Glutathionylated γG and γA subunits of hemoglobin F: a novel post‐translational modification found in extremely premature infants by LC‐MS and nanoLC‐MS/MS

Oxidative stress plays an important role in the development of various disease processes and is a putative mechanism in the development of bronchopulmonary dysplasia, the most common complication of extreme preterm birth. Glutathione, a major endogenous antioxidant and redox buffer, also mediates cellular functions through protein thiolation. We sought to determine if post‐translational thiol modification of hemoglobin F occurs in neonates by examining erythrocyte samples obtained during the first month of life from premature infants, born at 23 0/7 – 28 6/7 weeks gestational age, who were enrolled at our center in the Prematurity and Respiratory Outcomes Program (PROP). Using liquid chromatography‐mass spectrometry (LC‐MS), we report the novel finding of in vivo and in vitro glutathionylation of γG and γA subunits of Hgb F. Through tandem mass spectrometry (nanoLC‐MS/MS), we confirmed the adduction site as the Cys‐γ94 residue and through high‐resolution mass spectrometry determined that the modification occurs in both γ subunits. We also identified glutathionylation of the β subunit of Hgb A in our patient samples; we did not find modified α subunits of Hgb A or F. In conclusion, we are the first to report that glutathionylation of γG and γA of Hgb F occurs in premature infants. Additional studies of this post‐translational modification are needed to determine its physiologic impact on Hgb F function and if sG‐Hgb is a biomarker for clinical morbidities associated with oxidative stress in premature infants. Copyright © 2014 John Wiley & Sons, Ltd.     

Proteomic Analysis of Single Mammalian Cells Enabled by Microfluidic Nanodroplet Sample Preparation and Ultrasensitive NanoLC‐MS

We report on the quantitative proteomic analysis of single mammalian cells. Fluorescence‐activated cell sorting was employed to deposit cells into a newly developed nanodroplet sample processing chip, after which samples were analyzed by ultrasensitive nanoLC‐MS. An average of circa 670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. We demonstrate that the single‐cell proteomics platform can be used to differentiate cell types from enzyme‐dissociated human lung primary cells and identify specific protein markers for epithelial and mesenchymal cells.     

Study on the quality control of cell therapy products. Determination of N-glycolylneuraminic acid incorporated into human cells by nano-flow liquid chromatography/Fourier transformation ion cyclotron mass spectrometry

N-Glycolylneuraminic acid (NeuGc), an acidic nine-carbon sugar, is produced in several animals, such as cattle and mice. Since human cells cannot synthesize NeuGc, it is considered to be immunogenic in humans. Recently, NeuGc contamination was reported in human embryonic stem cells cultured with xenogeneic serum and cells, suggesting that possibly NeuGc may harm the efficacy and safety of cell therapy products. Sialic acids have been determined by derivatization with 1,2-diamino-4,5-methylenedioxybenzene (DMB) followed by liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS); however, the limited availability of cell therapy products requires more sensitive and specific methods for the quality test. Here we studied the use of nano-flow liquid chromatography/Fourier transformation ion cyclotron resonance mass spectrometry (nanoLC/FTMS) and nanoLC/MS/MS for NeuGc-specific determination at a low femtomole level. Using our method, we found NeuGc contamination of the human cell line (HL-60RG cells) cultured with human serum. Our method needs only 2.5x10(3) cells for one injection and would be applicable to the determination of NeuGc in cell therapy products.     

Identification of autoxidation oligomers of flavan‐3‐ols in model solutions by HPLC‐MS/MS

Autoxidation of flavan‐3‐ols was carried out in aqueous/methanol model solutions under mildly acidic conditions (pH 6.0), and these autoxidation products were analyzed by using high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS). The results showed that (+)‐catechins and (?)‐epicatechins generated autoxidation reaction with each other to form a series of oligomers that had the same [M ? H]^? molecular ions (MS¹) as those of natural procyanidins, but had completely different fragment ions (MS²). According to MS/MS analysis, the major fragments of these oligomers were derived not only from the retro‐Diels–Alder (RDA) dissociations on the C‐rings of the flavan‐3‐ol units, but also from the quinone‐methide (QM) cleavage of the interflavan linkages (IFL), and thus they were identified as B‐type dehydrodicatechins, B‐type dehydrotricatechins and A‐type dehydrotricatechins, respectively. The potential structures of their [M ? H]^? molecular ions and partial fragment ions were deduced on the basis of the MS/MS characterization and the oxidation of flavan‐3‐ols in previous reports. Some specific fragment ions were found to be very useful for identifying the autoxidation oligomers (the B‐type dehydrodicatechins at m/z 393, the B‐type dehydrotricatechins at m/z 681 and the A‐type dehydrotricatechins at m/z 725). Copyright © 2008 John Wiley & Sons, Ltd.     

Real time monitoring of drug metabolic enzyme response inside human hepatoma GS‐3A4‐HepG2 cells by means of electrochemical impedance measurement

Cytochrome P‐450s (CYPs) are important biopolymers for the maintenance of cellular function. If metabolic activity of the CYP in the cells can be estimated, so can the function of metabolism, which is closer to the organism. In this research, the method of measuring the drug metabolic activity inside the cell by making use of an electrochemical technique was examined. Human hepatoma GS‐3A4‐HepG2 cells of which the cytochrome P‐4503A4 (CYP3A4) drug metabolic activity is found to be the same as that of primary hepatocytes were used in the experiment. The GS‐3A4‐HepG2 cells were cultured on an indium‐tin oxide (ITO) electrode until they became confluent. Substrate testosterone and inhibitor ketoconazole of CYP3A4 were exposed to cells cultured on an ITO electrode, and the reaction was observed by noting the electrochemical impedance measurement. Impedance was decomposed into the resistance component and the reactance component, and each was examined in detail. As a result, according to testosterone concentration change, there was a remarkable time change in the reactance component. A similar impedance measurement was done by using human hepatoma HepG2 cells in which the drug metabolic activity had extremely decreased. Nevertheless, no time change in the reactance component that was noticed in GS‐3A4‐HepG2 cells was observed. Next, the amount of metabolite in the solution after impedance measurement was measured by means of liquid chromatography‐tandem mass spectroscopy (LC‐MS/MS). In the experiment with GS‐3A4‐HepG2 cells, a testosterone concentration‐dependent correlation was observed between the reactance component change and the amount of metabolite. But, in the impedance measurement by ketoconazole, the change in reactance components was not observed in either the GS‐3A4‐HepG2 cells or the HepG2 cells. Ketoconazole and the heme iron in CYP3A4 effect the coordination bond, but ketoconazole was not metabolized by CYP3A4. It was confirmed that the time change in the reactance component which was caused by the testosterone was detected neither in the cells that take up the substrate, nor in the coordination bond between the CYP enzyme and the drug. Therefore, the time change in the remarkable reactance component observed by this electrochemical impedance measurement is dependent on drug metabolic activity. An electrochemical drug metabolic activity measuring method with the human hepatoma GS‐3A4‐HepG2 cells was able to be established. Copyright © 2004 John Wiley & Sons, Ltd.     

Random poly(3‐hexylthiophene‐co‐3‐cyanothiophene‐co‐3‐(2‐ethylhexyl)thiophene) copolymers with high open‐circuit voltage in polymer solar cells

For the purpose of developing poly(3‐hexylthiophene) (P3HT) based copolymers with deep‐lying highest occupied molecular orbital (HOMO) levels for polymer solar cells with high open‐circuit voltage (V_oc), we report a combined approach of random incorporation of 3‐cyanothiophene (CNT) and 3‐(2‐ethylhexyl)thiophene (EHT) units into the P3HT backbone. This strategy is designed to overcome CNT content limitations in recently reported P3HT‐CNT copolymers, where incorporation of more than 15% of CNT into the polymer backbone leads to impaired polymer solubility and raises the HOMO level. This new approach allows incorporation of a larger CNT content, reaching even lower‐lying HOMO levels. Importantly, a very low HOMO level of ?5.78 eV was obtained, representing one of the lowest HOMO values for exclusively thiophene‐based polymers. Lower HOMO levels result in higher V_oc and higher power conversion efficiencies (PCE) compared to the previously reported P3HT‐CNT copolymers containing only 3‐hexylthiophene and CNT units. As a result, solar cells based on P3HT‐CNT‐EHT(15:15) , which contains 70% of P3HT, 15% of CNT and 15% of EHT, yield a V_oc of 0.83 V in blends with PC₆₁BM while preserving high fill factor (FF) and high short‐circuit current density (J_sc), resulting in 3.6% PCE. Additionally, we explored the effect of polymer number‐average molecular weight (M_n) on the optoelectronic properties and solar cell performance for the example of P3HT‐CNT‐EHT(15:15). The organic photovoltaic (OPV) performance improves with polymer M_n increasing from 3.4 to 6.7 to 9.6 kDa and then it declines as M_n further increases to 9.9 and to 16.2 kDa. The molecular weight study highlights the importance of not only the solar cell optimization, but also the significance of individual polymer properties optimization, in order to fully explore the potential of any given polymer in OPVs. The broader ramification of this study lies in potential application of these high band gap copolymers with low‐lying HOMO level in the development of ternary blend photovoltaics as well as tandem OPV. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 1526–1536     

Synthesis, Crystal Structure and Biological Activities of O, O-Dialkyl α-[1-（2-Chlorothiazol-5-ylmethyl）-5-methyl- 1 H-1,2,3-triazol-4-ylcarbonyloxy]alkylphosphonates

In order to search for novel agrochemicals with high activity and low toxicity, a series of phosphonate derivatives containing 1,2,3-triazole and thiazole rings were designed and synthesized using 2-chloro-5-（chloromethyl）- thiazole as the starting material. Their structures were confirmed by IR, ^1H NMR, ^31p NMR, EI-MS or ESI-MS and elemental analyses. The crystal structure of 7a was determined by single crystal X-ray diffraction. Preliminary bioassays indicated that most of the target compounds did not display insecticidal activities, but a fraction of them possessed herbicidal and fungicidal activities to some extent.     

Light‐Driven Coordination‐Induced Spin‐State Switching: Rational Design of Photodissociable Ligands

The bistability of spin states (e.g., spin crossover) in bulk materials is well investigated and understood. We recently extended spin‐state switching to isolated molecules at room temperature (light‐driven coordination‐induced spin‐state switching, or LD‐CISSS). Whereas bistability and hysteresis in conventional spin‐crossover materials are caused by cooperative effects in the crystal lattice, spin switching in LD‐CISSS is achieved by reversibly changing the coordination number of a metal complex by means of a photochromic ligand that binds in one configuration but dissociates in the other form. We present mathematical proof that the maximum efficiency in property switching by such a photodissociable ligand (PDL) is only dependent on the ratio of the association constants of both configurations. Rational design by using DFT calculations was applied to develop a photoswitchable ligand with a high switching efficiency. The starting point was a nickel–porphyrin as the transition‐metal complex and 3‐phenylazopyridine as the photodissociable ligand. Calculations and experiments were performed in two iterative steps to find a substitution pattern at the phenylazopyridine ligand that provided optimum performance. Following this strategy, we synthesized an improved photodissociable ligand that binds to the Ni–porphyrin with an association constant that is 5.36 times higher in its trans form than in the cis form. The switching efficiency between the diamagnetic and paramagnetic state is efficient as well (72 % paramagnetic Ni–porphyrin after irradiation at 365 nm, 32 % paramagnetic species after irradiation at 440 nm). Potential applications arise from the fact that the LD‐CISSS approach for the first time allows reversible switching of the magnetic susceptibility of a homogeneous solution. Photoswitchable contrast agents for magnetic resonance imaging and light‐controlled magnetic levitation are conceivable applications.     

An Air‐breathing Carbon Cloth‐based Screen‐printed Electrode for Applications in Enzymatic Biofuel Cells

We report a prototype air‐breathing carbon cloth‐based electrode that was fabricated starting from a commercially available screen‐printed electrode equipped with a transparent ITO working electrode (DropSens, ref. ITO10). The fabrication of the air‐breathing electrodes is straightforward, shows satisfactory reproducibility and a good electrochemical response as evaluated by means of [Fe(CN)₆]^3?/4? voltammetry. The gas‐diffusion electrodes were successfully modified with the O₂ reducing enzyme bilirubin oxidase from Myrothecium verrucaria in a direct electron transfer regime. The enzyme modified electrodes showed a remarkable high current density for O₂ reduction in passive air‐breathing mode of up to 5 mA cm^?2. Moreover, the enzyme modified electrodes were applied as O₂ reducing biocathodes in a glucose/air enzymatic biofuel cell in combination with a high current density glucose oxidase/redox polymer bioanode. The biofuel cell provides a high maximum power density of (0.34±0.02) mW cm^?2 at 0.25 V. The straightforward design, low cost and the high reproducibility of these electrodes are considered as basis for standardized measurements under gas‐breathing conditions and for high throughput screening of gas converting (bio‐)catalysts.     

Hyaluronic acid‐dependent protection against alkali‐burned human corneal cells

Hyaluronic acid (HA) is a high‐molecular‐weight glycosaminoglycan and extracellular matrix component that promotes cell proliferation. This study aimed to evaluate the effects of HA on alkali‐injured human corneal epithelial cells in vitro, and to elucidate the mechanisms by which HA mediates corneal cell protection. A human corneal epithelial cell line (HCE‐2) was treated with sodium hydroxide before incubation with low‐molecular‐weight HA (LMW‐HA, 127 kDa) or high‐molecular‐weight HA (HMW‐HA, 1525 kDa). A global proteomic analysis was then performed. Our data indicated that HA treatment protects corneal epithelial cells from alkali injury, and that the molecular weight of HA is a crucial factor in determining its effects. Only HMW‐HA reduced NaOH‐induced cytotoxic effects in corneal cells significantly and increased their migratory and wound healing ability. Results from 2D‐DIGE and MALDI‐TOF/TOF MS analyses indicated that HMW‐HA modulates biosynthetic pathways, cell migration, cell outgrowth, and protein degradation to stimulate wound healing and prevent cell death. To our knowledge, our study is the first to report the possible mechanisms by which HMW‐HA promotes repair in alkali‐injured human corneal epithelial cells.     

Design and Synthesis of α,β‐Epoxyketones as New Anticancer Agents

As epoxy functional group has high anticancer activity, α,β‐epoxyketones were designed and synthesized as new anticancer agents, and their structures were confirmed by UV, ¹H NMR, IR, MS technigeces and elemental analysis. Their in vitro anticancer activities were evaluated by MTT method and the results showed that the compound 4c exhibited good activity with IC₅₀ of 17.8, 22.0 and 24.1 µg/mL against A‐549, Hela and HepG2 cells, respectively. The dose of LD50 of the mice by intragastric administration was 1864.4 mg/kg. Therefore, the α,β‐epoxyketones could potentially provide as new anticancer agents.     

Rapid identification of differentiation markers from whole epithelial cells by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and statistical analysis

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) was applied to identify markers for cellular differentiation. The differentiation of a human colon epithelial carcinoma T84 cell line was monitored over a period of 28 days by transepithelial electrical resistance (TER) measurements, alkaline phosphatase (AP) assay, and MALDI-TOF mass spectral fingerprints combined with statistical analysis. MALDI-MS generated specific mass spectral fingerprints characteristic of cell differentiation. Twenty-two ions were selected as diagnostic signals of fully differentiated T84 cells. Ten protein ion signals, detected by MALDI-MS and validated by statistical analysis, were proposed as T84 cell differentiation markers. Among these signals, ubiquitin was identified as a T84 cell differentiation marker by nanospray liquid chromatography/tandem mass spectrometry (nanoLC/MS/MS). Moreover, depending on the concentration of the cells seeded on the growth support, it was possible to predict the timing of the exponential phase and of cellular differentiation by MALDI-MS-derived marker ions. MALDI-TOFMS was compared to other methods for the determination of cellular differentiation: TER measurements are rapid but yield limited information as to the cellular differentiation state. AP assays are more specific for the differentiation state but take more time. By contrast, MALDI-MS has been found to be a fast, sensitive and precise method for cell differentiation assessment and provides the opportunity for multiplexing and high throughput. Moreover, the consumable costs per assay are very low.     

Application of π‐Extended Ferrocene with Varied Anchoring Groups as Photosensitizers in TiO2‐Based Dye‐Sensitized Solar Cells (DSSCs)

Two new compounds, FcCHNC₆H₄COOH ( 1 ) and FcCHNCH₂CH₂OH ( 2 ) (Fc=C₅H₄FeC₅H₅), have been synthesized and characterized by elemental analyses, IR and ¹H NMR spectroscopy, and ESI‐MS. Attempt has been made to explain their quasi‐reversible redox behavior evidenced by cyclic voltammetry using density functional theory (DFT) calculations. Light‐harvesting properties of both the compounds and also the starting material, FcCHO ( 3 ), have been studied using these compounds as photosensitizers in TiO₂‐based dye‐sensitized solar cells having either a propylene carbonate‐based electrolyte or ionic liquid electrolyte, namely, 1‐propyl‐3‐methyl imidazolium iodide (PMII). Long‐term stability of the photocurrent output of the cell using compound 1 as photosensitizer has been monitored periodically over 1400 h.     

High‐throughput ultra‐high‐performance liquid chromatography/tandem mass spectrometry quantitation of insulin‐like growth factor‐I and leucine‐rich α‐2‐glycoprotein in serum as biomarkers of recombinant human growth hormone administration

Insulin‐like growth factor‐I (IGF‐I) is a known biomarker of recombinant human growth hormone (rhGH) abuse, and is also used clinically to confirm acromegaly. The protein leucine‐rich α‐2‐glycoprotein (LRG) was recently identified as a putative biomarker of rhGH administration. The combination of an ACN depletion method and a 5‐min ultra‐high‐performance liquid chromatography/tandem mass spectrometry (uHPLC/MS/MS)‐based selected reaction monitoring (SRM) assay detected both IGF‐I and LRG at endogenous concentrations. Four eight‐point standard addition curves of IGF‐I (16–2000 ng/mL) demonstrated good linearity (r² = 0.9991 and coefficients of variance (CVs) <13%). Serum samples from two rhGH administrations were extracted and their uHPLC/MS/MS‐derived IGF‐I concentrations correlated well against immunochemistry‐derived values. Combining IGF‐I and LRG data improved the separation of treated and placebo states compared with IGF‐I alone, further strengthening the hypothesis that LRG is a biomarker of rhGH administration. Artificial neural networks (ANNs) analysis of the LRG and IGF‐I data demonstrated an improved model over that developed using IGF‐I alone, with a predictive accuracy of 97%, specificity of 96% and sensitivity of 100%. Receiver operator characteristic (ROC) analysis gave an AUC value of 0.98. This study demonstrates the first large scale and high throughput uHPLC/MS/MS‐based quantitation of a medium abundance protein (IGF‐I) in human serum. Furthermore, the data we have presented for the quantitative analysis of IGF‐I suggest that, in this case, monitoring a single SRM transition to a trypsin peptide surrogate is a valid approach to protein quantitation by LC/MS/MS. Copyright © 2009 John Wiley & Sons, Ltd.     

Application of the ETD/PTR reactions in top‐down proteomics as a faster alternative to bottom‐up nanoLC‐MS/MS protein identification

Our goal was to compare two popular analytical techniques used nowadays in proteomic investigations for proteins/peptides sequencing and identification, a widely used nanoLC‐MS/MS approach applied in the bottom‐up proteomics and electron transfer dissociation/proton transfer reaction fragmentation preferably used when top‐down strategy is applied. Comparison was carried out with the aid of the ESI‐quadrupole ion‐trap instrument using the following criteria: total time of analysis including sample preparation, sequence coverage, Mascot scoring, capability to detect modifications, quality of the results as a function of protein molecular weight and sample consumption. Copyright © 2012 John Wiley & Sons, Ltd.     

Mass spectrometry‐based proteomics of oxidative stress: Identification of 4‐hydroxy‐2‐nonenal (HNE) adducts of amino acids using lysozyme and bovine serum albumin as model proteins

Modification of proteins by 4‐hydroxy‐2‐nonenal (HNE), a reactive by‐product of ω6 polyunsaturated fatty acid oxidation, on specific amino acid residues is considered a biomarker for oxidative stress, as occurs in many metabolic, hereditary, and age‐related diseases. HNE modification of amino acids can occur either via Michael addition or by formation of Schiff‐base adducts. These modifications typically occur on cysteine (Cys), histidine (His), and/or lysine (Lys) residues, resulting in an increase of 156 Da (Michael addition) or 138 Da (Schiff‐base adducts), respectively, in the mass of the residue. Here, we employed biochemical and mass spectrometry (MS) approaches to determine the MS “signatures” of HNE‐modified amino acids, using lysozyme and BSA as model proteins. Using direct infusion of unmodified and HNE‐modified lysozyme into an electrospray quadrupole time‐of‐flight mass spectrometer, we were able to detect up to seven HNE modifications per molecule of lysozyme. Using nanoLC‐MS/MS, we found that, in addition to N‐terminal amino acids, Cys, His, and Lys residues, HNE modification of arginine (Arg), threonine (Thr), tryptophan (Trp), and histidine (His) residues can also occur. These sensitive and specific methods can be applied to the study of oxidative stress to evaluate HNE modification of proteins in complex mixtures from cells and tissues under diseased versus normal conditions.     

In Situ Proteome Profiling and Bioimaging Applications of Small‐Molecule Affinity‐Based Probes Derived From DOT1L Inhibitors

DOT1L is the sole protein methyltransferase that methylates histone H3 on lysine 79 (H3K79), and is a promising drug target against cancers. Small‐molecule inhibitors of DOT1L such as FED1 are potential anti‐cancer agents and useful tools to investigate the biological roles of DOT1L in human diseases. FED1 showed excellent in vitro inhibitory activity against DOT1L, but its cellular effect was relatively poor. In this study, we designed and synthesized photo‐reactive and “clickable” affinity‐based probes (AfBPs), P1 and P2 , which were cell‐permeable and structural mimics of FED1 . The binding and inhibitory effects of these two probes against DOT1L protein were extensively investigated in vitro and in live mammalian cells (in situ). The cellular uptake and sub‐cellular localization properties of the probes were subsequently studied in live‐cell imaging experiments, and our results revealed that, whereas both P1 and P2 readily entered mammalian cells, most of them were not able to reach the cell nucleus where functional DOT1L resides. This offers a plausible explanation for the poor cellular activity of FED1 . Finally with P1 / P2 , large‐scale cell‐based proteome profiling, followed by quantitative LC‐MS/MS, was carried out to identify potential cellular off‐targets of FED1 . Amongst the more than 100 candidate off‐targets identified, NOP2 (a putative ribosomal RNA methyltransferase) was further confirmed to be likely a genuine off‐target of FED1 by preliminary validation experiments including pull‐down/Western blotting (PD/WB) and cellular thermal shift assay (CETSA).     

Scrutinizing Defects and Defect Density of Selenium‐Doped Graphene for High‐Efficiency Triiodide Reduction in Dye‐Sensitized Solar Cells

Understanding the impact of the defects/defect density of electrocatalysts on the activity in the triiodide (I₃^?) reduction reaction of dye‐sensitized solar cells (DSSCs) is indispensable for the design and construction of high‐efficiency counter electrodes (CEs). Active‐site‐enriched selenium‐doped graphene (SeG) was crafted by ball‐milling followed by high‐temperature annealing to yield abundant edge sites and fully activated basal planes. The density of defects within SeG can be tuned by adjusting the annealing temperature. The sample synthesized at an annealing temperature of 900 °C exhibited a superior response to the I₃^? reduction with a high conversion efficiency of 8.42 %, outperforming the Pt reference (7.88 %). Improved stability is also observed. DFT calculations showed the high catalytic activity of SeG over pure graphene is a result of the reduced ionization energy owing to incorporation of Se species, facilitating electron transfer at the electrode–electrolyte interface.     

Determination of l‐tryptophan and l‐kynurenine derivatized with (R)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole by LC‐MS/MS on a triazole‐bonded column and their quantification in human serum

The concentrations of l ‐tryptophan (Trp) and the metabolite l ‐kynurenine (KYN) can be used to evaluate the in‐vivo activity of indoleamine 2,3‐dioxygenase (IDO) and tryptophan 2,3‐dioxygenase (TDO). As such, a novel method involving derivatization of l ‐Trp and l ‐KYN with (R)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS) and separation by high‐performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection on a triazole‐bonded column (Cosmosil HILIC®) was developed to determine their concentrations. The optimized mobile phase, CH₃CN/10 mm ammonium formate in H₂O (pH 5.0) (90:10, v/v) eluted isocratically, resulted in satisfactory separation and MS/MS detection of the analytes. The detection limits of l ‐Trp and l ‐KYN were approximately 50 and 4.0 pm , respectively. The column temperature affected the retention behaviour of the Trp and KYN derivatives, with increased column temperatures leading to increased capacity factors; positive enthalpy changes were revealed by van't Hoff plot analyses. Using the proposed LC‐MS/MS method, l ‐Trp and l ‐KYN were successfully determined in 10 μL human serum using 1‐methyl‐l ‐Trp as an internal standard. The precision and recovery of l ‐Trp were in the ranges 2.85–9.29 and 95.8–113%, respectively, while those of l ‐KYN were 2.51–16.0 and 80.8–98.2%, respectively. The proposed LC‐MS/MS method will be useful for evaluating the in vivo activity of IDO or TDO. Copyright © 2016 John Wiley & Sons, Ltd.