CE‐ESI‐MS coupled with dynamic pH junction online concentration for analysis of peptides in human urine samples

In this article, an approach has been developed for the analysis of some small peptides with similar pI values by CE‐ESI‐MS based on the online concentration strategy of dynamic pH junction. The factors affected on the separation, detection and online enrichment, such as BGE, injection pressure, sheath flow liquid and separation voltage have been investigated in detail. Under the optimum conditions, i.e. using 0.5 mol/L formic acid (pH 2.15) as the BGE, preparing the sample in 50 mM ammonium acetate solution (pH 7.5), 50 mbar of injection pressure for 300 s, using 7.5 mM of acetic acid in methanol–water (80% v/v) solution as the sheath flow liquid and 20 kV as the separation voltage, four peptides with similar pI values, such as L ‐Ala‐L ‐Ala (pI=5.57), L ‐Leu‐D ‐Leu (pI=5.52), Gly‐D ‐Phe (pI=5.52) and Gly‐Gly‐L ‐Leu (pI=5.52) achieved baseline separation within 18.3 min with detection limits in the range of 0.2–2.0 nmol/L. RSDs of peak migration time and peak area were in the range of 1.45–3.57 and 4.93–6.32%, respectively. This method has been applied to the analysis of the four peptides in the spiked urine sample with satisfactory results.     

新型除草剂及其杂质的LC-MS和GC-MS分析

High performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) have been utilized to analyze the synthesized 2-(2-arylaminomethylphenoxy)pyrimidine derivatives, which are a new kind of environmentally benign herbicides and have passed the temporary pesticide registration. The identification of main product and impurities has been achieved according to the UV and mass spectra. Moreover, one impurity, introduced by the raw material in the last step of the synthetic route, was identified by GC-MS analysis. It can be concluded that the combination of chromatography and mass spectrometry, including LC-MS and GC-MS, provided a vital tool of the pesticide science.     

Selective detection of phosphatidylethanol homologues in blood as biomarkers for alcohol consumption by LC‐ESI‐MS/MS

A new validated method for the quantitation of the abnormal phospholipid phosphatidylethanol (PEth)—a biomarker for ethanol uptake—has been developed by LC‐ESI‐MS/MS following miniaturised organic solvent extraction and reversed phase chromatography with phosphatidylbutanol (PBut) as internal standard. PEth homologues with two fatty acid substituents—PEth 18 : 1/18 : 1, PEth 16 : 0/16 : 0—were determined in post‐mortem blood collected from heavy drinkers at autopsy and also in whole blood samples from a volunteer after a single 60 g‐dose of ethanol. Furthermore, PEth 18 : 1/16 : 0 or its isobaric isomer PEth—16 : 0/18 : 1 was detected. In comparison to previous high‐performance liquid chromatography (HPLC) methods with evaporative light scattering detection (ELSD), the LC‐MS/MS‐method is more sensitive—with a limit of detection below 20 ng/ml—and more selective for single PEth homologues, while ELSD has been used for detection of the sum of PEth homologues with approximately 10 times less sensitivity. LC‐MS/MS enables monitoring of PEth homologues as biomarkers for harmful and prolonged alcohol consumption as with HPLC/ELSD earlier, where PEth is measurable in blood only after more than 50 g ethanol daily intake for more than 2 weeks. Because of its higher sensitivity, there is a potential to detect single heavy drinking by LC‐MS/MS, when PEth is formed in very low concentrations. This opens a new field of application of PEth to uncover single or multiple heavy drinking at a lower frequency and with a larger window of detection in blood than before by HPLC/ELSD or by use of other direct markers, e.g. ethyl glucuronide or ethyl sulfate. Copyright © 2009 John Wiley & Sons, Ltd.     

Laser ablation sample transfer for localized LC‐MS/MS proteomic analysis of tissue

We have developed a mid‐infrared laser ablation sampling technique for nano‐flow liquid chromatography coupled with tandem mass spectrometry proteomic profiling of discrete regions from biological samples. Laser ablation performed in transmission geometry was used to transfer material from 50‐µm thick tissue sections mounted on a glass microscope slide to a capturing solvent. Captured samples were processed using filter‐aided sample preparation and enzymatically digested to produce tryptic peptides for data‐dependent analysis with an ion trap mass spectrometer. Comparison with ultraviolet laser capture microdissection from neighboring regions on the same tissue section revealed that infrared laser ablation transfer has higher reproducibility between samples from different consecutive sections. Both techniques allowed for proteomics investigation of different organelles without the addition of surfactants. Copyright © 2016 John Wiley & Sons, Ltd.     

Metabolic profile of glyburide in human liver microsomes using LC‐DAD‐Q‐TRAP‐MS/MS

The sulfonylurea urea drug glyburide (glibenclamide) is widely used for the treatment of diabetes milletus and gestational diabetes. In previous studies monohydroxylated metabolites were identified and characterized for glyburide in different species, but the metabolite owing to the loss of cyclohexyl ring was identified only in mouse. Glyburide upon incubation with hepatic microsomes resulted in 10 metabolites for human. The current study identifies new metabolites of glyburide along with the hydroxylated metabolites that were reported earlier. The newly identified drug metabolites are dihydroxylated metabolites, a metabolite owing to the loss of cyclohexyl ring and one owing to hydroxylation with dehydrogenation. Among the 10 identified metabolites, there were six monohydroxylated metabolites, one dihydroxylated metabolite, two metabolites owing to hydroxylation and dehydrogenation, and one metabolite owing to the loss of cyclohexyl ring. New metabolites of glyburide were identified and characterized using liquid chromatography–diode array detector–quadruple‐ion trap–mass spectrometry/mass spectrometry (LC‐DAD‐Q‐TRAP‐MS/MS). An enhanced mass scan–enhanced product ion scan with information‐dependent acquisition mode in a Q‐TRAP‐MS/MS system was used to characterize the metabolites. Liquid chromatography with diode array detection was used as a complimentary technique to confirm and identify the metabolites. Metabolites formed in higher amounts were detected in both diode array detection and mass spectrometry detection. Copyright © 2012 John Wiley & Sons, Ltd.     

Doping control analysis of desmopressin in human urine by LC‐ESI‐MS/MS after urine delipidation

The World Anti‐Doping Agency (WADA) has recently added desmopressin, a synthetic analogue of the endogenous peptide hormone arginine vasopressin, to the Prohibited List, owing to the potential masking effects of this drug on hematic parameters useful to detect blood doping. A qualitative method for detection of desmopressin in human urine by high‐performance liquid chromatography–electrospray tandem mass spectrometry (LC‐ESI‐MS/MS) has been developed and validated. Desmopressin purification from urine was achieved by means of delipidation with a 60:40 di‐isopropyl ether/n‐butanol and solid‐phase extraction with WCX cartridges. The lower limit of detection was 25 pg/mL. Extraction recovery was determined as 59.3% (SD 29.4), and signal reduction owing to ion suppression was estimated to be 42.7% (SD 12.9). The applicability of the method was proven by the analysis of real urine samples obtained after intravenous, oral and intranasal administration of desmopressin, achieving unambiguous detection of the peptide in all the cases. Copyright © 2012 John Wiley & Sons, Ltd.     

Bioanalysis of pentoxifylline and related metabolites in plasma samples through LC‐MS/MS

Analytical aspects related to the assay of pentoxifylline (PTX), lisofylline (M1) and carboxypropyl dimethylxanthine (M5) metabolites are discussed through comparison of two alternative analytical methods based on liquid chromatography separation and atmospheric pressure electrospray ionization tandem mass spectrometry detection. One method is based on a ‘pure’ reversed‐phase liquid chromatography mechanism, while the second one uses the additional polar interactions with embedded amide spacers linking octadecyl moieties to the silicagel surface (C‐18 Aqua stationary phase). In both cases, elution is isocratic. Both methods are equally selective and allows separation of unknowns (four species associated to PTX, two species associated to M1) detected through specific mass transitions of the parent compounds and owning respective structural confirmation. Plasma concentration–time patterns of these compounds follow typical metabolic profiles. It has been advanced that in‐vivo formation of conjugates of PTX and M1 is possible, such compounds being cleaved back to the parent ones within the ion source. The first method was associated with a sample preparation procedure based on plasma protein precipitation by strong organic acid addition. The second method used protein precipitation by addition of a water miscible organic solvent. Both analytical methods were fully validated and used to assess bioequivalence between a prolonged release generic formulation and the reference product, under multidose and single dose approaches. Copyright © 2009 John Wiley & Sons, Ltd.     

Studies on the metabolism of the Δ9‐tetrahydrocannabinol precursor Δ9‐tetrahydrocannabinolic acid A (Δ9‐THCA‐A) in rat using LC‐MS/MS,LC‐QTOF MS and GC‐MS techniques

In Cannabis sativa, Δ9‐Tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A) is the non‐psychoactive precursor of Δ9‐tetrahydrocannabinol (Δ9‐THC). In fresh plant material, about 90% of the total Δ9‐THC is available as Δ9‐THCA‐A. When heated (smoked or baked), Δ9‐THCA‐A is only partially converted to Δ9‐THC and therefore, Δ9‐THCA‐A can be detected in serum and urine of cannabis consumers. The aim of the presented study was to identify the metabolites of Δ9‐THCA‐A and to examine particularly whether oral intake of Δ9‐THCA‐A leads to in vivo formation of Δ9‐THC in a rat model. After oral application of pure Δ9‐THCA‐A to rats (15 mg/kg body mass), urine samples were collected and metabolites were isolated and identified by liquid chromatography‐mass spectrometry (LC‐MS), liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and high resolution LC‐MS using time of flight‐mass spectrometry (TOF‐MS) for accurate mass measurement. For detection of Δ9‐THC and its metabolites, urine extracts were analyzed by gas chromatography‐mass spectrometry (GC‐MS). The identified metabolites show that Δ9‐THCA‐A undergoes a hydroxylation in position 11 to 11‐hydroxy‐Δ9‐tetrahydrocannabinolic acid‐A (11‐OH‐Δ9‐THCA‐A), which is further oxidized via the intermediate aldehyde 11‐oxo‐Δ9‐THCA‐A to 11‐nor‐9‐carboxy‐Δ9‐tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A‐COOH). Glucuronides of the parent compound and both main metabolites were identified in the rat urine as well. Furthermore, Δ9‐THCA‐A undergoes hydroxylation in position 8 to 8‐alpha‐ and 8‐beta‐hydroxy‐Δ9‐tetrahydrocannabinolic acid‐A, respectively, (8α‐Hydroxy‐Δ9‐THCA‐A and 8β‐Hydroxy‐Δ9‐THCA‐A, respectively) followed by dehydration. Both monohydroxylated metabolites were further oxidized to their bishydroxylated forms. Several glucuronidation conjugates of these metabolites were identified. In vivo conversion of Δ9‐THCA‐A to Δ9‐THC was not observed. Copyright © 2009 John Wiley & Sons, Ltd.     

Qualitative and quantitative analysis of major constituents from Dazhu Hongjingtian capsule by UPLC/Q‐TOF‐MS/MS combined with UPLC/QQQ‐MS/MS

In this work, a sensitive and efficient method was established and validated for qualitative and quantitative analysis of major bioactive constituents in Dazhu Hongjingtian capsule by liquid chromatography tandem mass spectrometry. A total of 32 compounds were tentatively identified using ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. Furthermore, 12 constituents, namely gallic acid, 3,4‐dihydroxybenzoic acid, salidroside, p‐ coumaric acid‐4‐O ‐β ‐d ‐glucopyranoside, bergeninum, 4‐hydroxybenzoic acid, 4‐hydroxyphenylacetic acid, syringate, 6′′‐O ‐galloylsalidroside, rhodiosin, rhodionin and kaempferol‐7‐O ‐α ‐l ‐rhamnoside, were simultaneously quantified by the developed ultra‐performance liquid chromatography coupled with a triple quadrupole mass spectrometry method in 9 min. All of them were analyzed on an Agilent ZorBax SB‐C₁₈ column (3.0 × 100 mm, 1.8 μm) with linear gradient elution of methanol–0.1% formic acid water. The proposed method was applied to analyze three batches of samples with acceptable linearity (R , 0.9979–0.9997), precision (RSD, 1.3–4.7%), repeatability (RSD, 1.7–4.9%), stability (RSD, 2.2–4.9%) and recovery (RSD, 0.6–4.4%) of the 12 compounds. As a result, the analytical method possessing high throughput and sensitivity is suitable for the quality control of Dazhu Hongjingtian capsule.     

Two‐dimensional LC‐MS/MS to enhance ceramide and phosphatidylcholine species profiling in mouse liver

A two‐dimensional (2D) hydrophilic interaction liquid chromatography (HILIC) and reverse‐phase (RP) liquid chromatography (LC) system coupled with triple‐quadrupole mass spectrometry (MS) was developed to comprehensively profile ceramides and phosphatidylcholine in extracted biological samples. Briefly, the 2D HILIC‐RPLC system used a silica HILIC column operated in the first dimension to distinguish the lipid classes and a BEH C₁₈ column operated in the second dimension to separate the lipid species of the same class. The regression linearity of each lipid was satisfactory in both systems; however, the absolute matrix effect factor was reduced in 2D LC‐MS/MS system. Limits of detection of 2D LC‐MS/MS system were 2‐ to 3‐fold lower compared with one‐dimensional RPLC‐MS/MS. The recovery from the sample ranged from 84.5 to 110%. To summarize, the developed method was proven to be accurate and producible, as relative standard deviations remained <20% at three spiked levels. The efficiency of this newly developed system was applied to measure changes of lipids in the liver of mice after naphthalene treatment. Orthogonal projection to latent structures‐discriminant analysis discriminated the lipids from control and the treatment group. We concluded that 2D LC‐MS/MS is a promising method to assist lipidomic studies of complex biological samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Application of Z‐sinapinic matrix in peptide MALDI‐MS analysis

Since introduction of sinapinic acid (SA) and α‐cyano‐4‐hydroxycinnamic acid as matrices, successful application of matrix‐assisted laser desorption/ionization mass spectrometry started for protein/polypeptides. Both show some limitations in short peptide analysis because matrix clusters are quite abundant. Cinnamics currently used are E‐cinnamics. Here, Z‐SA as matrix for peptides is studied and compared with E‐SA and α‐cyano‐4‐hydroxycinnamic acid. Minor number of clusters is always observed in the low m/z region allowing the detection of short peptides. The results here described show that this novel matrix is a tool of choice for direct, rapid and sensitive detection of hydrophilic and hydrophobic peptides. Copyright © 2017 John Wiley & Sons, Ltd.     

Hydrophilic interaction and reversed‐phase ultra‐performance liquid chromatography TOF‐MS for metabolomic analysis of Veratrum nigrum‐induced cardiotoxicity

The acute cardiotoxicity induced by Veratrum nigrum (VN) is explored by analyzing heart tissue metabolic profiles in mouse models and applying reversed‐phase liquid chromatography mass spectrometry and hydrophilic interaction liquid chromatography mass spectrometry that are based on ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry. An animal model of acute heart injury was established in mice via intra‐gastric administration of VN. Then, electrocardiogram and echocardiograph monitoring of cardiac function and pathological examination were performed on mice in both the control and VN groups, and it was verified that acute heart injury was caused. Meanwhile, comparing the results of the control and VN groups, we detected 36 differential endogenous metabolites of heart tissue, including taurine, riboflavin, purine and lipids, which are related to many possible pathways such as purine metabolism, taurine and hypotaurine metabolism and energy metabolism. Our study provides a scientific approach for evaluating and revealing the mechanisms of VN‐induced cardiotoxicity via the metabolomic strategy.     

Identification and determination of the major constituents in Kai‐Xin‐San by UPLC‐Q/TOF MS and UFLC‐MS/MS method

In order to have overall chemical material information of Kai‐Xin‐San (KXS), the reliable ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometer (UHPLC–Q‐TOF‐MS) and ultra‐fast liquid chromatography mass spectrometer (UFLC‐MS/MS) methods were developed for the identification and determination of the major constituents in KXS. Moreover, the UHPLC–Q‐TOF‐MS method was also applied to screen for multiple absorbed components in rat plasma after oral administration of KXS. The UHPLC–Q‐TOF‐MS method was achieved on Agilent 6520 Q‐TOF mass and operated in the negative ion mode. Good separation was performed on a ZORBAX Eclipse Plus C18 column with a gradient elution at a flow rate of 0.2 ml/min. A total of 92 compounds in KXS were identified or tentatively characterized based on their exact molecular weights, fragmentation patterns, and literature data. A total of 26 compounds including 23 prototype components and three metabolites were identified in rat plasma after oral administration of KXS. Then, 16 major bioactive constituents were chosen as the benchmark substances to evaluate the quality of KXS. Their quantitative analyses were performed by a triple quadrupole tandem mass spectrometer (MS/MS) operating in multiple‐reaction monitoring mode(MRM). The analysis was completed with a gradient elution at a flow rate of 0.4 ml/min within 35 min. The simple and fast method was validated and showed good linearity, precision, and recovery. Furthermore, the method was successful applied for the determination of 16 compounds in KXS. All results would provide essential data for identification and quality control of active chemical constituents in KXS. Copyright © 2016 John Wiley & Sons, Ltd.     

Bacillus licheniformis peptidoglycan characterization by CZE–MS: Assessment with the benchmark RP‐HPLC‐MS method

Peptidoglycan or murein is an essential polymer found in bacterial cell wall. It is a dynamic structure that is continuously remodeled or modified during bacterial cell growth or in presence of cell wall stresses. These modifications are still poorly understood mainly due to the peptidoglycan, which is rather non‐soluble, and the difficulties to separate the hydrophilic glycopeptides (muropeptides) by reversed phase liquid chromatography, generated by the enzymatic digestion using mutanolysin, an N‐acetyl‐muramidase, cleaving the β_1→4 bound between N‐acetylglucosamine and N‐acetylmuramic acid. Here, we report the use of CZE–MS for an easy and fast screening of muropeptides generated by the action of muramidase on the Bacillus licheniformis cell wall. Electron transfer and CID–MS were also used to unambiguously identify and localize the presence or the absence of amidation and acetylation moieties on muropeptide variants. The reference method to analyse muropeptides by reversed phase chromatography was also tested and the advantages and disadvantages of both methods were evaluated.     

Identification of ilaprazole metabolites in human urine by HPLC‐ESI‐MS/MS and HPLC‐NMR experiments

Ilaprazole is a new proton pump inhibitor designed for the treatment of gastric ulcers, and limited data is available on the metabolism of the drug. In this article, the structural elucidation of urinary metabolites of ilaprazole in human was described by HPLC‐ESI‐MS/MS and stopped‐flow HPLC‐NMR experiments. Urinary samples were precipitated by sodium carbonate solution, and then extracted by liquid–liquid extraction after adding ammonium acetate buffer solution. The enriched sample was separated using a C₁₈ reversed‐phase column with the mobile phase composed of acetonitrile and 0.05 mol/L ammonium acetate buffer solution in a gradient solution, and then directly coupled to ESI‐MS/MS detection in an on‐line mode or ¹H‐NMR (500 MHz) spectroscopic detection in a stopped‐flow mode. As a result, four sulfide metabolites, ilaprazole sulfide (M1), 12‐hydroxy‐ilaprazole sulfide (M2), 11,12‐dihydroxy‐ilaprazole sulfide (M3) and ilaprazole sulfide A (M4), were identified by comparing their MS/MS and NMR data with those of the parent drug and available standard compounds. The main biotransformation reactions of ilaprazole were reduction and the aromatic hydroxylation of the parent drug and its relative metabolites. The result testified that HPLC‐ESI‐MS/MS and HPLC‐NMR could be widely applied in detection and identification of novel metabolites. Copyright © 2010 John Wiley & Sons, Ltd.     

Studies on the metabolism and detectability of the emerging drug of abuse diphenyl‐2‐pyrrolidinemethanol (D2PM) in rat urine using GC‐MS and LC‐HR‐MS/MS

In the last years, the number of new psychoactive substances, so‐called ‘legal highs’, has enormously increased. They are sold via online shops often with inaccurate and false information about the content. The aim of this work was to study the metabolism and the detectability of the drug of abuse diphenyl‐2‐pyrrolidinemethanol (D2PM) in rat urine using gas chromatography‐mass spectrometry and liquid chromatography‐high resolution‐tandem mass spectrometry. Five phase I and two phase II metabolites were identified suggesting hydroxylation at the pyrrolidine and diphenyl part as the main metabolic steps. Assuming similar kinetics, an intake of D2PM should be detectable in human urine mainly via its metabolites. Copyright © 2013 John Wiley & Sons, Ltd.     

Identification and structural characterization by LC‐ESI‐IONTRAP and LC‐ESI‐TOF of some metabolic conjugation products of homovanillic acid in urine of neuroblastoma patients

The levels of urinary catecholamine metabolites, such as homovanillic acid (HVA) and vanillylmandelic acid, are routinely used as a clinical tool in the diagnosis and follow‐up of neuroblastoma (NB) patients. Recently, in the Clinical Pathology Laboratory Unit of G. Gaslini Children Hospital, a commercial method that employs liquid chromatography coupled to electrochemical detection (LC‐EC) has been introduced for the measurement of these metabolites in the routine laboratory practice. Using this LC‐EC method, an unknown peak could be observed only in samples derived from NB patients. To investigate the nature of this peak, we used a combination of liquid chromatography‐time‐of‐flight mass spectrometry (LC‐TOF‐MS) and liquid chromatography‐ion trap tandem mass spectrometry (LC‐IT‐MS). The first approach was used to obtain the elemental composition of the ions present in this new signal. To get additional structural information useful for the elucidation of unknown compounds, the ion trap analyzer was exploited. We were able to identify not just one, but three unknown signals in urine samples from NB patients which corresponded to three conjugated products of HVA: HVA sulfate and two glucuronoconjugate isomers. The enzymatic hydrolysis with β‐glucuronidase confirmed the proposed structures, while the selective alkaline hydrolysis allowed us to distinguish the difference between phenol‐ and acyl‐glucuronide of HVA. The latter was the unknown peak observed in LC‐EC separations of urine samples from NB patients. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous determination of six C21 steroids of Xiao‐Ai‐Ping injection in rat plasma by LC‐MS/MS

Xiao‐Ai‐Ping injection (XAPI) is a traditional Chinese medicine that has been widely used to treat cancer. Modern pharmacological studies have demonstrated that C₂₁ steroids are the main active compounds in XAPI. In this study, a sensitive and specific liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed and validated the first time for simultanenous determination of three isomeric pregnane genins (17β‐tenacigenin B, tenacigenin B and tenacigenin A) and their corresponding glycosides (tenacigenoside A, tenacissoside F and marsdenoside I) from XAPI in rat plasma. A simple liquid–liquid extraction technique was used after the addition of dexamethasone acetate as internal standard. The chromatography separation of analytes was achieved on an Agilent Zorbax Eclipse XDB‐C₁₈ column (3.5 µm, 150 × 3 mm i.d.) using methanol–water as mobile phase in a gradient elution program. Detection was performed in multiple reaction monitoring mode using electrospray ionization in the negative ion mode. The method showed satisfactory linearity over a concentration range 5.00–2000.00 ng/mL for tenacigenin B, tenacigenin A, marsdenoside I and tenacissoside F (r² > 0.99), 10.00–4000.00 ng/mL for 17β‐tenacigenin B and tenacigenoside A (r² > 0.99). Intra‐ and inter‐day precisions (valued as relative standard deviation) were <9.00% and accuracies (as relative error) in the range ?6.31 to 7.23%. Finally, this validated method was successfully applied to the pharmacokinetic study of XAPI after intravenous administration to rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Analysis of hydroxyl radical‐induced oxidation process of glucagon by reversed‐phase HPLC and ESI‐MS/MS

Structural modification of a polypeptide hormone, glucagon, by a hydroxyl radical in vitro was investigated by reversed‐phase high‐performance liquid chromatography (RP‐HPLC), and the oxidized site of glucagon was detected by electrospray ionization tandem mass spectrometry (ESI‐MS/MS). It was shown that ²⁷methionine (Met) was oxidized to ²⁷Met sulfoxide by hydroxyl radical, and the production rate of ²⁷Met sulfoxide was faster than that by hydrogen peroxide. In addition, production of ²⁷Met sulfoxide enantiomer was confirmed by RP‐HPLC analysis. cAMP production in a HepG2 cell induced by ²⁷Met sulfoxide glucagon was reduced to approximately 75% as compared with that induced by the native form of glucagon. Copyright © 2009 John Wiley & Sons, Ltd.