Nonderivatized Method for the Detection of Perfluoroalkane Sulfonyl Fluorides by Liquid Chromatography-Microwave Plasma Torch Ionization Mass Spectrometry.

Per- and polyfluoroalkyl substances (PFAS) have caused widespread environmental concern in recent years. Among them, the levels of perfluoroalkane sulfonyl fluorides (PFASFs) in the environment have rarely been reported due to the lack of sensitive analytical methods. Herein, a novel liquid chromatography-microwave plasma torch ionization-mass spectrometry (LC-MPTI-MS) technique was designed for the direct analysis of PFASFs in the environment. The collaborative action of reactive oxygen species (such as hydroxyl radicals) and the elevated temperature within the ambient MPTI environment results in the replacement of the fluorine atom in sulfonyl fluoride by oxygen, leading to the detection of perfluoroalkanesulfonic acid (PFSA) ions by MS. Concurrently, LC was employed to separate other PFSAs that are present in the environment. Three PFASFs exhibited good linearity within the range of 1-500 µg/L with R2 > 0.994. The limit of detections (LODs) and the limit of quantifications (LOQs) were measured at 39.32-87.87 and 131.07-292.90 ng/L, respectively. The method was utilized for the direct detection of spiked perfluorooctane sulfonyl fluoride (PFOSF) in wastewater with recoveries of 77.16 to 124.81%. Our approach circumvents the laborious process of chemical derivatization and is anticipated to serve as a robust tool for determining the levels and behaviors of PFASFs in the environment.

Whole­exome sequencing reveals Lewis lung carcinoma is a hypermutated Kras/Nras-mutant cancer with extensive regional mutation clusters in its genome.

Lewis lung carcinoma (LLC), as a widely used preclinical cancer model, has still not been genetically and genomically characterized. Here, we performed a whole-exome sequencing analysis on the LLC cell line to elucidate its molecular characteristics and etiologies. Our data showed that LLC originated from a male mouse belonging to C57BL/6L (a transitional strain between C57BL/6J and C57BL/6N) and contains substantial somatic SNV and InDel mutations (> 20,000). Extensive regional mutation clusters are present in its genome, which were caused mainly by the mutational processes underlying the SBS1, SBS5, SBS15, SBS17a, and SBS21 signatures during frequent structural rearrangements. Thirty three deleterious mutations are present in 30 cancer genes including Kras, Nras, Trp53, Dcc, and Cacna1d. Cdkn2a and Cdkn2b are biallelically deleted from the genome. Five pathways (RTK/RAS, p53, cell cycle, TGFB, and Hippo) are oncogenically deregulated or affected. The major mutational processes in LLC include chromosomal instability, exposure to metabolic mutagens, spontaneous 5-methylcytosine deamination, defective DNA mismatch repair, and reactive oxygen species. Our data also suggest that LLC is a lung cancer similar to human lung adenocarcinoma. This study lays a molecular basis for the more targeted application of LLC in preclinical research.

Separation, identification, and structural characterization of sucrose ester isomers from tobacco.

Sucrose esters (SEs) are crucial tobacco smoke flavor precursors and play a significant role in tobacco's functionality. Due to their structural complexity, the separation and analysis of SEs in tobacco remain a major challenge, and massive structures of SEs have not yet been fully identified. In this study, the fractions enriched in SEs were obtained from oriental and flue-cured tobacco through a series of pretreatments, and two types of SEs (Types I and II) were distinguished by liquid chromatography-tandem mass spectrometry (LC-MSn ) analysis, with Type II SEs newly characterized in tobacco. Five groups of main SEs were further purified using preparative high-performance LC (HPLC) coupled to an evaporative light scattering detector, and their structures were characterized by nuclear magnetic resonance spectrometry techniques including 1 H, 13 C, correlation spectroscopy, heteronuclear single quantum correlation, and heteronuclear multiple bond correlation. By combining LC-MSn and nuclear magnetic resonance spectrometry, the structures of eight SE isomers were finally proposed, of which four were newly identified. These findings further enhance the understanding of the structural diversity of SEs in tobacco, serving as a valuable reference for future research on the elucidation, synthesis, and metabolism of SEs.

Discovery of 35 novel classes of per- and polyfluoroalkyl substances in representative commercial fluorinated products in China.

Per- and polyfluoroalkyl substances (PFAS) have received increasing scientific and regulatory attention due to their global distribution and health hazards. However, little is known about the PFAS composition of fluorinated products commercially available in China. In this study, a sensitive and robust analytical method was proposed for the comprehensive characterization of PFAS in aqueous film-forming foam and fluorocarbon surfactants in the domestic market based on liquid chromatography-high resolution mass spectrometry in full scan acquisition mode followed by parallel reaction monitoring mode. Consequently, a total of 102 PFAS from 59 classes were elucidated, of which 35 classes are reported for the first time, including 27 classes of anionic, seven classes of zwitterionic, and one class of cationic PFAS. The anionic-type products are mainly C6 fluorotelomerization-based (FT-based) PFAS. Perfluorooctanoic acid and perfluorooctane sulfonate are negligible, while some known electrochemical fluorination-based long-chain precursors in zwitterionic products are worthy of concern because of their high abundance and potential degradation. New precursors detected in zwitterionic products are FT-based PFAS, for example, 6:2 FTSAPr-AHOE and 6:2 FTSAPr-diMeAmPrC. The structural elucidation of PFAS in commercial products facilitates a better assessment of human exposure and environmental release.

Determination of Perfluorooctane Sulfonyl Fluoride and Perfluorohexane Sulfonyl Fluoride in Soil by Chemical Derivatization and Liquid Chromatography-Tandem Mass Spectrometry.

Perfluorooctane sulfonyl fluoride (PFOSF) and perfluorohexane sulfonyl fluoride (PFHxSF) were listed as persistent organic pollutants by the Stockholm Convention in 2009 and 2022, respectively. To date, their concentrations in environmental samples have not been reported due to the lack of sensitive methods. Herein, a novel chemical derivatization was developed for quantitative analysis of trace PFOSF and PFHxSF in soil by derivatizing them to the corresponding perfluoroalkane sulfinic acids. The method showed good linearity in the range from 25 to 500 ng L-1 with correlation coefficients (R2) better than 0.99. The detection limit of PFOSF in soil was 0.066 ng g-1 with recoveries in the range of 96-111%. Meanwhile, the detection limit of PFHxSF was 0.072 ng g-1 with recoveries in the range of 72-89%. Simultaneously, perfluorooctane sulfonic acid (PFOS) and perfluorohexane sulfonic acid (PFHxS) were also detected accurately without being affected by the derivative reaction. By applying this method in an abandoned fluorochemical manufacturing facility, PFOSF and PFHxSF were successfully detected at concentrations ranging from 2.7 to 357 ng g-1 and 0.23 to 26 ng g-1 dry weight, respectively. It is very interesting that 2 years after factory relocation, there still exists high concentrations of PFOSF and PFHxSF, which is of concern.

Forced degradation studies of elagolix sodium with the implementation of high resolution LC-UV-PDA-MSn (n = 1,2,3 ) and NMR structural elucidation.

Elagolix sodium (ELS) is a marketed product using to release moderate to severe endometriosis-associated pain. It contains functional groups such as carboxyl group, secondary amino group, 2,4-dioxo pyrimidinyl and several benzyl or benzyl-like position hydrogen atom that are susceptive to occur stress degradation. Forced degradation studies of ELS reveal different degradation profiles of the drug substance which are conducted under photo, thermal, acidic, neutral, alkaline and hydrogen peroxide oxidative conditions in the direction of the ICH guidances. With structural elucidation of LC-PDA/UV-MSn and NMR, the degradants were identified, and seven new degradants are reported in this study. It is confirmed that most of the degradation behaviors of ELS are related to the carboxyl group and secondary amino group in the 3-carboxyl propylamine side chain. Under the oxidative condition using hydrogen peroxide as the oxidant, the secondary amine was oxidized to form an N-hydrogen amine degradant and two further degradants of amine and carbonyl analogs were generated. Under the alkaline degradation condition, the ELS is proven to be stable and no obvious degradants are produced. On the other hand, under the acidic and neutral degradation condition, the 2,4-dioxo pyrimidinyl core of elagolix sodium is stable but the carboxyl group and secondary amine will occur ring cyclization to form the δ-lactam analogs of elagolix sodium. The plausible mechanisms for the degradation of acidic, thermal, photo-degradative and hydrogen peroxide mediated oxidative of elagolix sodium are proposed. It is worth to note that DP-3-4 are the potential degradants which are only found in the solution degradation and are not the real impurities of elagolix sodium.

Detection, isolation, and characterization of a novel impurity from several folic acid products.

Folic acid belongs to the group of water-soluble B vitamins and naturally exists in multiple forms in a wide variety of foods such as legumes, vegetables, liver, and milk (Iyer and Tomar, 2009; Lyon et al., 2020). It is involved in many biochemical reactions critical for cell division, such as purine and pyrimidine biosynthesis, DNA/RNA biosynthesis, and amino acid metabolism (Iyer and Tomar, 2009). Mammals cannot synthesize folic acid and thus they must acquire it from food. Although folic acid is ubiquitous in foods, folic acid deficiency still often occurs due to various causes such as unhealthy diet (Hildebrand et al., 2021; Iimura et al., 2022), disease-related malabsorption (Arcot and Shrestha, 2005), medication-related depletion (Arcot and Shrestha, 2005), or vitamin B12 deficiency (Fishman et al., 2000). Folic acid deficiency has been associated with several health problems, such as anemia (Carmel, 2005; Bailey and Caudill, 2012), cancer (Duthie, 1999), cardiovascular diseases (Wald et al., 2002), neural tube defects in newborns (van der Put et al., 2001), neuropsychiatric dysfunction (Shea et al., 2002), depression (Falade et al., 2021), inflammatory diseases (Suzuki and Kunisawa, 2015; Jones et al., 2019), and eye diseases (Sijilmassi, 2019). To prevent folic acid deficiency, its daily intake (400 µg/d) has been recommended for adults in the European Union, and its increased intake (600 µg/d) is advised for women before and during pregnancy (FAO/WHO, 2002; IOM, 2004). The New Zealand government mandated the fortification of non-organic wheat flour with folic acid in July 2021, and the UK government mandated the fortification of non-wholemeal wheat flour with folic acid in September 2021 (Haggarty, 2021).

Detection, identification, characterization, and high-performance liquid chromatography quantification of five impurities from a methazolamide product.

Methazolamide is an important carbonic anhydrase inhibitor and is mainly used for the treatment of glaucoma. Studies are extremely rare regarding the impurities in methazolamide products. In this work, the high-performance liquid chromatography/high-performance liquid chromatography-mass spectrometry methods were established for the analysis of impurities in methazolamide products. Five impurities (A, B, C, D, and E) were detected using the established high-performance liquid chromatography/high-performance liquid chromatography-mass spectrometry methods. Of these impurities, impurities A, B, and D are known compounds, and impurities C and E are novel compounds that have never been reported before. The identities of impurities A, B, D, and E were recognized by comparing their retention times and mass spectra with those of synthesized standard compounds under the same high-performance liquid chromatography-mass spectrometry conditions. Moreover, the structures of impurities C and E were characterized using a variety of analytical techniques including multidimensional nuclear magnetic resonance spectroscopy, Fourier transforming infrared spectroscopy, ultraviolet-visible absorption spectroscopy, and high-resolution quadrupole time-of-flight mass spectrometry. All of the five impurities are structural analogs of methazolamide. The formation mechanisms of these impurities were discussed.

Long-term music adjuvant therapy enhances the efficacy of sub-dose antiepileptic drugs in temporal lobe epilepsy.

AIMS: Noninvasive music adjuvant therapy shows great potential in improving seizure control when combined with routine antiepileptic drugs. However, the diversity of previous music protocols has resulted in disparate outcomes. The optimized protocol and features for music adjuvant therapy are still not fully understood which limits its feasibility. METHODS: By applying different regimens of music therapy in various temporal lobe epilepsy models, we evaluated the effect of music in combination with sub-dose drugs on epileptic seizures to determine the optimized protocol. RESULTS: A subgroup of kindled mice that were responsive to music adjuvant therapy was screened. In those mice, sub-dose drugs which were noneffective on kindled seizures, alleviated seizure severity after 12 h/day Mozart K.448 for 14 days. Shorter durations of music therapy (2 and 6 h/day) were ineffective. Furthermore, only full-length Mozart K.448, not its episodes or other music varieties, was capable of enhancing the efficacy of sub-dose drugs. This music therapeutic effect was not due to increasing cerebral drug concentration, but instead was related with the modulation of seizure electroencephalogram (EEG) spectral powers in the hippocampus. CONCLUSION: These results indicate that long-term full-length Mozart K.448 could enhance the anti-seizure efficacy of sub-dose drugs and may be a promising noninvasive adjuvant therapy for temporal lobe epilepsy.

Metabolic Profiling of Urinary Chiral Amino-Containing Biomarkers for Gastric Cancer Using a Sensitive Chiral Chlorine-Labeled Probe by HPLC-MS/MS.

Screening of characteristic biomarkers from chiral amino-containing metabolites in biological samples is difficult and important for the noninvasive diagnosis of gastric cancer (GC). Here, an enantiomeric pair of chlorine-labeled probes d-BPCl and l-BPCl was synthesized to selectively label d- and l-amino-containing metabolites in biological samples, respectively. Incorrect structural annotations were excluded according to the characteristic 3:1 abundance ratio of natural chlorine isotopes (35Cl and 37Cl) derived from the probes. A sensitive C18 HPLC-QQQ-MS/MS method in combination with the probes was then developed and applied in metabolomic analysis of amino-containing metabolites in urine samples. A total of 161 amino-containing metabolites were rapidly separated and determined, and 28 chiral amino acids and achiral glycine were quantified with good precision and accuracy. A total of 18 differential variables were discriminated by analyzing chiral amino-containing metabolites in urine samples of the GC patient and healthy person using the probe-based HPLC-MS/MS-MRM method combined with the orthogonal partial least squares discriminant analysis and Mann-Whitney U test with false discovery rate correction for multiple hypotheses. A diagnostic regression model including d-isoleucine, d-serine, and ß-(pyrazol-1-yl)-l-alanine and age was then constructed with an average prediction correctness of 88.9% in the validation set. This work established a close connection between gastric cancer and chiral amino-containing metabolites. The mass spectrometry data analyzed in the study are publicly available via Mendeley Data (DOI: 10.17632/4bd93j9yrr.1).

Separation and characterization of impurity P in azithromycin product.

Azithromycin is a macrolide antibiotic which is used to treat a wide variety of bacterial infections. Impurity P is one of the impurities in azithromycin product, which is registered in Pharmacopoeias of Europe and USA. However, to date, the structure of this impurity has still not been elucidated. In this work, we separated impurity P from azithromycin product using preparative chromatography and successfully identified its chemical structure using multiple analytical techniques. First, high-resolution ion trap-time-of-flight mass spectrometry (IT-TOF MS) was used to determine the accurate molecular mass ([M+H]+m/z 777.5121) and the chemical formula (C39H72N2O13) of the impurity. Second, Fourier transforming infrared spectroscopy (FT-IR), ultraviolet-visible absorption spectroscopy (UV-vis) and tandem mass spectrometry (MS/MS) analyses were performed to probe into the key functional groups of the impurity to aid the NMR analysis. Finally, the structure of the impurity was successfully resolved using multidimensional NMR. In addition, a mechanism for the formation of this impurity was proposed.

Sensitive Bromine-Labeled Probe D-BPBr for Simultaneous Identification and Quantification of Chiral Amino Acids and Amino-Containing Metabolites Profiling in Human Biofluid by HPLC/MS.

A novel bromine-isotope probe named D-BPBr with stereodynamic chiral recognition characteristics was developed for the labeling, separation, and detection of trace chiral amino acids and amino-containing metabolites. Fourteen enantiomeric pairs of amino acids could be successfully separated and quantified on a reverse-phase C18 column with an HPLC-MS/MS system after D-BPBr labeling. The chromatographic resolution for d,l-amino acid enantiomers ranged from 1.14 to 8.83 with the l-amino acid derivative always eluting prior to the corresponding d-enantiomer. Meanwhile, D-BPBr showed strong chiral selectivity on d-amino acids, and the ratio of mass spectrometric response for D-BPBr labeled d-amino acids to that of l-enantiomers ranged from 1.31 to 12.87 under the same condition. The D-BPBr labeling method was also demonstrated to be highly efficient and selective in separation and quantification of chiral amino acids especially for trace-level d-amino acids in human biofluids including urine and plasma, and in total, 11 l-amino acids and 10 d-amino acids in urine and 11 l-amino acids and 6 d-amino acids in plasma were detected and quantified. Based on the characteristic 2-Da mass difference of precursor ions and the nearly 1:1 peak intensity ratio originated from79Br and 81Br natural isotopes, as well as their dissociation features, 119 amino-containing metabolites were also rapidly detected in urine and plasma samples. Our work indicated that D-BPBr may be a potentially promising tool for the detection of d-amino acid-type biomarkers in disease diagnosis.

The oxidation of cysteine-containing peptides caused by perfluoroalkane sulfonyl fluorides.

Perfluorooctane sulfonyl fluoride (PFOSF) is the precursor of many fluorochemicals that are ubiquitous in the environment. However, its distribution and toxicology are rarely studied. In this work, the oxidability of PFOSF was found. PFOSF can accelerate oxidation of glutathione (GSH) to its oxidized form GSSG, and itself is reduced to a sulfinic acid. The yielded sulfinic acid was prepared and identified with high resolution mass spectrometry and NMR. Similar redox reactions were observed for PFOSF's short chain alternatives. The reduction potentials of perfluoroalkane sulfonyl fluorides (PFASFs) were determined to be -2.13 V vs. SCE with cyclic voltammetry, further demonstrating their oxidability. The peptide mixtures of GSH plus another cysteine-containing peptide were also oxidized by PFASFs to GSSG and an asymmetric disulfide GS-S-PEP. A single short-sequence PEP-SH could be oxidized to the symmetric disulfide PEP-S-S-PEP as the final product. In vitro experiments were carried out by adding PFASFs into rat liver S9 fractions. The turnover ratio of PFASFs were calculated to be about 4-10% by quantification of sulfinic acid with LC-MS/MS. Our work illustrates one of the potential metabolic pathways of PFASFs and demonstrates the oxidation of PEP-SHs by PFASFs, thus providing a preliminary exploration in the toxicology of these fluorochemicals.

Rapid characterization of compounds in fupo ganmao granules by high-performance liquid chromatography tandem mass spectrometry.

Fupo Ganmao Granules (FP) is a classic traditional Chinese medicine (TCM) formula, composed of 4 herbal medicines, Folium Hibisci Mutabilis, Magnolia officinalis, Pericarpium Citri Reticulatae, and Fructus arctii. It is regularly used in the treatment of common cold with wind-heat syndrome. The biologically active constituents in FP, especially those in trace amount, remain unclear. In this study, the components of FP were profiled and characterized using HPLC-ESI-MS2. A total of 58 compounds were identified according to their fragmentation features in IT-TOF-MS and IT-MS2, including 13 phenolic acids, 17 flavonoids, 21 lignans, 2 alkaloids, and 5 other compounds. Among them, the identities of 8 compounds were explicitly confirmed by comparing them with standard compounds, and their contents were determined. Our study provided a comprehensive understanding of the diverse chemical profile in FP, and the approach we developed is useful for the analysis of other TCM formulas.

Perfluoroalkane sulfonyl fluorides non-covalently bind to human serum albumin at Sudlow's sites.

Perfluorooctane sulfonyl fluoride (PFOSF) has been defined as persistent organic pollutant in the Stockholm Convention in 2009. Currently PFOSF and its substitutes (structural analogues in which octyl group is substituted for other aliphatic chains) are still scarcely studied. HSA is a main carrier for drugs in blood, and the influence of exogenous compounds including pollutants on HSA is of particular interest. In this work, the binding sites of HSA to Perfluoroalkane sulfonyl fluoride (PFASFs) were determined by fluorescence technique. The results demonstrated that PFASFs competitively bind to HSA at Sudlow's site I against warfarin and at Sudlow's site II against dansyl-proline and the related association constants were determined. The association constants of PFOSF, perfluorohexane sulfonyl fluoride (PFHSF) and perfluorobutane sulfonyl fluoride (PFBSF) were determined to be 2.59 × 10-3 µM-1, 4.65 × 10-3 µM-1 and 2.85 × 10-3 µM-1 at Sudlow's site I and 8.68 × 10-4 µM-1, 3.43 × 10-2 µM-1 and 1.92 × 10-2 µM-1 at Sudlow's site II, respectively. The results showed that PFASFs can bind tightly to HSA and thus migrate to all parts of the body through vascular system. Non-covalent interaction between HSA and PFASFs was confirmed with tryptic digestion experiment. The mass spectra results indicated that other binding sites of HSA are also involved in the binding of PFHSF and PFBSF. The total binding numbers of PFOSF, PFHSF and PFBSF on HSA are 2, 6 and 3, respectively.

Direct differentiation of stereoisomers of ezetimibe/ambrisentan/atorvastatin and their mechanism study by electrospray ionization quadrupole time-of-flight mass spectrometry.

A mass spectrometric method is introduced for rapid and accurate chiral quantification by examining a trimeric metal complex into which a chiral reference is incorporated with the analyte. Several metal ions (CuII , NiII , MgII , MnII , CoII , and ZnII ) were selected as the central metal ion, and chiral drugs ezetimibe (EZM) and ambrisentan (AMB) were used as the reference to each other for isomeric differentiation by using electrospray ionization quadrupole time-of-flight mass spectrometry. Doubly charged trimeric cluster ions instead of the singly charged clusters were applied in this study. Kinetic method (KM) and chiral recognition (CR) method were used for construction of a calibration curve for chiral quantitation. The results from the two methods were found to be complementary to each other, which improved quantitative analysis of stereoisomers for EZM. Furthermore, we have successfully used S-AMB as reference for the chiral differentiation of enantiomeric atorvastatin (ATO), which is frequently combined with EZM as a codrug. Experimental results showed that the binary mixture of EZM and ATO enantiomers can be determined simultaneously without prior separation steps. The direct measurement of chiral purity within 5% was demonstrated. This mass spectrometric method represents an effective alternative to commonly used chromatographic techniques as means of chiral purity determination and is of potential use in rapid screening experiments.

Chiral detection of entecavir stereoisomeric impurities through coordination with R-besivance and ZnII using mass spectrometry.

In this study, a mass spectrometry (MS)-based kinetic method (KM) is shown to be successful at analyzing a multichiral center drug stereoisomer, entecavir (ETV), both qualitatively and quantitatively. On the basis of the KM, the bivalent complex ion [MII (A)(ref*)2 ]2+ (MII  = divalent metal ion, A = analyte, and ref* = chiral reference) was set as precursor ion in MS/MS. The experiment results suggest strong chiral selectivity between ETV and its isomers when using ZnII coordinated with the chiral reference R-besivance (R-B). The logarithm of the fragment ion abundance ratio and the enantiomeric percentage (%) exhibits a strong linear relation because of the competitive loss of the reference and analyte. The product ion pair [ZnII (R-B)A-H]+ (m/z 733) and [ZnII (R-B)2 -H]+ (m/z 849), together with [R-B + H]+ (m/z 394) and [A + H]+ (m/z 278), can realize the identification of ETV and all of its chiral isomers. Theoretical calculation were also performed using the B3LYP functional with the 6-31G* and LanL2DZ basis set to clarify the mechanism of structural difference of these bivalent complex ions. The results reveal that MS-KM can be used to detect optical impurities without a chiral chromatographic column and fussy sample pretreatment. The established method has been used to determine stereoisomeric impurities of less than 0.1% in ETV crude drug, a demonstration of its simple and effective nature for rapid detection of stereoisomeric impurities.

Pair of Stereodynamic Chiral Benzylicaldehyde Probes for Determination of Absolute Configuration of Amino Acid Residues in Peptides by Mass Spectrometry.

This paper describes a simple method to determine the absolute configuration of amino acids residues in peptides by mass spectrometry using a newly developed pair of mass-tagged chiral probes without the requirement of reference standards. A pair of benzylicaldehyde probes, 1-(S)-1H in S configuration and 2-(R)-2D in deuterium-labeled R configuration with the ratio of 1:1, were synthesized for in situ condensation with amino acid residues and transformed into a pair of stereodynamic imine products. The characteristic intensity difference observed in mass spectrometry can be used to determine the absolute configuration and to quantify the enantiomeric composition of chiral amino acid residues. Significant chiral recognition ability was achieved for 18 natural chiral amino acids and for one ß-amino acid by comparing the ion intensity ratio of imine products I[1-(S)-1H-AA]- to I[2-(R)-2D-AA]-. For 16 kinds of amino acids, the L form of the amino acids was more reactive with 1-(S)-1H, while D configuration amino acids preferred to react with 2-(R)-2D. However, for three kinds of amino acid, the opposite result was obtained. The configurations of the residues in the peptides, Phe-Tyr-Ala, D-Phe-Tyr-Ala, Val-Pro-Phe-D-Leu-Met, Val-Pro-Phe-Leu-D-Met, as well as in a natural peptide with unknown chirality were determined by acid hydrolysis followed by the present method. In addition, molecular modeling results illustrate that the recognition process is mainly controlled by kinetic factors. Using the new probes coupled with a mass spectrometry approach avoids time-consuming workup and separation steps. We expect that the probes could be applied as tools to determine the absolute configuration of amino acid residues in proteins in future research.

Doubly charged trimeric cluster ions: effective in mutual chiral recognition of tadalafil and three proton pump inhibitors.

Mutual chiral recognition of four stereoisomers of tadalafil and three pairs of enantiomers of proton pump inhibitors (PPIs) including pantoprazole, lansoprazole, and omeprazole, as well as quantitative analysis of enantiomeric excess is achieved on the basis of the competitive fragmentation of doubly charged trimeric NiII cluster ions. Compared with a singly charged trimeric cluster ion, a doubly charged trimeric cluster ion was proved efficient in the recognition of chiral drugs with one or multiple chiral centers, due to its rich fragmentation ions. Upon collision-induced dissociation (CID), the cluster ion [NiII(PPIs)(tadalafil)2]2+ yielded two diagnostic ions [tadalafil + H]+ and [tadalafil - benzo[d][1,3]dixoloe]+ through electrospray ionization quadrupole time-of-flight mass spectrometry. The abundance ratio of the two fragment ions relied mainly on the configuration of PPIs and tadalafil, and therefore the chiral selectivity (Rchiral) of one enantiomer relative to the others is different. The chiral recognition of all four stereoisomers of tadalafil was achieved by using S configuration PPIs as references, and S-omeprazole showed the best selectivity. The Rchiral values for R,R/S,S, R,S/S,R, R,R/R,S and R,R/S,R-tadalafils were 1.47, 1.17, 2.37, and 2.10, respectively. In a reciprocal process, the Rchiral was 1.36 and 1.31 for R/S-pantoprazole and R/S-lansoprazole, respectively, by using R,R-tadalafil as a reference. Although omeprazole is a racemic drug, it can also be discriminated with S-omeprazole. Calibration curves were constructed with favorable correlation coefficients (r2 > 0.991) by relating the ln(Rchiral) values to the isomeric composition in a mixture. The sensitivity of the methodology allows mixtures to be analyzed for the enantiomeric excess (ee) by recording the ratios of fragment ion abundances in a mass spectrum. The sensitivity of the methodology allowed the determination of enantiomeric impurities of 5% molar composition in individual compounds present in mixtures; the diastereoisomeric impurity of R,R-tadalafil could be quantified even at 1%. We believe that the developed method not only has scientific significance in qualitative and quantitative chiral analyses of tadalafil and PPIs, but also provides great opportunity for enabling the discrimination on a wide range of chiral drugs.

Rapid identification of miglitol and its isomers by electrospray ionization tandem mass spectrometry.

RATIONALE: Miglitol (1) derived from 1-deoxynojirimycin is an iminosugar that is useful in the treatment of type 2 diabetes mellitus. Isomers (2, 3, 4) that differ at the C2 and C3 positions of hydroxyl groups from miglitol are impurities resulting from the synthesis of miglitol. The impurity profile of a drug substance is critical to its safety assessment and is important for monitoring the manufacturing process. Therefore, developing a fast and simple method that can rapidly identify the configuration of miglitol and its isomers (2, 3, 4) is necessary. METHODS: Miglitol (1) and its isomers 2-4 were derivatized with benzoboroxole (o-hydroxymethyl phenylboronic acid) at room temperature, and the cyclic boronate esters of different configurations were generated. Protonated miglitol and its isomers 2-4, as well as their derivatives, were subjected to collision-induced dissociation (CID) experiments by using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Elemental compositions of all the ions were verified by electrospray ion-trap time-of-flight mass spectrometry. RESULTS: Fragmentation of the protonated miglitol and its isomers gave the same fragment ions at m/z 190 and m/z 146. Both their fragmentation behavior and abundances were similar. Whereas the CID mass spectra of the precursor ions (m/z 322) of cyclic boronate esters showed four characteristic fragment ions, m/z 214 ([M-C7 H8 O](-) ), m/z 196 ([M-C7 H8 O-H2 O](-) ), m/z 151 ([M-C8 H13 NO3 ](-) ), and m/z 133 ([M-C8 H15 NO4 ](-) ). The abundances of these fragments are different which are related to the stereostructure of miglitol and its isomers. CONCLUSIONS: A facile method was established for the differentiation of the spatial configuration of miglitol and its isomers using the relative abundances of the fragment ions of boronate esters generated from in-situ reaction between analytes and benzoboroxole by ESI-MS/MS. This approach could be used to rapidly identify the stereoisomers and monitor the epimerization of miglitol and its isomers in chemical reactions and manufacturing processes. Copyright © 2016 John Wiley & Sons, Ltd.