by comparing the peak area ratios (quantifier/qualifier) with those of the calibration standards. As an acceptance criterion, the ratios in the urine samples should not deviate by more than ±25% from the mean ratios in the calibration standards. The precision was determined by measuring three urine samples with low, medium, and high concentrations and repeatedly measuring the 3-NTYR and NHPA in these urine samples. The intraday and interday CVs were 2.3−3.4% and 3.9−8.4% for 3-NTYR, respectively, while they were 1.6−4.2% and 1.7−3.8% for NHPA, respectively, as shown in Table 3. The relative recovery of 3-NTYR and NHPA in urine was estimated by the addition of an unlabeled 3-NTYR/NHPA standard mixture at three different concentrations to a pooled

crude urine sample. The relative recovery was calculated from the increase in the measured concentration after the addition of 3-NTYR and NHPA divided by the concentration that was added. The mean relative recoveries were 89−98% and 90− 98% for 3-NTYR and NHPA, respectively (Table 4). The absolute recoveries for the manual SPE and HPLC fractionation were estimated by comparing the peak areas of the internal standards (5 ng each of d3-3-NTYR and d3-NHPA) spiked before pretreatment with the peak areas of the internal standards spiked after the pretreatment but prior to online SPE LC-MS/MS measurement. The obtained mean absolute recoveries of 3-NTYR and NHPA were 82% and 72%, respectively. Matrix effects were calculated from the peak areas of the internal standards added to the neat solution and compared with the peak areas of the internal standards added to each urinary sample after the pretreatment but prior to online SPE LC-MS/MS measurement. In this study, the mean matrix effects for 3-NTYR and NHPA were 12% and 45%, respectively. Urinary Excretion of 3-NTYR, NHPA, 8-OxoGua, 8OxodGuo, N7-MeG, N3-MeA, and NDMA Concentrations in Healthy Adults. The characteristics of 65 participants and the urinary concentrations of 3-NTYR, NHPA, 8-oxoGua, 8oxodGuo, N7-MeG, N3-MeA, and NDMA are summarized in Table 5. The mean age and BMI values were 33.7 ± 10.2 and 1001


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23.9 ± 5.5, respectively. The mean urinary concentrations of 3NTYR and NHPA were 63.2 ± 51.5 and 77.4 ± 60.8 pg/mL, respectively, while the mean urinary concentrations of 8oxoGua and 8-oxodGuo in healthy subjects were 18.3 ± 11.2 and 3.87 ± 2.87 ng/mL, respectively. The mean urinary levels of N7-MeG and N3-MeA were 6016 ± 3049 and 6.94 ± 14.6 ng/mL, respectively. The mean urinary NDMA concentration was 0.75 ± 1.01 ng/mL. Correlations among 3-NTYR, NHPA, Oxidative/Methylated DNA Lesions, and NDMA. Possible correlations among the concentrations of 3-NTYR, NHPA, 8-oxoGua, 8-oxodGuo, N7-MeG, N3-MeA, and NDMA in the urine of healthy subjects were investigated using the Spearman correlation coefficient, and the results are shown in Table 6. The urinary 3-NTYR concentrations were individually positively associated with urinary NHPA (r = 0.411; P = 0.001), 8-oxoGua (r = 0.301; P = 0.015), 8-oxodGuo (r = 0.362; P = 0.003), N7-MeG (r = 0.316; P = 0.010), and N3-MeA (r = 0.320; P = 0.009) but were not associated with NDMA (r = 0.043; P = 0.738). Similarly, urinary NHPA concentrations were highly associated with urinary 8-oxoGua (r = 0.335; P = 0.006), 8-oxodGuo (r = 0.515; P < 0.001), N7-MeG (r = 0.423; P < 0.001), and N3MeA (r = 0.376; P = 0.002) but were not associated with NDMA (r = 0.082; P = 0.526). Multiple linear regressions revealed that the above correlations between urinary 3-NTYR or NHPA and 8-oxodGuo, N7-MeG, or N3-MeA were not affected by other variables (i.e., age, BMI and smoking status, P < 0.05). Furthermore, the concentrations of urinary oxidative DNA lesions (i.e., 8-oxoGua or 8-oxodGuo) were significantly associated with methylated DNA lesions (i.e., N7-MeG or N3MeA, Table 6). Neither oxidative nor methylated DNA lesions were associated with urinary NDMA (P > 0.05).

Table 4. Relative Recoveries in Human Urine for 3-NTYR and NHPA concn (pg/mL) measured compd

spiked

(mean ± SD, n = 3)

3-NTYR

0 25 50 100 0 42 84 168

47.3 ± 1.78 71.2 ± 3.65 87.7 ± 2.03 133.3 ± 7.62 72.6 ± 1.73 109.2 ± 1.29 148.7 ± 2.73 235.7 ± 2.79

NHPA

recoverya % 98 ± 7.9b 89 ± 7.3 93 ± 5.6 90 ± 1.8 94 ± 2.7 98 ± 0.7

a

Recovery of the analytes in urine was estimated by the addition of an unlabeled 3-NTYR/NHPA standard mixture at 3 different concentrations to a pooled sample. The recovery was estimated from the increase in the measured concentration after the addition of the analyte divided by the concentration that was added. bMean relative recovery.

Table 5. Overall Characteristics of the Study Participants (n = 65) variables age (years) BMI (kg/m2) 3-NTYR (pg/mL) (pg/mg creatinine) NHPA (pg/mL) (pg/mg creatinine) 8-oxoGua (ng/mL) (ng/mg creatinine) 8-oxodGuo (ng/mL) (ng/mg creatinine) N7-MeG (ng/mL) (ng/mg creatinine) N3-MeA (ng/mL) (ng/mg creatinine) NDMA (ng/mL) (ng/mg creatinine)

mean ± SD

range

33.7 ± 10.2 23.9 ± 5.5 63.2 ± 51.5 (51.0 ± 37.6) 77.4 ± 60.8 (67.5 ± 50.6) 18.3 ± 11.2 (14.3 ± 4.89) 3.87 ± 2.87 (2.90 ± 1.19) 6016 ± 3049 (4736 ± 985) 6.94 ± 14.6 (5.61 ± 9.19) 0.75 ± 1.01 (0.72 ± 0.78)

21−59 17.3−34.1 NDa,b − 291 (ND − 219) NDc − 255 (ND − 238) 1.99−69.8 (6.06−33.3) 0.38−17.1 (0.89−8.16) 1122−13,702 (3292−8723) 0.49−101 (0.45−53.6) NDd − 7.86 (ND − 4.67)

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DISCUSSION In this study, we developed a selective and sensitive LC-MS/ MS method for the simultaneous determination of urinary 3NTYR and NHPA in humans. Because of the use of an efficient pretreatment and online SPE, this method had low LOQ values of 9.2 pg and 7.5 pg for 3-NTYR and NHPA, respectively (LOD values: 2.0 pg for 3-NTYR and 1.0 pg for NHPA). Previously, Du et al.27 developed an LC-UV method that involved a derivatization step and HPLC purification to determine 3-NTYR in urine that had an LOD of 68 pg. Kato et al.28 developed an LC-MS/MS method to determine urinary 3-NTYR involving an off-line SPE cleanup and derivatization procedure and reported an LOD of 23 pg, while Radabaugh et

a

ND, not detected. bSeven of 65 (10.8%) urine samples had nondetectable levels of 3-NTYR. cOne of 65 (1.5%) urine samples had a nondetectable level of NHPA. dOne of 65 (1.5%) urine samples had a nondetectable level of NDMA.

Table 6. Correlation among Urinary Biomarkers Measured in Subjects (n = 65)

NHPA 8-oxoGua 8-oxodGuo N7-MeG N3-MeA NDMA

3-NTYR

NHPA

8-oxoGua

8-oxodGuo

N7-MeG

N3-MeA

r = 0.411 P = 0.001 r = 0.301 P = 0.015 r = 0.362 P = 0.003 r = 0.316 P = 0.010 r = 0.320 P = 0.009 r = 0.043 P = 0.738

r = 0.335 P = 0.006 r = 0.515 P < 0.001 r = 0.423 P < 0.001 r = 0.376 P = 0.002 r = 0.082 P = 0.526

r = 0.773 P < 0.001 r = 0.807 P < 0.001 r = 0.398 P = 0.001 r = −0.009 P = 0.942

r = 0.858 P < 0.001 r = 0.435 P < 0.001 r = 0.137 P = 0.289

r = 0.395 P = 0.001 r = 0.192 P = 0.135

r = 0.133 P = 0.302

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Table 7. Urinary Concentrations of Free 3-NTYR and NHPA for Healthy Subjects in the Literature (Mean ± SD, pg/mg Creatinine) urinary concentration methods

pretreatment

sample size

3-NTYR

GC-FID GC-MS/MS LC-MS/MS LC-MS/MS GC-TEA GC-MS/MS online SPE LC-MS/MS

manual SPE/derivatization HPLC fractionation/derivatization manual SPE/derivatization manual SPE by immunoaffinity column purification liquid−liquid extraction/HPLC fractionation/derivatization derivatization manual SPE/HPLC fractionation

10 10 23 18 9 20 65

37,300 ± 2270a 920 ± 980 2800 ± 800 31−367c

51.0 ± 37.6

NHPA

ref

2267 ± 1400 3940d 67.5 ± 50.6

Schwemmer et al.31 Tsikas et al.32 Kato et al.28 Radabaugh et al.24 Ohshima et al.9 Keimer et al.33 this study

b

a

The value was obtained by adjusting the urinary creatinine by 1.5 g per day for an average adult. bNot measured. cThe value was obtained by adjusting the urinary creatinine by 1.6 g per liter for an average adult; the values were determined from a group including normal and osteoarthritic subjects. dMedian.

al. 24 also described an LC-MS/MS method following immunoaffinity column purification to quantify urinary 3NTYR that had an LOD of 9.9 pg. With regard to urinary NHPA quantification, Ohshima et al.9 described a GC-TEA method involving an ethyl acetate extraction and HPLC purification with an LOD of 3 pg. Our developed online SPE LC-MS/MS method was more sensitive than the mentioned methods. To the best of our knowledge, this is the first report describing an assay that simultaneously determines the levels of 3-NTYR and NHPA in urine, which could be a useful tool in biomedical research to evaluate the whole-body burden of nitrative stress. As shown in Figure 2, the deuterated internal standards eluted slightly prior to the analytes, which could be attributed to the altered hydrophilic nature of the internal standards labeled with three deuterium atoms; this is known as the “deuterium isotope effect” during reversed-phase LC separation. A previous study29 reported that substantial isotope effects in the ionization efficacy could be observed with LC-MS/MS and that hydrogen−deuterium exchange of deuterated internal standard compounds could occur during the ionization process. In our method, the urine samples were purified with manual SPE, preparative HPLC, and an additional online SPE system. After three purification steps, the interferences were largely eliminated; thus, any isotope effects during the ionization process were small and could be neglected. This was evidenced by satisfactory relative recoveries obtained in this study (89− 98% for 3-NTYR and 90−98% for NHPA, Table 4). Additionally, we confirmed that no hydrogen−deuterium exchange occurred in our study by infusing the pure d3-3NTYR and d3-NPHPA internal standards into ESI-MS/MS. No signals of the precursor ions of (H3)-3-NTYR [or (DH2)-3NTYR or (D2H)-3-NTYR] or (H3)-NHPA [or (DH2)-NHPA or (D2H)-NHPA] were observed. A possible methodological problem encountered during the determination of 3-NTYR and NHPA involves the artifactual formation of 3-NTYR and NHPA from endogenous tyrosine (e.g., L-tyrosine), PHPA, and nitrite/nitrate, which are naturally and abundantly present in human urine.5,10,30 Sample pretreatments, such as acid hydrolysis of proteins or sample derivatization reactions under acidic conditions, have been reported to induce the artifactual nitration of L-tyrosine,5 which could lead to an overestimation of basal levels of 3-NTYR and NHPA. In our study, the potential for artifact generation during manual SPE was evaluated, and the results showed that no apparent artifactual nitration occurred during our pretreatment (see Supporting Information, Table S2). This observation

stemmed from the fact that the nitrite/nitrate is washed away during the washing step of the manual SPE procedure (data not shown). However, the artifact test revealed that PHPA is more prone to nitrative damage than L-tyrosine in the solution containing nitrite/nitrate. The basal urinary levels of 3-NTYR and NHPA measured in this study were 63.2 ± 51.5 pg/mL (51.0 ± 37.6 pg/mg creatinine) and 77.4 ± 60.8 pg/mL (67.5 ± 50.6 pg/mg creatinine), respectively, which were substantially lower than those obtained by GC-FID, GC-TEA, GC-MS/MS, and LCMS/MS combined with derivatization (920−37,300 pg/mg creatinine for 3-NTYR and 2267−3940 pg/mg creatinine for NHPA; Table 7).9,28,31−33 These previously reported values were at a minimum 18 times and 34 times higher for 3-NTYR and NHPA, respectively, than our measured levels. A possible explanation for these results could be that the previous measurements listed involved derivatization reactions that were performed in acidic media. These processes can induce artifactual nitration of endogenous tyrosine and PHPA in the presence of nitrite/nitrate, which would further overestimate the urinary concentrations of 3-NTYR and NHPA. Conversely, our newly developed online SPE LC-MS/MS method following manual SPE and HPLC fractionation without derivatization avoided the artifactual formation of 3-NTYR and NHPA. In support of this statement, our basal urinary 3-NTYR concentrations were comparable to previous measurements obtained by the LC-MS/MS method following an immunoaffinity column purification without derivatization (31−367 pg/ mg creatinine, Table 7).24 In addition to the artifacts that were generated during the derivatization reaction, the wide range of concentrations reported in published studies could also be explained by several reasons. First, incomplete 3-NTYR isolation from tyrosine could occur during pretreatment. For example, some studies28,31 have included only manual SPE to separate 3NTYR from tyrosine prior to derivatization. However, residual tyrosine always remains following manual SPE treatment.34 Therefore, the chromatographic isolation of 3-NTYR from tyrosine, nitrite, and nitrate using HPLC is recommended.5 Second, the quality of the previously reported analytical data is not known, as highlighted in the recent review by Tsikas and Duncan.5 They noted that method validation in previous studies9,28,31,33 was seriously compromised or altogether missing (e.g., specificity, linearity, accuracy, precision, detection limit, and quantitation limit). Poorly validated methods may lead to erroneous findings reported in the literature; therefore, significant discrepancies in basal levels of 3-NTYR and NHPA 1003


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Figure 3. Hypothetical association among nitratively damaged protein and oxidative and methylated DNA lesions.

through the reaction with ROS that are produced within cells, which mediates nitrative and oxidative damage to cellular components (e.g., 3-NTYR/NHPA and 8-oxodGuo).35,36 Meanwhile, RNS can react with secondary amines/amides (e.g., dimethylamine and N-methylurea) to form methylating NOCs,15,37 which can further induce methylated DNA lesions (e.g., N7-MeG and N3-MeA). NDMA is a known methylating NOC that is formed by the nitrosation of dimethylamine with N2O3 and is also a wellknown carcinogen. The metabolic transformation of NDMA generates reactive electrophilic metabolites (e.g., the methanediazonium ion) that can cause methylated DNA lesions (e.g., N7-MeG or N3-MeA).38,39 However, in this study, we found that the NDMA concentrations were not correlated with either the 3-NTYR (or NHPA) or N7-MeG (or N3-MeA) concentrations. The lack of correlation between NDMA and N7-MeG (or N3-MeA) could be because NDMA is not the only compound among the NOCs that can cause methylated DNA lesions. Other NOCs, such as N-nitrosamides (e.g., MNU), which are derived from the nitrosation (reaction with nitrite) of amides,15 are also known to produce a number of methylated DNA lesions.40 Moreover, the sources of preformed NDMA from dietary41 and unidentified endogenous sources may have interfered with the correlation between NDMA and 3-NTYR (or NHPA) and methylated DNA lesions.

could be expected. Finally, other confounding factors (e.g., antioxidants and lifestyle) were not always specified or considered in the literature for the study subjects, which may have interfered with the results. In this study, urinary samples were initially concentrated 10fold by manual SPE; however, 3-NTYR and NHPA could not be detected in 10-fold concentrated urine in all of the cases (see Supporting Information, Figure S7). This inability was likely due to their low concentrations in urine and because severe matrix interferences/effects were not efficiently removed by the manual SPE process. When the urine samples were purified/ enriched 30-times by manual SPE and HPLC fractionation, ∼90% of the urinary samples displayed detectable levels of 3NTYR and NHPA (Table 5), suggesting that our newly developed online SPE coupled with the isotope dilution LCMS/MS method following manual SPE and HPLC purification could effectively overcome the matrix interferences for the successful quantification of 3-NTYR and NHPA in urine. This study further showed that, in human urine, nitrated protein products (i.e., 3-NTYR and NHPA) are highly correlated with oxidative (i.e., 8-oxoGua and 8-oxodGuo) and methylated DNA lesions (i.e., N7-MeG and N3-MeA) (Table 6). On the basis of a review of the relevant literature and our findings, a possible hypothetical mechanism was proposed and is shown in Figure 3. When inflammation or nitrative stress develops in the body, RNS (e.g., ONOO−) are generated 1004


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(4) Thanan, R., Pairojkul, C., Pinlaor, S., Khuntikeo, N., Wongkham, C., Sripa, B., Ma, N., Vaeteewoottacharn, K., Furukawa, A., Kobayashi, H., Hiraku, Y., Oikawa, S., Kawanishi, S., Yongvanit, P., and Murata, M. (2013) Inflammation-related DNA damage and expression of CD133 and Oct3/4 in cholangiocarcinoma patients with poor prognosis. Free Radical Biol. Med. 65, 1464−1472. (5) Tsikas, D., and Duncan, M. W. (2014) Mass spectrometry and 3nitrotyrosine: strategies, controversies, and our current perspective. Mass Spectrom. Rev. 33, 237−276. (6) Pacher, P., Beckman, J. S., and Liaudet, L. (2007) Nitric oxide and peroxynitrite in health and disease. Physiol. Rev. 87, 315−424. (7) Yeo, W. S., Kim, Y. J., Kabir, M. H., Kang, J. W., and Kim, K. P. (2015) Mass spectrometric analysis of protein tyrosine nitration in aging and neurodegenerative disease. Mass Spectrom. Rev. 34, 166− 183. (8) Chen, H. J., and Chiu, W. L. (2008) Simultaneous detection and quantification of 3-nitrotyrosine and 3-bromotyrosine in human urine by stable isotope dilution liquid chromatography tandem mass spectrometry. Toxicol. Lett. 181, 31−39. (9) Ohshima, H., Friesen, M., Brouet, I., and Bartsch, H. (1990) Nitrotyrosine as a new marker for endogenous nitrosation and nitration of proteins. Food Chem. Toxicol. 28, 647−652. (10) Mani, A. R., Pannala, A. S., Orie, N. N., Ollosson, R., Harry, D., Rice-Evans, C. A., and Moore, K. P. (2003) Nitration of endogenous para-hydroxyphenylacetic acid and the metabolism of nitrotyrosine. Biochem. J. 374, 521−527. (11) Yu, H., Venkatarangan, L., Wishnok, J. S., and Tannenbaum, S. R. (2005) Quantitation of four guanine oxidation products from reaction of DNA with varying doses of peroxynitrite. Chem. Res. Toxicol. 18, 1849−1857. (12) Fraga, C. G., Shigenaga, M. K., Park, J. W., Degan, P., and Ames, B. N. (1990) Oxidative damage to DNA during aging: 8-hydroxy-2′deoxyguanosine in rat organ DNA and urine. Proc. Natl. Acad. Sci. U.S.A. 87, 4533−4537. (13) Hu, C. W., Cooke, M. S., Tsai, Y. H., and Chao, M. R. (2014) 8Oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine concentrations in various human body fluids: implications for their measurement and interpretation. Arch. Toxicol. 89, 201−210. (14) Evans, M. D., Saparbaev, M., and Cooke, M. S. (2010) DNA repair and the origins of urinary oxidized 2′-deoxyribonucleosides. Mutagenesis 25, 433−442. (15) Dietrich, M., Block, G., Pogoda, J. M., Buffler, P., Hecht, S., and Preston-Martin, S. (2005) A review: dietary and endogenously formed N-nitroso compounds and risk of childhood brain tumors. Cancer Causes Control 16, 619−635. (16) Monti, P., Traverso, I., Casolari, L., Menichini, P., Inga, A., Ottaggio, L., Russo, D., Iyer, P., Gold, B., and Fronza, G. (2010) Mutagenicity of N3-methyladenine: a multi-translesion polymerase affair. Mutat. Res. 683, 50−56. (17) Shrivastav, N., Li, D., and Essigmann, J. M. (2010) Chemical biology of mutagenesis and DNA repair: cellular responses to DNA alkylation. Carcinogenesis 31, 59−70. (18) Drablos, F., Feyzi, E., Aas, P. A., Vaagbo, C. B., Kavli, B., Bratlie, M. S., Pena-Diaz, J., Otterlei, M., Slupphaug, G., and Krokan, H. E. (2004) Alkylation damage in DNA and RNA–repair mechanisms and medical significance. DNA Repair 3, 1389−1407. (19) Hu, C. W., Chao, M. R., and Sie, C. H. (2010) Urinary analysis of 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine by isotope-dilution LC-MS/MS with automated solid-phase extraction: Study of 8-oxo-7,8-dihydroguanine stability. Free Radical Biol. Med. 48, 89−97. (20) Hu, C. W., Lin, B. H., and Chao, M. R. (2011) Quantitative determination of urinary N3-methyladenine by isotope-dilution LC− MS/MS with automated solid-phase extraction. Int. J. Mass Spectrom. 304, 68−73. (21) Chao, M. R., Wang, C. J., Yang, H. H., Chang, L. W., and Hu, C. W. (2005) Rapid and sensitive quantification of urinary N7methylguanine by isotope-dilution liquid chromatography/electro-

In conclusion, we reported a sensitive and reliable LC-MS/ MS method for the simultaneous quantification of urinary 3NTYR and NHPA. This method exhibited low LOQs, which enabled the detection of 3-NTYR and NHPA in urine. Because the concentrations of 3-NTYR are highly correlated with NHPA in urine, both of the products should be considered good biomarkers of nitrative stress in humans, although the higher NHPA sensitivity in urine is an additional advantage for its accurate measurement. More importantly, this study first demonstrated in human urine that nitrated protein products (i.e., 3-NTYR and NHPA) are highly correlated to oxidative (i.e., 8-oxoGua and 8-oxodGuo) and methylated DNA lesions (i.e., N7-MeG and N3-MeA). Our findings may help to elucidate the interactions between RNS and ROS.

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ASSOCIATED CONTENT

S Supporting Information *

HPLC gradient used for the 3-NTYR and NHPA fractionation of the urine extracts, potential artifactual formation of 3-NTYR and NHPA during manual SPE purification, LC-MS/MS chromatograms of 8-oxoGua, 8-oxodGuo, N7-MeG, N3-MeA, and NDMA in urine, fragmentation patterns of the precursors 3-NTYR and NHPA, and LC-MS/MS chromatograms of 3NTYR/NHPA in neat solution and in a 10-fold concentrated urine sample. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Fax: +886 4 23248179. E-mail: [email protected] or [email protected]. Author Contributions ⊥

M.-R.C. and Y.-W.H. contributed equally to this work.

Funding

We thank the National Science Council, Taiwan (Grants NSC 102-2314-B-040-016-MY3 and NSC 102-2632-B-040-001MY3) for financial support. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS 3-NTYR, 3-nitrotyrosine; 8-oxodGuo, 8-oxo-7,8-dihydro-2′deoxyguanosine; 8-oxoGua, 8-oxo-7,8-dihydroguanine; BER, base excision repair; BMI, body mass index; ESI, electrospray ionization; LC-MS/MS, liquid chromatography−tandem mass spectrometry; LOD, limit of detection; LOQ, limit of quantification; N3-MeA, N3-methyladenine; N7-MeG, N7methylguanine; NDMA, N-nitrosodimethylamine; NHPA, 3nitro-4-hydroxyphenylacetic acid; NOCs, N-nitroso compounds; PHPA, para-hydroxyphenylacetic acid; RNS, reactive nitrogen species; ROS, reactive oxygen species; and SPE, solidphase extraction

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REFERENCES

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