Characterization and quantification of the Chinese medical formula Zhi-Zi-Chi decoction, a systematic strategy for the attenuation and synergy of compatibility mechanism.

Zhi-Zi-Chi decoction (ZZCD), comprising of Gardenia jasminoides Ellis (GJE) and Semen sojae preparatum (SSP), is a classical Chinese medicine formula. A novel analysis strategy was set up to obtain an evaluation of ZZCD on attenuation and synergy of compatibility. High-resolution ion trap/time-of-flight mass spectrometry (LC-IT-TOF-MS) was used for qualitative analysis. Variant ingredients were analyzed to compare the componential differences between ZZCD formula and single herbs. Based on our previous fingerprint studies that combined with chemometric methods, 13 remarkable chemical markers were selected and evaluated for quantitative determination by high performance liquid chromatography (HPLC) in three different ratios of ZZCD. 62 compounds in ZZCD, 55 compounds in GJE and 16 compounds in SSP were characterized. The compatibility of GJE and SSP may lead to the undetection of hepatotoxic components such as genipin and the emergence of protective components such as jasminoside A, which was not found in single herbs. Meanwhile, 13 selected chemical markers were successfully determined in three ratios of ZZCD. The compatibility may lead to the decrease of toxic ingredients and the increase of beneficial ingredients. By comparing the dissolution of chemical markers, iridoids in GJE and flavonoids in SSP had the best dissolution when the compatibility ratio was 1:1. This strategy would be a valuable reference for further study on the compatibility of traditional Chinese medicine formula.

Structure identification and content analysis of active components in Xiaoyao pills / 药学学报

Xiaoyao pills are a famous traditional Chinese medicine collected in Welfare Pharmacy, which is a classic prescription for treating liver depression and spleen deficiency. However, its composition is complex. In order to better control the quality of Xiaoyao pills, in this study, HPLC-ion-trap time-of-flight mass spectrometry (LC-IT-TOF/MS) was used to identify the main ingredients of Xiaoyao pills, paeoniflorin, albiflorin, glycyrrhizic acid, saikosaponin A and saikosaponin B2. Then a liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed for simultaneous determination and quantification of the main compounds. Fragmentation pathways of five active components were obtained. The method was validated. Five active ingredients in Xiaoyao pills had a good linear relationship, and the values of RSD (%) of repeatability were all less than 5%, the recovery ranges were between 90% and 115%, and the values of RSD (%) of each substance were less than 10% after the sample solution is placed for 24 hours. Three batches of Xiaoyao pills (concentrated pellets) and two batches of Xiaoyao pills (water pellets) were determined, the contents of paeoniflorin in concentrated pills were more than 4.0 mg·g-1, and those in water pills were more than 2.5 mg·g-1, which was accordance with Chinese Pharmacopoeia. However, other compounds behave differently. This method has high sensitivity and reliable measurement results, which provides basis for quality control of Xiaoyao pills and material basis for pharmacology research.

Global analysis of qualitative and quantitative metabolism of Notoginsenoside R1 in rat liver-brain-gut axis based on LC-IT-TOF/MS combing mMDF strategy.

BACKGROUND: The metabolism study of active components for traditional Chinese medicine (TCM) in target organs is conducive to clarify the authentic active ingredients. Notoginsenoside R1 (NG-R1), one of the bioactive components of Panax notoginsenoside (PNS), is commonly acknowledged as the characteristic marker of PNS. However, the metabolism of NG-R1 in target organs has not been clarified yet due to the lack of robust technique and approach. PURPOSE: The present study aimed to develop a reliable and efficient strategy and technology for revealing the qualitative and quantitative metabolism of active components of TCMs in target organs, and to clarify the biotransformation of NG-R1 in liver-brain-intestinal axis. METHODS: The metabolic transformation of NG-R1 in the brain gut axis was investigated in the in vitro incubation system of fresh rat brain, liver homogenate, and intestinal flora. To quickly lock the target metabolites, we set the mass defect filter (MDF) in different ranges to screen metabolites with different molecular weight (MW). This strategy was defined as multi-stage MDF (mMDF). In addition, we performed relative quantitative analysis on all metabolites according to the peak area acquired by LC-IT-TOF/MS to overcome the challenge that metabolites are difficult to be quantified due to the lack of standards. RESULTS: When MDF was set at 0.50 to 0.65 to screen metabolites with MW of 900 to 1200 Da, 6 novel metabolites were quickly found, and then identified as glucuronic acid binding, oxidation, dehydrogenation, methylation and hydrogenation products according to their LC and MS characteristics. When setting MDF at 0.42 - 0.52, 6 metabolites with MW of 600 to 900 Da were effectively screened and identified as Rg1, NG-R2, Rh1, Rg1+CH2+2H and Rg1+CH2. To screen the metabolites with MW of 300 to 600 Da, MDF was set at 0.25 - 0.42, and 4 novel metabolites were screened rapidly. The results of quantitative metabolism suggested that intestinal flora was the main metabolic site of NG-R1 in rat, and more than 60% of NG-R1 was converted to Rg1 by deglycosylation in the intestinal flora. CONCLUSION: The mMDF strategy can significantly improve the research efficiency of qualitative metabolism of saponins. Although NG-R1 could be transformed into a variety of metabolites in rat liver and brain homogenate, it still existed mainly in prototype form. In the rat flora, NG-R1 mainly existed in the form of deglycosylated metabolite Rg1.

Analysis of the polymerized impurities in cefmetazole sodium based on novel separation principle by liquid chromatography tandem ion trap/time-of-flight mass spectrometry.

To effectively control the polymerized impurities in cefmetazole sodium, novel high performance gel filtration chromatography (HPSEC) with TSK-gel G2000SWxl column and RP-HPLC method with C18 column were used in replace of classical gel filtration chromatography with Sephadex G-10 gel. By studying the chromatographic behavior of polymerized impurities in both chromatographic systems with different chromatographic separation principles, the polymerized impurities in cefmetazole sodium were separated and detected effectively. The two-dimensional liquid chromatography tandem ion trap/time-of-flight mass spectrometry (2D LC-IT-TOF MS) was applied to characterize the structures of polymerized impurities eluted from HPSEC method, and liquid chromatography tandem ion trap/time-of-flight mass spectrometry was applied to characterize the structures of polymerized impurities and other unknown impurities eluted from RP-HPLC method. The structures of fourteen unknown impurities in cefmetazole sodium were deduced based on the MS n data, nine of which were polymerized impurities. The corresponding relationship between impurities in the HPSEC method and RP-HPLC method was established, and the specificity of the two methods was evaluated. The RP-HPLC method for analysis of the polymerized impurities has higher column efficiency and specificity than the HPSEC method. The RP-HPLC method is suitable for quality control of the polymerized impurities in cefmetazole sodium. The forming mechanisms of degradation impurities in cefmetazole sodium were also studied.

Analysis of polymerized impurities in mezlocillin sodium and sulbenicillin sodium using two chromatographic separation mechanisms coupled to tandem mass spectrometry.

To effectively control the polymerized impurities in mezlocillin sodium and sulbenicillin sodium, a HPSEC method with TSK-gel G2000SWxl column and a RP-HPLC method with C18 analytical column were established to replace the classical gel filtration chromatography with Sephadex G-10 gel as stationary phase. By studying the chromatographic behavior of polymerized impurities in both methods with different chromatographic separation mechanisms, the polymerized impurities in mezlocillin sodium and sulbenicillin sodium were separated and detected effectively. The column switching two-dimension liquid chromatography ion trap/time-of-flight mass spectrometry was applied to characterize the structures of polymerized impurities eluted from the HPSEC method and RP-HPLC method for both drugs. The structures of the polymerized impurities in mezlocillin sodium and sulbenicillin sodium were deduced based on the MSn data. The results showed that the polymerized impurities detected by HPSEC method and RP-HPLC method were completely different. Therefore, two methods should be used meanwhile to control the polymerized impurities in mezlocillin sodium and sulbenicillin sodium.

Study of the polymerized impurities in cefotaxime sodium and cefepime by applying various chromatographic modes coupled with ion trap/time-of-flight mass spectrometry.

Polymerized impurities in ß-lactam antibiotics can induce allergic reaction, which seriously threaten the health of patients. In order to study the polymerized impurities in cefotaxime sodium and cefepime, a HPSEC method with TSK-gel G2000SWxl column and a RP-HPLC method with C18 column were established to replace the classical gel filtration chromatography with Sephadex G-10 gel as stationary phase. Two-dimensional (2D) liquid chromatography was employed to further investigate the HPSEC and RP-HPLC method and ion trap/time-of-flight mass spectrometry was applied to characterize the structures of polymerized impurities eluted from the two methods. The structures of the polymerized impurities in cefotaxime sodium and cefepime were deduced based on the MSn data, six of which were previously unknown. The corresponding relationship of impurities between the two methods was established, and the specificity of the two methods was compared. The results showed that the polymerized impurities in cefotaxime sodium and cefepime were co-eluted with other small molecular weight impurities with high polarity in HPSEC, leading to a poor specificity. The newly established RP-HPLC methods could effectively separate and detect polymerized impurities and were suitable for the quality control in daily analysis.

[Research on in vivo metabolites of Longxue Tongluo Capsules by liquid chromatography coupled with hybrid ion trap/time-of-flight mass spectrometry].

Longxue Tongluo Capsules(LTC) has good efficacy against blood stasis syndrome during the recovery period of ischemic stroke. Its main active ingredient is the phenolic extract of Chinese dragon's blood. In our previous study, the primary mass fragmentation pathways of phenolic derivatives from LTC were clarified. Herein, the metabolites in rat plasma were characterized following the oral administration of loureirin A and loureirin C using liquid chromatography coupled with hybrid ion trap/time-of-flight mass spectro-metry(LC-IT-TOF-MS), with 18 and 55 metabolites identified, respectively. On this basis, with the help of the obtained accurate molecular weight, characteristic fragment ions, reference comparison, combined with LTC database and natural products database self-created in our group, 18 prototypes and 106 metabolites were tentatively identified in rat plasma after oral gavage of LTC at a dose of 500 mg·kg~(-1). Glucuronidation, sulfonation, and methylation were major biotransformation pathways of LTC. This study preliminarily clarified the LTC constituents absorbed into blood and laid the foundation for clarifying the effective substances of LTC.

Characterization of eight unknown impurities and analysis of their source in xinfujunsu and injection by liquid chromatography coupled with ion trap/time-of-flight mass spectrometry.

Eight unknown impurities in xinfujunsu and its injection were characterized by liquid chromatography coupled with ion trap/time-of-flight mass spectrometry (LC-IT-TOF MS). In order to determine the m/z values of the molecular ions and predict the formulas of all detected impurities, full scan LC-MS in positive ion mode was firstly executed to obtain the m/z value of the molecules. Then LC-MS2, LC-MS3 and LC-MS4 were carried out on target compounds to obtain as much structural information as possible. Based on MSn spectral data and exact mass measurements, the chemical structures of eight unknown impurities were characterized, among which three impurities were degradation impurities and five impurities were process impurities. In addition, the source of impurities and the correlation between process and impurities were also studied. The production of degraded impurities was caused in the high pressure sterilization process of xinfujunsu injection. Based on characterization of impurities, this study revealed the cause of impurity production and provided guidance for enterprises to improve the process to reduce impurity content. Furthermore, this study also provided scientific basis for the further improvement of official monographs in pharmacopoeias.

Preparation and characterization of partial de-O-sulfation of heparin oligosaccharide library.

The O-sulfation, including 2-O- and 6-O-sulfation, in heparan sulfate (HS) have important biological and pathophysiological roles. Therefore, the ability to chemically generate a series of oligosaccharides, which have a similar structure to the naturally-occurring, 2-O- and 6-O-sulfating oligosaccharides from HS, would greatly contribute to investigating their natural role in HS. In this study, a heparin oligosaccharide library, including dp2, dp4 and dp6, were prepared from the chemical modification of the fully sulfated dp2, dp4 and dp6. Chemical reaction conditions were optimized to generate different patterns of 2-O- and 6-O-sulfated oligosaccharides, then the disaccharide composition and structure of the library was detected by high-performance liquid chromatography-ion trap/time-of-flight mass spectrometry (LC-IT-TOF/MS) analysis. This provides a foundation for further structural and functional studies of O-sulfated groups in HS.

Research on in vivo metabolites of Longxue Tongluo Capsules by liquid chromatography coupled with hybrid ion trap/time-of-flight mass spectrometry / 中国中药杂志

Longxue Tongluo Capsules(LTC) has good efficacy against blood stasis syndrome during the recovery period of ischemic stroke. Its main active ingredient is the phenolic extract of Chinese dragon's blood. In our previous study, the primary mass fragmentation pathways of phenolic derivatives from LTC were clarified. Herein, the metabolites in rat plasma were characterized following the oral administration of loureirin A and loureirin C using liquid chromatography coupled with hybrid ion trap/time-of-flight mass spectro-metry(LC-IT-TOF-MS), with 18 and 55 metabolites identified, respectively. On this basis, with the help of the obtained accurate molecular weight, characteristic fragment ions, reference comparison, combined with LTC database and natural products database self-created in our group, 18 prototypes and 106 metabolites were tentatively identified in rat plasma after oral gavage of LTC at a dose of 500 mg·kg~(-1). Glucuronidation, sulfonation, and methylation were major biotransformation pathways of LTC. This study preliminarily clarified the LTC constituents absorbed into blood and laid the foundation for clarifying the effective substances of LTC.

Metabolomic profiling of Saposhnikoviae Radix from Mongolia by LC-IT-TOF-MS/MS and multivariate statistical analysis.

Saposhnikoviae Radix (SR) is a commonly used crude drug that is obtained from the root and rhizome of Saposhnikovia divaricata which is distributed throughout China, Korea, Mongolia, and Russia. To evaluate the quality of Mongolian S. divaricata, metabolomic profiling of 43 plant specimens from different regions of Mongolia, as well as 8 SR samples and 2 plant specimens from China, were conducted by liquid chromatography-ion-trap-time-of-flight-mass spectrometer (LC-IT-TOF-MS). LC-MS profiles of the specimens showed uniformity and 30 compounds were tentatively identified, including 13 chromones and 17 coumarins. Among them, 16 compounds were isolated and unambiguously verified by comparing them with the spectroscopic data of standard compounds. Orthogonal partial least squares-discriminant analysis (OPLS-DA) based on LC-MS data from 7 Mongolian specimens and 8 Chinese SR samples as well as 2 plant specimens revealed that these 2 groups were clearly distinguishable and that Mongolian specimens were characterized by an abundance of prim-O-glucosylcimifugin (1). Moreover, the OPLS-DA of the Mongolian specimens showed that they can be discriminated by their growing regions based on the content of 8 chromones. The total content of dihydrofurochromones 1-3 was relatively higher in the specimens from Khalkhgol in the far eastern part of Mongolia, while contents of 10, 11, 15, and 16 were higher in those from Holonbuir in the eastern part. Based on this research, the roots of S. divaricata from Mongolia have potential as a new resource of SR in Kampo medicine.

Mass Spectrometry-Based Untargeted Metabolomics and α-Glucosidase Inhibitory Activity of Lingzhi (Ganoderma lingzhi) During the Developmental Stages.

Lingzhi is a Ganoderma mushroom species which has a wide range of bioactivities. Analysis of the changes in metabolites during the developmental stages of lingzhi is important to understand the underlying mechanism of its biosynthesis, as well as its bioactivity. It may also provide valuable information for the cultivation efficiency of lingzhi. In this study, mass spectrometry based untargeted metabolomics was carried out to analyze the alteration of metabolites during developmental stages of lingzhi. Eight developmental stages were categorized on the basis of morphological changes; starting from mycelium stage to post-mature stage. GC/MS and LC/MS analyses along with multivariate analysis of lingzhi developmental stages were performed. Amino acids, organic acids, sugars, polyols, fatty acids, fatty alcohols, and some small polar metabolites were extracted as marker metabolites from GC/MS analysis, while, lanostane-type triterpenoids were observed in LC/MS analysis of lingzhi. The marker metabolites from untargeted analysis of lingzhi developmental stages were correlated with the α-glucosidase inhibitory activity. Two metabolites, compounds 34 and 35, were identified as potential contributors of the α-glucosidase inhibitory activity. The current result shows that some metabolites are involved in the developmental process and α-glucosidase inhibitory activity of lingzhi.

Simultaneous LC-MS quantification of anthocyanins and non-anthocyanin phenolics from blueberries with widely divergent profiles and biological activities.

A sensitive and straightforward LC-IT-TOF-MS method was validated for the profiling and simultaneous quantification of anthocyanins, flavan-3-ols, flavonols, phenolic acids, and resveratrol in blueberry genotypes with fruit color ranging from deep purple (Vaccinium angustifolium) to various shades of pink (crosses of V. corymbosum, V. darrowii, and V. ashei). Standard calibration curves were linear for all analytes with correlation coefficients >0.99. The relative standard deviation for intra- and inter-day precision was lower than 10%. The method allowed an easy and selective identification and quantification of phenolics in blueberries with divergent profiles. The in vitro antioxidant assay results were strongly correlated with total phenolics and total anthocyanin content. Lowbush blueberry extracts (50 µg/mL) reduced ROS and NO production, and inhibited the transcription of the proinflammatory cytokines IL-6ß, COX2, iNOS, and IL-6 in the in vitro assays at much lower concentrations than pink fruited berries (250 µg/mL).

Development of a novel HPLC method for the determination of the impurities in desonide cream and characterization of its impurities by 2D LC-IT-TOF MS.

A novel HPLC method for the determination of the impurities in desonide cream was established and validated for the further improvement of the official monograph in USP. Desonide was well resolved from the photodegradation impurity, which overlapped with desonide in USP method. The method was validated in accordance to the regulatory guidelines recommended by the International Conference on Harmonisation and this validation included specificity, limit of detection, limit of quantification, linearity and accuracy. Four degradation impurities in desonide cream were characterized by a trap-free two-dimensional liquid chromatography coupled to high resolution ion trap/time-of-flight mass spectrometry (2D LC-IT-TOF MS) in positive mode of electrospray ionization. Through the multiple heart-cutting 2D LC approach and online demineralization technique, the problem of incompatibility between non-volatile salt mobile phase and mass spectrometry was solved completely, and the TIC chromatogram of LC-MS could be in conformity with the LC chromatogram of the official analytical method in the peak sequence of impurities. In the first dimension, the column was Phenomenex Kinetex C8 (4.6 mm × 150 mm, 2.6 µm) with a non-volatile salt mobile phase. In the second dimension, the column was Shimadzu Shim-pack GISS C18 (50 mm × 2.1 mm, 1.9 µm) with a volatile salt mobile phase. The structures of four degradation impurities in desonide cream were deduced based on the HPLC-MSn data. The established method in this study was simple and reliable for routine quality control of desonide cream.

A hybrid method for the determination of tubeimoside I in rat plasma after oral administration by LC-IT-TOF and UPLC-MS/MS.

Tubeimoside I (Tub) is a triterpenoid saponin isolated from Bolbostemma paniculatum[Maxim]Franquet. A sensitive and validated method was developed to determine Tub in rat plasma. This method combined the qualitative and quantitative advantages from liquid chromatography coupled with hybrid ion trap-time of flight mass spectrometer (HPLC-DAD-IT-TOF-MS) and a triple quadrupole-linear ion trap mass spectrometer 5500 (Qtrap 5500), owing to the narrow molecular range of Qtrap 5500 relative to the molecular weight of Tub. Initially, ion detection was achieved using negative ionization mode along with full scan on IT-TOF-MS. The detected precursor and product ions of Tub with the optimal mass parameters were determined on Qtrap 5500 by an online stepped optimization strategy and operated in negative multiple reaction monitoring mode. A simple methanol precipitation was employed with saikosaponin A as internal standard. The method was validated over the range from 20 to 2000 ng/mL with a lower limit of quantification of 20 ng/mL for Tub in plasma. The developed method was successfully applied to the pharmacokinetic study of Tub in rats following oral administration. Moreover, this method has some directive significance for the determination of other drugs whose parent ions exceeding the upper detection limit in Qtrap 5500.

A fragmentation study of isoflavones by IT-TOF-MS using biosynthesized isotopes.

To aid in the identification and quantification of biologically and agriculturally significant natural products, tandem mass spectrometry can provide accurate structural information with high selectivity and sensitivity. In this study, diagnostic fragmentation patterns of isoflavonoids were examined by liquid chromatography-ion trap-time of flight-mass spectrometry (LC-IT-TOF-MS). The fragmentation scheme for [M+H-2CO]+ ions derived from isoflavones and [M+H-B-ring-CO]+ ions derived from 5-hydroxyisoflavones, were investigated using different isotopically labeled isoflavones, specifically [1',2',3',4',5',6',2,3,4-13C9] and [2',3',5',6',2-D5] isoflavones. Specific isotopically labeled isoflavones were prepared through the biosynthetic incorporation of pharmacologically applied 13C- and D-labelled L-phenylalanine precursors in soybean plants following the application of insect elicitors. Using this approach, we empirically demonstrate that the [M+H-2CO]+ ion is generated by an intramolecular proton rearrangement during fragmentation. Furthermore, [M+H-B-ring-CO]+ ion is demonstrated to contain a C2H moiety derived from C-ring of 5-hydroxyisoflavones. A mechanistic understanding of characteristic isoflavone fragmentation patterns contributes to the efficacy and confidence in identifying related isoflavones by LC-MSn.

Separation and characterization of allergenic polymerized impurities from cephalosporin for injection by trap free two-dimensional high performance size exclusion chromatography × reversed phase liquid chromatography coupled with ion trap time-of-flight mass spectrometry.

As requested by regulatory authorities, polymerized impurities are an important issue of quality control. In this study, we presented the utilization of a trap-free two-dimensional chromatography, which was consisted by a high performance size exclusion chromatography (HPSEC) and a reversed-phase liquid chromatography (RP-LC) coupled to ion trap time-of-flight mass spectrometry with positive mode of electrospray ionization, to separate and characterize ten allergenic impurities in ceftazidime for injection, cefazolin sodium for injection, cefoperazone sodium and sulbactam sodium for injection and cefamandole nafate for injection. An effective method for characterizing the polymerized impurities in ß-lactam antibiotics was established on the basis of column-switching technique which effectively combined the advantages of HPSEC and the ability of RP-HPLC to identify the special impurities. In the first dimension, the column was the Xtimate SEC-120 analytical column (7.8 mm × 30 cm, 5 µm) and the flow rate was 1.0 mL min-1 with gradient elution using 0.005 mol L-1 phosphate buffer solution at pH 7.0 and acetonitrile as mobile phase. In the second dimension, the analytical column was ZORBAX SB-C18 (4.6 × 150 mm, 3.5 µm) using ammonium formate solution (10 mM) and ammonium formate (8 mM) in [acetonitrile-water (4:1, v/v)] solution as mobile phase at a flow rate of 0.4 mL min-1. Eluent associate with each peak separated in the first dimension was trapped by a 20 µL quantitative loop and then transferred (via a six-port valve) into the second dimension system with volatile mobile phase. Through the multiple heart-cutting 2D-LC approach and online desalting technique, the problem of incompatibility between non-volatile mobile phase and mass spectrometry was solved completely. The fragmentation behaviors of ten allergenic impurities were studied. The structures of ten allergenic impurities in cephalosporin drug substance were deduced based on the HPLC-MSn data, in which four impurities were polymerized impurities. The forming factors of polymerized impurity in cephalosporins were also studied.

Application of a trap-free two-dimensional liquid chromatography combined with ion trap/time-of-flight mass spectrometry for separation and characterization of impurities and isomers in cefpiramide.

High-resolution mass spectrometry had been routinely used for structure identification of impurity. However, all LC-MS methods were based on a volatile mobile phase, and a non-volatile system is used in the official analytical method of United States Pharmacopoeia for cefpiramide which limited the use of mass spectrometry for structure characterization of the impurities. Here we presented the utilization of a trap-free two-dimensional liquid chromatography coupled to high resolution ion trap/time-of-flight mass spectrometry (2D LC-IT-TOF MS) with positive and negative modes of electrospray ionization for characterization of eight impurities in cefpiramide. Trap-free two-dimensional liquid chromatography and online desalting technique made it possible to characterize the impurity in cefpiramide in the condition of official standard, and the TIC chromatogram of LC-MS was in conformity with the LC chromatogram of the official analytical method in the peak sequence of impurities, which could further improve the method of official monographs in pharmacopoeias. Each peak separated by the non-volatile mobile phase was trapped by a 20 µL quantitative loop then transferred into a system with a volatile mobile phase connected to a MS detector. In the first dimension, the column was Kromasil C8 analytical column (250 mm × 4.6 mm, 5 µm) with a non-volatile salt mobile phase at the flow rate of 0.8 mL min-1. In the second dimension, the column was Shimadzu Shim-pack GISS C18 (50 mm × 2.1 mm, 1.9 µm) with a volatile salt mobile phase at the flow rate of 0.3 mL min-1. Through the multiple heart-cutting 2D-LC approach and online desalting technique, the problem of incompatibility between non-volatile salt mobile phase and mass spectrometry was solved completely. The fragmentation behavior of cefpiramide and its eight impurities were studied. The structures of eight impurities in cefpiramide drug substance were deduced based on the HPLC-MSn data, in which seven impurities were novel impurities. The forming mechanisms of degradation products in cefpiramide were also studied.

Separation and characterization of allergic polymerized impurities in cephalosporins by 2D-HPSEC×LC-IT-TOF MS.

Eleven unknown allergic impurities in cefodizime, cefmenoxime and cefonicid were separated and characterized by a trap-free two-dimensional high performance size exclusion chromatography (HPSEC) and reversed phase liquid chromatography (RP-HPLC) coupled to high resolution ion trap/time-of-flight mass spectrometry (2D-HPSEC×LC-IT-TOF MS) with positive and negative modes of electrospray ionization method. Separation and characterization the allergic polymerized impurities in ß-lactam antibiotics were on the basis of column-switching technique which effectively combined the advantages of HPSEC and the ability of RP-HPLC to identify the special impurities. In the first dimension HPSEC, the column was Xtimate SEC-120 analytical column (7.8mm×30cm, 5µm), and the gradient elution used pH 7.0 buffer-acetonitrile as mobile phase And the second dimension analytical column was ZORBAX SB-C18 (4.6×150mm, 3.5µm) with ammonium formate solution (10mM) and ammonium formate (8mM) in [acetonitrile-water (4:1, v/v)] solution as mobile phase. Structures of eleven unknown impurities were deduced based on the high resolution MSn data with both positive and negative modes, in which nine impurities were polymerized impurities. The forming mechanism of ß-lactam antibiotic polymerization in cephalosporins was also studied. The question on incompatibility between non-volatile salt mobile phase and mass spectrometry was solved completely by multidimensional heart-cutting approaches and online demineralization technique, which was worthy of widespread use and application for the advantages of stability and repeatability.

Separation and characterization of unknown impurities and isomers in flomoxef sodium by LC-IT-TOF MS and study of their negative-ion fragmentation regularities.

Thirteen unknown impurities in flomoxef sodium were separated and characterized by liquid chromatography coupled with high resolution ion trap/time-of-flight mass spectrometry (LC-IT-TOF MS)with positive and negative modes of electrospray ionization method for further improvement of official monographs in pharmacopoeias. The fragmentation patterns of impurities in flomoxef in the negative ion mode were studied in detail, and new negative-ion fragmentation regularities were discovered. Chromatographic separation was performed on a Kromasil C18 column (250mm×4.6mm, 5µm). The mobile phase consisted of (A) ammonium formate aqueous solution (10mM)-methanol (84:16, v/v) and (B) ammonium formate aqueous solution (10mM)-methanol (47:53, v/v). In order to determine the m/z values of the molecular ions and formulas of all detected impurities, full scan LC-MS in both positive and negative ion modes was firstly executed to obtain the m/z value of the molecules. Then LC-MS2 and LC-MS3 were carried out on target compounds to obtain as much structural information as possible. Complete fragmentation patterns of impurities were studied and used to obtain information about the structures of these impurities. Structures of thirteen unknown degradation products in flomoxef sodium were deduced based on the high resolution MSn data with both positive and negative modes. The forming mechanisms of degradation products in flomoxef sodium were also studied.