MS

Journal of Chromatography B, 1029 (2016) 213–221

Contents lists available at ScienceDirect

Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Simultaneous assessment of endogenous thiol compounds by LC–MS/MS Yao Sun a,1 , Tong Yao a,1 , Xiucai Guo a , Ying Peng (PhD) a,∗∗ , Jiang Zheng (PhD) b,c,∗ a

School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, PR China Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, PR China c Center for Developmental Therapeutics, Seattle Children’s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington School of Medicine, Seattle, WA 98101, United States b

a r t i c l e

i n f o

Article history: Received 27 May 2016 Received in revised form 14 June 2016 Accepted 15 June 2016 Available online 12 July 2016 Keywords: LC–MS/MS Simultaneous assessment Thiol compounds

a b s t r a c t Biological thiol compounds are very important molecules that participate in various physiological events. Alteration in levels of endogenous thiols has been suggested as a biomarker of early stage of pathological changes. We reported a chemical derivatization- and LC–MS/MS-based approach to simultaneously determine thiol compounds including glutathione (GSH), cysteine (Cys), N-acetyl cysteine (NAC), homocysteine (Hcy), and cysteinylglycine (CysGly) in biological samples. Thiol-containing samples were derivatized with monobromobimane (mBrB) at room temperature, followed by LC–MS/MS analysis. Assessment of the analytes with baseline separation was completed within 10 min, using a gradient elution on a C18 reversed-phase column. Excellent linearity was observed for all analytes over their concentration ranges of 1–400 ␮M. The lowest limits of detection (S/N = 3) in a range from 0.31 fmol (for NAC) to 4.98 fmol (for CysGly) were achieved. The results indicate that this approach was sensitive, selective, and well suited for high-throughput quantitative determination of the biological thiols. © 2016 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: BSA, bovine serum albumin; BSO, l-buthionine sulfoximine; CE, collision energy; CV, coefficient of variation; CXP, collision cell exit potential; Cys, cysteine; Cys-mBrB, mBrB-derivatized Cys conjugate; CysGly, cysteinylglycine; CysGly-mBrB, mBrB-derivatized CysGly conjugate; DP, declustering potential; Em, emission wavelength; Ex, excitation wavelength; HPLC, high performance liquid chromatography; IS, internal standard; LC–MS/MS, liquid chromatography tandem mass spectrometry; FL, fluorometric; GSH, glutathione; GSH-mBrB, mBrBderivatized GSH conjugate; Hcy, homocysteine; Hcy-mBrB, mBrB-derivatized Hcy conjugate; LOD, limit of detection; mBrB, monobromobimane; MRM, multiplereaction monitoring; MS, mass spectrometry; NAC, N-acetyl cysteine; NAC-mBrB, mBrB-derivatized NAC conjugate; QC, quality control; S/N, signal-to-noise ratio; TEM, temperature. ∗ Corresponding author at: Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, PR China; Center for Developmental Therapeutics, Seattle Children’s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington School of Medicine, Seattle, WA 98101, United States. ∗ ∗ Corresponding author at: School of Pharmacy, Shenyang Pharmaceutical University, PO Box 50, 103 Wenhua Road, Shenyang 110016, PR China. E-mail addresses: [email protected] (Y. Peng), [email protected] (J. Zheng). 1 These authors contributed equally. http://dx.doi.org/10.1016/j.jchromb.2016.06.024 1570-0232/© 2016 Elsevier B.V. All rights reserved.

Thiols are chemically and biochemically active components of the sulfur cycle of the natural environment. Low molecular-mass thiols, such as glutathione (GSH), cysteine (Cys), N-acetylcysteine (NAC), homocysteine (Hcy), and cysteinylglycine (CysGly) are critical cellular components that play numerous roles in metabolism and homeostasis, and are important in a variety of physiological and pathological processes [1–4]. For instance, GSH is the main antioxidant and nucleophile with crucial roles as a scavenger of toxic free radicals and electrophilic species in detoxification of xenobiotics [5–7]. GSH content is thought to contribute to the development of many common diseases including cancer [8], heart attack, stroke [9], alcoholic liver diseases [10], and Parkinson’s disease [11]. Cys is one of the twenty amino acids used for protein biosynthesis. Cys deficiency is involved in hematopoiesis decrease, leukocyte loss, skin lesions, and weakness [12–14]. Elevated levels of Cys have been associated with neurotoxicity [15,16]. Sulfhydryl group is nearly the most reactive chemical functionality in organism and has poor stability. Thus, a simple and rapid approach for accurate and interference-free measurement of thiol compounds in biological samples is of great biological, clinical, and pharmacological importance. As the consequence, a wide variety

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Y. Sun et al. / J. Chromatogr. B 1029 (2016) 213–221 Table 1 MRM parameters for determination of GSH-mBrB, Cys-mBrB, NAC-mBrB, Hcy-mBrB, CysGly-mBrB, and S-hexylglutathione.

Scheme 1. Synthesis of mBrB-thiols by reaction of thiols with monobromobimane (mBrB).

of analytical methodologies have been developed for the determination of thiols in biological samples employing various detection and separation techniques. These methods include enzymatic assay [17], high-pressure liquid chromatography (HPLC) [18–20], gas chromatograph [21], or capillary electrophoresis [22,23]. HPLC methods were developed with a variety of detection techniques, such as sample derivatization followed by UV detection [24] or fluorometric (FL) detection [18,25], electrochemical detection without sample derivatization [26], and mass spectrometry (MS) detection [27–29]. Each of these methods has their own limitations, in terms of performance, complexity, sample processing, and validation parameters assessed, which creates challenges or renders them impractical for high-throughput routine clinical or research purposes. Due to high sensitivity, relative simplicity, ease of automation, and high-throughput capability, LC–MS/MS is already becoming the most commonly employed method for determination of most of compounds that we knew. Here we reported a derivatizationand LC–MS/MS-based method for simultaneous measurement of biological thiol compounds, including GSH, Cys, NAC, Hcy, and CysGly. The derivatization of thiols was established by reaction with monobromobimane (mBrB, Scheme 1).

2. Experimental 2.1. Chemicals and materials Glutathione (GSH, ≥98%), cysteine (Cys, ≥98%), N-acetyl cysteine (NAC, ≥98%), homocysteine (Hcy, ≥95%), cysteinylglycine (CysGly, ≥85%), bovine serum albumin (BSA, >99%), l-buthionine sulfoximine (BSO, >99%), S-hexylglutathione (>97%), and monobromobimane (mBrB, >97%) were obtained from Sigma-Aldrich (St. Louis, MO). Formic acid and acetonitrile were from Fisher Scientific (Springfield, NJ). All reagents and solvents were of either analytical or HPLC grade.

Name

m/z

EP

DP

CE

CXP

GSH-mBrB Cys-mBrB NAC-mBrB Hcy-mBrB CysGly-mBrB S-Hexylglutathione

498.1 → 435.1 312.3 → 225.3 354.1 → 225.1 326.0 → 225.2 369.2 → 192.1 392.2 → 246.3

10 10 10 10 10 10

98 150 130 208 160 86

35 25 25 25 27 24

13 13 13 13 13 13

2.3. LC/MS/MS instrumentation LC/MS/MS analyses were carried out using an AB SCIEX Triple QuadTM 5500 mass spectrometer (Applied Biosystems, Foster City, CA, USA) interfaced online with an Agilent 1260 series HPLC system, consisting of two pumps, a degasser, and an auto-sampler (Agilent Technologies, Biblingen, Germany). Data were analyzed using Applied Biosystems/SCIEX AnalystTM software (versions 1.6.1). The chromatographic separation was performed on an Agilent ZORBAX SB-C18 (5.0 ␮m, 150 × 4.6 mm) at 25 ◦ C, using 0.1% formic acid in acetonitrile and double-distilled water as mobile phases A and B, respectively. A programmed mobile phase gradient was used for separation of the analytes. The mobile phase consisted of acetonitrile with 0.1% formic acid (A) and 0.1% formic acid in water (B) with a gradient elution of 10% A at 0–1 min, 10–90% A at 1–7 min, 90–10% A at 7–8 min, and 10% A at 8–10 min. Flow rate was 1.0 mL/min. Nitrogen was used as nebulizing, cone, desolvation gas, and collision gas. Samples were analyzed by multiple-reaction monitoring (MRM) scanning in positive ion mode. The optimized MS instrument parameters obtained after tuning were as follows: ion spray voltage (IS) was set at 5500 V, and ion source temperature (TEM) was at 650 ◦ C. Curtain gas, gas 1, and gas 2 were set to 35, 50, and 50 psi, respectively. Thereafter, optimization of MS/MS conditions was performed for the thiol conjugate and internal standard analyses, by infusing the individual solutions into the electrospray source. The parameters were optimized in MRM mode in order to achieve the highest sensitivity possible. The characteristics of ion pairs (corresponding to declustering potential DP, collision energy CE, and collision cell exit potential CXP) for thiol-mBrB conjugate and internal standard analyses is listed in Table 1. 2.4. LC/Fluorescence analysis The corresponding thiol-mBrB conjugates were also analyzed by an Agilent 1260 HPLC system equipped with a fluorescence (FL) detector for comparison. The same column and eluting system were applied for the separation of the analytes. The fluorescence detector was employed for the detection of the analytes, and wavelength was set at Ex: 380 nm/Em: 470 nm.

2.2. Derivatization of thiols 2.5. Method validation Aliquots of thiol solutions or thiol-containing biological samples (30 ␮L) were placed in Eppendorf vials containing 75 ␮L of phosphate-buffered saline (PBS, pH 7.4) and 30 ␮L of mBrB solution (1.5 mg/mL in acetonitrile). The derivatization reaction was conducted at room temperature in the dark for 20 min to produce the corresponding mBrB-derived thiol conjugates (GSH-mBrB, Cys-mBrB, NAC-mBrB, Hcy-mBrB, and CysGly-mBrB), followed by addition of 150 ␮L 10% 5-sulfosalicylic acid to precipitate proteins (biological samples). The resulting samples were spiked with 15 ␮L of S-hexylglutathione dissolved in water (0.15 ␮M, internal standard). The resulting mixtures were centrifuged at 16,000g for 10 min, and 5.0 ␮L of the resulting supernatant was injected into a liquid chromatography–tandem mass spectrometry (LC/MS/MS) system for analysis.

In order to minimize experimental errors resulting from autooxidation, the thiol stock solutions were freshly prepared before use. For the establishment of a standard curve, individual thiol stock solutions were serially diluted with PBS containing BSA (5.0 mg/mL) to the final concentrations of 1.0, 2.0, 5.0, 25, 100, 200, and 400 ␮M, followed by derivatization with mBrB under the same condition as described above. Seven-point calibration curves were evaluated by plotting the absolute peak-area ratios of the analyte to the internal standard area (thiol-mBrB/IS) against the corresponding thiol concentrations. The linearity of the thiol-mBrB conjugate to IS peak area ratio versus the theoretical concentration was verified using 1/x-weighted linear regression. To assess intra-day precision and accuracy, six replicates were prepared and

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Fig. 1. MS/MS spectra of GSH-mBrB (A), Cys-mBrB (B), NAC-mBrB (C), Hcy-mBrB (D), and CysGly-mBrB (E).

Table 2 Intra- and inter-day variabilities for the assays of GSH, Cys, NAC, Hcy, and CysGly in BSA solution. -SH level

Intra-day (n = 6)

Inter-day (n = 6)

Mean (␮M)

RSD (%)

Accuracy (%)

Mean (␮M)

RSD (%)

Accuracy (%)

GSH

2 25 320

2.02 24 320

2.04 1.71 0.92

101 96.1 99.9

2.06 25.2 319

14.12 12.17 10.15

103 101 99.5

Cys

2 25 320

1.93 25.6 310

4.94 2.28 1.57

96.3 103 96.9

2.06 25.7 312

12.79 13.83 14.79

103 103 97.4

NAC

2 25 320

1.95 26.7 322

1.39 0.67 0.7

97.4 107 101

1.93 25.3 325

7.07 3.73 5.74

96.7 101 102

Hcy

2 25 320

1.96 24.9 323

2.38 1.48 2.06

98.2 99.4 101

1.94 26.8 322

13.37 12.54 10.64

97.2 107 101

CysGly

2 25 320

1.94 25.7 319

1.7 1.14 1.6

97 103 99.6

1.96 25.2 322

6 7.05 1.92

98.2 101 101

analyzed on the same day. To assess inter-day precision, samples were analyzed for three consecutive days. The precision and accuracy for intra- and inter-day analyses of the thiols were performed by quality control (QC) samples at concentrations of 2.0, 25, and 320 ␮M. Sensitivity was assessed by examining the limit of detection (LOD).

2.6. Animal handling and sample collection The investigations were performed in eight male Kunming mice (20 ± 2 g) provided by the Department of Experimental Animals of Shenyang Pharmaceutical University (Shenyang, China) and remained on standard mice chow. Environmental controls for the

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Fig. 2. Fluorescent chromatograms (Ex: 380 nm/Em: 470 nm) obtained from analysis of mBrB-derived GSH (A), Cys (B), NAC (C), Hcy (D), and CysGly (E). Thiol concentration: 1.0 ␮M; volume of injection: 5 ␮L.

animal room were set as follows: temperature at 22 ± 3 ◦ C, relative humidity at 55 ± 5%, and a 12 h light/dark cycle. The animal studies were approved by the Animal Ethics Committee of Shenyang Pharmaceutical University. The mice were fasted for 12 h prior to the study and allowed to access to water. The mice were randomly divided into two groups, and each group contained 4 mice: mice in group I were exposed to saline; mice in group II were exposed to l-buthionine sulfoximine (BSO, 300 mg/kg) prepared with saline for intraperitoneal injection.

7.4) using a homogenizer. Afterwards, the homogenates were centrifuged at 16,000g for 10 min at 4 ◦ C. The resulting supernatants were immediately treated with the mBrB solution as described above and submitted to LC/MS/MS for analysis.

2.7. Plasma sample preparation

Characterization of mBrB-derived thiol conjugates (GSH-mBrB, Cys-mBrB, NAC-mBrB, Hcy-mBrB, and CysGly-mBrB) was performed by LC–MS/MS. Products, resulting from the derivatization reactions, with protonated molecular ions [M+H]+ at m/z 498, 312, 354, 326, and 369 were observed in positive mode, respectively. The observed protonated molecular ions matched the molecular masses of the corresponding thiol-mBrB conjugates. The MS/MS spectra of the products obtained through product ions (MS/MS) scanning showed the indicative characteristic fragment ions associated with the cleavage of the thiol-mBrB conjugates (Fig. 1). For GSH-mBrB, the ion at m/z 435 was assigned to the one produced by loss of the carboxyl and H2 O moieties from GSH. For NAC-mBrB, Cys-mBrB, Hcy-mBrB, and CysGly-mBrB, the ion at m/z 225 was

Blood samples (about 0.3 mL from each mouse) were harvested by cardiac puncture 2 h after BSO injection and placed in test tubes coated with heparin. Each sample was centrifuged at 9000g for 10 min. In order to minimize experimental errors resulting from auto-oxidation, the resulting plasma samples were immediately treated with mBrB as described above. 2.8. Liver sample preparation Mouse liver tissues harvested from the above animals were finely minced and homogenized in five volumes of ice-cold PBS (pH

3. Results 3.1. Identification of derivatization products by mass spectrometry

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217

Fig. 3. Extracted ion chromatograms obtained from LC–MS/MS analysis of mBrB-derived GSH (A), Cys (B), NAC (C), Hcy (D), and CysGly (D). Thiol concentration: 1.0 ␮M; volume of injection: 5 ␮L.

corresponded to the [bimane+SH+H]+ . The product ion at m/z 192 originated from the bimane moiety of the thiol-mBrB conjugates. In order to achieve the highest sensitivity as far as possible, the ion pairs with the most intensity, i.e. m/z 498.1 → 435.1 for GSH-mBrB, m/z 354.1 → 225.1 for NAC-mBrB, m/z 312.3 → 225.3 for Cys-mBrB, m/z 326.0 → 225.2 for Hcy-mBrB, and m/z 369.2 → 192.1 for CysGlymBrB, were selected for MRM acquisitions.

3.2. Method validation 3.2.1. Sensitivity of detection The limits of detection for the thiols were calculated as the amounts of the thiol-mBrB conjugates that resulted in a peak area three times larger than those of the baseline noise. The limits of detection for GSH, Cys, NAC, Hcy, and CysGly by the approach (LC–MS/MS) developed were found to be 0.91, 1.97, 0.31, 1.08, and 4.98 fmol, respectively. Thiol-mBrB conjugates are known to show characteristic fluorescence at Ex: 380 nm/Em: 470 nm. For comparison, the sensitivity of LC-FL for the detection of the thiolmBrB conjugates was determined. Little signals responsible for the conjugates were detected by LC-FL applied with 5.0 pmol equivalence of the thiols after derivatization with mBrB (Fig. 2). The same samples with the same amount were submitted to LC–MS/MS for analysis. All five thiol-mBrB conjugates were detected by mass spectrometry, and the peak height of the analytes was found with

the intensity in a range of 2.2 × 103 –3.2 × 104 cps (Fig. 3). Furthermore, a mouse plasma sample was treated with mBrB, followed by LC–MS/MS and LC-FL analysis (Fig. 4). As expected, all the five conjugates were detected by LC–MS/MS. However, it appears that LC-FL failed to detect any conjugates derived from the five thiol compounds (Fig. 4A). Obviously, the MS approach is much more sensitive for the detection of the thiol-mBrB conjugates than the FL method. 3.2.2. Selectivity of detection Detection selectivity was evaluated by examining the interference from co-eluted components. Massive interference to the analysis of NAC-mBrB, Cys-mBrB, Hcy-mBrB, and CysGly-mBrB was found in the fluorescence-based analysis of a mouse plasma sample treated with mBrB (Fig. 4A). As expected, the analysis of the same sample by LC–MS/MS showed clean and sharp peaks without any overlapped components (Fig. 4B). 3.2.3. Linearity of measurement Seven-point calibration curves in a range of 1.0–400 ␮M of the thiols were established and evaluated under the optimized separation conditions. The linear regression equations for GSH, Cys, NAC, Hcy, and CysGly were y = 0.0212x + 0.0405 (r2 = 0.9984), y = 0.0455x + 0.1129 (r2 = 0.9986), y = 0.004x − 0.0049 (r2 = 0.9994), y = 0.0213x − 0.0495 (r2 = 0.9991), and y = 0.0326x + 0.0568

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Fig. 4. Selectivity in detection of thiol-mBrB by LC-FL and LC–MS/MS. (A) Fluorescence detection-based (Ex: 380 nm/Em: 470 nm) chromatogram obtained from analysis of mouse plasma treated with mBrB. (B) Total ion chromatograms obtained from LC–MS/MS analysis of the same sample analyzed by LC-FL. Inserted: zoomed ion chromatogram.

(r2 = 0.9988), respectively (x: thiol concentration, ␮M). Fig. 5 illustrates typical chromatograms obtained from the derivatized thiol samples, which showed two chromatographic peaks with a sharp and narrow shape without any interfering signals. The peaks with early retention time (4.46, 3.19, 5.11, 4.37, and 3.62 min) were responsible for the five thiol-mBrB conjugates, and the later one with retention time at 5.69 min responded to S-hexylglutathione (internal standard). These analytes were eluted from the column with a great reproducibility. 3.2.4. Intra- and inter-assay accuracy and precision The precision and accuracy of the method for intra- (n = 6) and inter-day (n = 6) analyses of the thiols were evaluated with designed quality control (QC) samples at low, middle, and high concentrations of 2.0, 25, and 320 ␮M. The intra- and inter-day precision and accuracy obtained are summarized in Table 2. The coefficient of variation (CV) ranged from 0.67% to 4.94% for intraday analysis and from 3.73% to 14.79% for inter-day analysis. The accuracy was in the range of 96.1–107% (intra-day) and 96.7–107% (inter-day). The stability of the thiol-mBrB conjugates was evaluated by monitoring the ratio of the thiol-mBrB conjugates over IS. No significant change in the conjugate/IS ratio was found over 15 days, when stored at room temperature. 3.3. Thiol depletion test In order to examine the practicability of the method, thiol depletion studies were performed in mice. Mice were treated (i.p.) with vehicle or l-buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The levels of the five thiols in plasma and liver were

monitored by the established approach. Decreases in plasma and hepatic GSH, Cys, and CysGly were observed in mice after 2 h treatment with BSO (Fig. 6). 4. Discussion Thiol compounds are critical physiological components and widely occur in cells, tissues, and biological fluids. These compounds serve numerous vital functions in cellular biology, biochemistry, and pharmacology. A great attention has been paid to development of analytical approaches to detect biological thiols. Thiols could be directly analyzed, but there are some problems for direct analysis of thiol compounds. For example, the sensitivity of direct analysis of thiols by mass spectrometry was low, due to poor ionization in mass spectrometry. Additionally, thiol compounds are labile to air (oxygen) and are readily oxidized to disulfides. GSH concentration was reportedly decreased by about 33% within 1 h at room temperature in whole blood [30], and the half-life of GSH is less than 2 min in blood [31]. mBrB as a derivatization reagent reacts with the −SH group of the analytes rapidly and specifically to form the corresponding thioethers that are much more stable than thiols. The irreversible derivatization reaction allows us to protect thiols from auto-oxidation during storage and to increase the reproducibility of the assessment. The mass spectra of the five analytes and IS were recorded in positive ion mode. MRM scan mode was used as the acquisition mode in order to achieve high selectivity and sensitivity. To obtain chromatograms with better resolution and appropriate retention time, various columns were tested, including Agilent ZORBAX SB-C18 (150 × 4.6 mm, 5.0 ␮m) (Agilent, Santa Clara, CA), Kinetex Hilic column (50 × 2.1 mm, 2.6 ␮m) (Phenomenex, Torrance, CA),

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219

Fig. 5. Linearity of measurement of the thiol-mBrB conjugates by LC–MS/MS. Extracted ion chromatograms obtained from LC–MS/MS analysis of GSH-mBrB (A, m/z 498.1 → 435.1), Cys-mBrB (B, m/z 312.3 → 225.3), NAC-mBrB (C, m/z 354.1 → 225.1), Hcy-mBrB (D, m/z 326.0 → 225.2), and CysGly-mBrB (E, m/z 369.2 → 192.1). Inserted: seven-point calibration curve of GSH, Cys, NAC, Hcy, and CysGly (1.0–400 ␮M) after derivatization with mBrB.

Kinetex F5 column (50 × 2.1 mm, 2.6 ␮m) (Phenomenex), and Ultimate XB-C18 column (2.1 × 100 mm, 3 mm) (Welch Scientific, Inc., Shanghai, China). The Agilent ZORBAX SB-C18 column offered the best response and resolution for the separation of the analytes (data not shown). Futhermore, various mobile phases were evaluated to optimize the analytical performances. Acetonitrile was found to achieve better resolution than methanol. When a small amount of formic acid was included in the mobile phases as a modifier, the sensitivities, retention behaviors, and peak shapes of the analytes were markedly improved. Various proportions of formic acid were tested, and the best result was achieved with a mobile phase consisting of 0.1% formic acid in acetonitrile and water, which provided satisfactory peak shapes and adequate sensitivities for all analytes. Moreover, in order to achieve a well separation of the analytes and IS in a shorter run time, a gradient elution mode was utilized. The retention times of GSH-mBrB, Cys-mBrB, NAC-mBrB, Hcy-mBrB, CysGly-mBrB, and IS were 4.46, 3.19, 5.11, 4.37, 3.62, and 5.69 min, respectively, and the total run time was 10 min. Thus, the method may be applied to high-throughput assays. In addition, simulta-

neous analysis of the thiols allows us to reduce the experimental errors and labor in comparison with one-by-one analysis. The mBrB-based thiol analysis was originally developed on the basis of the characteristic fluorescence of thiol-mBrB. The present study expanded the application of the derivatization procedure and provided an alternative to detect thiol-mBrB conjugates. Little fluorescent signal was observed in analysis of mBrB-derivatized NAC in amount of 5.0 pmol applied (Fig. 2), while the limit of detection (LOD) by LC–MS/MS-MRM was about 0.3 fmol. Clearly, mass spectrometry is much (>17,000-fold) more sensitive than fluorescence detectors for the detection of the thiols-mBrB conjugates. A number of mass spectrometry-based approaches have been documented for assessment of thiol compounds. Among them, the most sensitive one (LOD = 12 fmol) reported was achieved by derivatization of GSH with p-hydroxymercuribenzoate [32]. The potential problem with mercury is that the metal may catalyze auto-oxidation of thiols and potentially causes toxicity. To our knowledge, the approach we developed is the most sensitive for the detection of the thiols among mass spectrometry-based methods reported.

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gram for Innovative Research Team of the Ministry of Education and Program for Liaoning Innovative Research Team in University.

References

Fig. 6. Levels of GSH, Cys, NAC, Hcy, and CysGly in plasma (A) and liver (B) of mice treated with vehicle (white) and BSO (black) (mean ± SD, n = 4). * p < 0.05 and ** p < 0.01 were considered significantly different.

Selectivity is another important issue of thiol measurement. Alkylation of thiols by mBrB results in characteristic fluorescence at Ex: 380 nm/Em: 470 nm. All mBrB modified cellular thiol-containing molecules can be detected by fluorescence detectors without any discrimination, which results in a potential for unwanted background and interference. The fluorescencebased analysis showed remarkable fluorescent background over the course of chromatography in which unwanted fluorescence interferes the analysis of the thiol-mBrB conjugates (Fig. 4A). The observed poor selectivity of detection made it impossible to analyze the analytes by LC-FL. Unlike fluorescence detection, the MRM acquisition monitors a pair of ions sequentially generated, which avoids interfering ions and tremendously increases selectivity of detection by decreasing the background (Fig. 4B). A thiol depletion study was performed in mice to examine the practicability of the approach developed. The levels of the five thiols in plasma and liver were monitored before and after treatment with BSO. The results of the biological sample analyses indicate that the established method allowed us not only to simultaneously determine the thiols with a huge difference in concentration but also to monitor the changes in the levels of the analytes. In conclusion, a method based on mBrB derivatization and mass spectrometry has been developed for simultaneous assessment of GSH, CysGly, Hcy, Cys, and NAC. The derivatization process increases the reproducibility of analysis by slowing down autooxidation of the thiols. This approach has proven sensitive and selective with a potential for high-throughput analysis of biological thiols.

Acknowledgments This work was supported by 81373471 and 81430086 (in part) of the National Natural Science Foundation of China, and the pro-

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