On Interactions of Sulfamerazine with Cyclodextrins from Coupled Diffusometry and Toxicity Tests.

This scientific study employs the Taylor dispersion technique for diffusion measurements to investigate the interaction between sulfamerazine (NaSMR) and macromolecular cyclodextrins (ß-CD and HP-ß-CD). The results reveal that the presence of ß-CD influences the diffusion of the solution component, NaSMR, indicating a counterflow of this drug due to solute interaction. However, diffusion data indicate no inclusion of NaSMR within the sterically hindered HP-ß-CD cavity. Additionally, toxicity tests were conducted, including pollen germination (Actinidia deliciosa) and growth curve assays in BY-2 cells. The pollen germination tests demonstrate a reduction in sulfamerazine toxicity, suggesting potential applications for this antimicrobial agent with diminished adverse effects. This comprehensive investigation contributes to a deeper understanding of sulfamerazine-cyclodextrin interactions and their implications for pharmaceutical and biological systems.

In situ discrimination of polymorphs and phase transformation of sulfamerazine using quartz crystal microbalance.

A novel strategy utilizing the quartz crystal microbalance (QCM) was developed for the in situ discrimination of polymorphic nucleation (form-I and form-II) and phase transformation of sulfamerazine (SMZ) in cooling crystallization. According to Ostwald's rule of stages, metastable form-I of SMZ is first nucleated and then shifted to stable form-II by solution-mediated phase transformation. Through surface modification with the self-assembled monolayer technique of a functional group, QCM distinctively detects the formation of the two polymorphs. The results indicated that -NH2 (among the several functional groups tested) selectively accommodated stable form-II on the QCM sensor's surface and completely prevented the adsorption of metastable form-I on the surface. Therefore, the-NH2-terminated QCM detected the formation of form-I only using the solution viscosity variation on the surface. However, it monitored the nucleation and growth of form-II via the solid mass change on the surface during the phase transformation of form-I to form-II. This strategy suggests a new and precise solution for in situ discrimination of SMZ polymorphs and their phase transformation.

Synthesis and in silico studies of triazene-substituted sulfamerazine derivatives as acetylcholinesterase and carbonic anhydrases inhibitors.

A novel series of sulfonamides, 4-(3-phenyltriaz-1-en-1-yl)-N-(4-methyl-2-pyrimidinyl)benzenesulfonamides (1-9), was designed and synthesized by the diazo reaction between sulfamerazine and substituted aromatic amines for the first time. Their chemical structures were characterized by 1 H nuclear magnetic resonance (NMR), 13 C NMR, and high-resolution mass spectra. The newly synthesized compounds were evaluated in terms of acetylcholineasterase (AChE) and human carbonic anhydrases (hCA) I and II isoenzymes inhibitory activities. According to the AChE inhibition results, the Ki values of the compounds 1-9 were in the range of 19.9 ± 1.5 to 96.5 ± 20.7 nM against AChE. Tacrine was used as the reference drug and its Ki value was 49.2 ± 2.7 nM against AChE. The Ki values of the compounds 1-9 were in the range of 10.2 ± 2.6 to 101.4 ± 27.8 nM against hCA I, whereas they were 18.3 ± 4.4 to 48.1 ± 4.5 nM against hCA II. Acetazolamide was used as a reference drug and its Ki values were 72.2 ± 5.4 and 52.2 ± 5.7 nM against hCA I and hCA II, respectively. The most active compounds, 1 (nonsubstituted) against AChE, 5 (4-ethoxy-substituted) against hCA I, and 8 (4-bromo-substituted) against hCA II, were chosen and docked at the binding sites of these enzymes to explain the inhibitory activities of the series. The newly synthesized compounds presented satisfactory pharmacokinetic properties via the estimation of ADME properties.

An overview of carbohydrate-based carbonic anhydrase inhibitors.

Carbonic anhydrases (CAs) are metalloenzymes responsible for the reversible hydration of carbon dioxide to bicarbonate, a fundamental reaction involved in various physiological and pathological processes. In the last decades, CAs have been considered as important drug targets for different pathologies such as glaucoma, epilepsy and cancer. The design of potent and selective inhibitors has been an outstanding goal leading to the discovery of new drugs. Among the different strategies developed to date, the design of carbohydrate-based CA inhibitors (CAIs) has emerged as a versatile tool in order to selectively target CAs. The insertion of a glycosyl moiety as a hydrophilic tail in sulfonamide, sulfenamide, sulfamate or coumarin scaffolds allowed the discovery of many different series of sugar-based CAIs, with relevant inhibitory results. This review will focus on carbohydrate-based CAIs developed so far, classifying them in glycosidic and glycoconjugated inhibitors based on the conjugation chemistry adopted.

Catalytic, Enantioselective Sulfenofunctionalization of Alkenes: Development and Recent Advances.

The last decade has witnessed a burgeoning of new methods for the enantioselective vicinal difunctionalization of alkenes initiated by electrophilic sulfenyl group transfer. The addition of sulfenium ions to alkenes results in the generation of chiral, non-racemic thiiranium ions. These highly reactive intermediates are susceptible to attack by a myriad of nucleophiles in a stereospecific ring-opening event to afford anti 1,2-sulfenofunctionalized products. The practical application of sulfenium ion transfer has been enabled by advances in the field of Lewis base catalysis. This Review will chronicle the initial discovery and characterization of thiiranium ion intermediates followed by the determination of their configurational stability and the challenges of developing enantioselective variants. Once the framework for the reactivity and stability of thiiranium ions has been established, a critical analysis of pioneering studies will be presented. Finally, a comprehensive discussion of modern synthetic applications will be categorized around the type of nucleophile employed for sulfenofunctionalization.

An amide-based sulfenamide prodrug of gamma secretase inhibitor BMS-708163 delivers parent drug from an oral conventional solid dosage form in male beagle dog.

The objective of this Letter is to report the first (to our knowledge) in vivo proof of concept for a sulfenamide prodrug to orally deliver a poorly soluble drug containing a weakly-acidic NH-acid from a conventional solid dosage formulation. This proof of concept was established using BMS-708163 (1), a gamma secretase inhibitor containing a weakly acidic primary amide NH-acid as the chemical handle for attaching a series of thiol-based promoieties via a sulfenamide linkage. Aqueous stabilities and solubilities are reported for a series of six sulfenamide prodrugs (2-7) of 1. The sulfenamide prodrug containing the cysteine methyl ester promoiety (5) was chosen for a orally-dosed PK study in male beagle dog comparing a solubilized formulation of 1 against a solid dosage form of 5 in a cross-over fashion at an equivalent molar dose of 3 mg/kg. Prodrug 5 delivered essentially a superimposable PK profile of 1 compared to the solubilized formulation of 1, without any detectable exposure of 5 in systemic circulation.

Connected Subgraph Fingerprints: Representing Molecules Using Exhaustive Subgraph Enumeration.

Molecular fingerprints are an efficient and widely used method for similarity-driven virtual screening. Most fingerprint methods can be distinguished by the class of structural features considered. The Connected Subgraph Fingerprint (CSFP) overcomes this limitation and regards all structural features of a compound. This results in a more complete feature space and high adaptive potential to certain application scenarios. The novel descriptor surpasses widely used fingerprint methods in some cases and opens the way for topological search in combinatorial fragment spaces.

DFT based engineering of N-heterocyclic carbenes to exacerbate its activity for SO2 fixation and storage.

Carbene compounds are very reactive to SO2 which assert their candidature to seize this greenhouse gas. Unfortunately, most of the carbenes which produce S,S-dioxide with SO2, undergo dissociation to yield ketone derivative and SO through an intermediate formation of oxathiirane S-oxide derivative. Thus, carbenes are excluded from the list of catalyst for SO2 fixation and storage technology. To eradicate this retardation, the stability of different oxathiirane S-oxide derivatives obtained from SO2 and 56 carbenes of various structures are studied using Density Functional Theory (DFT). Through our study, we are able to find out three oxathiirane S-oxide derivatives which have positive ΔG values for their decomposition to the respective ketone derivatives. This study corroborates that proper engineering of carbene leads to produce a stable oxathiirane S-oxide derivative as a stable product. We observed that carbenes are highly efficient to nab SO2 at room temperature. This finding should necessitate the recommencement of the use of carbene for SO2 fixation technology. We also found three carbenes which are able to produce sulfene derivative (C - S bond length is less than 1.7 Å) on reaction with SO2.

Synthesis of Sulfonimidamides from Sulfenamides via an Alkoxy-amino-λ6 -sulfanenitrile Intermediate.

Sulfonimidamides are intriguing new motifs for medicinal and agrochemistry, and provide attractive bioisosteres for sulfonamides. However, there remain few operationally simple methods for their preparation. Here, the synthesis of NH-sulfonimidamides is achieved directly from sulfenamides, themselves readily formed in one step from amines and disulfides. A highly chemoselective and one-pot NH and O transfer is developed, mediated by PhIO in iPrOH, using ammonium carbamate as the NH source, and in the presence of 1 equivalent of acetic acid. A wide range of functional groups are tolerated under the developed reaction conditions, which also enables the functionalization of the antidepressants desipramine and fluoxetine and the preparation of an aza analogue of the drug probenecid. The reaction is shown to proceed via different and concurrent mechanistic pathways, including the formation of novel S≡N sulfanenitrile species as intermediates. Several alkoxy-amino-λ6 -sulfanenitriles are prepared with different alcohols, and shown to be alkylating agents to a range of nucleophiles.

Oxygen vacancy enhancing the Fe2O3-CeO2 catalysts in Fenton-like reaction for the sulfamerazine degradation under O2 atmosphere.

To develop an efficient and reusable heterogeneous Fenton-like catalyst is a great challenge for its application in practical water treatment. Effective oxygen vacancy (OVs)-promoted Fe2O3-CeO2 catalyst was prepared by a sol-gel method, and applied in the heterogeneous Fenton-like reaction of the sulfamerazine (SMR) degradation. The Fe2O3-CeO2 catalyst showed good activity and stability, and total SMR conversion was achieved in the Fenton-like reaction after 75 min at pH 3.0 and 45 °C under O2 atmosphere. Moreover, the SMR removal was significantly enhanced under O2 atmosphere. The surface-bounded OH radicals played a dominant role for the SMR degradation. The Fe2O3-CeO2 catalyst remarkably promoted the generation of OH in the Fenton-like reaction under O2 atmosphere, mostly because abundant OVs on the catalyst surface not only accelerated electron transfer to promote the H2O2 decomposition, but also oxygen molecules, adsorbed on OVs, formed O2-/HO2 and promoted the Fe2+/Fe3+ redox cycle.

Sulfenamide and Sulfonamide Derivatives of Metformin - A New Option to Improve Endothelial Function and Plasma Haemostasis.

Type 2 diabetes mellitus (T2DM) is a multi-factorial disease which can cause multiple organ dysfunction, including that of the vascular endothelium. The aim of the present study was to evaluate the effects of metformin, and its sulfenamide and sulfonamide derivatives (compounds 1-8) on the selected markers of endothelial function and blood coagulation. The integrity of endothelial cells(ECs) was examined using the real-time cell electric impedance system. Tissue Factor(TF) production, the release of von Willebrand Factor (vWF) and tissue plasminogen activator(t-PA) from ECs were determined using immunoenzymatic assays, while the process of platelet thrombus formation using the Total Thrombus-Formation Analysis System. Sulfenamide with n-butyl alkyl chain(3) does not interfere with ECs integrity, and viability (nCI(24h) = 1.03 ± 0.03 vs. 1.06 ± 0.11 for control), but possesses anticoagulation properties manifested by prolonged platelet-dependent thrombus formation (Occlusion Time 370.3 ± 77.0 s vs. 286.7 ± 65.5 s for control) in semi-physiological conditions. Both p- and o-nitro-benzenesulfonamides (compounds7,8) exhibit anti-coagulant properties demonstrated by decreased vWF release and prolonged parameters of platelet thrombus formation and total blood thrombogenicity. In conclusion, chemical modification of metformin scaffold into sulfenamides or sulfonamides might be regarded as a good starting point for the design and synthesis of novel biguanide-based compounds with anticoagulant properties and valuable features regarding endothelial function.

Sulfenamide derivatives can improve transporter-mediated cellular uptake of metformin and induce cytotoxicity in human breast adenocarcinoma cell lines.

Metformin, the most frequently administered oral anti-diabetic drug, is a substrate for organic cation transporters (OCTs). This determines not only its pharmacokinetic properties but also its biochemical effects in humans, including its recently-discovered antiproliferative properties. The aim of the study was to verify the hypothesis whether chemical modification of its biguanide backbone may increase the cellular uptake and antiproliferative efficacy of metformin. The study examines five sulfenamide derivatives of metformin with differing lengths of alkyl chains. It determines their cellular uptake and the role of OCTs in their transport in human breast adenocarcinoma cells (epithelial-like MCF-7, and MDA-MB-231). It also evaluates whether increased cellular uptake of metformin derivatives is associated with their cytotoxic properties. Sulfenamide derivatives were characterized by a greater ability to bind to OCTs than metformin. Compound 2 with n-octyl alkyl chain was found to possess the greatest affinity towards OCTs, as measured by determination of [14C]choline uptake inhibition (IC50 = 236.1 ±â€¯1.28 µmol/L, and 217.4 ±â€¯1.33 µmol/L, for MCF-7 and MDA-MB-231 respectively). Sulfenamides were also found to exhibit better cellular uptake in comparison with the parent drug, metformin. For instance, the uptake of cyclohexyl derivative 1 was 1.28 ±â€¯0.19 nmol/min/mg of proteins and thus was 12-fold higher than the metformin in MCF-7 cells. Furthermore, higher uptake was associated with the greatest antiproliferative properties expressed as the lowest IC50 value i.e. inhibiting the growth of 50% of the cells (IC50 = 0.72 ±â€¯1.31 µmol/L). Collectively, chemical modification of metformin into sulfenamides with different alkyl substituents obtains better substrates for OCTs, and subsequently higher cellular uptake in MCF-7 and MDA-MB-231 cells. Additionally, the length of alkyl chain introduced to the sulfenamides was found to influence selectivity and transport efficiency via OCT1 compared to other possible transporters, as well as potential intracellular activity and cytotoxicity.

Application of affinity microspheres for effective SPE cleanup before the determination of sulfamerazine by HPLC.

This paper describes the application of poly (ethylene glycol dimethacrylate-N-methacryloyl-L-tryptophane methyl ester) [p(EGDMA-MATrp)] microspheres as an affinity sorbent for the SPE (solid phase extraction) cleanup of sulfamerazine (SMR) from food samples of animal origin before high performance liquid chromatography (HPLC) analysis. The microspheres were prepared by suspension polymerization of ethylene glycol dimethacrylate (EGDMA) and N-methacryloyl-L-tryptophane methyl ester (MATrp) as a crosslinker and a monomer, respectively. Various parameters affecting the SPE cleanup efficiency of the p(EGDMA-MATrp) microspheres (contact time, pH, initial SMR concentration, ionic strength etc.) were optimized. Under the optimized conditions, maximum adsorption capacity was found to be 8.55 ±â€¯0.44 mg/g sorbent at pH 5.0. 90% of the adsorbed SMR was desorbed by using ACN:MeOH (5:5) mixture as a desorption agent. Applicability of the microspheres for the SPE cleanup of SMR residues in the food samples such as egg and milk with HPLC was also determined. It was demonstrated that the prepared p(EGDMA-MATrp) microspheres could be repeatedly applied for SPE cleanup of sulfamerazine before chromatographic analysis. Also, the recoveries of SMR in milk and egg samples were reasonably satisfactory and in the range of 71 to 90%.

Density fluctuations in amorphous pharmaceutical solids. Can SAXS help to predict stability?

The analytical potential of X-ray small-angle scattering (SAXS) combined with simultaneous wide-angle diffraction (WAXS) has been explored on the example of three active pharmaceutical ingredients, (desvenlofaxine, simvastatin, and sulfamerazine, resp.) with the aim of identifying quantitative parameters obtained from SAXS that allow to describe the nano-structural characteristics of different amorphous forms and to monitor the processes of amorphisation and ageing. Cryo-milling, co-milling with polymer, melting and melt-quenching have been used for amorphisation of initially crystalline powders. In parallel to SAXS, the WAXS patterns have been obtained to fingerprint the crystalline state. The SAXS results demonstrate strong, systematic nanostructure variations in amorphous samples obtained by different milling conditions, or by melt-quenching. It has been found that the mean-square density fluctuation, directly obtained from the SAXS invariant, is a sensitive and robust parameter to characterize the degree of nano-heterogeneity, which is related to entropy and hence thermodynamic stability. The SAXS curves also allow estimates of amorphous domain sizes of different density. The propensity to recrystallize or to remain amorphous, respectively, upon ageing has been found to depend on the existence of domains in the starting amorphous materials.

Quantitative models for predicting adsorption of oxytetracycline, ciprofloxacin and sulfamerazine to swine manures with contrasting properties.

Understanding antibiotic adsorption in livestock manures is crucial to assess the fate and risk of antibiotics in the environment. In this study, three quantitative models developed with swine manure-water distribution coefficients (LgKd) for oxytetracycline (OTC), ciprofloxacin (CIP) and sulfamerazine (SM1) in swine manures. Physicochemical parameters (n=12) of the swine manure were used as independent variables using partial least-squares (PLSs) analysis. The cumulative cross-validated regression coefficients (Q2cum) values, standard deviations (SDs) and external validation coefficient (Q2ext) ranged from 0.761 to 0.868, 0.027 to 0.064, and 0.743 to 0.827 for the three models; as such, internal and external predictability of the models were strong. The pH, soluble organic carbon (SOC) and nitrogen (SON), and Ca were important explanatory variables for the OTC-Model, pH, SOC, and SON for the CIP-model, and pH, total organic nitrogen (TON), and SOC for the SM1-model. The high VIPs (variable importance in the projections) of pH (1.178-1.396), SOC (0.968-1.034), and SON (0.822 and 0.865) established these physicochemical parameters as likely being dominant (associatively) in affecting transport of antibiotics in swine manures.

Sulfenamide and sulfonamide derivatives of metformin can exert anticoagulant and profibrinolytic properties.

Type 2 diabetes mellitus (T2DM) is characterised not only by hyperglycaemia and insulin resistance but also an impaired balance between the processes of coagulation and fibrinolysis. The aim of this study was to examine the effects of metformin, a widely-used oral anti-diabetic drug, phenformin and eight sulfenamide and sulfonamide derivatives of metformin on several haemostasis parameters. Thrombin Time (TT) tests were performed according to the available commercial method. The activity of factor X was conducted based on deficient plasma factor X. The activity of two main enzymes involved in haemostasis, thrombin and plasmin, was measured spectrophotometrically with chromogenic substrates. Protein C and antithrombin III (AT) activity assays using chromogenic substrates were conducted to determine the effect of the derivatives of metformin on these both naturally occurring anticoagulants. Two of the compounds, sulfenamide with hexyl tail and para-nitro-benzenesulfonamide significantly shortened TT. ortho-nitro sulfonamide at a concentration of 0.3-1.5 µmol/mL contributed to a significant decrease in the activity of factor X. However, sulfenamides with cyclohexyl, butyl and branched ethyl-hexyl tails at 1.5 of µmol/mL increased its activity, and simultaneously shortened PT. Additionally, ortho-nitro-benzenesulfonamide at concentrations of 1.5 µmol/mL was found to significantly decrease reaction velocity (↓ dA/dt) in the thrombin activity assay. On contrary, it was noticed that branched sulfenamide at the concentration of 1.5 µmol/mL significantly increased the enzymatic activity of plasmin. Metformin, phenformin and octyl and butyl sulfenamides were associated with a significant increase in the activity of AT. Hexyl sulfenamide and para-nitro- as well as para-trifluoro-ortho-nitro-benzenesulfonamide contributed to the decrease in the activity of protein C, while the other tested compounds did not affect its activity. In conclusion, 2-nitro-benzenesulfonamide derivative of metformin presents highly beneficial anticoagulant properties. This compound is therefore promising candidate for further in vitro and in vivo studies.

Biocompatible sulfenamide and sulfonamide derivatives of metformin can exert beneficial effects on plasma haemostasis.

As the pharmacokinetic properties of metformin are unfavourable, several analogues and prodrugs have been synthesised to improve its bioavailability. The aim of this study was to assess the plasma stability of sulfenamide and sulfonamide derivatives of metformin and establish their effects on plasma haemostasis and integrity of red blood cells (RBCs). The overall haemostasis potential was evaluated spectrophotometrically by clot formation and lysis test (CL-test). PT (Prothrombin Time) and APTT (Activated Partial Tromboplastin Time) were used to evaluate the effects if the compounds on the extrinsic and intrinsic coagulation pathway. Haemolysis assay, microscopy and flow cytometry studies were conducted to determine the effect of the compounds on RBCs. Two sulfonamide and one sulfenamide derivatives of metformin were associated with a statistically significant decrease in the overall potential of clot formation and fibrinolysis (↓ CLAUC), suggesting that these compounds may exert beneficial effects regarding plasma haemostasis, which is frequently impaired in diabetic patients. p- and o-Nitrobenzene sulfonamides contributed to the beneficial change in kinetic parameters of clot formation and fibrinolysis. o-Nitrobenzene sulfonamide significantly increased thrombin generation time (↑ TGt) and was also found to prolong both APTT and PT. All compounds did not exert any effects on the integrity of RBCs over the concentration range 0.006-0.6 µmol/mL which constitutes the expected therapeutic concentration. In conclusion, sulfonamide derivatives of metformin present potentially beneficial properties in terms of plasma haemostasis which is frequently impaired in T2DM patients. Therefore, metformin sulfonamides may become a prototype for further design and synthesis of novel metformin analogues and prodrugs with improved pharmacokinetic properties.

Novel sulfenamides and sulfonamides based on pyridazinone and pyridazine scaffolds as CB1 receptor ligand antagonists.

A series of sulfenamide and sulfonamide derivatives was synthesized and evaluated for the affinity at CB1 and CB2 receptors. The N-bornyl-S-(5,6-di-p-tolylpyridazin-3-yl)-sulfenamide, compound 11, displayed good affinity and high selectivity for CB1 receptors (Ki values of 44.6 nM for CB1 receptors and >40 µM for CB2 receptors, respectively). The N-isopinocampheyl-sulfenamide 12 and its sulfonamide analogue 22 showed similar selectivity for CB1 receptors with Ki values of 75.5 and 73.2 nM, respectively. These novel compounds behave as antagonists/inverse agonists at CB1 receptor in the [35S]-GTPγS binding assays, and none showed adequate predictive blood-brain barrier permeation, exhibiting low estimated LD50. However, testing compound 12 in a supraspinal analgesic test (hot-plate) revealed that it was as effective as the classic CB1 receptor antagonist rimonabant, in reversing the analgesic effect of a cannabinoid agonist.

Metformin and Its Sulfenamide Prodrugs Inhibit Human Cholinesterase Activity.

The results of epidemiological and pathophysiological studies suggest that type 2 diabetes mellitus (T2DM) may predispose to Alzheimer's disease (AD). The two conditions present similar glucose levels, insulin resistance, and biochemical etiologies such as inflammation and oxidative stress. The diabetic state also contributes to increased acetylcholinesterase (AChE) activity, which is one of the factors leading to neurodegeneration in AD. The aim of this study was to assess in vitro the effects of metformin, phenformin, and metformin sulfenamide prodrugs on the activity of human AChE and butyrylcholinesterase (BuChE) and establish the type of inhibition. Metformin inhibited 50% of the AChE activity at micromolar concentrations (2.35 µmol/mL, mixed type of inhibition) and seemed to be selective towards AChE since it presented low anti-BuChE activity. The tested metformin prodrugs inhibited cholinesterases (ChE) at nanomolar range and thus were more active than metformin or phenformin. The cyclohexyl sulfenamide prodrug demonstrated the highest activity towards both AChE (IC50 = 890 nmol/mL, noncompetitive inhibition) and BuChE (IC50 = 28 nmol/mL, mixed type inhibition), while the octyl sulfenamide prodrug did not present anti-AChE activity, but exhibited mixed inhibition towards BuChE (IC50 = 184 nmol/mL). Therefore, these two bulkier prodrugs were concluded to be the most selective compounds for BuChE over AChE. In conclusion, it was demonstrated that biguanides present a novel class of inhibitors for AChE and BuChE and encourages further studies of these compounds for developing both selective and nonselective inhibitors of ChEs in the future.

Quantification of Hemoglobin and White Blood Cell DNA Adducts of the Tobacco Carcinogens 2-Amino-9H-pyrido[2,3-b]indole and 4-Aminobiphenyl Formed in Humans by Nanoflow Liquid Chromatography/Ion Trap Multistage Mass Spectrometry.

Aromatic amines covalently bound to hemoglobin (Hb) as sulfinamide adducts at the cysteine 93 residue of the Hb ß chain have served as biomarkers to assess exposure to this class of human carcinogens for the past 30 years. In this study, we report that 2-amino-9H-pyrido[2,3-b]indole (AαC), an abundant carcinogenic heterocyclic aromatic amine formed in tobacco smoke and charred cooked meats, also reacts with Hb to form a sulfinamide adduct. A novel nanoflow liquid chromatography/ion trap multistage mass spectrometry (nanoLC-IT/MS3) method was established to assess exposure to AαC and the tobacco-associated bladder carcinogen 4-aminobiphenyl (4-ABP) through their Hb sulfinamide adducts. Following mild acid hydrolysis of Hb in vitro, the liberated AαC and 4-ABP were derivatized with acetic anhydride to form the N-acetylated amines, which were measured by nanoLC-IT/MS3. The limits of quantification (LOQ) for AαC- and 4-ABP-Hb sulfinamide adducts were ≤7.1 pg/g Hb. In a pilot study, the mean level of Hb sulfinamide adducts of AαC and 4-ABP were, respectively, 3.4-fold and 4.8-fold higher in smokers (>20 cigarettes/day) than nonsmokers. In contrast, the major DNA adducts of 4-ABP, N-(2'-deoxyguanosin-8-yl)-4-aminobiphenyl, and AαC, N-(2'-deoxyguanosin-8-yl)-2-amino-9H-pyrido[2,3-b]indole, were below the LOQ (3 adducts per 109 bases) in white blood cell (WBC) DNA of smokers and nonsmokers. These findings reaffirm that tobacco smoke is a major source of exposure to AαC. Hb sulfinamide adducts are suitable biomarkers to biomonitor 4-ABP and AαC; however, neither carcinogen binds to DNA in WBC, even in heavy smokers, at levels sufficient for biomonitoring.