4-Plex Chemical Labeling Strategy Based on Cinchona Alkaloid

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4-Plex Chemical Labeling Strategy Based on Cinchona Alkaloid-derived Primary Amines for the Analysis of Chiral Carboxylic Acids by Liquid Chromatography-Mass Spectrometry Jie Zheng, Shu-Jian Zheng, Chu-Bo Qi, Dong-Mei Wu, and Yu-Qi Feng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02909 • Publication Date (Web): 09 Aug 2019 Downloaded from pubs.acs.org on August 10, 2019

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Analytical Chemistry

4-Plex Chemical Labeling Strategy Based on Cinchona Alkaloidderived Primary Amines for the Analysis of Chiral Carboxylic Acids by Liquid Chromatography-Mass Spectrometry

Jie Zheng,1 Shu-Jian Zheng,1 Chu-Bo Qi,2 Dong-Mei Wu,3 Yu-Qi Feng1,*

1 Key

Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of

Chemistry, Wuhan University, Wuhan 430072, P.R. China 2 Department

3

of Pathology, Hubei Cancer Hospital, Wuhan, 430079, P.R. China

Department of Oncology, Zhongnan Hospital of Wuhan University, Wuhan 430071, PR China

* To whom correspondence should be addressed. Tel: +86-27-68755595; fax: +86-27-68755595. E-mail address: [email protected]

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ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT Chiral carboxylic acids play important roles in energy metabolism and signal transduction in human body. These enantiomers usually possess different bioactivities and are also associated with the development of some diseases. Therefore, the simultaneous determination of multiple chiral carboxylic acids is vital for the study of the pathogenesis of related diseases. However, it is still challenging to simultaneously detect the enantiomers of multiple chiral carboxylic acids in biological samples. Here, we developed a novel 4-plex chemical labeling strategy based on 4 analogues of cinchona alkaloid-derived primary amines (CAPAs) for the simultaneous determination of 16 enantiomers of 8 chiral carboxylic acids by liquid chromatography-mass spectrometry (LC-MS). To achieve high-throughput analysis, one CAPA analogue was used to label chiral carboxylic acid standards and served as internal standards (ISs); while the other 3 CAPA analogues were used to label endogenous chiral carboxylic acids in 3 different biological samples, respectively. After CAPAs labeling, the 16 chiral carboxylic acid enantiomers could be detected by LC-MS and their detection sensitivity was greatly enhanced by up to 3 orders of magnitude comparing to intact analytes. Further, the developed method for the determination of 16 chiral carboxylic acid enantiomers was validated in human serums and mammalian cells. Finally, the proposed method was applied to the determination of chiral carboxylic acids in the serum samples from Type 2 diabetes mellitus (T2DM) and colorectal cancer (CRC) patients. We found that 5 chiral carboxylic acid enantiomers in T2DM serum samples and 4 chiral carboxylic acid enantiomers in CRC serum samples exhibited significant change comparing to the healthy control (HC).

Key words: Chiral carboxylic acids; Cinchona alkaloid-derived primary amines; 4-Plex chemical labeling; Liquid chromatography-mass spectrometry; Type 2 diabetes mellitus and colorectal cancer.

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INTRODUCTION Carboxylic acids are a vital class of metabolites and widely found in various tissues and organs.1-4 The existence of chiral carbon atoms in some carboxylic acids makes them become chiral carboxylic acids accompanied with different biological activities. These carboxylic acid enantiomers enter into the designated metabolic pathways after generated by specific enzymes to participate in cellular signal transduction and energy metabolism.5 At the same time, the changes in the contents of endogenous chiral carboxylic acids reflect the state of human health to some extent.6 For example, R-3hydroxybutyric acid (R-3-HBA), one of the ketone bodies, not only provides energy, but also acts as G protein-coupled receptors activator and histone deacetylases inhibitor,7 while S-3-hydroxybutyric acid (S-3-HBA) possesses anticonvulsant activity.8 L-lactic acid (L-LA) is produced by glycolysis and found to be up-regulated in cancer cells because of Warburg effect,9 whereas D-lactic acid (D-LA) is born in methylglyoxal pathway.10 Furthermore, it has been reported that the level of D-LA is significantly higher in the urine of type 2 diabetes mellitus (T2DM) patients than that in healthy control (HC).11 Therefore, the development of analytical methods for accurate quantification of multiple endogenic chiral carboxylic acids would be helpful to understand the mechanisms underlying the related metabolic diseases. So far, several analytical methods have been developed for the separation and quantification of carboxylic acid enantiomers.12-14 For example, Cha et al.12 developed a gas chromatography-mass spectrometry (GC-MS) method to separate chiral 2-hydroxy carboxylic acids by transforming the enantiomers into O-(−)-menthoxycarbonylated diastereoisomers. However, only thermally stable and easily volatile analytes are suitable for GC-MS analysis, and the detection sensitivity is limited in some cases. Recently, chiral reagent labeling assisted LC-MS have emerged as an alternative technique for the analysis of chiral carboxylic acids. For instance, Takayama et al.13 developed a method for the separation of D/L-LA and R/S-3-HBA using a chiral labeling regent, (S)-1-(4, 6-dimethoxy-1, 3, 5-triazin-2-yl) pyrrolidin-3-amine, and then applied to the determination of the above enantiomers in saliva samples by LC-MS. Cheng et al.14 used (S)-(+)-1-(2-pyrrolidinylmethyl)-pyrrolidine to label R/S-2-hydroxybutyric acid (R/S-2-HBA) and R/S-3HBA in human tissues followed by LC-MS analysis. However, the studies as mentioned above mainly focused on a limited number of chiral carboxylic acids, which is not conducive to the understanding of mechanisms underlying the chiral carboxylic acids associated diseases. Cinchona alkaloids are a kind of natural products that have been widely used in biomedicine,15 asymmetric catalysis,16, 17 and chromatographic separation.18-20 In the chromatographic separation applications, cinchona alkaloids have been demonstrated to be powerful chiral selectors as ligands of chiral stationary phases and mobile phase chiral 3



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additives in LC. For example, Calderón et al.18 realized the LC separation of short chain aliphatic hydroxy carboxylic acid enantiomers on cinchona alkaloids (quinine and quinidine)-bonded silica stationary phase. Pettersson et al.19 added cinchona alkaloid (quinine) into mobile phase to separate 10-camphorsulphonic acid enantiomers by LC. Based on the successful applications of cinchona alkaloids as ligands of chiral stationary phases and mobile phase chiral additives, we speculated that cinchona alkaloid-derived primary amine (CAPA) might be an efficient chiral labeling reagent for the analysis of chiral carboxylic acids by LC-MS; it could not only improve the chiral separation of the chiral carboxylic acids on achiral column, but also increase the detection sensitivity of them because cinchona alkaloids have good MS response.21 In the present work, we established a method for the simultaneous analysis of multiple chiral carboxylic acids by CAPA labeling assisted LC-MS. After CAPA labeling, 16 enantiomers of 8 chiral carboxylic acids could be separated with good resolution and detected with high sensitivity by LC-MS. The detection sensitivity of the CAPA labeled chiral carboxylic acids was greatly enhanced comparing to the corresponding intact analytes. Furthermore, a strategy of 4-plex chemical labeling based on 4 CAPA analogues was proposed for high-throughput analysis of endogenous chiral carboxylic acids. In the proposed strategy, we chose chiral carboxylic acid standards labeled by one CAPA analogue as internal standards (ISs); while the other 3 CAPA analogues were utilized to label endogenous chiral carboxylic acids in 3 different samples, respectively. The developed method was validated by the simultaneous determination of multiple chiral carboxylic acids in human serum and mammalian cells, and finally used to investigate the contents of the chiral carboxylic acids in the serum samples from T2DM and CRC patients.

EXPERIMENTAL SECTION Chemical Reagents R-glyceric acid (R-GA), S-glyceric acid (S-GA), D-lactic acid (D-LA), L-lactic acid (L-LA), R-2-hydroxybutyric acid (R-2-HBA), S-2-hydroxybutyric acid (S-2-HBA), R-3-hydroxybutyric acid (R-3-HBA), S-3-hydroxyisobutyric acid (S-3-HBA), R-3-hydroxyisobutyric acid (R-3-HIB), S-3-hydroxybutyric acid (S-3-HIB), R-mandelic acid (R-MA), Smandelic acid (S-MA), R-phenyllactic acid (R-PLA), S-phenyllactic acid (S-PLA), N-acetyl-D-tryptophan (N-A-D-T), and N-acetyl-L-tryptophan (N-A-L-T) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The detailed information was listed in Table S1. (8α, 9S)-10, 11-dihydro-6’-methoxy-cinchonan-9-amine (CAPA analogue a), (8α, 9S)-6’-methoxy-cinchonan-9-amine (CAPA analogue b), (8α, 9S)-10, 11-dihydro-cinchonan-9-amine (CAPA analogue 4


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c), and (8α, 9S)-cinchonan-9-amine (CAPA analogue d) (Figure 1) were purchased from DAICEL Chiral Technologies (China) CO., LTD (Shanghai, China). Triphenylphosphine (TPP) and 2, 2’-dithiodipyridine (DPDS) were purchased from Bide Pharmatech (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo-Fisher Scientific (MA, USA). HPLC-grade acetonitrile (ACN) was purchased from TEDIA Co. (Fairfield, OH, USA). Biological and Clinical Samples Serum samples of 25 HC and 22 T2DM patients were collected from Zhongnan Hospital of Wuhan University (Wuhan, China) and 20 CRC patients were from Hubei Cancer Hospital (Wuhan, China). Detailed information was listed in Table S2. The study met the requirements of the declaration of Helsinki. All the experiments were performed in accordance with Ethics Committee’s guidelines and regulations in Hubei. Mammalian cells (HepG2) was obtained from China Center for Type Culture Collection and maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. HepG2 were collected at a density of approximately 3.0 × 106 cells per mL (5 mL). Sample Pretreatment Sample preparation was performed according to the previously reported method.22 The treatment of human serum and mammalian cells were similar, except that cells needed to be disintegrated at first.23 For human serum, a droplet of blood from finger prick was enough for the analysis. Briefly, 10 µL of human serum and 100 μL of ACN were mixed, and then the mixture was exposed to ultrasound for 5 min. Then, the mixture was centrifuged at 13, 600 g for 3 min, and the supernatant was collected. The extraction procedure for one sample was repeated 3 times and the obtained supernatants were mixed followed by CAPA labeling. For cells, each sample was added to a 1.5-mL polypropylene tube containing 100 μL of ACN. The mixture was treated with ultrasonic cell crusher at 4°C to accelerate the analytes releasing from cells. The following steps were the same with the preparation process of human serum. Finally, 280 μL of each sample supernatant was mixed with 20 µL of 10 µmol/mL TPP/DPDS and 10 μL of 2 μmol/mL CAPAs, and the mixture was incubated at 40°C for 30 min (Figure S1). Instrumentation and Analytical Conditions We conducted LC-MS analysis on Shimadzu MS-8045 mass spectrometer (Tokyo, Japan). The chromatographic separation was achieved on an Acquity UPLC BEH C18 column (100 × 2.1 mm i.d., 1.7 μm; Waters, Milford, USA) at 5



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40°C. FA in water (0.1%, v/v, solvent A) and ACN (solvent B) were employed as mobile phases for the analysis of CAPAs labeled chiral carboxylic acids with a flow rate of 0.4 mL/min. A post-gradient with the following proportions (v/v) of solvent B was applied: 0-3 min at 10% B, 3-13 min 10% to 20% B, 13-15 min 20% to 30% B, 15-15.5 min 30% to 80% B, 15.5-16.5 min 80% B, and 16.5-17 min 80% to 10% B. The injection volume was 2 μL. Autosampler was kept at 4°C during the analysis. Triplicate measurements were performed. All chiral carboxylic acid derivatives were analyzed by multiple reaction monitoring (MRM) in the positive mode. Detailed parameters of UHPLC-MRM-ESIMS/MS were in Supporting Information. The optimized MRM parameters of CAPAs labeled chiral carboxylic acids were summarized in Table S3.

RESULTS AND DISCUSSION Optimization of Chemical Labeling Conditions The study was aimed to develop a high-throughput method for the simultaneous analysis of multiple chiral carboxylic acids based on chemical labeling by LC-MS, in which 4 CAPA analogues were selected as 4-plex chiral labeling reagents. At first, we verified the feasibility of the labeling reaction. As shown in Table S3, the cation quasimolecular ions ([M + H]+) of all chiral carboxylic acids investigated in this study could be obtained, indicating that all chiral carboxylic acids could be labeled effectively by 4 CAPA analogues. Then, we used typical chiral carboxylic acids (D-LA, R-3-HBA, S-PLA, and N-A-L-T) at 50 ng/mL to optimize the labeling conditions including the concentrations of catalysts (TPP/DPDS) and chemical labeling reagents (4 CAPA analogues a, b, c, and d), reaction time and reaction temperature. Taking CAPA analogue a as the example, the labeling reaction reached to a plateau when the concentration of TPP/DPDS was 0.6 μmol/mL (20 μL) (Figure S2a). The peak areas of all derivatives reached to the maximum when the concentration of CAPA analogue a was set at 0.3 μmol/mL (20 μL) as shown in Figure S2b. The reaction temperature was found to have slight influence on the labeling reactions in the range of 25°C to 60°C (Figure S2c), which was probably due to the high reaction activity.24 The labeling reactions could be completed in 30 min (Figure S2d). Similarly, we also optimized the labeling conditions for the other 3 CAPA analogues (Figure S3: CAPA analogue b, Figure S4: CAPA analogue c, and Figure S5: CAPA analogue d) and obtained similar results: TPP/DPDS, 0.6 μmol/mL; labeling reagent, 0.3 μmol/mL; reaction temperature, 40°C; reaction time, 30 min. Labeling efficiency is one of the most important parameters that affect detection sensitivity and stability of derivatives. Under the optimal labeling conditions, the labeling efficiency of 4 CAPA analogues was evaluated (Figure 6


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S6) and was found to be greater than 95%. In addition, we examined the stability of the derivatives within 24 h. The peak areas of the CAPAs labeled chiral carboxylic acids had no significant difference (Figure S7), suggesting that the resulting CAPAs derivatives were stable within 24 h. Establishment of MRM Method To obtain qualitative and quantitative product ions in MRM method, we explored the fragmentation pathway of 4 CAPAs labeled chiral carboxylic acids under CID. It was indicated that the fragmentation of all derivatives could produce one kind of characteristic product ions deriving from the broken of C-N bond along with the elimination of one neutral fragment. Taking 4 CAPAs labeled R-2-HBA as examples, all derivatives could loss 103.0 (C4H9NO2) and produce one certain kind of product ions (CAPA analogue a: m/z 309.3, CAPA analogue b: m/z 307.3, CAPA analogue c: m/z 277.3, and CAPA analogue d: m/z 275.3). In this respect, we selected these above characteristic product ions as quantitative ions due to their high relative intensity. Furthermore, the fragmentation of quinine group led to the generation of characteristic product ions at m/z 184.2 (Figure 1b and 1c) and m/z 168.1 (Figure 1d and 1e), which were selected to ensure qualitative accuracy. Overall, all CAPAs labeled chiral carboxylic acids could produce characteristic fragmentation ions, which met the needs of both qualitative and quantitative analysis assisted LC-MS. The detailed fragmentation pathway of 4 CAPAs labeled chiral carboxylic acids was presented in Figure S8. Next, we compared the detection sensitivities of chiral carboxylic acids before and after CAPAs labeling under the optimal labeling conditions. After CAPAs labeling, the detection sensitivity of chiral carboxylic acids was generally improved by up to 3 orders of magnitude comparing to their intact analytes (Table S4). The reason why the detection sensitivity of chiral carboxylic acids was improved after CAPAs labeling could be explained by the facts: on the one hand, CAPAs possessed good MS intensity, so the MS intensity of corresponding chiral carboxylic acid derivatives was enhanced accordingly; on the other hand, the hydrophobicity of chiral carboxylic acids was increased after CAPA labeling and the ionization efficiency was improved, which was contributed to their high MS response. 4-Plex Chemical Labeling Strategy for the Analysis of Multiple Chiral Carboxylic Acids Simultaneous analysis of multiple chiral carboxylic acids is vital for understanding the pathogenesis of related diseases. However, it is difficult to distinguish carboxylic acid enantiomers under achiral conditions due to their identical physical and chemical properties. To separate enantiomers on achiral column, enantiomers need to be converted to diastereoisomers. Chiral chemical labeling plays an important role in the chiral separation of carboxylic acid enantiomers. In the current study, we used CAPA as the chiral labeling reagent to label chiral carboxylic acids, which 7



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converted carboxylic acid enantiomers to diastereoisomers. In addition, to realize high-throughput analysis of multiple chiral carboxylic acids in biological samples, we proposed a 4-plex chemical labeling strategy using 4 CAPA analogues to label chiral carboxylic acids in parallel: one CAPA analogue was used to label chiral carboxylic acid standards as ISs for accurate quantification; while the other 3 were utilized to label endogenous chiral carboxylic acids in 3 biological samples, respectively. The 3 labeled samples and ISs were mixed prior to LC-MS analysis. The general procedure of 4plex chemical labeling strategy was illustrated in Figure 2. To achieve high-throughput analysis in one-run analysis, there were two points needed to be verified: (1) The chromatographic separation conditions of 4 CAPAs labeled 16 chiral carboxylic acid enantiomers; (2) The influence of chiral carboxylic acids labeled by different CAPA analogues on accurate quantification. The chromatograms of 16 chiral carboxylic acid enantiomers after labeled with 4 CAPAs, respectively, are shown in Figure 2. To make the chromatograms clear, the peaks of CAPA labeled analytes are separately displayed according to their MRM transitions. Obviously, our developed method could be applied to the separation of not only different isomers (2-HBA, 3-HBA, and 3-HIB), but also the enantiomers of isomers (R/S-2-HBA and R/S-3-HBA) on achiral column. Overall, 16 enantiomers of 8 chiral carboxylic acids labeled by 4 CAPA analogues were completely separated under the same chromatographic condition (R > 1.93, Table S5), which was better than the reported work.14, 25-27 Since 4 CAPA analogues possessed the same spatial configuration, all labeled chiral carboxylic acids were eluted in the same order (Figure 2). Then, we carried out a series of experiments to verify the quantitative accuracy of the 4-plex chemical labeling strategy. As shown in Table S6, we labeled S-PLA with a series of concentrations (1, 2, 5, 10, 20, 50, and 100 ng/mL) by CAPA analogues a and b, respectively. Next, we mixed them into 9 samples prior to LC-MS analysis. The theoretical ratios of the concentration of S-PLA labeled by CAPA analogues a and b were as x-axis; while the measured ratios of the MS intensity of corresponding CAPA derivatives were as y-axis. Then, we got a unary linear regression equation concerning the quantitative accuracy of CAPA analogues a and b with determination coefficient (R2) = 0.9801 (Figure 3a). The slope of the equation represented the ratio of the sensitivity of CAPA analogues a and b. The closer R2 was to 1, the more consistent labeling results of CAPA analogues a and b could be obtained. Similarly, we evaluated the labeling effects of the other combinations of different CAPA analogues. In summary, R2 of all combinations were greater than 0.9702, indicating that we could obtain consistent results after the same sample labeled by different CAPA analogues (Figure 3 and S9). Therefore, all CAPA analogues labeled chiral carboxylic acids could act as ISs for each other. In addition, the slope of the equation represented the ratio of the sensitivity of two CAPA analogues. According 8


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to Figure 3, the slopes of all curves involving CAPA analogue b were < 1, which suggested that the sensitivity of CAPA analogue b was lower than that of a, c, and d. Finally, the CAPA analogue b labeled chiral carboxylic acids were determined as the ISs of this method for accurate quantification, and the other 3 CAPA analogues for the labeling of samples. Method Validation Sensitivity and linearity The detection sensitivity of chiral carboxylic acids labeled by 4 CAPA analogues was evaluated by determining the limits of detection (LOD) and limits of quantification (LOQ) at their signal to noise ratios (S/N) of 3 and 10, respectively. In summary, all analytes had LODs from 0.006 to 0.2 pg and LOQs from 0.02 to 0.6 pg (Table S7). To evaluate the linearity of the method, 16 chiral carboxylic acid standards were labeled with CAPA analogues a, c, and d, respectively, and fixed amount of ISs were added prior to LC-MS analysis. The calibration curves were constructed by plotting the peak area ratio (analyte/IS) versus the concentrations of chiral carboxylic acids. Finally, we obtained the calibration curves of 16 chiral carboxylic acid enantiomers labeled by CAPA analogues a, c, and d. Good linearity for all analytes were obtained with R2 > 0.9866 (Table S7). Extraction efficiency and matrix effect Extraction efficiency was defined as the percentage of chiral carboxylic acids extracted from a spiked sample to that of an extracted sample with the same concentration of standards spiked.28 As shown in Figure S10a, the extraction efficiency of the method in human serum was 66.2-96.0%. The aim of investigating matrix effect was to assess possible variation of MS intensity caused by co-eluents from the biological matrix. Due to the difference in the retention time of 4 CAPA analogues derivatives, we explored the matrix effect of the method by CAPA analogues a, c, and d. According to the Figure S10, the matrix effect on MS detection was 75.3-123.1% indicating that human serum matrix interferents had minor influence on ionization efficiency of CAPA analogues derivatives. Similarly, we investigated the extraction efficiency and matrix effect in mammalian cells (1.0 × 106 cells). As shown in Figure S11, the extraction efficiency was 72.6-103.2% and the matrix effect on MS detection was 71.9-118.4%. Furthermore, we investigated the matrix effect on the chemical labeling reaction. For human serum, the matrix effect on labeling was 75.1-120.4%, suggesting that human serum matrix interferents had minor influence on the labeling efficiency of CAPA analogues. For mammalian cells, the matrix effect on labeling was 69.3-111.6%. Detailed 9



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experimental procedures were in Supporting Information Accuracy and precision The precision and accuracy of the developed method were evaluated by the recoveries and intra- and inter-day RSDs. As shown in Table S8, the method possessed a satisfactory accuracy for all analytes of interest in human serum (64.6-108.7%). Furthermore, the RSDs of intra- and inter-day precision ranged from 1.6% to 13.7% and 1.3% to 12.6%, respectively. All these results suggested that the developed method was reliable. Method Application Serum samples of HC, T2DM, and CRC patients were prepared and subsequently labeled by CAPA analogues a, c, and d, respectively. Using the established 4-plex chemical labeling method, we obtained the contents of chiral carboxylic acids in the serums of HC, T2DM, and CRC patients in one-run analysis. Nine chiral carboxylic acid enantiomers were detected, and the detailed contents were listed in Table S9, S10, and S11. Among these chiral carboxylic acids detected in the serum, 8 enantiomers from 4 chiral carboxylic acids, including D-LA/L-LA, R-3HBA/S-3-HBA, R-3-HIB/S-3-HIB, and N-A-D-T/N-A-L-T, could be detected. For 2-HBA enantiomers, only S-2-HBA was detected, suggesting that R-2-HBA may not exist in human serum14 or its concentration in the serum was lower than the LOD of method. In addition, we also calculated the ratios of chiral carboxylic acid enantiomers to provide a further judgement on whether human metabolism was normal or not (Figure S12). Then, PLS-DA was performed to assess the classification performance between HC, T2DM, and CRC patients. As shown in Figure 4, HC (black boxes), T2DM (blue dots), and CRC (red diamonds) groups could be distinctly separated (R2X = 0.570, R2Y = 0.701, Q2 (cum) = 0.698). Detected analytes of N-A-L-T, D-LA, S-2-HBA, S-3-HIB, and N-A-D-T were found to have significant difference between HC and T2DM samples based on the VIP > 1 in PLS-DA analysis and p < 0.05 in t-test analysis. In the serum of T2DM patients, the contents of N-A-L-T, D-LA, S-2-HBA, and S-3-HIB were increased; while the content of N-AD-T was decreased comparing to that of HC (Figure S13). The metabolism of glucose and fatty acids were blocked in T2DM patients, so protein degradation served as supplementary energy source for human body. Therefore, the content of free amino acids (N-A-L-T) increased. As the downstream metabolites of glutamate and valine, S-2-HBA and S-3HIB also showed an increasing trend.29, 30 In the previous work, Mardinoglu et al.31 found that 3-HIB increased in the serum of T2DM patients. However, it was not clear about the relation between spatial configuration (R/S) of 3-HIB and T2DM. In the current study, we found that the potential relationship between the enantiomer ratio of 3-HIB and T2DM. 10


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On the other hand, due to the imbalance of various metabolites in T2DM, the sources of methylglyoxal became more and more, which led to increased D-LA. However, L-LA dehydrogenase in the body could not degrade the accumulated D-LA11. In our study, the content of D-LA in the serum of T2DM patients was twice than that of in HC. Currently, there are few reports on N-A-D-T 32, 33 and its metabolic pathways. It is still needed to explore their biological functions. D-LA, L-LA, S-3-HBA and N-A-L-T were found to have significant difference between HC and CRC samples. In the serum of CRC patients, D-LA, L-LA, and S-3-HBA presented an increasing trend and N-A-L-T decreased comparing to that of HC (Figure S14). In normal cells, glycolysis was effectively restrained when oxygen was sufficient. So the content of L-LA usually remained stable. However, cancer cells produced much more L-LA because of Warburg effect.34, 35 At the same time, the activity of glyoxalase 1 in methylglyoxal pathway was much higher in cancer cells, which led to accumulated D-LA.36 So the contents of D-LA and L-LA increased in the serums of CRC patients. Generally, the proliferation of cancer cells is faster than normal cells. The excessive pyruvate produced by glycolysis in cancer cells was converted into acetyl CoA followed by the formation of S-3-HBA.5 Therefore, the content of S-3-HBA increased. In addition, more proteins were needed to be synthesized in cancer cells. N-A-L-T was involved as substrate.37, 38

Therefore, the content of N-A-L-T decreased.

CONCLUSIONS In the study, 4 CAPA analogues were used to develop a 4-plex chemical labeling strategy for the simultaneous analysis of multiple chiral carboxylic acids in biological samples. The detection sensitivity of chiral carboxylic acids was greatly improved after CAPAs labeling. Method validation about 4 CAPAs labeled 16 chiral carboxylic acid enantiomers showed acceptable results. With the developed method, we detected 9 chiral carboxylic acid enantiomers in human serum. Furthermore, 4-plex chemical labeling strategy achieved high-throughput analysis. We obtained the contents of chiral carboxylic acids in the serums of HC, T2DM, and CRC patients in one-run analysis. Noticeably, 5 chiral carboxylic acid enantiomers were considered to be potential biomarkers of T2DM; while 4 chiral carboxylic acid enantiomers were related to CRC. Taken together, the developed method was a powerful auxiliary tool for the analysis of endogenic chiral carboxylic acids.

ASSOCIATE CONTENT 11



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Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional experimental methods; Information on 16 chiral carboxylic acid enantiomers; Information on HC, T2DM, and CRC patients; MRM parameters of 4 CAPAs labeled chiral carboxylic acids; Sensitivity of chiral carboxylic acids before and after labeled by 4 CAPAs; Resolution of 4 CAPAs labeled chiral carboxylic acids; Comparison of measured and theoretical ratios of S-PLA labeled by CAPA analogues a and b; LODs, LOQs, and calibration curves of CAPAs labeled chiral carboxylic acids; Recoveries and intra- and inter-day precisions; Contents of the detected chiral carboxylic acids in the serums of HC, T2DM, and CRC patients; Optimization of labeling reaction; Labeling efficiency; Stability of 4 CAPAs labeled chiral carboxylic acids; General fragmentation pathway of 4 CAPAs labeled chiral carboxylic acids; Verification of accurate quantification of R-2-HBA labeled by 4 CAPAs; Extraction efficiency and matrix effect; Ratios of carboxylic acid enantiomers in the serums of HC, T2DM, and CRC patients; Chiral carboxylic acids that differed significantly in the serums of between HC, T2DM, and CRC.

AUTHOR INFORMATION Corresponding Authors * E-mail for Y.-Q.F.: [email protected], Tel.: +86-27-68755595, Fax: +86-27-68755595. ORCID Yu-Qi Feng: 0000-0003-1107-5385 Author Contributions All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The work is supported by the National Key R&D Program of China (2017YFC0906800), and the National Natural Science Foundation of China (21635006, 31670373, 21721005).

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Figure legends Figure 1. (a) Structures of 4 CAPA analogues. (b) Fragmentation of CAPA analogue a labeled R-2-HBA. (b) Fragmentation of CAPA analogue b labeled R-2-HBA. (c) Fragmentation of CAPA analogue c labeled R-2-HBA. (d) Fragmentation of CAPA analogue d labeled R-2-HBA.

Figure 2. Work flow of the 4-plex chemical labeling strategy. (a) 1, S-GA-CAPAs; 2, R-GA-CAPAs. (b) 3, D-LACAPAs; 4, L-LA-CAPAs. (c) 5, R-3-HIB-CAPAs; 6, R-3-HBA-CAPAs; 7, S-3-HBA-CAPAs; 8, S-3-HIB-CAPAs; 9, R-2-HBA-CAPAs; 10, S-2-HBA-CAPAs. (d) 11, R-MA-CAPAs; 12, S-MA-CAPAs. (e) 13, R-PLA-CAPAs; 14, SPLA-CAPAs. (f) 15, N-A-D-T-CAPAs; 16, N-A-L-T-CAPAs.

Figure 3. Verification of accurate quantification of S-PLA labeled by 4 CAPA analogues. (a) Combination of a and b. (b) Combination of a and c. (c) Combination of a and d. (d) Combination of b and c. (e) Combination of b and d. (f) Combination of c and d.

Figure 4. PLS-DA score plot of all human serum samples.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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4-Plex Chemical Labeling Strategy Based on Cinchona Alkaloid

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