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Simultaneous Determination of 34 Amino Acids in Tumor Tissues from Colorectal Cancer Patients Based on the Targeted UHPLC-MS/MS Method

Simultaneous Determination of 34 Amino Acids in Tumor Tissues from Colorectal Cancer Patients... Hindawi Journal of Analytical Methods in Chemistry Volume 2020, Article ID 4641709, 12 pages https://doi.org/10.1155/2020/4641709 Research Article Simultaneous Determination of 34 Amino Acids in Tumor Tissues from Colorectal Cancer Patients Based on the Targeted UHPLC-MS/MS Method 1,2,3 1 1 1 1 1 Yang Yang, Feng Zhang, Shouhong Gao, Zhipeng Wang, Mingming Li, Hua Wei, 3 1 Renqian Zhong , and Wansheng Chen Department of Pharmacy, Changzheng Hospital, e Second Military Medical University of CPLA, Shanghai 200003, China Department of Pharmacy, e 71st Group Army Hospital of CPLA Army, e Affiliated Huaihai Hospital of Xuzhou Medical University, Xuzhou 221004, China Department of Laboratory Diagnostics, Changzheng Hospital, e Second Military Medical University of CPLA, Shanghai 200003, China Correspondence should be addressed to Renqian Zhong; zhongrq@smmu.edu.cn and Wansheng Chen; chenwansheng@ smmu.edu.cn Received 17 February 2020; Revised 22 April 2020; Accepted 1 May 2020; Published 1 August 2020 Academic Editor: Boryana M. Nikolova Damyanova Copyright © 2020 Yang Yang et al. -is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A targeted ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was established and validated for the simultaneous determination of 34 amino acids in tissue samples from colorectal cancer (CRC) patients. -e chromatographic separation was achieved on an Agilent ZORBAX SB-C column (3.0 ×150 mm, 5 μm) with a binary gradient elution system (A, 0.02% heptafluorobutyric acid and 0.2% formic acid in water, v/v; B, methanol). -e run time was 10 min. -e multiple reaction monitoring mode was chosen with an electrospray ionization source operating in the positive ionization mode for data acquisition. -e linear correlation coefficients were >0.99 for all the analytes in their corresponding calibration ranges. -e sample was pretreated based on tissue homogenate and protein precipitation with a 100 mg aliquot sample. -e average recovery and matrix effect for 34 amino acids and 3 internal standards were 39.00%∼146.95% and 49.45%∼173.63%, respectively. -e intra- and interday accuracy for all the analytes ranged from −13.52% to 14.21% (RSD ≤8.57%) and from −14.52% to 12.59% (RSD≤10.31%), respectively. Deviations of stability under different conditions were within±15% for all the analytes. -is method was applied to simultaneous quantification of 34 amino acids in tissue samples from 94 CRC patients. in blood, urine, and feces between CRC patients and healthy 1. Introduction volunteers or between CRC tissue and normal tissue. Recent Amino acids play an essential role in both metabolism and findings are related to (1) glycemic and/or ketogenic amino proteome. Proper amino acid management is critical for acids that are involved in the energy metabolism by entering maintaining cell function and organismal viability, partic- tricarboxylic acid cycle pathway; (2) amino acids that are ularly under metabolic disturbance. With the alterations in synthetic precursors of pyrimidine bases or provide nitrogen tumor cell metabolism recognized as a hallmark of cancer, sources for the synthesis of purine bases; and (3) amino acids there have been more research studies on amino acids, which that are the decomposition products from cytosine, uracil, are involved in the primary tumor formation, growth, and and thymine nucleotides and that can reflect the severity of metabolism. As shown in Table 1 [1–18], studies in the field malignant tumor and the therapeutic effect of chemotherapy of colorectal cancer (CRC) metabolism were routinely for malignant tumor [19]. To sum up, details about the performed to elucidate the difference of amino acid profiles association of amino acids with the formation, growth, and 2 Journal of Analytical Methods in Chemistry Table 1: -e summarized variations of amino acids in blood, urine, metabolomic study with increasing levels of sophistication feces, and cancerous tissue of CRC patients. [28–30]. Out of these considerations, thirty-four endoge- nous amino acids in human biological samples were in- Variations Amino volved in our study by using the LC-MS technique. References In In In In acids Amino acid metabolomics in human serum has been ∗ ∗ ∗ blood urine feces tissue studied on a larger scale due to its potential diagnostic value Gly ↓ ↑ [1, 2] in patients with breast, lung, ovarian, head and neck, gastric, Ala ↑ ↑ [3–5] and pancreatic cancers or in CRC patients [31], suggesting Ser ↑ ↓ [3, 6, 7] that the amino acid profiling in plasma/serum or in other Val ↓ ↑ [2, 6, 7] body fluids or selected tissue samples might be a new tool for -r ↓ [2] early diagnosis of cancers [32]. As different cancer subtypes Leu ↓↑ ↑ [3, 5–7] Ile ↑ [6–8] show distinct metabolic phenotypes, the present paper Asp ↓↑ ↓ ↑ [3, 4, 6, 7, 9] aimed to evaluate the amino acid profile in clinical samples Gln ↓ ↑ [3, 8] from CRC patients by means of a targeted metabolomic Glu ↑ ↑ [4, 8, 9] assay based on a UHPLC-MS/MS method, which was Phe ↓ ↑ [3, 10] established and validated for the quantitative analysis of 34 Arg ↑ [6, 7] amino acids in tumor tissues. So far, no research has ever Tyr ↑ ↑ [3, 10] evaluated the ability of the amino acid metabolic phenotype Pro ↓ ↑ ↑ [3, 11, 12] of CRC patients. Additionally, this was the first experiment Met ↓ ↓ [5–7] to show the amino acid profile differences between can- Trp ↑ [3] cerous, paracancerous, and normal tissue of CRC patients, Cys ↑ ↑ [4, 11] His ↓ ↓ [3, 6, 7] which may shed light on the metabolic stability of amino Cit ↑ [3] acids in humans and provide biological information to find ADMA ↑ [13–15] and evaluate a new diagnostic tool for the further study of Cyss ↑ [9] CRC. Aba ↑ [16, 17] Opr ↑ [4] 2. Materials and Methods Hpr ↓ [3] Orn ↑ [3] 2.1. Chemicals and Reagents. -irty-four amino acids, in- Ahd ↓ [5] cluding glycine (Gly), L-alanine (Ala), L-serine (Ser), Hia ↑ ↓ [5, 18] L-valine (Val), L-threonine (-r), L-leucine (Leu), L-iso- SDMA ↑ [14, 15] Kyn ↑ ↓ [9, 18] leucine (Ile), L-aspartic acid (Asp), L-lysine (Lys), L-gluta- mine (Gln), L-glutamic acid (Glu), L-phenylalanine (Phe), Note. CRC patients vs. healthy volunteers; ↑: increase; ↓: decrease; ↓↑: increase and decrease. -e cancerous tissue of CRC patients vs. the normal L-arginine (Arg), L-tyrosine (Tyr), L-proline (Pro), L-as- tissue of CRC patients. paragine (Asn), L-methionine (Met), L-tryptophan (Trp), L-cysteine (Cys), L-histidine (His), L-citrulline (Cit), asymmetric dimethylarginine (ADMA), L-cystine (Cyss), metabolism of CRC are presented in Figure 1. Besides, as a sarcosine (Sar), β-alanine (3-aminopropanoic acid, Apa), higher level of sarcosine was observed in the urine of patients β-aminoisobutyric acid (3-amino-2-methylpropanoic acid, Amp), c-aminobutyric acid (4-aminobutyric acid, Aba), 5- with prostate cancer [20–22], sarcosine was believed to be closely related to cancer and also investigated in our study. oxo-L-proline (Opr), 4-hydroxy-L-proline (Hpr), L-orni- Despite the overlap between the results of the targeted thine (Orn), 2-amino-L-hexanoic diacid (α-aminoadipic and nontargeted metabolomic assays, there were also sub- acid, Ahd), hippuric acid (Hia), symmetric dimethylarginine stantial inconsistencies. Whenever the assessment of a (SDMA), and L-kynurenine (Kyn) were purchased from the specific pathway such as amino acids became the focus of National Institutes for Food and Drug Control of China interest, a targeted metabolomic assay would seem prefer- (Beijing, China), Dalian Meilun Biotech Co., Ltd., (Dalian, able to a nontargeted one [23]. Generally, gas chromatog- China), Shanghai Yuanye Biotech Co., Ltd., (Shanghai, raphy-mass spectrometry (GC-MS) was available for the China), and Sigma-Aldrich LLC. (Darmstadt, Germany). L-Alanine-d4 (Ala-d4), L-methionine-d3 (Met-d3), and amino acid analysis in some laboratories [24–26], but it required a derivatization step for nonvolatile analytes, which L-phenylalanine-d5 (Phe-d5) were chosen as internal was important for the analyte detection and quantification, standards (ISs), which were all provided by Toronto Re- but could be avoided when a liquid chromatography-mass search Chemicals Inc., (Toronto, Canada). -e information spectrometry (LC-MS) method was adopted. Amino acids, about the 34 standards and 3 ISs is shown in Supplementary as a group of polar chemical compounds, should also be Material Table S1. modified by using a derivatization reagent to make them Methanol and acetonitrile of HPLC grade were pur- volatile and thus accessible for GC-MS [27]. Over the past chased from Merck Inc., (Darmstadt, Germany). Phosphate- two decades, many researchers had described the ultrahigh- buffered solution (PBS) (10x, namely, 0.1 mol/L) of the cell performance liquid chromatography-tandem mass spec- culture grade (Lot. MA0016-May-19B) was purchased from trometry- (UHPLC-MS/MS-) based method for a targeted Dalian Meilun Biotech Co., Ltd., (Dalian, China). Journal of Analytical Methods in Chemistry 3 Glycemic and ketogenic amino acids r, Ile, Phe, Tyr, Trp Glycemic amino acids Gly, Ala, Ser, Val, Asn, Asp, Gln, Glu, Arg, Cys, Mat, Pro, His Participation Gly, Gln, Asp Ketogenic amino acids Leu, Lys Synthesis Apa, Amp Decomposition Tricarboxylic acid cycle Nucleotides Material Energy supplementary supplementary Feces CRC tissue Increase or decrease Pro, Cyss Differeces Pro, Ala, Cys, Glu, Opr, Asp, Phe, Tyr, Gly, Aba Urine Normal tissue Increase or decrease Increase or decrease Leu, His, Pro, Asp, Hpr, Gln, Phe, Ser, Ile, Val, Arg, Leu, His, Met, Cit, Orn, Kyn, Cyss, Glu, Val, r, Gly, Ser, Asp, Trp, Ala, Gln, Tyr, Blood Met, Ahd, Hia, ADMA, SDMA Hia, Kyn Figure 1: -e association of amino acids with the formation, growth, and metabolism of CRC. Chromatographic separation was performed on an Agilent Heptafluorobutyric acid (HFBA) (Lot. P11933) was obtained from Adamas Reagents Co., Ltd., (Basel, Switzerland). ZORBAX SB-C column (3.0 × 150 mm, 5 μm), whose Formic acid (FA) (Lot. C10009619) was gained from temperature was maintained at 50 C. Binary gradient elution Shanghai Macklin Biochemical Co., Ltd., (Shanghai, China). was used by mixing the mobile phase A (0.02% HFBA and Hydrochloric acid (HCl) of analytical grade was the product 0.2% FA in water, v/v) and B (methanol) at a flow rate at of Sinopharm Chemical Reagent Co., Ltd., (Shanghai, 0.4 mL/min. -e mobile phase elution procedure was as China). Sodium chloride injection (0.9% saline, 1000 ml/bag, follows (A/B, v/v): 0 min, 98 : 2; 1 min, 85 :15; 4 min, 85 : 15; Lot. 160613) was produced by the pharmaceutical prepa- 5 min, 80 : 20; and 10 min, 20 : 80. -e run time was 10 min ration factory of Shanghai Changzheng Hospital, the Second for each sample, and the post time was set at 4.0 min to Military Medical University (Shanghai, China). Water was equilibrate the column pressure. -e autosampler temper- deionized and further purified by a Milli-Q Plus water ature was maintained at 4 C. -e injected volume was 2 μL purification system (Darmstadt, Germany) in our labora- with needle wash. tory. -e other reagents and solvents were of analytical -e ionization of analytes was performed based on an grade. electrospray ionization (ESI) source under the positive ionization mode, and the data acquisition was carried out in a multiple reaction monitoring (MRM) mode. Optimized 2.2. Liquid Chromatography and Mass Spectrometry mass spectrometer conditions were as follows: capillary Conditions. An Agilent 1290 UHPLC coupled to an Agilent voltage, 5.0 kV; dwell time, 40 ms; collision gas (high-purity 6460 triple-quadrupole tandem mass spectrometer (Agilent nitrogen) pressure, 0.2 MPa; and nebulizer gas (nitrogen) Inc, USA) was used to establish the method. pressure, 50 psi. -e dry gas temperature was 325 C and Colorectal wall 4 Journal of Analytical Methods in Chemistry delivered at 10 L/min. -e sheath gas temperature was 350 C aliquot of the supernatant was transferred to a micro- at the flow rate of 12 L/min. centrifuge tube, and 150 μL of 0.2% FA acetonitrile solution All data were acquired and processed using Agilent Mass (containing 400 ng/mL ISs) was added. -e mixture was Hunter workstation software (version B.07.00). -e opti- centrifuged again under the same conditions after being mized MRM parameters of 34 amino acids and 3 ISs are rested for 3 min and vortex-mixed for 2 min, and 2 μL of summarized in Table 2. supernatant was injected into the UHPLC-MS/MS system for analysis. 2.3. Preparation of Calibration Standards and Quality Control Samples. Stock solutions (2.5 mg/mL) for each analyte were 2.5. Method Validation. Method validation was performed prepared separately and stored at −80 C. Gly, Ala, Ser, Val, according to Chinese Pharmacopoeia [36] and US Food and -r, Leu, Ile, Lys, Phe, Arg, Pro, Met, His, Cit, Sar, Apa, Drug Administration (FDA) guidance [37] and with ref- Amp, Aba, Opr, Hpr, Orn, Hia, and Kyn were dissolved in erence to our previous report [38]. 5% methanol aqueous solutions, and Gln, Glu, and Trp were -e selectivity was evaluated by comparing six different prepared in 0.2% FA aqueous solutions. Asp, Tyr, Asn, Cys, batches of the blank matrix to the corresponding spiked Cyss, Ahd, ADMA, and SDMA were in 4% HCl aqueous samples, and the responses of interferents in the blank solutions. Stock solutions were further diluted with 5% matrix less than 20% of the low limit of quantitation (LLOQ) methanol aqueous solution to obtain the following 4 groups samples and 5% of ISs were considered acceptable. of working solutions. Group A included Ala, Val, -r, Leu, -e calibration standards were prepared in triplicates Ile, Lys, Glu, Phe, Arg, and Tyr (250 μg/mL for every ana- and measured three times on different days (at least 2 days). lyte). Group B included Gly, Ser, Asp, and Gln (250 μg/mL -e calibration curve was regressed from the IS-adjusted for every analyte) and Pro, Asn, Met, and Trp (125 μg/mL for peak area versus the nominal concentration under a 1/X every analyte). Group C included Cys, His, Cit, ADMA, and weighting factor. LLOQ was defined as the lowest concen- Cyss (250 μg/mL for every analyte). Group D included Sar, tration point of the calibration curve. A deviation of Apa, Amp, Aba, Opr, Hpr, Orn, Ahd, Hia, SDMA, and Kyn backcalculation for each calibration standard within ±15% (125 μg/mL for each analyte). was thought to be acceptable, and for LLOQ, the deviation -e highest calibration standard solution was prepared should be within ±20%. by adding appropriate volumes of working solutions group -e recovery and matrix effect were assessed by pre- A∼D into PBS (1x, namely, 0.01 mol/L) using previously paring six replicates of the QC sample at low and high reported methods [33–35]. -en, the other 8 calibration concentration levels. -e matrix effect was the ratio of the standard solutions were obtained by diluting the highest peak area in the spiked postextraction samples to solvent- calibration standard solution with PBS. -e final concen- substituted samples at the same concentration, and the trations of calibration standard solutions were 1000, 2000, recovery was the ratio of the peak area in the spiked samples 4000, 8000, 10000, 20000, 40000, 60000, and 80000 ng/mL to spiked postextraction samples at the same concentration. for Gly, Ala, Ser, Val, -r, Leu, Ile, Asp, Lys, Gln, Glu, Phe, -e intra- and interday accuracy and precision were Arg, and Tyr; 500, 1000, 2000, 4000, 5000, 10000, 20000, assessed using the QC samples at LLOQ, low, medium, and 30000, and 40000 ng/mL for Pro, Asn, Met, and Trp; 100, high concentration levels (n � 5). Samples were analyzed in 200, 400, 800, 1000, 2000, 4000, 6000, and 8000 ng/mL for three analytical lots on separate days (at least 2 days), and the Cys, His, Cit, ADMA, and Cyss; and 50, 100, 200, 400, 500, relative standard deviation (RSD) % for inter- and intraday 1000, 2000, 3000, and 4000 ng/mL for Sar, Apa, Amp, Aba, precision not more than 15% was regarded as acceptable (for Opr, Hpr, Orn, Ahd, Hia, SDMA, and Kyn, respectively. LLOQ, not more than 20%). For intra- and interday ac- Quality control (QC) samples were also separately pre- curacy, the relative error (RE) % within ±15% (for LLOQ, pared in the same way and at low, medium, and high con- within ±20%) was considered reasonable. centrations (QC1∼3). -e low, medium, and high -e stability of each analyte was assessed at three con- concentrations of the QC samples were 2000, 10000, and centration levels (low, medium, and high) using the QC 60000 ng/mL for Gly, Ala, Ser, Val, -r, Leu, Ile, Asp, Lys, samples (n � 3) under four different conditions: room tem- Gln, Glu, Phe, Arg, and Tyr; 1000, 5000, and 30000 ng/mL for perature stability was evaluated after exposing samples at room Pro, Asn, Met, and Trp; 200, 1000, and 6000 ng/mL for Cys, temperature for 6 h; three freeze-thaw cycles stability was His, Cit, ADMA, and Cyss; and 100, 500, and 3000 ng/mL for evaluated after freeze and thaw of samples from −20 C to room Sar, Apa, Amp, Aba, Opr, Hpr, Orn, Ahd, Hia, SDMA, and temperature three times; short-term stability was assessed by Kyn, respectively. All solutions were stored at −20 C. analyzing samples kept in the autosampler (4 C) for 24 h; and long-term stability was evaluated after the samples were stored 2.4. Sample Pretreatment. Each tissue sample with a mass of at −20 C for 90 days. -e dilution effect of all the analytes was assessed by about 100 mg was precisely weighed and added with a 5-fold mass of 0.9% saline before being homogenized by a superfine diluting the sample with a blank matrix into the calibration range and comparing the measured concentrations to the homogenizer at 15000 r/min for 2 min in the ice water bath to obtain tissue homogenate, and then the mixture was nominal concentrations. Each dilution factor should be assessed at least five times, and the RSD% and RE% should centrifuged for 15 min at 19060 ×g at 4 C after five minutes of ultrasonic treatment in the ice water bath. -en, a 50 μL be less than 15% and within ±15%, respectively. Journal of Analytical Methods in Chemistry 5 Table 2: -e optimized MRM parameters of 34 amino acids and 3 ISs (ESI positive). Analyte Molecular weight Precursor ion Product ion Fragmentor (V) Collision energy (eV) Gly 75.07 76 30 50 13 Ala 89.09 90 44 50 8 Ser 105.09 106 60 65 9 Val 117.15 118 72 60 7 -r 119.12 120 74 65 9 Leu 131.17 132 86 65 7 Ile 131.17 132 86 65 7 Asp 133.10 134 74 65 12 Lys 146.19 147 84 70 11 Gln 146.14 147 84 65 12 Glu 147.13 148 84 70 12 Phe 165.19 166 120 65 10 Arg 174.20 175 70 90 16 Tyr 181.19 182 136 70 11 Pro 115.13 116 70 70 15 Asn 132.12 133 74 60 11 Met 149.21 150 56 65 13 Trp 204.23 205 188 70 5 Cys 121.16 122 59 60 21 His 155.15 156 110 80 13 Cit 175.19 176 159 70 7 ADMA 202.25 203 46 90 16 Cyss 240.30 241 152 80 12 Sar 89.09 90 44 55 10 Apa 89.09 90 30 60 12 Amp 103.12 104 30 60 11 Aba 103.12 104 87 65 9 Opr 129.11 130 84 75 12 Hpr 131.13 132 86 75 13 Orn 132.16 133 70 65 10 Ahd 161.16 162 98 65 11 Hia 179.17 180 105 65 6 SDMA 202.25 203 172 90 14 Kyn 208.21 209 192 70 7 Ala-d4 93.12 94 48 50 9 Met-d3 152.23 153 56 65 13 Phe-d5 170.22 171 125 70 11 2.6. Study Population and Sample Collection. -e experi- filter tissue. -e samples were stored at −80 C within cry- mental protocol was reviewed and approved by the Ethical otubes until analysis. Committee of Changzheng Hospital prior to specimen collection, and it was conducted in accordance with the 2.7. Data Analysis. Data were analyzed statistically, and Helsinki Declaration of 1964, as revised in 2013, and according to regulatory guidance. Informed consent was graphs were generated by GraphPad Prism 6.01 for Win- dows (GraphPad Software, Inc., La Jolla, CA, USA). A obtained from all participants enrolled in this study. Between July 2016 and December 2017, 94 patients (male nonparametric test (Friedman test) was performed to compare the content differences of 34 amino acids between 56, female 38) with CRC were enrolled from Changzheng sample sets. -e p value less than 0.05 was considered Hospital. None of the patients received neoadjuvant treat- ment. -e median age of these patients was 60 (ranging from statistically significant. 32 to 87). 10 patients were diagnosed with stage I CRC, 33 patients with stage II, 45 patients with stage III, and 6 pa- 3. Results and Discussion tients with stage IV. -e demographic and clinical chemistry characteristics of these CRC patients are shown in Table 3. 3.1. Method Development. Many studies have published the -e sample set including cancerous, paracancerous, and quantitative analyses of amino acids in plasma normal tissue samples was collected from each of these CRC [23, 24, 28, 31, 32], but these methods have never been patients and was named Tc, Tp, and Tn, respectively. All applied in tissue homogenate. In this study, we developed a samples were immediately washed using 0.9% icy saline robust method for the quantitative analysis of underivatized solution, and the surfaces were subsequently dried by the amino acids in human tissue by UHPLC-MS/MS. 6 Journal of Analytical Methods in Chemistry Table 3: -e demographic and clinical chemistry characteristics of CRC patients. Items Total Male Female Number of patients 94 56 38 Age (median, range) 60, (32∼87) 58, (32∼87) 61, (38∼80) Number of patients with TNM stage Stage I 10 3 7 Stage II 33 18 15 Stage III 45 33 12 Stage IV 6 2 4 As for optimization of ESI-MS/MS conditions, this study high ionization efficiency were obtained in the positive highlighted the importance of quantifying the isomeric ionization mode for all the analytes. analytes using two strategies. For compounds that shared the -e selection of an appropriate matrix for calibration samples and the QC samples preparation was an important same MRM transitions, such as Ala and Sar, modifications of the mobile phase and its gradient, as well as the column part of methodological development when LC-MS was used for the quantitative analysis of endogenous compounds in optimization, were tried to make sure they were completely separated in chromatography. -e coelution of the other two biological samples. -ere were two main approaches to this pairs of standards, such as Leu, Ile, and Hpr and Lys and Gln, problem: the first was to dissolve alternative analytes in the was also avoided at the same time. If the isomeric analytes real matrix and the other was to use real analytes in an had a similar retention time, another MRM transition was alternative matrix [35]. -e ideal substitutive matrix should chosen to separate them on different mass spectrometer be completely analyte-free and identical to the real matrix in channels. For example, the MRM transition for ADMA was terms of analyte solubility and extractability, but it was set at 203/46 instead of 203/70 and 203/172 for SDMA. -e unpractical for the detection of endogenous compounds. In representative MRM chromatograms of Ala, Sar, Leu, Ile, our experiment, plasma was processed using neutral de- colorizing carbon for stripping some endogenous carbo- Hpr, Lys, Gln, ADMA, and SDMA are shown in Figure 2. During the selection of ISs, the ideal condition was an hydrates [40]. -is approach was effective for carbohydrates but not for all amino acids because the prepared plasma still isotope-labeled internal standard for each analyte, but the major problem was the high expense and longer delivery contained a high concentration of amino acids. -erefore, time. Besides, those amino acids were of the same structure, the calibration and the QC samples could be prepared in an so it was acceptable to use a structural analog as the internal artificial matrix only as it was impossible to make an standard for analytes. Furthermore, method verification in “analyte-free” matrix. As human biofluid usually contains a our experiment was currently acceptable as specified by variety of proteins, fatty acids, and electrolytes, which is hard Chinese Pharmacopoeia and FDA guidelines. Hence, the for simulation, some research studies documented that the PBS [33, 34] or mobile phase [41] could be treated as the practical value was the availability of three isotopically la- beled amino acids, which could facilitate application while matrix when the calibration and the QC samples were prepared. In addition, the pH value, osmotic pressure, and making research less expensive. In terms of optimization of chromatographic conditions, ion concentration of PBS were closer to those of the biofluid of humans than the mobile phase [35]. As such, PBS was generally, analysis using sub-2 μm columns yielded a greater (S/N) due to the reduction in band broadening and thus an applied as the “mimic tissue fluid” to prepare both the increase in sensitivity. We actually conducted the analysis by calibration and the QC samples. using reverse-phase LC columns with 1.8 and 5.0 μm When it came to optimization of sample preparation, packing materials, which generated a similar chromato- treatment of the tissue homogenate commonly involved graphic peak resolution. Moreover, ion suppression from the protein precipitation (PPT), liquid-liquid extraction, and coeluting peak was alleviated when the 5.0 μm column was solid phase extraction. -e PPT method was considered the best method in that it was user-friendly, inexpensive, and used. Since gradual accumulation of small amounts of protein and/or particulates might occur and become no- suitable for high-throughput biological sample pretreat- ment. It was also validated in our previous study for the assay ticeable after injection of a large number of samples owing to the incomplete efficiency of protein removal (only about of 18 plasma amino acids by a UHPLC-MS/MS method [38]. By adding a 3-fold volume of precipitator (0.2% FA aceto- 95%∼99%) [39], a wash step of the column was set between different analysis batches. Besides, the use of a column of nitrile solution containing each IS of 400 ng/mL), stable and 5 μm particle size was considered to be less vulnerable optimal recovery as well as matrix effect was achieved, so this pollution than one of 1.8 μm particle size. Based on our pretreatment method was applied in this improved method. previous reports [38], the adding of HFBA could lead to the In all, the quantification of amino acids in human plasma best performance separation for amino acids. -erefore, we by both GC-MS- and LC-MS-based mass spectroscopy was found that 0.02% HFBA and 0.2% FA aqueous solution with well established [23, 24, 28, 31, 32] and would ideally suit our validation study after improvement. As the key differences methanol could result in better separation of compounds and chromatographic peaks shapes and higher signal re- for LC-MS were often in terms of sample preparation and separation parameters, the comparison of those published sponse (S/N) for most analytes. Also, great abundance and Journal of Analytical Methods in Chemistry 7 3 6 ×10 ×10 + MRM (90.00000 -> 44.00000) blank matrix.d + MRM (90.00000 -> 44.00000) blank matrix spiked with amino acids.d ×10 + MRM (90.00000 -> 44.00000) carcinoma tissue.d 2 2 2 2 2 2 2 1.5 2 ←Ala 1.25 ←Ala 1.5 1.5 0.75 1 0.5 0.5 0.5 0.25 0 0 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. acquisition time (min) Counts vs. acquisition time (min) Counts vs. acquisition time (min) 3 6 ×10 ×10 + MRM (90.00000 -> 44.00000) blank matrix.d + MRM (90.00000 -> 44.00000) blank matrix spiked with amino acids.d ×10 + MRM (90.00000 -> 44.00000) carcinoma tissue.d 2 2 2 2 2 2 2 1.25 1.5 1.5 0.75 0.5 0.5 0.5 0.25 sar→ 0 0 0 sar→ 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. acquisition time (min) Counts vs. acquisition time (min) Counts vs. acquisition time (min) 3 6 ×10 ×10 + MRM (132.00000 -> 86.00000) blank matrix.d + MRM (132.00000 -> 86.00000) blank matrix spiked with amino acids.d ×10 + MRM (132.00000 -> 86.00000) carcinoma tissue.d 2 2 2 2 1.2 4 2 2 ←Leu 1 3 ←Leu 0.8 2 0.6 0.4 0.2 0 0 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. acquisition time (min) Counts vs. acquisition time (min) Counts vs. acquisition time (min) 3 6 ×10 ×10 + MRM (132.00000 -> 86.00000) blank matrix.d + MRM (132.00000 -> 86.00000) blank matrix spiked with amino acids.d ×10 + MRM (132.00000 -> 86.00000) carcinoma tissue.d 2 2 2 2 1.2 2 2 3 Ile→ 1 0.8 Ile→ 2 0.6 0.4 0.2 0 0 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. acquisition time (min) Counts vs. acquisition time (min) Counts vs. acquisition time (min) 3 6 ×10 ×10 + MRM (132.00000 -> 86.00000) blank matrix.d + MRM (132.00000 -> 86.00000) blank matrix spiked with amino acids.d ×10 + MRM (132.00000 -> 86.00000) carcinoma tissue.d 2 2 2 2 2 2 2 1.5 1.5 1 3 ←Hpr 0.5 0.5 ←Hpr 0 0 0 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. acquisition time (min) Counts vs. acquisition time (min) Counts vs. acquisition time (min) 2 6 6 ×10 + MRM (147.00000 -> 84.00000) blank matrix.d ×10 + MRM (147.00000 -> 84.00000) blank matrix spiked with amino acids.d ×10 + MRM (147.00000 -> 84.00000) carcinoma tissue.d 2 2 2 2 2 2 6 8 ←Lys 1.5 1 ←Lys 0.5 0 0 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. acquisition time (min) Counts vs. acquisition time (min) Counts vs. acquisition time (min) 2 6 6 ×10 + MRM (147.00000 -> 84.00000) blank matrix.d ×10 + MRM (147.00000 -> 84.00000) blank matrix spiked with amino acids.d ×10 + MRM (147.00000 -> 84.00000) carcinoma tissue.d 2 2 2 2 2 2 6 2 ←Gln 8 1.5 ←Gln 0.5 0 0 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. acquisition time (min) Counts vs. acquisition time (min) Counts vs. acquisition time (min) 2 5 ×10 ×10 + MRM (203.00000 -> 46.00000) blank matrix.d + MRM (203.00000 -> 46.00000) blank matrix spiked with amino acids.d ×10 + MRM (203.00000 -> 46.00000) carcinoma tissue.d 2.5 2 2 2 2 2 2 1.5 3 ←ADMA 2 ←ADMA 1.5 1 0.5 0.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. acquisition time (min) Counts vs. acquisition time (min) Counts vs. acquisition time (min) 2 4 4 ×10 + MRM (203.00000 -> 172.00000) blank matrix.d ×10 + MRM (203.00000 -> 172.00000) blank matrix spiked with amino acids.d ×10 + MRM (203.00000 -> 172.00000) carcinoma tissue.d 2 2 2 2 2 2 2.5 ←SDMA 1.5 ←SDMA 1.5 0.5 1 0.5 0 0 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. acquisition time (min) Counts vs. acquisition time (min) Counts vs. acquisition time (min) (a) (b) (c) Figure 2: -e representative MRM chromatograms of Ala, Sar, Leu, Ile, Hpr, Lys, Gln, ADMA, and SDMA: (a) blank matrix; (b) blank matrix spiked with 34 amino acid and 3 ISs; (c) cancerous tissue sample. 8 Journal of Analytical Methods in Chemistry Table 4: -e retention times, regression equations, coefficients, calibration ranges, and LLOQ for 34 amino acids and each corresponding IS. Calibration range LLOQ Analyte Retention time (min) Regression equation (n � 9) Coefficient R IS (ng/ml) (ng/ml) −5 Gly 1.866 y � 1.973405 ×10 x + 0.013171 0.99434 1000∼80000 1000 Phe-d5 Ala 2.118 y � 0.002292x − 0.471134 0.99538 1000∼80000 1000 Ala-d4 −4 Ser 1.809 y � 1.120434 × 10 x + 0.405218 0.99220 1000∼80000 1000 Phe-d5 Val 3.607 y � 0.003533x − 0.598212 0.99472 1000∼80000 1000 Ala-d4 -r 2.026 y � 0.001422x + 0.129849 0.99604 1000∼80000 1000 Met-d3 Leu 6.402 y � 0.021977x − 7.784039 0.99375 1000∼80000 1000 Met-d3 Ile 5.863 y � 0.022788x − 6.270995 0.99535 1000∼80000 1000 Met-d3 −4 Asp 1.787 y � 4.507537 × 10 x + 0.522395 0.98923 1000∼80000 1000 Met-d3 −4 Lys 2.994 y � 5.106616 × 10 x + 0.021605 0.98157 1000∼80000 1000 Phe-d5 Gln 1.882 y � 0.005491x + 4.126505 0.99513 1000∼80000 1000 Met-d3 Glu 2.066 y � 0.001872x + 0.886773 0.99897 1000∼80000 1000 Met-d3 Phe 7.554 y � 0.006928x − 3.149161 0.99258 1000∼80000 1000 Phe-d5 Arg 3.329 y � 0.001577x + 0.298584 0.99370 1000∼80000 1000 Met-d3 −4 Tyr 4.427 y � 1.241443 × 10 x + 0.056822 0.99250 1000∼80000 1000 Phe-d5 Pro 2.146 y � 0.004142x + 0.052886 0.99646 500∼40000 500 Ala-d4 −4 Asn 1.770 y � 9.372446 × 10 x + 0.471997 0.99304 500∼40000 500 Met-d3 Met 3.636 y � 0.001404x − 0.179621 0.99310 500∼40000 500 Met-d3 −4 Trp 8.755 y � 3.881832 ×10 x − 0.034688 0.99746 500∼40000 500 Phe-d5 −4 Cys 2.056 y � 6.151545 ×10 x − 0.024106 0.99544 100∼8000 100 Met-d3 His 2.695 y � 0.005120x + 0.129674 0.98902 100∼8000 100 Met-d3 Cit 2.139 y � 0.00316x + 0.123514 0.99490 100∼8000 100 Met-d3 −4 ADMA 3.610 y � 2.22466 ×10 x − 0.008033 0.99537 100∼8000 100 Phe-d5 −5 Cyss 1.843 y � 3.122902 × 10 x + 0.007852 0.99611 100∼8000 100 Phe-d5 −4 Sar 1.856 y � 7.269948 ×10 x + 0.017312 0.99724 50∼4000 50 Phe-d5 −4 Apa 2.432 y � 7.290745 ×10 x − 0.009428 0.99649 50∼4000 50 Met-d3 −4 −4 Amp 3.181 y � 1.459612 ×10 x + 8.722727 × 10 0.98729 50∼4000 50 Phe-d5 Aba 2.771 y � 0.005955x − 0.085380 0.99898 50∼4000 50 Met-d3 −4 Opr 2.567 y � 1.048082 × 10 x + 0.035973 0.99634 50∼4000 50 Phe-d5 Hpr 1.759 y � 0.017388x + 0.017864 0.99533 50∼4000 50 Met-d3 Orn 2.676 y � 0.001756x + 0.236473 0.99406 50∼4000 50 Met-d3 Ahd 2.577 y � 0.003022x − 0.039314 0.99902 50∼4000 50 Ala-d4 Hia 7.210 y � 0.001064x − 0.002872 0.99309 50∼4000 50 Phe-d5 −5 SDMA 3.619 y � 7.281465 × 10 x − 0.001614 0.99514 50∼4000 50 Phe-d5 −4 −4 Kyn 6.298 y � 3.698408 ×10 x − 4.537424 ×10 0.99744 50∼4000 50 Phe-d5 LC-MS methods with the UHPLC-MS/MS method estab- concentrations in all samples within the analytical runs. Good correlation coefficients (r> 0.99) were observed for all lished by us could find that (a) our analysis was time-saving and economical and without any derivatization process; (b) analytes in their corresponding calibration ranges. -e re- a unique chromatographic configuration minimized ion gression equations, coefficients, calibration ranges, and suppression and yielded excellent analytic performance, LLOQ for the 34 amino acids and ISs are shown in Table 4. especially for the isomers; and (c) this method could easily be extended to different sample matrices, such as plasma (data 3.2.3. Recovery and Matrix Effect. -e average recovery not shown). results of the 34 amino acids and 3 ISs using QC1 and QC3 samples ranged from 39.00% to 146.95% (RSD 0.44%∼ 3.2. Method Validation Results 7.40%). -e average matrix effect results using the same two samples ranged from 49.45% to 173.63% (RSD 0.61%∼ 3.2.1. Specificity. -e representative total ion current chro- 12.97%), indicating that the extraction procedure was matograms and MRM chromatograms of blank sample, blank consistent and stable (Supplementary Material Table S2). sample spiked with 34 amino acids and 3 ISs, and real CRC samples are shown in Supplementary Material Figure S1. -e retention time of the 34 amino acids is shown in Table 4. No 3.2.4. Inter- and Intraday Accuracy and Precision. -e intra- interfering peaks from endogenous matrix substances were and interday accuracy and precision of this method were shown at the retention time of 34 amino acids and 3 ISs, assessed using the LLOQ, QC1, QC2, and QC3 samples. -e suggesting satisfactory separation and selectivity. deviations (RE%) of intraday ranged from −13.52% to 14.21%, and the RSD were less than 8.57%, while the de- 3.2.2. Linearity of Calibration Curves and LLOQ. -e linear viations of interday ranged from −14.52% to 12.59%, and the equations were regressed to calculate the measured RSD was not more than 10.31%. -e intra- and interday Journal of Analytical Methods in Chemistry 9 ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗ ∗∗∗∗ 700 900 ∗∗∗∗ 600 ∗∗∗∗ ∗∗ ∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ 400 ∗∗∗∗ ∗∗∗∗ ∗∗ ∗∗∗ 400 ∗∗∗ ∗∗ ∗∗ Val Leu Ile Lys Phe Arg Tyr Tc Gly Ala Ser r Asp Gln Glu Tp Tc Tn Tp Tn (a) (b) ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ 600 160 ∗∗∗∗ 320 70 320 70 ∗∗∗∗ 60 ∗∗∗∗ ∗∗∗∗ 240 ∗ 40 ∗∗∗∗ ∗∗∗ 160 ∗∗∗ 0 0 Cys His Cit ADMA Cyss Pro Asn Met Trp Tc Tc Tp Tp Tn Tn (c) (d) ∗∗∗∗ ∗∗ ∗∗ ∗∗∗∗ ∗∗ ∗∗∗∗ 6.0 40 ∗∗∗∗ 4.5 27 ∗∗∗∗ 3.0 ∗∗∗∗ ∗∗∗ 3.0 ∗ ∗∗∗ ∗∗ 2.5 ∗∗∗∗ ∗∗∗∗ 2.0 ∗∗∗∗ ∗∗∗∗ 1.5 1.0 0.5 0.0 SDMA Sar Ahd Amp Hia Kyn Opr Orn Aba Apa Hpr Tc Tc Tp Tp Tn Tn (e) (f) ∗ ∗∗ Figure 3: -e contents of 34 amino acids in cancerous, paracancerous, and normal tissue from 94 CRC patients ( p< 0.05, p< 0.01, ∗∗∗ ∗∗∗∗ p< 0.001, and p< 0.0001). Measured content (μg/g) Measured content (μg/g) Measured content (μg/g) Measured content (μg/g) Measured content (μg/g) Measured content (μg/g) 10 Journal of Analytical Methods in Chemistry Table 5: -e median and SD values of 34 amino acids in cancerous, tumor tissue samples. Figure 3 and Table 5 depict the paracancerous, and normal tissue from 94 CRC patients. contents, median, and SD values of 34 amino acids in cancerous, paracancerous, and normal tissue from 94 CRC Cancerous tissue Paracancerous Normal tissue patients. As expected, the contents of some amino acids were (μg/g) tissue (μg/g) (μg/g) Analyte significantly different between sample types. For example, Median SD Median SD Median SD Gly in Tc sample was markedly higher than that in Tp and Tn Gly 244.454 110.611 156.906 91.526 141.401 79.605 samples (each p< 0.0001). In addition, significantly in- Ala 330.693 163.616 244.684 145.235 177.389 137.482 creased contents were also found in Tc sample for the Asp, Ser 143.072 92.541 124.721 84.070 109.057 78.118 Glu, Pro, Cys, ADMA, Cyss, Kyn, Orn, Aba, Apa, and Hpr Val 141.541 58.259 107.203 56.744 102.171 54.720 (each p< 0.0001). -is supported the trait of altered -r 104.922 63.748 73.603 59.520 67.197 54.334 metabolism of cancer, and it was encouraging that the Leu 181.020 94.785 122.750 84.062 118.304 79.573 Ile 97.020 58.074 71.391 43.525 70.712 41.863 candidate metabolites varied so much between cancer and Asp 107.197 67.386 69.694 44.618 70.323 42.289 normal tissue. However, it was not enough to diagnose the Lys 284.995 231.061 278.033 248.794 255.968 217.480 CRC by the amino acid profiles alone because the samples in Gln 159.779 77.801 170.369 70.657 173.578 64.244 our study were limited, and further research was needed to Glu 335.986 145.846 268.418 116.329 264.143 100.596 evaluate their potential as biomarkers for CRC diagnosis and Phe 127.176 70.195 85.905 58.385 84.676 57.804 treatment. Arg 150.227 97.943 127.102 79.598 119.700 72.975 Tyr 113.370 60.918 88.310 47.617 87.389 46.861 Pro 141.950 152.552 68.731 110.943 65.552 99.701 4. Conclusion Asn 54.833 42.362 42.362 28.168 39.203 25.920 Met 76.329 45.541 63.348 31.676 61.494 32.014 A simple, rapid, sensitive, and efficient targeted UHPLC- Trp 32.690 19.826 27.032 17.460 23.207 17.925 MS/MS method was developed for the determination of 34 Cys 15.593 16.903 10.482 6.979 9.233 6.233 amino acids with analytical time less than 10 min in tumor His 6.044 5.366 4.033 4.267 3.050 4.008 tissues, which was validated for selectivity, linearity, ex- Cit 16.222 16.414 16.281 14.671 17.802 13.330 traction recovery, matrix effect, intra- and interday accuracy ADMA 17.082 11.799 7.841 9.571 6.117 8.310 and precision, and stability. -e use of diluted PBS as the Cyss 26.370 33.970 10.720 14.34 6.713 11.742 “mimic tissue fluid” could prevent serious interference from Sar 0.611 0.490 0.520 0.384 0.491 0.281 endogenous amino acids, which was proved to be simple and Apa 1.934 1.456 0.529 0.266 0.474 0.192 efficient. Enough retention was achieved for the highly polar Amp 0.525 0.474 0.469 0.284 0.478 0.286 amino acid analytes in the C column by using the HFBA, Aba 2.341 2.356 2.069 1.857 1.246 1.745 Opr 2.982 3.705 2.637 2.047 2.514 1.955 an ion-pairing reagent, as the mobile phase addictive, Hpr 2.390 1.385 1.425 0.831 1.282 0.791 without any derivatization procedure. -e one step PPT Orn 1.551 2.266 1.043 1.634 1.101 1.735 method for 100 mg cancer tissue supported the high- Ahd 0.481 0.390 0.745 0.514 0.862 0.458 throughput testing. In summary, this UHPLC-MS/MS Hia 0.656 0.103 0.546 0.220 0.625 0.358 method was successfully applied to plot the profiles of 34 SDMA 1.525 1.093 1.116 1.139 1.145 1.017 amino acids in cancerous, paracancerous, and normal tissue Kyn 7.065 8.108 2.043 1.553 1.536 1.488 from CRC patients, which may be of help for the diagnosis and treatment of CRC in the future. accuracy of the LLOQ sample ranged from −18.03% to Abbreviations 16.99%, and precision was less than 13.25%. -e results are shown in Supplementary Material Table S3. Aba: 4-Aminobutyric acid ADMA: Asymmetric dimethylarginine Ahd: 2-Amino-L-hexanoic diacid 3.2.5. Stability. -e stability results of different conditions are shown in Supplementary Material Table S4. It demon- Ala: L-Alanine Ala-d4: L-Alanine-d4 strated that all the analytes were stable with the accuracy Amp: 3-Amino-2-methylpropanoic acid within ±15% under different conditions. Apa: 3-Aminopropanoic acid Arg: L-Arginine 3.2.6. Dilution Effect. -e dilution effect results showed that Asn: L-Asparagine the accuracy and precision for 8-time dilution were ac- Asp: L-Aspartic acid ceptable (RE ranged from −12.86% to 14.42%, RSD ≤5.46% Cit: L-Citrulline CRC: Colorectal cancer for all the analytes). -e results are shown in Supplementary Material Table S5. Cys: L-Cysteine Cyss: L-Cystine ESI: Electrospray ionization 3.3. Application in Determination of Clinical Samples. FA: Formic acid -is targeted UHPLC-MS/MS method was successfully FDA: Food and drug administration applied to simultaneously determine 34 amino acids in GC-MS: Gas chromatography-mass spectrometry Journal of Analytical Methods in Chemistry 11 authors would like to acknowledge the kind support of Huan Gln: L-Glutamine Man, Jing Chen, Weixiang Liu, Jianhua He, and Dongli Li and Glu: L-Glutamic acid Agilent Technologies Co., Ltd., (Shanghai, China). Gly: Glycine HCl: Hydrochloric acid Supplementary Materials HFBA: Heptafluorobutyric acid Hia: Hippuric acid TABLE S1: the control substances of 34 amino acids and 3 His: L-Histidine ISs. TABLE S2: the extraction recovery and matrix effect of Hpr: 4-Hydroxy-L-proline 34 amino acids and 3 ISs (n = 6). TABLE S3: the intra- and HPLC: High-performance liquid chromatography interday accuracy and precision of 34 amino acids (n = 5). Ile: L-Isoleucine TABLE S4: the stability results of 34 amino acids (n = 3). IS: Internal standard TABLE S5: the dilution effect results of 34 amino acids Kyn: L-Kynurenine (n = 3). FIGURE S1: the representative total ion current LC-MS: Liquid chromatography-mass spectrometry chromatograms and MRM chromatograms of 34 amino Leu: L-Leucine acids and 3 ISs—(a) blank matrix; (b) blank matrix spiked LLOQ: Low limit of quantitation with 34 amino acid and 3 ISs; (c) cancerous tissue sample. 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Simultaneous Determination of 34 Amino Acids in Tumor Tissues from Colorectal Cancer Patients Based on the Targeted UHPLC-MS/MS Method

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Hindawi Journal of Analytical Methods in Chemistry Volume 2020, Article ID 4641709, 12 pages https://doi.org/10.1155/2020/4641709 Research Article Simultaneous Determination of 34 Amino Acids in Tumor Tissues from Colorectal Cancer Patients Based on the Targeted UHPLC-MS/MS Method 1,2,3 1 1 1 1 1 Yang Yang, Feng Zhang, Shouhong Gao, Zhipeng Wang, Mingming Li, Hua Wei, 3 1 Renqian Zhong , and Wansheng Chen Department of Pharmacy, Changzheng Hospital, e Second Military Medical University of CPLA, Shanghai 200003, China Department of Pharmacy, e 71st Group Army Hospital of CPLA Army, e Affiliated Huaihai Hospital of Xuzhou Medical University, Xuzhou 221004, China Department of Laboratory Diagnostics, Changzheng Hospital, e Second Military Medical University of CPLA, Shanghai 200003, China Correspondence should be addressed to Renqian Zhong; zhongrq@smmu.edu.cn and Wansheng Chen; chenwansheng@ smmu.edu.cn Received 17 February 2020; Revised 22 April 2020; Accepted 1 May 2020; Published 1 August 2020 Academic Editor: Boryana M. Nikolova Damyanova Copyright © 2020 Yang Yang et al. -is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A targeted ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was established and validated for the simultaneous determination of 34 amino acids in tissue samples from colorectal cancer (CRC) patients. -e chromatographic separation was achieved on an Agilent ZORBAX SB-C column (3.0 ×150 mm, 5 μm) with a binary gradient elution system (A, 0.02% heptafluorobutyric acid and 0.2% formic acid in water, v/v; B, methanol). -e run time was 10 min. -e multiple reaction monitoring mode was chosen with an electrospray ionization source operating in the positive ionization mode for data acquisition. -e linear correlation coefficients were >0.99 for all the analytes in their corresponding calibration ranges. -e sample was pretreated based on tissue homogenate and protein precipitation with a 100 mg aliquot sample. -e average recovery and matrix effect for 34 amino acids and 3 internal standards were 39.00%∼146.95% and 49.45%∼173.63%, respectively. -e intra- and interday accuracy for all the analytes ranged from −13.52% to 14.21% (RSD ≤8.57%) and from −14.52% to 12.59% (RSD≤10.31%), respectively. Deviations of stability under different conditions were within±15% for all the analytes. -is method was applied to simultaneous quantification of 34 amino acids in tissue samples from 94 CRC patients. in blood, urine, and feces between CRC patients and healthy 1. Introduction volunteers or between CRC tissue and normal tissue. Recent Amino acids play an essential role in both metabolism and findings are related to (1) glycemic and/or ketogenic amino proteome. Proper amino acid management is critical for acids that are involved in the energy metabolism by entering maintaining cell function and organismal viability, partic- tricarboxylic acid cycle pathway; (2) amino acids that are ularly under metabolic disturbance. With the alterations in synthetic precursors of pyrimidine bases or provide nitrogen tumor cell metabolism recognized as a hallmark of cancer, sources for the synthesis of purine bases; and (3) amino acids there have been more research studies on amino acids, which that are the decomposition products from cytosine, uracil, are involved in the primary tumor formation, growth, and and thymine nucleotides and that can reflect the severity of metabolism. As shown in Table 1 [1–18], studies in the field malignant tumor and the therapeutic effect of chemotherapy of colorectal cancer (CRC) metabolism were routinely for malignant tumor [19]. To sum up, details about the performed to elucidate the difference of amino acid profiles association of amino acids with the formation, growth, and 2 Journal of Analytical Methods in Chemistry Table 1: -e summarized variations of amino acids in blood, urine, metabolomic study with increasing levels of sophistication feces, and cancerous tissue of CRC patients. [28–30]. Out of these considerations, thirty-four endoge- nous amino acids in human biological samples were in- Variations Amino volved in our study by using the LC-MS technique. References In In In In acids Amino acid metabolomics in human serum has been ∗ ∗ ∗ blood urine feces tissue studied on a larger scale due to its potential diagnostic value Gly ↓ ↑ [1, 2] in patients with breast, lung, ovarian, head and neck, gastric, Ala ↑ ↑ [3–5] and pancreatic cancers or in CRC patients [31], suggesting Ser ↑ ↓ [3, 6, 7] that the amino acid profiling in plasma/serum or in other Val ↓ ↑ [2, 6, 7] body fluids or selected tissue samples might be a new tool for -r ↓ [2] early diagnosis of cancers [32]. As different cancer subtypes Leu ↓↑ ↑ [3, 5–7] Ile ↑ [6–8] show distinct metabolic phenotypes, the present paper Asp ↓↑ ↓ ↑ [3, 4, 6, 7, 9] aimed to evaluate the amino acid profile in clinical samples Gln ↓ ↑ [3, 8] from CRC patients by means of a targeted metabolomic Glu ↑ ↑ [4, 8, 9] assay based on a UHPLC-MS/MS method, which was Phe ↓ ↑ [3, 10] established and validated for the quantitative analysis of 34 Arg ↑ [6, 7] amino acids in tumor tissues. So far, no research has ever Tyr ↑ ↑ [3, 10] evaluated the ability of the amino acid metabolic phenotype Pro ↓ ↑ ↑ [3, 11, 12] of CRC patients. Additionally, this was the first experiment Met ↓ ↓ [5–7] to show the amino acid profile differences between can- Trp ↑ [3] cerous, paracancerous, and normal tissue of CRC patients, Cys ↑ ↑ [4, 11] His ↓ ↓ [3, 6, 7] which may shed light on the metabolic stability of amino Cit ↑ [3] acids in humans and provide biological information to find ADMA ↑ [13–15] and evaluate a new diagnostic tool for the further study of Cyss ↑ [9] CRC. Aba ↑ [16, 17] Opr ↑ [4] 2. Materials and Methods Hpr ↓ [3] Orn ↑ [3] 2.1. Chemicals and Reagents. -irty-four amino acids, in- Ahd ↓ [5] cluding glycine (Gly), L-alanine (Ala), L-serine (Ser), Hia ↑ ↓ [5, 18] L-valine (Val), L-threonine (-r), L-leucine (Leu), L-iso- SDMA ↑ [14, 15] Kyn ↑ ↓ [9, 18] leucine (Ile), L-aspartic acid (Asp), L-lysine (Lys), L-gluta- mine (Gln), L-glutamic acid (Glu), L-phenylalanine (Phe), Note. CRC patients vs. healthy volunteers; ↑: increase; ↓: decrease; ↓↑: increase and decrease. -e cancerous tissue of CRC patients vs. the normal L-arginine (Arg), L-tyrosine (Tyr), L-proline (Pro), L-as- tissue of CRC patients. paragine (Asn), L-methionine (Met), L-tryptophan (Trp), L-cysteine (Cys), L-histidine (His), L-citrulline (Cit), asymmetric dimethylarginine (ADMA), L-cystine (Cyss), metabolism of CRC are presented in Figure 1. Besides, as a sarcosine (Sar), β-alanine (3-aminopropanoic acid, Apa), higher level of sarcosine was observed in the urine of patients β-aminoisobutyric acid (3-amino-2-methylpropanoic acid, Amp), c-aminobutyric acid (4-aminobutyric acid, Aba), 5- with prostate cancer [20–22], sarcosine was believed to be closely related to cancer and also investigated in our study. oxo-L-proline (Opr), 4-hydroxy-L-proline (Hpr), L-orni- Despite the overlap between the results of the targeted thine (Orn), 2-amino-L-hexanoic diacid (α-aminoadipic and nontargeted metabolomic assays, there were also sub- acid, Ahd), hippuric acid (Hia), symmetric dimethylarginine stantial inconsistencies. Whenever the assessment of a (SDMA), and L-kynurenine (Kyn) were purchased from the specific pathway such as amino acids became the focus of National Institutes for Food and Drug Control of China interest, a targeted metabolomic assay would seem prefer- (Beijing, China), Dalian Meilun Biotech Co., Ltd., (Dalian, able to a nontargeted one [23]. Generally, gas chromatog- China), Shanghai Yuanye Biotech Co., Ltd., (Shanghai, raphy-mass spectrometry (GC-MS) was available for the China), and Sigma-Aldrich LLC. (Darmstadt, Germany). L-Alanine-d4 (Ala-d4), L-methionine-d3 (Met-d3), and amino acid analysis in some laboratories [24–26], but it required a derivatization step for nonvolatile analytes, which L-phenylalanine-d5 (Phe-d5) were chosen as internal was important for the analyte detection and quantification, standards (ISs), which were all provided by Toronto Re- but could be avoided when a liquid chromatography-mass search Chemicals Inc., (Toronto, Canada). -e information spectrometry (LC-MS) method was adopted. Amino acids, about the 34 standards and 3 ISs is shown in Supplementary as a group of polar chemical compounds, should also be Material Table S1. modified by using a derivatization reagent to make them Methanol and acetonitrile of HPLC grade were pur- volatile and thus accessible for GC-MS [27]. Over the past chased from Merck Inc., (Darmstadt, Germany). Phosphate- two decades, many researchers had described the ultrahigh- buffered solution (PBS) (10x, namely, 0.1 mol/L) of the cell performance liquid chromatography-tandem mass spec- culture grade (Lot. MA0016-May-19B) was purchased from trometry- (UHPLC-MS/MS-) based method for a targeted Dalian Meilun Biotech Co., Ltd., (Dalian, China). Journal of Analytical Methods in Chemistry 3 Glycemic and ketogenic amino acids r, Ile, Phe, Tyr, Trp Glycemic amino acids Gly, Ala, Ser, Val, Asn, Asp, Gln, Glu, Arg, Cys, Mat, Pro, His Participation Gly, Gln, Asp Ketogenic amino acids Leu, Lys Synthesis Apa, Amp Decomposition Tricarboxylic acid cycle Nucleotides Material Energy supplementary supplementary Feces CRC tissue Increase or decrease Pro, Cyss Differeces Pro, Ala, Cys, Glu, Opr, Asp, Phe, Tyr, Gly, Aba Urine Normal tissue Increase or decrease Increase or decrease Leu, His, Pro, Asp, Hpr, Gln, Phe, Ser, Ile, Val, Arg, Leu, His, Met, Cit, Orn, Kyn, Cyss, Glu, Val, r, Gly, Ser, Asp, Trp, Ala, Gln, Tyr, Blood Met, Ahd, Hia, ADMA, SDMA Hia, Kyn Figure 1: -e association of amino acids with the formation, growth, and metabolism of CRC. Chromatographic separation was performed on an Agilent Heptafluorobutyric acid (HFBA) (Lot. P11933) was obtained from Adamas Reagents Co., Ltd., (Basel, Switzerland). ZORBAX SB-C column (3.0 × 150 mm, 5 μm), whose Formic acid (FA) (Lot. C10009619) was gained from temperature was maintained at 50 C. Binary gradient elution Shanghai Macklin Biochemical Co., Ltd., (Shanghai, China). was used by mixing the mobile phase A (0.02% HFBA and Hydrochloric acid (HCl) of analytical grade was the product 0.2% FA in water, v/v) and B (methanol) at a flow rate at of Sinopharm Chemical Reagent Co., Ltd., (Shanghai, 0.4 mL/min. -e mobile phase elution procedure was as China). Sodium chloride injection (0.9% saline, 1000 ml/bag, follows (A/B, v/v): 0 min, 98 : 2; 1 min, 85 :15; 4 min, 85 : 15; Lot. 160613) was produced by the pharmaceutical prepa- 5 min, 80 : 20; and 10 min, 20 : 80. -e run time was 10 min ration factory of Shanghai Changzheng Hospital, the Second for each sample, and the post time was set at 4.0 min to Military Medical University (Shanghai, China). Water was equilibrate the column pressure. -e autosampler temper- deionized and further purified by a Milli-Q Plus water ature was maintained at 4 C. -e injected volume was 2 μL purification system (Darmstadt, Germany) in our labora- with needle wash. tory. -e other reagents and solvents were of analytical -e ionization of analytes was performed based on an grade. electrospray ionization (ESI) source under the positive ionization mode, and the data acquisition was carried out in a multiple reaction monitoring (MRM) mode. Optimized 2.2. Liquid Chromatography and Mass Spectrometry mass spectrometer conditions were as follows: capillary Conditions. An Agilent 1290 UHPLC coupled to an Agilent voltage, 5.0 kV; dwell time, 40 ms; collision gas (high-purity 6460 triple-quadrupole tandem mass spectrometer (Agilent nitrogen) pressure, 0.2 MPa; and nebulizer gas (nitrogen) Inc, USA) was used to establish the method. pressure, 50 psi. -e dry gas temperature was 325 C and Colorectal wall 4 Journal of Analytical Methods in Chemistry delivered at 10 L/min. -e sheath gas temperature was 350 C aliquot of the supernatant was transferred to a micro- at the flow rate of 12 L/min. centrifuge tube, and 150 μL of 0.2% FA acetonitrile solution All data were acquired and processed using Agilent Mass (containing 400 ng/mL ISs) was added. -e mixture was Hunter workstation software (version B.07.00). -e opti- centrifuged again under the same conditions after being mized MRM parameters of 34 amino acids and 3 ISs are rested for 3 min and vortex-mixed for 2 min, and 2 μL of summarized in Table 2. supernatant was injected into the UHPLC-MS/MS system for analysis. 2.3. Preparation of Calibration Standards and Quality Control Samples. Stock solutions (2.5 mg/mL) for each analyte were 2.5. Method Validation. Method validation was performed prepared separately and stored at −80 C. Gly, Ala, Ser, Val, according to Chinese Pharmacopoeia [36] and US Food and -r, Leu, Ile, Lys, Phe, Arg, Pro, Met, His, Cit, Sar, Apa, Drug Administration (FDA) guidance [37] and with ref- Amp, Aba, Opr, Hpr, Orn, Hia, and Kyn were dissolved in erence to our previous report [38]. 5% methanol aqueous solutions, and Gln, Glu, and Trp were -e selectivity was evaluated by comparing six different prepared in 0.2% FA aqueous solutions. Asp, Tyr, Asn, Cys, batches of the blank matrix to the corresponding spiked Cyss, Ahd, ADMA, and SDMA were in 4% HCl aqueous samples, and the responses of interferents in the blank solutions. Stock solutions were further diluted with 5% matrix less than 20% of the low limit of quantitation (LLOQ) methanol aqueous solution to obtain the following 4 groups samples and 5% of ISs were considered acceptable. of working solutions. Group A included Ala, Val, -r, Leu, -e calibration standards were prepared in triplicates Ile, Lys, Glu, Phe, Arg, and Tyr (250 μg/mL for every ana- and measured three times on different days (at least 2 days). lyte). Group B included Gly, Ser, Asp, and Gln (250 μg/mL -e calibration curve was regressed from the IS-adjusted for every analyte) and Pro, Asn, Met, and Trp (125 μg/mL for peak area versus the nominal concentration under a 1/X every analyte). Group C included Cys, His, Cit, ADMA, and weighting factor. LLOQ was defined as the lowest concen- Cyss (250 μg/mL for every analyte). Group D included Sar, tration point of the calibration curve. A deviation of Apa, Amp, Aba, Opr, Hpr, Orn, Ahd, Hia, SDMA, and Kyn backcalculation for each calibration standard within ±15% (125 μg/mL for each analyte). was thought to be acceptable, and for LLOQ, the deviation -e highest calibration standard solution was prepared should be within ±20%. by adding appropriate volumes of working solutions group -e recovery and matrix effect were assessed by pre- A∼D into PBS (1x, namely, 0.01 mol/L) using previously paring six replicates of the QC sample at low and high reported methods [33–35]. -en, the other 8 calibration concentration levels. -e matrix effect was the ratio of the standard solutions were obtained by diluting the highest peak area in the spiked postextraction samples to solvent- calibration standard solution with PBS. -e final concen- substituted samples at the same concentration, and the trations of calibration standard solutions were 1000, 2000, recovery was the ratio of the peak area in the spiked samples 4000, 8000, 10000, 20000, 40000, 60000, and 80000 ng/mL to spiked postextraction samples at the same concentration. for Gly, Ala, Ser, Val, -r, Leu, Ile, Asp, Lys, Gln, Glu, Phe, -e intra- and interday accuracy and precision were Arg, and Tyr; 500, 1000, 2000, 4000, 5000, 10000, 20000, assessed using the QC samples at LLOQ, low, medium, and 30000, and 40000 ng/mL for Pro, Asn, Met, and Trp; 100, high concentration levels (n � 5). Samples were analyzed in 200, 400, 800, 1000, 2000, 4000, 6000, and 8000 ng/mL for three analytical lots on separate days (at least 2 days), and the Cys, His, Cit, ADMA, and Cyss; and 50, 100, 200, 400, 500, relative standard deviation (RSD) % for inter- and intraday 1000, 2000, 3000, and 4000 ng/mL for Sar, Apa, Amp, Aba, precision not more than 15% was regarded as acceptable (for Opr, Hpr, Orn, Ahd, Hia, SDMA, and Kyn, respectively. LLOQ, not more than 20%). For intra- and interday ac- Quality control (QC) samples were also separately pre- curacy, the relative error (RE) % within ±15% (for LLOQ, pared in the same way and at low, medium, and high con- within ±20%) was considered reasonable. centrations (QC1∼3). -e low, medium, and high -e stability of each analyte was assessed at three con- concentrations of the QC samples were 2000, 10000, and centration levels (low, medium, and high) using the QC 60000 ng/mL for Gly, Ala, Ser, Val, -r, Leu, Ile, Asp, Lys, samples (n � 3) under four different conditions: room tem- Gln, Glu, Phe, Arg, and Tyr; 1000, 5000, and 30000 ng/mL for perature stability was evaluated after exposing samples at room Pro, Asn, Met, and Trp; 200, 1000, and 6000 ng/mL for Cys, temperature for 6 h; three freeze-thaw cycles stability was His, Cit, ADMA, and Cyss; and 100, 500, and 3000 ng/mL for evaluated after freeze and thaw of samples from −20 C to room Sar, Apa, Amp, Aba, Opr, Hpr, Orn, Ahd, Hia, SDMA, and temperature three times; short-term stability was assessed by Kyn, respectively. All solutions were stored at −20 C. analyzing samples kept in the autosampler (4 C) for 24 h; and long-term stability was evaluated after the samples were stored 2.4. Sample Pretreatment. Each tissue sample with a mass of at −20 C for 90 days. -e dilution effect of all the analytes was assessed by about 100 mg was precisely weighed and added with a 5-fold mass of 0.9% saline before being homogenized by a superfine diluting the sample with a blank matrix into the calibration range and comparing the measured concentrations to the homogenizer at 15000 r/min for 2 min in the ice water bath to obtain tissue homogenate, and then the mixture was nominal concentrations. Each dilution factor should be assessed at least five times, and the RSD% and RE% should centrifuged for 15 min at 19060 ×g at 4 C after five minutes of ultrasonic treatment in the ice water bath. -en, a 50 μL be less than 15% and within ±15%, respectively. Journal of Analytical Methods in Chemistry 5 Table 2: -e optimized MRM parameters of 34 amino acids and 3 ISs (ESI positive). Analyte Molecular weight Precursor ion Product ion Fragmentor (V) Collision energy (eV) Gly 75.07 76 30 50 13 Ala 89.09 90 44 50 8 Ser 105.09 106 60 65 9 Val 117.15 118 72 60 7 -r 119.12 120 74 65 9 Leu 131.17 132 86 65 7 Ile 131.17 132 86 65 7 Asp 133.10 134 74 65 12 Lys 146.19 147 84 70 11 Gln 146.14 147 84 65 12 Glu 147.13 148 84 70 12 Phe 165.19 166 120 65 10 Arg 174.20 175 70 90 16 Tyr 181.19 182 136 70 11 Pro 115.13 116 70 70 15 Asn 132.12 133 74 60 11 Met 149.21 150 56 65 13 Trp 204.23 205 188 70 5 Cys 121.16 122 59 60 21 His 155.15 156 110 80 13 Cit 175.19 176 159 70 7 ADMA 202.25 203 46 90 16 Cyss 240.30 241 152 80 12 Sar 89.09 90 44 55 10 Apa 89.09 90 30 60 12 Amp 103.12 104 30 60 11 Aba 103.12 104 87 65 9 Opr 129.11 130 84 75 12 Hpr 131.13 132 86 75 13 Orn 132.16 133 70 65 10 Ahd 161.16 162 98 65 11 Hia 179.17 180 105 65 6 SDMA 202.25 203 172 90 14 Kyn 208.21 209 192 70 7 Ala-d4 93.12 94 48 50 9 Met-d3 152.23 153 56 65 13 Phe-d5 170.22 171 125 70 11 2.6. Study Population and Sample Collection. -e experi- filter tissue. -e samples were stored at −80 C within cry- mental protocol was reviewed and approved by the Ethical otubes until analysis. Committee of Changzheng Hospital prior to specimen collection, and it was conducted in accordance with the 2.7. Data Analysis. Data were analyzed statistically, and Helsinki Declaration of 1964, as revised in 2013, and according to regulatory guidance. Informed consent was graphs were generated by GraphPad Prism 6.01 for Win- dows (GraphPad Software, Inc., La Jolla, CA, USA). A obtained from all participants enrolled in this study. Between July 2016 and December 2017, 94 patients (male nonparametric test (Friedman test) was performed to compare the content differences of 34 amino acids between 56, female 38) with CRC were enrolled from Changzheng sample sets. -e p value less than 0.05 was considered Hospital. None of the patients received neoadjuvant treat- ment. -e median age of these patients was 60 (ranging from statistically significant. 32 to 87). 10 patients were diagnosed with stage I CRC, 33 patients with stage II, 45 patients with stage III, and 6 pa- 3. Results and Discussion tients with stage IV. -e demographic and clinical chemistry characteristics of these CRC patients are shown in Table 3. 3.1. Method Development. Many studies have published the -e sample set including cancerous, paracancerous, and quantitative analyses of amino acids in plasma normal tissue samples was collected from each of these CRC [23, 24, 28, 31, 32], but these methods have never been patients and was named Tc, Tp, and Tn, respectively. All applied in tissue homogenate. In this study, we developed a samples were immediately washed using 0.9% icy saline robust method for the quantitative analysis of underivatized solution, and the surfaces were subsequently dried by the amino acids in human tissue by UHPLC-MS/MS. 6 Journal of Analytical Methods in Chemistry Table 3: -e demographic and clinical chemistry characteristics of CRC patients. Items Total Male Female Number of patients 94 56 38 Age (median, range) 60, (32∼87) 58, (32∼87) 61, (38∼80) Number of patients with TNM stage Stage I 10 3 7 Stage II 33 18 15 Stage III 45 33 12 Stage IV 6 2 4 As for optimization of ESI-MS/MS conditions, this study high ionization efficiency were obtained in the positive highlighted the importance of quantifying the isomeric ionization mode for all the analytes. analytes using two strategies. For compounds that shared the -e selection of an appropriate matrix for calibration samples and the QC samples preparation was an important same MRM transitions, such as Ala and Sar, modifications of the mobile phase and its gradient, as well as the column part of methodological development when LC-MS was used for the quantitative analysis of endogenous compounds in optimization, were tried to make sure they were completely separated in chromatography. -e coelution of the other two biological samples. -ere were two main approaches to this pairs of standards, such as Leu, Ile, and Hpr and Lys and Gln, problem: the first was to dissolve alternative analytes in the was also avoided at the same time. If the isomeric analytes real matrix and the other was to use real analytes in an had a similar retention time, another MRM transition was alternative matrix [35]. -e ideal substitutive matrix should chosen to separate them on different mass spectrometer be completely analyte-free and identical to the real matrix in channels. For example, the MRM transition for ADMA was terms of analyte solubility and extractability, but it was set at 203/46 instead of 203/70 and 203/172 for SDMA. -e unpractical for the detection of endogenous compounds. In representative MRM chromatograms of Ala, Sar, Leu, Ile, our experiment, plasma was processed using neutral de- colorizing carbon for stripping some endogenous carbo- Hpr, Lys, Gln, ADMA, and SDMA are shown in Figure 2. During the selection of ISs, the ideal condition was an hydrates [40]. -is approach was effective for carbohydrates but not for all amino acids because the prepared plasma still isotope-labeled internal standard for each analyte, but the major problem was the high expense and longer delivery contained a high concentration of amino acids. -erefore, time. Besides, those amino acids were of the same structure, the calibration and the QC samples could be prepared in an so it was acceptable to use a structural analog as the internal artificial matrix only as it was impossible to make an standard for analytes. Furthermore, method verification in “analyte-free” matrix. As human biofluid usually contains a our experiment was currently acceptable as specified by variety of proteins, fatty acids, and electrolytes, which is hard Chinese Pharmacopoeia and FDA guidelines. Hence, the for simulation, some research studies documented that the PBS [33, 34] or mobile phase [41] could be treated as the practical value was the availability of three isotopically la- beled amino acids, which could facilitate application while matrix when the calibration and the QC samples were prepared. In addition, the pH value, osmotic pressure, and making research less expensive. In terms of optimization of chromatographic conditions, ion concentration of PBS were closer to those of the biofluid of humans than the mobile phase [35]. As such, PBS was generally, analysis using sub-2 μm columns yielded a greater (S/N) due to the reduction in band broadening and thus an applied as the “mimic tissue fluid” to prepare both the increase in sensitivity. We actually conducted the analysis by calibration and the QC samples. using reverse-phase LC columns with 1.8 and 5.0 μm When it came to optimization of sample preparation, packing materials, which generated a similar chromato- treatment of the tissue homogenate commonly involved graphic peak resolution. Moreover, ion suppression from the protein precipitation (PPT), liquid-liquid extraction, and coeluting peak was alleviated when the 5.0 μm column was solid phase extraction. -e PPT method was considered the best method in that it was user-friendly, inexpensive, and used. Since gradual accumulation of small amounts of protein and/or particulates might occur and become no- suitable for high-throughput biological sample pretreat- ment. It was also validated in our previous study for the assay ticeable after injection of a large number of samples owing to the incomplete efficiency of protein removal (only about of 18 plasma amino acids by a UHPLC-MS/MS method [38]. By adding a 3-fold volume of precipitator (0.2% FA aceto- 95%∼99%) [39], a wash step of the column was set between different analysis batches. Besides, the use of a column of nitrile solution containing each IS of 400 ng/mL), stable and 5 μm particle size was considered to be less vulnerable optimal recovery as well as matrix effect was achieved, so this pollution than one of 1.8 μm particle size. Based on our pretreatment method was applied in this improved method. previous reports [38], the adding of HFBA could lead to the In all, the quantification of amino acids in human plasma best performance separation for amino acids. -erefore, we by both GC-MS- and LC-MS-based mass spectroscopy was found that 0.02% HFBA and 0.2% FA aqueous solution with well established [23, 24, 28, 31, 32] and would ideally suit our validation study after improvement. As the key differences methanol could result in better separation of compounds and chromatographic peaks shapes and higher signal re- for LC-MS were often in terms of sample preparation and separation parameters, the comparison of those published sponse (S/N) for most analytes. Also, great abundance and Journal of Analytical Methods in Chemistry 7 3 6 ×10 ×10 + MRM (90.00000 -> 44.00000) blank matrix.d + MRM (90.00000 -> 44.00000) blank matrix spiked with amino acids.d ×10 + MRM (90.00000 -> 44.00000) carcinoma tissue.d 2 2 2 2 2 2 2 1.5 2 ←Ala 1.25 ←Ala 1.5 1.5 0.75 1 0.5 0.5 0.5 0.25 0 0 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. acquisition time (min) Counts vs. acquisition time (min) Counts vs. acquisition time (min) 3 6 ×10 ×10 + MRM (90.00000 -> 44.00000) blank matrix.d + MRM (90.00000 -> 44.00000) blank matrix spiked with amino acids.d ×10 + MRM (90.00000 -> 44.00000) carcinoma tissue.d 2 2 2 2 2 2 2 1.25 1.5 1.5 0.75 0.5 0.5 0.5 0.25 sar→ 0 0 0 sar→ 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. acquisition time (min) Counts vs. acquisition time (min) Counts vs. acquisition time (min) 3 6 ×10 ×10 + MRM (132.00000 -> 86.00000) blank matrix.d + MRM (132.00000 -> 86.00000) blank matrix spiked with amino acids.d ×10 + MRM (132.00000 -> 86.00000) carcinoma tissue.d 2 2 2 2 1.2 4 2 2 ←Leu 1 3 ←Leu 0.8 2 0.6 0.4 0.2 0 0 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. acquisition time (min) Counts vs. acquisition time (min) Counts vs. acquisition time (min) 3 6 ×10 ×10 + MRM (132.00000 -> 86.00000) blank matrix.d + MRM (132.00000 -> 86.00000) blank matrix spiked with amino acids.d ×10 + MRM (132.00000 -> 86.00000) carcinoma tissue.d 2 2 2 2 1.2 2 2 3 Ile→ 1 0.8 Ile→ 2 0.6 0.4 0.2 0 0 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. acquisition time (min) Counts vs. acquisition time (min) Counts vs. acquisition time (min) 3 6 ×10 ×10 + MRM (132.00000 -> 86.00000) blank matrix.d + MRM (132.00000 -> 86.00000) blank matrix spiked with amino acids.d ×10 + MRM (132.00000 -> 86.00000) carcinoma tissue.d 2 2 2 2 2 2 2 1.5 1.5 1 3 ←Hpr 0.5 0.5 ←Hpr 0 0 0 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. acquisition time (min) Counts vs. acquisition time (min) Counts vs. acquisition time (min) 2 6 6 ×10 + MRM (147.00000 -> 84.00000) blank matrix.d ×10 + MRM (147.00000 -> 84.00000) blank matrix spiked with amino acids.d ×10 + MRM (147.00000 -> 84.00000) carcinoma tissue.d 2 2 2 2 2 2 6 8 ←Lys 1.5 1 ←Lys 0.5 0 0 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. acquisition time (min) Counts vs. acquisition time (min) Counts vs. acquisition time (min) 2 6 6 ×10 + MRM (147.00000 -> 84.00000) blank matrix.d ×10 + MRM (147.00000 -> 84.00000) blank matrix spiked with amino acids.d ×10 + MRM (147.00000 -> 84.00000) carcinoma tissue.d 2 2 2 2 2 2 6 2 ←Gln 8 1.5 ←Gln 0.5 0 0 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. acquisition time (min) Counts vs. acquisition time (min) Counts vs. acquisition time (min) 2 5 ×10 ×10 + MRM (203.00000 -> 46.00000) blank matrix.d + MRM (203.00000 -> 46.00000) blank matrix spiked with amino acids.d ×10 + MRM (203.00000 -> 46.00000) carcinoma tissue.d 2.5 2 2 2 2 2 2 1.5 3 ←ADMA 2 ←ADMA 1.5 1 0.5 0.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. acquisition time (min) Counts vs. acquisition time (min) Counts vs. acquisition time (min) 2 4 4 ×10 + MRM (203.00000 -> 172.00000) blank matrix.d ×10 + MRM (203.00000 -> 172.00000) blank matrix spiked with amino acids.d ×10 + MRM (203.00000 -> 172.00000) carcinoma tissue.d 2 2 2 2 2 2 2.5 ←SDMA 1.5 ←SDMA 1.5 0.5 1 0.5 0 0 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.56 6.5 7 7.5 8 8.5 9 9.5 1.5 2 2.5 3 3.5 4 4.55 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts vs. acquisition time (min) Counts vs. acquisition time (min) Counts vs. acquisition time (min) (a) (b) (c) Figure 2: -e representative MRM chromatograms of Ala, Sar, Leu, Ile, Hpr, Lys, Gln, ADMA, and SDMA: (a) blank matrix; (b) blank matrix spiked with 34 amino acid and 3 ISs; (c) cancerous tissue sample. 8 Journal of Analytical Methods in Chemistry Table 4: -e retention times, regression equations, coefficients, calibration ranges, and LLOQ for 34 amino acids and each corresponding IS. Calibration range LLOQ Analyte Retention time (min) Regression equation (n � 9) Coefficient R IS (ng/ml) (ng/ml) −5 Gly 1.866 y � 1.973405 ×10 x + 0.013171 0.99434 1000∼80000 1000 Phe-d5 Ala 2.118 y � 0.002292x − 0.471134 0.99538 1000∼80000 1000 Ala-d4 −4 Ser 1.809 y � 1.120434 × 10 x + 0.405218 0.99220 1000∼80000 1000 Phe-d5 Val 3.607 y � 0.003533x − 0.598212 0.99472 1000∼80000 1000 Ala-d4 -r 2.026 y � 0.001422x + 0.129849 0.99604 1000∼80000 1000 Met-d3 Leu 6.402 y � 0.021977x − 7.784039 0.99375 1000∼80000 1000 Met-d3 Ile 5.863 y � 0.022788x − 6.270995 0.99535 1000∼80000 1000 Met-d3 −4 Asp 1.787 y � 4.507537 × 10 x + 0.522395 0.98923 1000∼80000 1000 Met-d3 −4 Lys 2.994 y � 5.106616 × 10 x + 0.021605 0.98157 1000∼80000 1000 Phe-d5 Gln 1.882 y � 0.005491x + 4.126505 0.99513 1000∼80000 1000 Met-d3 Glu 2.066 y � 0.001872x + 0.886773 0.99897 1000∼80000 1000 Met-d3 Phe 7.554 y � 0.006928x − 3.149161 0.99258 1000∼80000 1000 Phe-d5 Arg 3.329 y � 0.001577x + 0.298584 0.99370 1000∼80000 1000 Met-d3 −4 Tyr 4.427 y � 1.241443 × 10 x + 0.056822 0.99250 1000∼80000 1000 Phe-d5 Pro 2.146 y � 0.004142x + 0.052886 0.99646 500∼40000 500 Ala-d4 −4 Asn 1.770 y � 9.372446 × 10 x + 0.471997 0.99304 500∼40000 500 Met-d3 Met 3.636 y � 0.001404x − 0.179621 0.99310 500∼40000 500 Met-d3 −4 Trp 8.755 y � 3.881832 ×10 x − 0.034688 0.99746 500∼40000 500 Phe-d5 −4 Cys 2.056 y � 6.151545 ×10 x − 0.024106 0.99544 100∼8000 100 Met-d3 His 2.695 y � 0.005120x + 0.129674 0.98902 100∼8000 100 Met-d3 Cit 2.139 y � 0.00316x + 0.123514 0.99490 100∼8000 100 Met-d3 −4 ADMA 3.610 y � 2.22466 ×10 x − 0.008033 0.99537 100∼8000 100 Phe-d5 −5 Cyss 1.843 y � 3.122902 × 10 x + 0.007852 0.99611 100∼8000 100 Phe-d5 −4 Sar 1.856 y � 7.269948 ×10 x + 0.017312 0.99724 50∼4000 50 Phe-d5 −4 Apa 2.432 y � 7.290745 ×10 x − 0.009428 0.99649 50∼4000 50 Met-d3 −4 −4 Amp 3.181 y � 1.459612 ×10 x + 8.722727 × 10 0.98729 50∼4000 50 Phe-d5 Aba 2.771 y � 0.005955x − 0.085380 0.99898 50∼4000 50 Met-d3 −4 Opr 2.567 y � 1.048082 × 10 x + 0.035973 0.99634 50∼4000 50 Phe-d5 Hpr 1.759 y � 0.017388x + 0.017864 0.99533 50∼4000 50 Met-d3 Orn 2.676 y � 0.001756x + 0.236473 0.99406 50∼4000 50 Met-d3 Ahd 2.577 y � 0.003022x − 0.039314 0.99902 50∼4000 50 Ala-d4 Hia 7.210 y � 0.001064x − 0.002872 0.99309 50∼4000 50 Phe-d5 −5 SDMA 3.619 y � 7.281465 × 10 x − 0.001614 0.99514 50∼4000 50 Phe-d5 −4 −4 Kyn 6.298 y � 3.698408 ×10 x − 4.537424 ×10 0.99744 50∼4000 50 Phe-d5 LC-MS methods with the UHPLC-MS/MS method estab- concentrations in all samples within the analytical runs. Good correlation coefficients (r> 0.99) were observed for all lished by us could find that (a) our analysis was time-saving and economical and without any derivatization process; (b) analytes in their corresponding calibration ranges. -e re- a unique chromatographic configuration minimized ion gression equations, coefficients, calibration ranges, and suppression and yielded excellent analytic performance, LLOQ for the 34 amino acids and ISs are shown in Table 4. especially for the isomers; and (c) this method could easily be extended to different sample matrices, such as plasma (data 3.2.3. Recovery and Matrix Effect. -e average recovery not shown). results of the 34 amino acids and 3 ISs using QC1 and QC3 samples ranged from 39.00% to 146.95% (RSD 0.44%∼ 3.2. Method Validation Results 7.40%). -e average matrix effect results using the same two samples ranged from 49.45% to 173.63% (RSD 0.61%∼ 3.2.1. Specificity. -e representative total ion current chro- 12.97%), indicating that the extraction procedure was matograms and MRM chromatograms of blank sample, blank consistent and stable (Supplementary Material Table S2). sample spiked with 34 amino acids and 3 ISs, and real CRC samples are shown in Supplementary Material Figure S1. -e retention time of the 34 amino acids is shown in Table 4. No 3.2.4. Inter- and Intraday Accuracy and Precision. -e intra- interfering peaks from endogenous matrix substances were and interday accuracy and precision of this method were shown at the retention time of 34 amino acids and 3 ISs, assessed using the LLOQ, QC1, QC2, and QC3 samples. -e suggesting satisfactory separation and selectivity. deviations (RE%) of intraday ranged from −13.52% to 14.21%, and the RSD were less than 8.57%, while the de- 3.2.2. Linearity of Calibration Curves and LLOQ. -e linear viations of interday ranged from −14.52% to 12.59%, and the equations were regressed to calculate the measured RSD was not more than 10.31%. -e intra- and interday Journal of Analytical Methods in Chemistry 9 ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗ ∗∗∗∗ 700 900 ∗∗∗∗ 600 ∗∗∗∗ ∗∗ ∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ 400 ∗∗∗∗ ∗∗∗∗ ∗∗ ∗∗∗ 400 ∗∗∗ ∗∗ ∗∗ Val Leu Ile Lys Phe Arg Tyr Tc Gly Ala Ser r Asp Gln Glu Tp Tc Tn Tp Tn (a) (b) ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ 600 160 ∗∗∗∗ 320 70 320 70 ∗∗∗∗ 60 ∗∗∗∗ ∗∗∗∗ 240 ∗ 40 ∗∗∗∗ ∗∗∗ 160 ∗∗∗ 0 0 Cys His Cit ADMA Cyss Pro Asn Met Trp Tc Tc Tp Tp Tn Tn (c) (d) ∗∗∗∗ ∗∗ ∗∗ ∗∗∗∗ ∗∗ ∗∗∗∗ 6.0 40 ∗∗∗∗ 4.5 27 ∗∗∗∗ 3.0 ∗∗∗∗ ∗∗∗ 3.0 ∗ ∗∗∗ ∗∗ 2.5 ∗∗∗∗ ∗∗∗∗ 2.0 ∗∗∗∗ ∗∗∗∗ 1.5 1.0 0.5 0.0 SDMA Sar Ahd Amp Hia Kyn Opr Orn Aba Apa Hpr Tc Tc Tp Tp Tn Tn (e) (f) ∗ ∗∗ Figure 3: -e contents of 34 amino acids in cancerous, paracancerous, and normal tissue from 94 CRC patients ( p< 0.05, p< 0.01, ∗∗∗ ∗∗∗∗ p< 0.001, and p< 0.0001). Measured content (μg/g) Measured content (μg/g) Measured content (μg/g) Measured content (μg/g) Measured content (μg/g) Measured content (μg/g) 10 Journal of Analytical Methods in Chemistry Table 5: -e median and SD values of 34 amino acids in cancerous, tumor tissue samples. Figure 3 and Table 5 depict the paracancerous, and normal tissue from 94 CRC patients. contents, median, and SD values of 34 amino acids in cancerous, paracancerous, and normal tissue from 94 CRC Cancerous tissue Paracancerous Normal tissue patients. As expected, the contents of some amino acids were (μg/g) tissue (μg/g) (μg/g) Analyte significantly different between sample types. For example, Median SD Median SD Median SD Gly in Tc sample was markedly higher than that in Tp and Tn Gly 244.454 110.611 156.906 91.526 141.401 79.605 samples (each p< 0.0001). In addition, significantly in- Ala 330.693 163.616 244.684 145.235 177.389 137.482 creased contents were also found in Tc sample for the Asp, Ser 143.072 92.541 124.721 84.070 109.057 78.118 Glu, Pro, Cys, ADMA, Cyss, Kyn, Orn, Aba, Apa, and Hpr Val 141.541 58.259 107.203 56.744 102.171 54.720 (each p< 0.0001). -is supported the trait of altered -r 104.922 63.748 73.603 59.520 67.197 54.334 metabolism of cancer, and it was encouraging that the Leu 181.020 94.785 122.750 84.062 118.304 79.573 Ile 97.020 58.074 71.391 43.525 70.712 41.863 candidate metabolites varied so much between cancer and Asp 107.197 67.386 69.694 44.618 70.323 42.289 normal tissue. However, it was not enough to diagnose the Lys 284.995 231.061 278.033 248.794 255.968 217.480 CRC by the amino acid profiles alone because the samples in Gln 159.779 77.801 170.369 70.657 173.578 64.244 our study were limited, and further research was needed to Glu 335.986 145.846 268.418 116.329 264.143 100.596 evaluate their potential as biomarkers for CRC diagnosis and Phe 127.176 70.195 85.905 58.385 84.676 57.804 treatment. Arg 150.227 97.943 127.102 79.598 119.700 72.975 Tyr 113.370 60.918 88.310 47.617 87.389 46.861 Pro 141.950 152.552 68.731 110.943 65.552 99.701 4. Conclusion Asn 54.833 42.362 42.362 28.168 39.203 25.920 Met 76.329 45.541 63.348 31.676 61.494 32.014 A simple, rapid, sensitive, and efficient targeted UHPLC- Trp 32.690 19.826 27.032 17.460 23.207 17.925 MS/MS method was developed for the determination of 34 Cys 15.593 16.903 10.482 6.979 9.233 6.233 amino acids with analytical time less than 10 min in tumor His 6.044 5.366 4.033 4.267 3.050 4.008 tissues, which was validated for selectivity, linearity, ex- Cit 16.222 16.414 16.281 14.671 17.802 13.330 traction recovery, matrix effect, intra- and interday accuracy ADMA 17.082 11.799 7.841 9.571 6.117 8.310 and precision, and stability. -e use of diluted PBS as the Cyss 26.370 33.970 10.720 14.34 6.713 11.742 “mimic tissue fluid” could prevent serious interference from Sar 0.611 0.490 0.520 0.384 0.491 0.281 endogenous amino acids, which was proved to be simple and Apa 1.934 1.456 0.529 0.266 0.474 0.192 efficient. Enough retention was achieved for the highly polar Amp 0.525 0.474 0.469 0.284 0.478 0.286 amino acid analytes in the C column by using the HFBA, Aba 2.341 2.356 2.069 1.857 1.246 1.745 Opr 2.982 3.705 2.637 2.047 2.514 1.955 an ion-pairing reagent, as the mobile phase addictive, Hpr 2.390 1.385 1.425 0.831 1.282 0.791 without any derivatization procedure. -e one step PPT Orn 1.551 2.266 1.043 1.634 1.101 1.735 method for 100 mg cancer tissue supported the high- Ahd 0.481 0.390 0.745 0.514 0.862 0.458 throughput testing. In summary, this UHPLC-MS/MS Hia 0.656 0.103 0.546 0.220 0.625 0.358 method was successfully applied to plot the profiles of 34 SDMA 1.525 1.093 1.116 1.139 1.145 1.017 amino acids in cancerous, paracancerous, and normal tissue Kyn 7.065 8.108 2.043 1.553 1.536 1.488 from CRC patients, which may be of help for the diagnosis and treatment of CRC in the future. accuracy of the LLOQ sample ranged from −18.03% to Abbreviations 16.99%, and precision was less than 13.25%. -e results are shown in Supplementary Material Table S3. Aba: 4-Aminobutyric acid ADMA: Asymmetric dimethylarginine Ahd: 2-Amino-L-hexanoic diacid 3.2.5. Stability. -e stability results of different conditions are shown in Supplementary Material Table S4. It demon- Ala: L-Alanine Ala-d4: L-Alanine-d4 strated that all the analytes were stable with the accuracy Amp: 3-Amino-2-methylpropanoic acid within ±15% under different conditions. Apa: 3-Aminopropanoic acid Arg: L-Arginine 3.2.6. Dilution Effect. -e dilution effect results showed that Asn: L-Asparagine the accuracy and precision for 8-time dilution were ac- Asp: L-Aspartic acid ceptable (RE ranged from −12.86% to 14.42%, RSD ≤5.46% Cit: L-Citrulline CRC: Colorectal cancer for all the analytes). -e results are shown in Supplementary Material Table S5. Cys: L-Cysteine Cyss: L-Cystine ESI: Electrospray ionization 3.3. Application in Determination of Clinical Samples. FA: Formic acid -is targeted UHPLC-MS/MS method was successfully FDA: Food and drug administration applied to simultaneously determine 34 amino acids in GC-MS: Gas chromatography-mass spectrometry Journal of Analytical Methods in Chemistry 11 authors would like to acknowledge the kind support of Huan Gln: L-Glutamine Man, Jing Chen, Weixiang Liu, Jianhua He, and Dongli Li and Glu: L-Glutamic acid Agilent Technologies Co., Ltd., (Shanghai, China). Gly: Glycine HCl: Hydrochloric acid Supplementary Materials HFBA: Heptafluorobutyric acid Hia: Hippuric acid TABLE S1: the control substances of 34 amino acids and 3 His: L-Histidine ISs. TABLE S2: the extraction recovery and matrix effect of Hpr: 4-Hydroxy-L-proline 34 amino acids and 3 ISs (n = 6). TABLE S3: the intra- and HPLC: High-performance liquid chromatography interday accuracy and precision of 34 amino acids (n = 5). Ile: L-Isoleucine TABLE S4: the stability results of 34 amino acids (n = 3). IS: Internal standard TABLE S5: the dilution effect results of 34 amino acids Kyn: L-Kynurenine (n = 3). FIGURE S1: the representative total ion current LC-MS: Liquid chromatography-mass spectrometry chromatograms and MRM chromatograms of 34 amino Leu: L-Leucine acids and 3 ISs—(a) blank matrix; (b) blank matrix spiked LLOQ: Low limit of quantitation with 34 amino acid and 3 ISs; (c) cancerous tissue sample. 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