Journal of Chromatography B (v.921-922, #C)
Editorial Board (i).
Contribution to aroma characteristics of mutton process flavor from the enzymatic hydrolysate of sheep bone protein assessed by descriptive sensory analysis and gas chromatography olfactometry by Ping Zhan; Honglei Tian; Xiaoming Zhang; Liping Wang (1-8).
► Sheep bone protein hydrolysates were used as precursor of mutton process flavor (MPF). ► Five sensory notes were selected to assess MPFs using descriptive sensory analysis. ► The corresponding volatile odor-active compounds were analyzed by GC–MS–O. ► We established the PLSR model to show the correlation between samples and others.Changes in the aroma characteristics of mutton process flavors (MPFs) prepared from sheep bone protein hydrolysates (SBPHs) with different degrees of hydrolysis (DH) were evaluated using gas chromatography–mass spectrometry (GC–MS), gas chromatography–olfactometry (GC–O), and descriptive sensory analysis (DSA). Five attributes (muttony, meaty, roasted, mouthful, and simulate) were selected to assess MPFs. The results of DSA showed a distinct difference among the control sample MPF0 and other MPF samples with added SBPHs for different DHs of almost all sensory attributes. MPF5 (DH 25.92%) was the strongest in the muttony, meaty, and roasted attributes, whereas MPF6 (DH 30.89%) was the strongest in the simulate and roasted attributes. Thirty-six compounds were identified as odor-active compounds for the evaluation of the sensory characteristics of MPFs via GC–MS–O analysis. The results of correlation analysis among odor-active compounds, molecular weight, and DSA further confirmed that the SBPH with a DH range of 25.92–30.89% may be a desirable precursor for the sensory characteristics of MPF.
Keywords: Mutton process flavor; Sheep bone protein hydrolysate; Descriptive sensory analysis; Gas chromatography–mass spectrometry–olfactometry analysis; Partial least squares regression;
High throughput LC–MS/MS method for simultaneous determination of zidovudine, lamivudine and nevirapine in human plasma by Valluru Rajani Kumar; B. Phani Bhushana Reddy; B. Ravi Kumar; K. Sreekanth; Kilaru Naveen Babu (9-14).
► LC–MS/MS method for simultaneous determination of zidovudine, lamivudine and nevirapine. ► Increased sensitivity of 5 ng/mL for zidovudine, lamivudine and 10 ng/mL for nevirapine. ► Method utilizes highly selective solid extraction technique with simple sample preparation protocol.A selective and sensitive high performance liquid chromatography–tandem mass spectrometry method has been developed and validated for simultaneous determination of zidovudine (ZDV), lamivudine (3TC) and nevirapine (NVP) in human plasma. After Solid phase extraction (SPE), analytes and ISTDs were run on Peerless Basic C18 column with an injection volume of 3 μL and run time of 3.0 min. An isocratic mobile phase of 0.1% formic acid in water:methanol (15:85, v/v) was used with positive mass spectrometric detection. The method was validated over a concentration range of 5–1500 ng/mL for ZDV and 3TC and over the concentration range of 10–3000 ng/mL for NVP. The intraday and interday precision and accuracy across four validation runs were ranged from 1.6 to 10.1% and 93.8 to 110.8% respectively.
Keywords: Zidovudine (ZDV); Lamivudine (3TC); Nevirapine (NVP); Solid phase extraction (SPE); Internal standard (ISTD);
Rapid and sensitive detection of Cronobacter spp. (previously Enterobacter sakazakii) in food by duplex PCR combined with capillary electrophoresis–laser-induced fluorescence detector by Jia Ruan; Ming Li; Ya-Pan Liu; Yuan-Qian Li; Yong-Xin Li (15-20).
► Cronobacter spp. in food was determined by CE–LIF. ► Multiplex PCR parameters were optimized by CE in a rapid and intuitive way. ► Molecular technique combined with CE–LIF improved specificity and sensitivity. ► 120 infant food formula in China were tested for the presence of Cronobacter spp. ► This method was cheap, environmental friendly and suitable for routine analysis. Cronobacter spp. (Enterobacter sakazakii) is an emerging opportunistic pathogen with a 40–80% mortality rate in infants and immunocompromised crowd resulting from the consumption of contaminated food. A novel method for detecting Cronobacter spp. in food samples by duplex polymerase chain reaction (PCR) in combination with capillary electrophoresis–laser induced fluorescence (CE–LIF) detector has been developed. The specific gene sequences of 16S–23S rDNA internal transcribed spacer (ITS) and the outer membrane protein A (OmpA) of Cronobacter spp. were amplified by duplex PCR. The PCR products were separated and determined sensitively by CE–LIF within 12 min. The relative standard deviations of migration time for the detected DNA fragments were 2.01–2.91%. The detection limit was as low as 1.6 × 101 cfu/mL of Cronobacter spp. Besides, the specificity of the method was verified by 24 non-Cronobacter bacterial strains. A total of 120 commercial infant food formula were tested for the presence of Cronobacter spp. by using the proposed method. This current study demonstrates that the combination of CE–LIF method with duplex PCR is rapid, sensitive and environmental friendly, and has the potential to be adapted for the routine detection of Cronobacter spp. in food samples. To the best of our knowledge, this is the first use of CE–LIF for the detection of Cronobacter spp.
Keywords: Capillary electrophoresis with laser-induced fluorescence detector; Duplex polymerase chain reaction; Cronobacter spp.; Food samples;
Synthesis and characterization of a molecularly imprinted polymer for the determination of trace tributyltin in seawater and seafood by liquid chromatography–tandem mass spectroscopy by Shanshan Zhu; Futao Hu; Ting Yang; Ning Gan; Daodong Pan; Yuting Cao; Dazhen Wu (21-26).
► Imprinted polymer with acrylamide as monomer for adsorbing tributyltin chloride. ► High retention capacity and selectivity for this adsorbent. ► Developing a new analysis approach by coupling this polymer to LC–MS/MS.Analysis of tributyltin chloride (TBT) in environmental samples, such as seawater, is important in order to evaluate the TBT contamination and accumulation in the trophic chain. The environmental impact of organotin compounds has been a particular focus of analytical studies. The present study reports the use of molecular imprinting technology coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS) to determine trace amounts of TBT in seawater and seafood (mussel tissue samples). The imprinted polymer was synthesized by a non-covalent free-radical approach using acrylamide (AM) as a monomer and TBT as a template molecule in acetonitrile solvent (polymerization media). The imprinted polymer synthesized by this approach exhibited good adsorptive capacity and allowed specific retention of TBT. Recoveries of TBT in seawater samples spiked with different TBT concentrations ranged from 67.2% to 81.1% with peak area precision (RSD) < 3.7%, and recoveries of TBT in mussel tissue samples ranged from 75.0% to 94.2% with RSD < 4.8%.
Keywords: Molecular imprinting technology; Tributyltin chloride; Liquid chromatography–tandem mass spectrometry; Seawater; Mussel tissue;
Simultaneous quantification of 22R and 22S epimers of budesonide in human plasma by ultra-high-performance liquid chromatography–tandem mass spectrometry: Application in a stereoselective pharmacokinetic study by Youming Lu; Zuoming Sun; Yifan Zhang; Xiaoyan Chen; Dafang Zhong (27-34).
► Simultaneously determine budesonide epimers in human plasma. ► Resolution and sensitivity of the analytes were highly improved by UHPLC system. ► The chromatography run time was significantly reduced to 7 min.Budesonide (BUD) is used as a mixture of 22R and 22S epimers for the topical treatment of asthma, rhinitis, and inflammatory bowel disease. To study stereoselectivity in the pharmacokinetics of each epimer, we developed a stereoselective and sensitive ultra-high-performance liquid chromatography–tandem mass spectrometry method for the quantitative determination of 22R and 22S epimers of BUD in human plasma. The epimers of BUD were extracted from plasma using n-hexane/dichloromethane/isopropanol (2:1:0.1, v/v/v) under alkaline conditions. Baseline separation was obtained within 7 min on an Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm) column using an isocratic mobile phase consisting of acetonitrile/5 mM ammonium acetate/acetic acid (29:71:0.142, v/v/v) at a flow rate of 0.7 mL/min. Mass spectrometric detection was performed in a multiple reaction monitoring mode using the m/z 489 → 357 transition for BUD epimers and the m/z 497 → 357 transition for the internal standard d8-BUD epimers. Calibration curves were linear over the concentration ranges of 5.0–500 and 5.0–3000 pg/mL for 22R-BUD and 22S-BUD, respectively. The lower limit of quantification was 5.0 pg/mL for both epimers. The method was successfully applied in a pharmacokinetic study of BUD controlled-release capsules in humans. Consistent differences in the pharmacokinetics of the 22R and 22S epimers were observed, the AUC(0–∞) of 22S-BUD was approximately six times higher than that of 22R-BUD, and the 22S-/22R-BUD ratio of total body clearance was 0.17.
Keywords: Ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS); Budesonide epimers; Stereoselective pharmacokinetics;
Simultaneous online SPE–HPLC–MS/MS analysis of docetaxel, temsirolimus and sirolimus in whole blood and human plasma by Alicia Navarrete; M. Paz Martínez-Alcázar; Ignacio Durán; Emiliano Calvo; Belén Valenzuela; Coral Barbas; Antonia García (35-42).
► Online SPE–HPLC–MS/MS method for three antitumor drugs in a combined anticancer therapy. ► Harmonized sample treatment for plasma and whole blood samples. ► Analytical method validated. ► Real samples of plasma and blood from patients under oncologic therapy were measured and results are included.Docetaxel and temsirolimus are some of the most used drugs in a wide range of solid tumors. In preclinical studies, mTOR inhibitors such as temsirolimus have demonstrated synergistic cytotoxic effects with taxanes providing the rationale for combination studies. These anticancer agents exhibit a narrow therapeutic concentration range and due to their high inter- and intra-individual pharmacokinetic variability, therapeutic dose monitoring by highly sensitive methods as LC–MS/MS are important for clinical research. Therefore, the aim of this study was to develop and validate a sensitive, fast and convenient method for the simultaneous identification and quantification of docetaxel, temsirolimus and its main metabolite, sirolimus, using paclitaxel, another anticancer drug, as the internal standard. These analytes were quantified by an integrated online solid phase extraction–high performance liquid chromatography–tandem mass spectrometry (SPE–HPLC–MS/MS) system. Separation was performed on a Zorbax eclipse XDB-C8 (150 mm × 4.6 mm, 5 μm) column. The mass spectrometer tandem quadruple detector was equipped with jet stream electrospray ionization, monitored in multiple reactions monitoring (MRM) and operated in positive mode. A combination of protein precipitation with methanol/zinc sulphate (70:30) (v/v) and online SPE using a Zorbax eclipse plus C8 (12.5 mm × 4.6 mm, 5 μm) cartridge was used to extract the compounds. This method allows the use of the same reagents, sample treatment and analytical technique independently of whether the samples are whole blood or plasma. The method has been successfully validated and applied to real samples. It is a suitable method for dose adjustment and for evaluating potential drug interactions during combined treatments.
Keywords: Solid tumor; Blood; Plasma; Anticancer; LC–MS;
Multicomponent high-performance liquid chromatography/tandem mass spectrometry analysis of ten chemotherapeutic drugs in wipe samples by Shinichiro Maeda; Yoshihiro Miwa (43-48).
► We developed a multicomponent analysis method for 10 common chemotherapy drugs. ► The lower limit of quantification for each drug was 5–50 ng per wipe. ► Our procedure has sufficient sensitivity and is convenient for regular monitoring.Progress in chemotherapy leads to increased numbers and variety of chemotherapeutic drugs, and multicomponent analysis of these drugs is a necessary step. We used liquid chromatography–tandem mass spectrometry and developed a multicomponent analysis of ten drugs used in chemotherapy: vindesine, vincristine, vinblastine, doxorubicin, epirubicin, ifosfamide, cyclophosphamide, irinotecan, docetaxel, and paclitaxel. We selected five internal standards for each category of drug, because the ionization efficiencies of product ions varied widely. The total run time was 22 min, applying a gradient elution of water and acetonitrile in the presence of 0.1% formic acid. The lower limit of quantification was 50 ng/wipe samples for vindesine, vincristine, and vinblastine, and 5 ng/wipe samples for the remaining seven drugs. Accuracy (88.6–112.9%, 85.2–111.7%) and precision (1.0–11.5%CV, 3.6–14.4%CV) in within-run and between-run assays of QC solutions were acceptable. Without outliers, in within-run and between-run assays of QC samples, accuracy was 90.6–113.9% and 91.1–130.4%, respectively, and precision was 2.2–19.0%CV and 4.8–14.9%CV, respectively. Accuracy and precision of High QC samples of irinotecan were deviated. Our analysis procedure has sufficient sensitivity and is convenient enough for regular monitoring.
Keywords: Multicomponent analysis; Chemotherapeutic drug; High-performance liquid chromatography/tandem mass spectrometry; Environmental monitoring; External exposure;
Simultaneous quantification of cefpodoxime proxetil and clavulanic acid in human plasma by LC–MS using solid phase extraction with application to pharmacokinetic studies by Anil Dubala; Janaki Sankarachari Krishnan Nagarajan; Chandran Sathish Vimal; Renjith George (49-55).
► There was no LC–MS method reported for the simultaneous determination of CP and CA in biological samples. ► We developed and validated a rapid and sensitive LC–MS method for simultaneous estimation of CP and CA in human plasma. ► We evaluated the pharmacokinetic variables of tablet (containing CP 200 mg and CA 125 mg) after a single oral dose administration in twelve healthy human volunteers.A simple, rapid and selective high performance liquid chromatography–atmospheric pressure chemical ionization-mass spectrometry (HPLC–APCI-MS) method was developed and validated for the simultaneous estimation of cefpodoxime proxetil (CDPX) and clavulanic acid (CA) in human plasma. Extraction of samples was done by solid phase extraction technique (SPE) and chloramphenicol used as internal standard. Chromatographic separation was carried out on a reverse phase Princeton SPHER C18 (150 mm × 4 mm i.d., 5 μm) column using mixture of methanol: acetonitrile: 2 mM ammonium acetate (25:25:50, v/v, pH 3.5) at 0.8 mL/min flow rate. Detection was performed on a single quadrupole MS by selected ion monitoring (SIM) mode via APCI source. The calibration curve was linear within the concentration range, 0.04–4.4 μg/mL and 0.1–10.0 μg/mL for CDPX and CA respectively. Pharmacokinetic parameters of tablet (CDPX 200 mg, CA 125 mg) were evaluated. C max, T max, T 1/2 , elimination rate constant (K el ), AUC 0–t , and AUC 0–∞ of tablet were 2.13 ± 0.06 μg/mL, 2 h, 3.05 ± 0.15 h, 0.24 ± 0.37 h−1, 6.81 ± 0.14 μg h/mL and 7.72 ± 0.23 μg h/mL respectively for cefpodoxime (CP), 5.34 ± 0.28 μg/mL, 2 h, 2.73 ± 0.25 h, 0.26 ± 0.31 h−1, 15.37 ± 0.16 μg h/mL and 16.59 ± 0.53 μg h/mL respectively for CA.
Keywords: Cefpodoxime proxetil; Clavulanic acid; LC–MS; APCI; Pharmacokinetics;
Comparative validations of non-aqueous capillary electrophoresis and high-performance liquid chromatography methods for the simultaneous determination of histamine H2 receptor antagonists in human urine by Juan José Berzas Nevado; Gregorio Castañeda Peñalvo; Rosa María Rodríguez Dorado; Virginia Rodríguez Robledo (56-63).
► Histamine H2 receptor antagonists are drugs used to block the action of histamine. ► A previously optimised electrophoretic method and HPLC standard method were used. ► Both analytical methods were compared using complete validation procedures. ► Both methods were used to the screening determination of the analytes in human urine.This paper reports a previously optimised method based on non-aqueous capillary electrophoresis (NACE) using UV detection for the separation and simultaneous determination of cimetidine (CIM), ranitidine (RAN), roxatidine (ROX), nizatidine (NIZ) and famotidine (FAM) in human urine. Separation is performed at 25 °C and at a separation voltage of 15 kV. Methanol containing 10 mM ammonium acetate and 0.2% acetic acid was used as background electrolyte, and detection at 214 nm. These conditions allow the five analytes to be separated within 4 min. In addition in the present paper a HPLC method using diode-array as well as detector, was proposed as standard analytical method, which chromatography conditions were following: a mobile phase consisting of 80:20 20 mM phosphate buffer (pH 7.5)/acetonitrile, and using 1 mL min−1 as flow rate of the mobile phase. Detection limits were evaluated on the basis of baseline noise and were establishing between 8 and 15 μg L−1 for NACE and between 16 and 162 μg L−1 for HPLC. The methods showed good precision with overall intra- and inter-day variations of 0.5–2.0% and 0.7–3.8%, respectively. Finally the proposed methods were successfully applied to the screening determination of the analytes in human urine, with recoveries between 97 and 105%, being able the use as pharmacokinetic data in clinical urine samples.
Keywords: Non-aqueous capillary zone electrophoresis; High-performance liquid chromatography; Histamine H2 receptor antagonists; Validation; Robustness/ruggedness; Human urine sample;
A simplified and completely automated workflow for regulated LC–MS/MS bioanalysis using cap-piercing direct sampling and evaporation-free solid phase extraction by Naiyu Zheng; Adela Buzescu; Stephanie Pasas-Farmer; Mark E. Arnold; Zheng Ouyang; Mohammed Jemal; Qianping Peng; Terry Van Vleet; Jianing Zeng (64-74).
► The workflow for regulated bioanalysis is simplified and fully automated. ► Cap-piercing direct sampling and evaporation-free solid phase extraction are used. ► It eliminates manual intervention steps and potential risk of sample spillage. ► Good extraction recoveries, assay sensitivities and raggedness were achieved. ► It has been applied to three regulated bioanalytical assays for incurred samples.Automated sample extraction for regulated bioanalysis by liquid chromatography/tandem mass spectrometry (LC–MS/MS) still presents significant challenges. A new sample preparation methodology with a simplified and completely automated workflow was developed to overcome these challenges using cap piercing for direct biofluid transfer and evaporation-free solid phase extraction (SPE). Using pierceable cap sample tubes, a robotic liquid handler was able to sample without uncapping or recapping during sample preparation. Evaporation for SPE was eliminated by using a mobile phase-compatible elution solvent followed by sample dilution prior to LC–MS/MS analysis. Presented here are three LC–MS/MS assays validated using this methodology to support three CNS drug development programs: (1) BMS-763534 and its metabolite, BMS-790318, in dog plasma; (2) BMS-694153 in monkey plasma; and (3) Pexacerfont (BMS-562086) and two metabolites, BMS-749241 and DPH-123554, in human plasma. These assays were linear from 1.00 to 1000 or 2.00 to 2000 ng/mL for each analyte with excellent assay accuracy, precision and reproducibility. These assays met acceptance criteria for regulated bioanalysis and have been successfully applied to drug development study samples. The methodology described here successfully eliminated all manual intervention steps achieving fully automated sample preparation without compromising assay performance. Importantly, this methodology eliminates the potential exposure of the bioanalyst to any infectious biofluids during sample preparation.
Keywords: Automated biofluid transfer; Evaporation-free SPE; Solid phase extraction; Pierceable caps; LC–MS/MS;
A universal, high recovery assay for protein quantitation through temperature programmed liquid chromatography (TPLC) by Dennis J. Orton; Alan A. Doucette (75-80).
► Temperature programming increases protein recovery above 90% in reversed phase LC. ► 214 nm provides consistent response (by mass) across the proteome. ► LC–UV assay more accurately measures protein concentration versus colorimetric assays. ► LC–UV is applicable to SDS-containing samples, following detergent depletion.As an alternative to direct UV absorbance measurements, estimation of total protein concentration is typically conducted through colorimetric reagent assays. However, for protein-limited applications, the proportion of the sample sacrificed to the assay becomes increasingly significant. This work demonstrates a method for quantitation of protein samples with high recovery. Temperature programmed liquid chromatography (TPLC) with absorbance detection at 214 nm permits accurate estimation of total protein concentration from samples containing as little as 0.75 μg. The method incorporates a temperature gradient from 25 to 80 °C to facilitate elution of total protein into a single fraction. Analyte recovery, as measured from 1 and 10 μg protein extracts of Escherichia coli, is shown to exceed 93%. Extinction coefficients at 214 nm were calculated across the human proteome, providing a relative standard deviation of 21% (versus 42% at 280 nm), suggesting absorbance values at 214 nm provide a more consistent measure of protein concentration. These results translate to a universal protein detection strategy exhibiting a coefficient of variation below 10%. Together with the sensitivity and tolerance to contaminants, TPLC with UV detection is a favorable alternative to colorimetric assay for total protein quantitation, particularly in sample-limited applications.
Keywords: Protein quantitation; UV absorbance; Intact protein; Reversed phase; Temperature programmed liquid chromatography;
A rugged and accurate liquid chromatography–tandem mass spectrometry method for the determination of asunaprevir, an NS3 protease inhibitor, in plasma by Long Yuan; Hao Jiang; Zheng Ouyang; Yuan-Qing Xia; Jianing Zeng; Qianping Peng; Robert W. Lange; Yuzhong Deng; Mark E. Arnold; Anne-Françoise Aubry (81-86).
► Developed and validated a rugged and accurate method for the determination of asunaprevir in plasma. ► A systematic method optimization strategy achieved optimized method and ensured assay quality. ► The validated method was successfully used to support pre-clinical toxicokinetic studies in different species.Asunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) non-structural protein protease inhibitor currently in Phase III clinical trials for the treatment of HCV infection. A rugged and accurate LC–MS/MS method was developed and validated for the quantitation of asunaprevir in rat, dog, monkey, rabbit and mouse plasma. A systematic method screening and optimization strategy was applied to achieve optimized mass spectrometry, chromatography, and sample extraction conditions. The validated method utilized stable-isotope labeled D9-asunaprevir as the internal standard. The samples were extracted by liquid–liquid extraction using 10% ethyl acetate in hexane. Chromatographic separation was achieved with gradient elution on a Waters Atlantis dC18 analytical column. Analyte and its internal standard were detected by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 5.00 to 2000 ng/mL for asunaprevir, was fitted to a 1/x 2 weighted linear regression model. The intra-assay precision was within ±3.6% CV, inter-assay precision was within ±4.0% CV, and the assay accuracy was within ±8.1% of the nominal values in all the species. The method was successfully applied to support multiple pre-clinical toxicokinetic studies in different species.
Keywords: Asunaprevir; BMS-650032; Hepatitis C virus (HCV); Quantitative; LC–MS/MS;
HPLC–UV method for measuring nicotinamide N-methyltransferase activity in biological samples: Evidence for substrate inhibition kinetics by Misha Patel; Muhammad M. Vasaya; Daniel Asker; Richard B. Parsons (87-95).
► 1-Methylnicotinamide is produced from nicotinamide by nicotinamide N-methyltransferase. ► We describe a novel HPLC–UV method for detecting 1-methylnicotinamide in biological samples. ► Requires no sample preparation bar simple deprotonation. ► Linear over two orders of magnitude with LOD and LOQ of 0.05 and 0.15/100 μL respectively. ► We show that nicotinamide N-methyltransferase demonstrates substrate inhibition kinetics.Nicotinamide N-methyltransferase (NNMT, E.C. 184.108.40.206) N-methylates nicotinamide to produce 1-methylnicotinamide. Enhanced NNMT activity is a feature of many types of cancer, and has been linked to processes such as tumour metastasis, resistance to radiotherapy and tumour drug resistance. As such, inhibition of NNMT activity is a promising therapeutic target for cancer therapy. To screen for NNMT inhibitors, there is a need for a standardised, rapid and cost-effective NNMT assay. Here, we describe a cell-free assay coupled with ion-pairing reverse-phase HPLC–UV detection of 1-methylnicotinamide which requires minimal sample manipulation, is linear over 2.5 orders of magnitude with limits of detection and quantification of 0.05 and 0.15 nmol 1-methylnicotinamide/100 μL injection respectively. The assay was sufficiently sensitive to measure basal hepatic 1-methylnicotinamide concentration and NNMT activity in mouse, rabbit and human liver. 1-Methylnicotinamide concentration and the NNMT kinetic parameters specific activity, V max and K m all demonstrated species differences. NNMT also demonstrated substrate inhibition kinetics in all three species, which again was species-specific in term of calculated K i . This assay demonstrates improved sensitivity over other previously published methods whilst lacking many of their drawbacks such as extensive sample preparation, use of non-physiological substrates and radioisotopic labelling.
Keywords: Aldehyde oxidase; HPLC–UV; Nicotinamide N-methyltransferase; 1-Methylnicotinamide; Substrate inhibition;