Elution behavior of insulin on high-performance size exclusion chromatography at neutral pH

The pharmacopoeia protocol for HP-SEC of insulin, using an acidic non-physiological eluent, does not represent insulin's association state in the formulation. This study aimed to evaluate insulin's elution behavior in HP-SEC in a “physiological” (aqueous, neutral pH) eluent, using on-line UV absorption and multi-angle laser light scattering detection. The effect of insulin concentration and association state in the formulation (monitored by circular dichroism) and eluent composition (zinc ion, arginine) on its elution behavior was assessed. We showed that the elution behavior of insulin in “physiological” HP-SEC is affected by both dynamic association–dissociation of insulin molecules and insulin–column interactions. Insulin molecules re-equilibrated in the HP-SEC eluent, making its elution behavior practically insensitive to the association state of insulin in the formulation. Zinc ions in the eluent promoted association of insulin to hexamers, whereas arginine overruled the effect of zinc ions and induced on-column dissociation of insulin to dimers and monomers. Combined results from “physiological” and compendial HP-SEC were shown to provide a better view of the aggregation state of heat-stressed insulin than either of the single methods. The insights obtained with this study are crucial for a proper evaluation of HP-SEC data of insulin.     

Molecular weights of poly(butyl cyanoacrylate) nanoparticles determined by mass spectrometry and size exclusion chromatography

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and size exclusion chromatography (SEC) were employed to elucidate the chemical composition, mean number average molecular weight (M_n), mean weight average molecular weight (M_w), and polydispersity (PD) of poly(butyl cyanoacrylate) (PBCA) manufactured by emulsion polymerisation. Both methods gave similar results for M_n, but substantial differences were observed for M_w and PD, with MALDI producing consistently lower values which could not be improved by off-line coupling of SEC and MALDI. MALDI gave a more detailed view on the chemical composition of the cyanoacrylate and revealed the presence of two additional polymer series with different end groups besides the expected PBCA series, which showed different retention in SEC. Their formation is explained by the secession/addition of formaldehyde from/to the regular polymer via (reverse) Knoevenagel reaction. In additional experiments, the influence of different pH on PBCA-NP during polymerisation was examined by comparison of polymerisation yield and particle diameter to their chemical composition as revealed by the MALDI spectra. The most uniform nanoparticles, with the highest polymerisation yield, narrowest particle size, and mass distribution were produced at pH 1.     

Absolute molecular weight determination of hypromellose acetate succinate by size exclusion chromatography: use of a multi angle laser light scattering detector and a mixed solvent

Molecular weight distribution is an important quality attribute for hypromellose acetate succinate (HPMCAS), a pharmaceutical excipient used in spray-dried dispersions. Our previous study showed that neither relative nor universal calibration method of size exclusion chromatography (SEC) works for HPMCAS polymers. We here report our effort to develop a SEC method using a mass sensitive multi angle laser light scattering detector (MALLS) to determine molecular weight distributions of HPMCAS polymers. A solvent screen study reveals that a mixed solvent (60:40%, v/v 50 mM NaH₂PO₄ with 0.1 M NaNO₃ buffer: acetonitrile, pH* 8.0) is the best for HPMCAS-LF and MF sub-classes. Use of a mixed solvent creates a challenging condition for the method that uses refractive index detector. Therefore, we thoroughly evaluated the method performance and robustness. The mean weight average molecular weight of a polyethylene oxide standard has a 95% confidence interval of (28,443-28,793) g/mol vs. 28,700 g/mol from the Certificate of Analysis. The relative standard deviations of average molecular weights for all polymers are 3-6%. These results and the Design of Experiments study demonstrate that the method is accurate and robust.     

聚乳酸分子量对利福平聚乳酸微球性质的影响

目的 :考察聚乳酸分子量对利福平聚乳酸微球性质的影响。方法 :采用分散一溶媒扩散法制备利福平聚乳酸微球 ,测定微球的粒径分布和包封率 ,进行体外释药和稳定性试验。结果 :在本制备方法中 ,聚乳酸分子量对微球粒径分布的影响作用不明显 ;药物包封率随聚乳酸分子量增大而增加 ;聚乳酸分子量减小 ,微球体外释药速度加快。稳定性试验表明 ,微球在 4℃和室温 (2 0～ 2 5℃ )条件下性质稳定 :3 7℃条件下因聚乳酸软化 ,微球发生粘连聚集。结论 :应根据实验目的选择适宜分子量的聚乳酸 ,以获得所需性质的微球。     

Simultaneous quantification of eight organic acid components in Artemisia capillaris Thunb (Yinchen) extract using high-performance liquid chromatography coupled with diode array detection and high-resolution mass spectrometry

We aim to determine the chemical constituents of Yinchen extract and Yinchen herbs using high-performance liquid chromatography coupled with diode array detection and high-resolution mass spectrometry. The method was developed to analyze of eight organic acid components of Yinchen extract (including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, 1,3-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid). The separation was conducted using an Agilent TC-C18 column with acetonitrile – 0.2% formic acid solution as the mobile phases under gradient elution. The analytical method was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery, and subsequently the method was performed for the quantitative assessment of Yinchen extracts and Yinchen herbs. In addition, the changes of selected markers were studied when Yinchen herbs decocting in water and isomerization occurred between the chlorogenic acids. The proposed method enables both qualitative and quantitative analyses and could be developed as a new tool for the quality evaluation of Yinchen extract and Yinchen herbs. The changes of selected markers in water decoction process could give us some novel idea when studying the link between substances and drug efficacy.     

分子排阻法测定国产注射用替卡西林钠克拉维酸钾中的高分子聚合物

目的 测定注射用替卡西林钠克拉维酸钾中的替卡西林聚合物.方法 采用Sephadex G-10色谱柱(300 mm×15mm),流动相A为pH8的50 mmol·L-1磷酸盐缓冲液,流动相B为超纯水,流速1.0 mL·min-1,检测波长254 nm,进样量100μL.结果 替卡西林中高分子聚合物与替卡西林药物单体能较好地分离,19.98 ～199.83 μg·mL-1替卡西林对照品与其峰面积呈良好的线性关系(r =0.9999),定量限为248 ng,方法精密度良好(RSD=0.52％,n=5),样品测定的重复性良好(RSD=2.33％,n＝3).结论 所用方法简便、准确、重复性好,适用于注射用替卡西林钠克拉维酸钾中高分子聚合物的检测.     

Development of a sensitive size exclusion HPLC method with fluorescence detection for the quantitation of recombinant human erythropoietin (r-HuEPO) aggregates

Human erythropoietin produced by recombinant DNA technology, is now marketed worldwide for the treatment of anemias associated with chronic renal failure and chemotherapy. No sensitive methods, which can determine r-HuEPO dimer or oligomer aggregate content in formulated products, have been published to date. This report describes the development and validation of a sensitive size exclusion high performance liquid chromatography (HPLC) method for the quantitation of r-HuEPO aggregates in formulations containing 0.03% polysorbate 80. A Waters Alliance 2690 HPLC system connected to a TosoHaas TSKgel G3000 SWxl (7.8 mm x 30 cm, 250 A pore size, 5 microm particle size) column and a Waters 474 fluorescence detector was used. The mobile phase for the SEC-HPLC method consists of isopropyl alcohol-potassium phosphate (0.1 M)/potassium chloride buffer (pH 6.8+/-0.1, 0.2 M) (25:75, v/v). The flow rate was 0.3 mL/min and the method run time was 60 min. The SEC-HPLC method presented here was shown to be specific for r-HuEPO total aggregates (dimer and oligomers) and allows for their quantitation at 80 ng/mL or 4 ngs/injection, in the presence of r-HuEPO monomer and the pharmaceutical excipients, glycine (5 mg/mL), sodium chloride (4.3 mg/mL), and 0.03% polysorbate 80. The finalized method is stability-indicating and is suitable for determining r-HuEPO aggregates between 0.2 and 0.5% levels in the formulated product of r-HuEPO. This method offers a robust way to measure total aggregates on a routine basis with a high sensitivity for use in product quality control.     

Size exclusion chromatography with evaporative light scattering detection as a method for speciation analysis of polydimethylsiloxanes. II. Validation of the method for analysis of pharmaceutical formulations

The aim of this study was to demonstrate the usefulness of the size exclusion chromatography with evaporative light scattering detection (SEC-ELSD) method in the identification and quantitative analysis of polydimethylsiloxanes (PDMS). The process of validation for the method was conducted, and the values obtained were compared with the acceptance criteria. Particularly important was the conclusion that SEC-ELSD method showed a high specificity for PDMS. PDMS is an organosilicon polymer and for this reason, it does not exist as a concrete chemical species. Depending on the length of the chain, PDMS can be toxic for organism. So far, the SEC-ELSD method has not been applied for the control of pharmaceutical products containing such PDMS as dimeticone or simeticone. The safety of use and effectiveness of such pharmaceutical products relies on the control of their quality. Therefore, the analytical methods and procedures that meet acceptance criteria for qualitative and quantitative analysis of the PDMS should be used. In the case of the analysis of pharmaceutical products, the acceptance criteria are established and recommended by, for example, the Pharmacopoeias, the U.S. Food and Drug Administration (FDA), the International Conference on Harmonisation (ICH) and the World Health Organization (WHO). The progress of knowledge, however, requires the development of new analytical tools which are able to solve incoming problems. In the case of pharmaceutical formulations containing PDMS, which are used not only by adults but also by children, it is necessary to use analytical methods which are characterized by a high specificity.     

Determination of surface-bound hydroxypropylcellulose (HPC) on drug particles in colloidal dispersions using size exclusion chromatography: a comparison of ELS and RI detection

Evaporative light scattering (ELS) and refractive index (RI) detection methods were evaluated for the determination of surface-bound hydroxypropylcellulose (HPC) on drug particles in colloidal dispersions. Size exclusion chromatography (SEC) was used to separate HPC from other components of the dispersions. The instrumental parameters of the ELS detector were optimized to obtain maximum peak intensity, adequate peak shape and minimal baseline noise by varying the mobile phase flow rate, nebulizer temperature, and evaporation temperature. The chromatographic method was validated using both detectors. The ELS detector response exhibited second order polynomial and linear double logarithmic correlation with concentration over a 10–300% range while the RI response was linear. The double logarithmic correlation simplified the calculation compared to using the polynomial fit, and it provided more accurate results compared to the linear fit approach. Total HPC was obtained by solubilizing all components of the dispersion and analyzing for HPC. Non-bound HPC was obtained by ultracentrifuging the dispersion and analyzing the supernatant for HPC concentration. Analysis for total- and non-bound HPC in a representative colloidal dispersion gave method precisions with R.S.D.s of 2.5 and 2.2% for ELS, and 4.5 and 2.4% for RI (n = 4). HPC bound to the surface of the drug particles was determined by difference: % bound HPC = 100% − % non-bound HPC. Resultant % bound HPC values ranged from 22.1 to 25.4% of available HPC. Both ELS and RI are satisfactory detection techniques for HPC quantitation and for determination of the proportion of HPC bound to drug colloid particles, and the assay results are comparable.     

Stability of flavone-8-acetic acid (LM975) in aqueous solutions by high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed to investigate the stability of solutions of flavone-8-acetic acid (LM975) during preparation and storage. LM975 (20 μg ml⁻¹ in PBS) was found to be completely stable for 10 days at 80°C as long as light was rigorously excluded. The drug showed no significant adsorption to containers of different materials or to two filtration units tested. Drug degradation did occur however, on exposure to light. In normal laboratory light the t_0.95 (5% degraded) was 30.3 min, in intense natural light (laboratory window sill) the t_0.95 was 3.3 min and in intense artificial light (100 W bulb at 10 cm) the t_0.95 was 13.8 min. NMR and mass spectral analysis of the isolated degradation product implied the formation of the decarboxylated product, 8-methyl flavone. It is suggested that care be taken to exclude light during the preparation, storage and infusion of solutions of flavone-8-acetic acid.     

Determination of total and lactone form of a new camptothecin derivative gimatecan (ST1481) and its metabolite ST1698 in human plasma by high-performance liquid chromatography with fluorimetric detection

A new sensitive high-performance liquid chromatographic (HPLC) method for the determination of gimatecan (ST1481), a new camptothecin derivative, and its metabolite (ST1698) in plasma sample has been developed. The method consisted of on-line column solid phase extraction of analytes from human plasma, chromatographic separation by isocratic elution, then fluorimetric detection. The limits of quantitation were 0.25 ng/mL for both the analytes. The recovery of the extraction procedure was in the range of 62.8–71.1% for all the compounds. Good linearity (R² > 0.999) was observed within the calibration ranges studied: 0.25–25 ng/mL for both ST1481 and ST1698. Precision was in the range 1.2–4.3%, and accuracy was always lower than 4.7%. Surprisingly, after administration of ST1481 to humans, plasma concentrations found were higher than expected, while metabolite plasma concentrations were negligible. For this reason, a second calibration curve range was validated to quantify ST1481 in human plasma, ranging from 5 to 200 ng/mL. A good accuracy and precision were obtained, confirming the usefulness of the procedure. By using neutral analytical condition the intact lactone form was estimated in plasma samples from a patient. The lactone form amounted to 80–100% of the total ST1481.     

二维液相色谱技术在药物分析中的应用

二维液相色谱联用(2D-LC)技术作为一项新的分析技术,已在复杂样品的分离分析中发挥了巨大作用。2D-LC提供了一个真正的正交分离系统,使其分辨率达到最大。它把分离机理不同而又互相独立的两支色谱柱以串联方式结合组成二维液相色谱,在这两支色谱柱之间的接口起捕集再传送的作用。本文简单介绍了该技术的原理和方法,并对其在天然药物和体内药物分析中的应用进行了总结分析。     

Tetradecylmaltoside (TDM) enhances in vitro and in vivo intestinal absorption of enoxaparin,a low molecular weight heparin

Tetradecylmaltoside (TDM) was evaluated as a potential gastrointestinal absorption enhancer for low molecular weight heparin (LMWH), enoxaparin. The in vitro efficacy of TDM (0.0625, 0.125 and 0.25% w/v) in enhancing transport of ³H-enoxaparin or ¹⁴C-mannitol was investigated in human colonic epithelial cells (C2_BBel). Metabolic stability of the drug was determined in C2_BBel cell extracts. Transepithelial electrical resistance (TEER) was measured before and after exposure of the cells to TDM. Enoxaparin was further administered to anesthetized Sprague–Dawley rats in oral formulations in the absence or presence of increasing concentrations of TDM and drug absorption was monitored by measuring anti-factor Xa activity in rat blood. In vitro permeability study shows that apparent permeability (P_app) of ³H-enoxaparin across C2_BBe1 cells was increased by 8-fold in the presence of 0.0625% TDM compared to untreated cells. The movement of ¹⁴C-mannitol across the cell monolayer followed a similar pattern in the presence of increasing concentrations of TDM. No degradation or depolymerization of enoxaparin was observed when the drug was incubated in C2_BBel cell extract. TEER was reversible after 60 min exposure of the cells to 0.0625% (w/v) TDM. Oral formulations of enoxaparin containing TDM administered to anesthetized rats significantly and rapidly increased gastrointestinal absorption as compared to those animals which received enoxaparin plus saline (p<0.05). In the presence of 0.125% TDM in the formulation, enoxaparin oral bioavailability was increased by 2.5-fold compared to the saline control group. Overall, the data on the effect of TDM on the in vitro and in vivo intestinal permeation of enoxaparin suggest that TDM may represent a promising excipient for use in oral LMWH formulations.     

Synthesis of N-tert.-butoxycarbonyl(α-phenyl)aminomethylphenoxyacetic acid for use as a handle in solid-phase synthesis of peptide α-carboxamides

N-tert.-butoxycarbonyl-aminomethyl(α-phenyl)phenoxyacetic acid was synthesized and found to be suitable for use as a handle in the solid-phase synthesis of peptide α-carboxamides. This handle was prepared with an 82% yield when N-tert.-butoxycarbonyl-(p-hydroxy)benzhydrylamine was treated with excess sodium iodoacetate under alkaline conditions. In stability studies the linkage between the C-terminal amino acid and the handle was found to be resistant to acidolysis in 50% TFA/CH₂Cl₂ (< 1% loss after 10h). Upon treatment for 30min with HF:anisole(9:1) at 0°, 92% cleavage of glycinamide from Glyhandle-resin was obtained. In a test synthesis of a peptide α-carboxamide, pyroglutamylhistidylprolinamide was synthesized in 83% yield. Two other handles, tert.-butoxycarbonyl-aminomethylphenoxyacetic acid and N-tert.-butoxycarbonyl-aminornethylphenyloxymethylphenoxyacetic acid, were also synthesized but found to be unsuitable for carboxamide synthesis under the same conditions of solid-phase synthesis.     

Poly(amido amine) dendrimers as absorption enhancers for oral delivery of camptothecin

Oral delivery of camptothecin has a treatment advantage but is limited by low bioavailability and gastrointestinal toxicity. Poly(amido amine) or PAMAM dendrimers have shown promise as intestinal penetration enhancers, drug solubilizers and drug carriers for oral delivery in vitro and in situ. There have been very limited studies in vivo to evaluate PAMAM dendrimers for oral drug delivery. In this study, camptothecin (5 mg/kg) was formulated and co-delivered with cationic, amine-terminated PAMAM dendrimer generation 4.0 (G4.0) (100 and 300 mg/kg) and anionic, carboxylate-terminated PAMAM generation 3.5 (G3.5) (300 and 1000 mg/kg) in CD-1 mice. Camptothecin associated to a higher extent with G4.0 than G3.5 in the formulation, attributed to an electrostatic interaction on the surface of G4.0. Both PAMAM G4.0 and G3.5 increased camptothecin solubilization in simulated gastric fluid and caused a 2–3 fold increase in oral absorption of camptothecin when delivered at 2 h. PAMAM G4.0 and G3.5 did not increase mannitol transport suggesting that the oral absorption of camptothecin was not due to tight junction modulation. Histologic observations of the epithelial layer of small intestinal segments of the gastrointestinal tract (GIT) at 4 h post dosing supported no evidence of toxicity at the evaluated doses of PAMAM dendrimers. This study demonstrates that both cationic (G.4) and anionic (G3.5) PAMAM dendrimers were effective in enhancing the oral absorption of camptothecin. Results suggest that drug inclusion in PAMAM interior controlled solubilization in simulated gastric and intestinal fluids, and increased oral bioavailability.     

Impact of molecular weight on the mechanism of cellular uptake of polyethylene glycols (PEGs) with particular reference to P-glycoprotein

Polyethylene glycols (PEGs) in general use are polydisperse molecules with molecular weight (MW) distributed around an average value applied in their designation e.g., PEG 4000. Previous research has shown that PEGs can act as P-glycoprotein (P-gp) inhibitors with the potential to affect the absorption and efflux of concomitantly administered drugs. However, questions related to the mechanism of cellular uptake of PEGs and the exact role played by P-gp has not been addressed. In this study, we examined the mechanism of uptake of PEGs by MDCK-mock cells, in particular, the effect of MW and interaction with P-gp by MDCK-hMDR1 and A549 cells. The results show that: (a) the uptake of PEGs by MDCK-hMDR1 cells is enhanced by P-gp inhibitors; (b) PEGs stimulate P-gp ATPase activity but to a much lesser extent than verapamil; and (c) uptake of PEGs of low MW (<2000 Da) occurs by passive diffusion whereas uptake of PEGs of high MW (>5000 Da) occurs by a combination of passive diffusion and caveolae-mediated endocytosis. These findings suggest that PEGs can engage in P-gp-based drug interactions which we believe should be taken into account when using PEGs as excipients and in PEGylated drugs and drug delivery systems.     

The transillumination technique as a method for the assessment of spermatogenesis using medicinal plants: the effect of extracts of black maca (Lepidium meyenii) and camu camu (Myrciaria dubia) on stages of the spermatogenic cycle in male rats

Abstract

Transillumination technique for assessment of stages of spermatogenic cycle is a useful tool for toxicological studies. This study was designed to determine the effect of two medicinal plants on spermatogenesis in male rats using the transillumination technique. For this, the effect of the combination of a fruit with highest content of ascorbic acid (Myrciaria dubia, camu camu) and extract of black maca (Lepidium meyenii) on seminiferous tubule stages scored by transillumination on intact tubules in adult male rats was assessed. Animals were treated during seven days with vehicle, black maca, camu camu or a mixture of black maca?+?camu camu and assessed for daily sperm production (DSP), stages of spermatogenic cycle as well as antioxidant activity and levels of flavonoids and polyphenols. Black maca increased stages of spermiation (VII–VIII) and mitosis of germ cells (IX–XI), whereas camu camu increased stages of mitosis (IX–XI) and meiosis (XII). Mixture of maca?+?camu camu increased stages of spermiation, mitosis and meiosis. All treatments increased DSP (p?<?0.05) and epididymal sperm count (p?<?0.05). Total polyphenols, flavonoids levels and antioxidant activity were higher in camu camu (p?<?0.001) than in black maca. In conclusion, M. dubia (camu camu) has potential effects improving spermatogenesis and co-administered with maca increase stages of mitosis, meiosis and spermiation of the spermatogenic cycle as assessed by the transillumination technique. This technique is becoming increasingly a useful tool for assessment spermatogenesis.     

Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non-competitive inhibition by the small molecule “crystallization chaperone” 5′-O-tritylinosine (KIN59)

Thymidine phosphorylase (TP) is a catabolic enzyme in thymidine metabolism that is frequently upregulated in many solid tumors. Elevated TP levels are associated with tumor angiogenesis, metastasis and poor prognosis. Therefore, the use of TP inhibitors might offer a promising strategy for cancer treatment. The tritylated inosine derivative 5′-O-tritylinosine (previously designated KIN59) is a non-competitive inhibitor of TP which was previously found to be instrumental for the crystallization of human TP. A combination of computational studies including normal mode analysis, automated ligand docking and molecular dynamics simulations were performed to define a plausible binding site for 5′-O-tritylinosine on human TP. A cavity in which 5′-O-tritylinosine could fit was identified in the vicinity of the Gly405-Val419 loop at a distance of about 11 Å from the substrate-binding site. In the X-ray crystal structure, this pocket is characterized by an intricate hydrogen-bonding network in which Asp203 was found to play an important role to afford the loop stabilization that is required for efficient enzyme catalysis. Site-directed mutagenesis of this amino acid residue afforded a mutant enzyme with a severely compromised catalytic efficiency (V_max/K_m of mutant enzyme ∼50-fold lower than for wild-type TP) and pronounced resistance to the inhibitory effect of 5′-O-tritylinosine. In contrast, the D203A mutant enzyme kept full sensitivity to the competitive inhibitors 6-aminothymine and 6-amino-5-bromouracil, which is in line with the kinetic properties of these inhibitors. Our findings reveal the existence of a previously unrecognized site in TP that can be targeted by small molecules to inhibit the catalytic activity of TP.     

Caution in the use of 2‐iminothiolane (Traut's reagent) as a cross‐linking agent for peptides. The formation of N‐peptidyl‐2‐iminothiolanes with bombesin (BN) antagonist (d‐Trp6,Leu13‐ψ[CH2NH]‐Phe14)BN6−14 and d‐Trp‐Gln‐Trp‐NH2

Abstract: During a study aimed at generating a bispecific molecule between BN antagonist (d ‐Trp⁶,Leu¹³‐ψ[CH₂NH]‐Phe¹⁴)BN_6‐14 (Antag1) and mAb22 (anti‐FcγRI), we attempted to cross‐link the two molecules by introducing a thiol group into Antag1 via 2‐iminothiolane (2‐IT, Traut's reagent). We found that reaction of Antag1 with 2‐IT, when observed using HPLC, affords two products, but that the later eluting peptide is rapidly transformed into the earlier eluting peptide. To understand what was occurring we synthesized a model peptide, d ‐Trp‐Gln‐Trp‐NH₂ (TP1), the N‐terminal tripeptide of Antag1. Reaction of TP1 with 2‐IT for 5 min gave products 1a and 3a ; the concentration of 1a decreased with reaction time, whereas that of 3a increased. Thiol 1a , the expected Traut product, was identified by collecting it in a vial containing N‐methylmaleimide and then isolating the resultant Michael addition product 2a , which was confirmed by mass spectrometry. Thiol 1a is stable at acidic pH, but is unstable at pH 7.8, cyclizes and loses NH₃ to give N‐TP1‐2‐iminothiolane ( 3a ), ES‐MS (m/z) [602.1 (M+H)⁺], as well as regenerating TP1. Repeat reaction with Antag1 and 2‐IT allowed us to isolate N‐Antag1–2‐iminothiolane ( 3b ), FAB‐MS (m/z) [1212.8 (M+H)⁺] and trap the normal Traut product 1b as its N‐methylmaleimide Michael addition product 2b , ES‐MS (m/z) [1340.8 (M+H)⁺]. Thiol 1b is also stable at acidic pH, but when neutralized is unstable and cyclizes, forming 3b and Antag1.