Rapid gas chromatography with flame photometric detection of multiple organophosphorus pesticides in Salvia miltiorrhizae after ultrasonication assisted one-step extraction

Publication date: Available online 14 October 2017
Source:Journal of Chromatography B
Author(s): Shanshan Zhang, Xiaofei Liu, Jia’an Qin, Meihua Yang, Hongzheng Zhao, Yong Wang, Weiying Guo, Zhijie Ma, Weijun Kong
A simple and rapid gas chromatography-flame photometric detection (GC-FPD) method was developed for the determination of 12 organophosphorus pesticides (OPPs) in Salvia miltiorrhizae by using ultrasonication assisted one-step extraction (USAE) without any clean-up steps. Some crucial parameters such as type of extraction solvent and temperature programming were optimized to improve the method performance for trace analysis. Any clean-up steps were negligent as no interferences were detected in the GC-FPD chromatograms for sensitive detection. Under the optimized conditions, limits of detection (LODs) and quantitation (LOQs) for all pesticides were in the range of 0.001–0.002mg/kg and 0.004–0.01mg/kg, respectively, which were all below the regulatory maximum residue limits suggested. RSDs for method precision (intra- and inter-day variations) were lower than 6.8% in approval with international regulations. Average recovery rates for all pesticides at three fortification levels (0.5, 1.0 and 5.0mg/kg) were in the range of 71.2–101.0% with relative standard deviations (RSDs) <13%. The developed method was evaluated for its feasibility in the simultaneous pre-concentration and determination of 12 OPPs in 32 batches of real S. miltiorrhizae samples. Only one pesticide (dimethoate) out of the 12 targets was simultaneously detected in four samples at concentrations of 0.016–0.02mg/kg. Dichlorvos and omethoate were found in the same sample from Sichuan province at 0.004 and 0.027mg/kg, respectively. Malathion and momocrotophos were determined in the other two samples at 0.014 and 0.028mg/kg, respectively. All the positive samples were confirmed by LC–MS/MS. The simple, reliable and rapid USAE-GC-FPD method with many advantages over traditional techniques would be preferred for trace analysis of multiple pesticides in more complex matrices.

Publication date: Available online 14 October 2017
Source:Journal of Chromatography B

Author(s): Shanshan Zhang, Xiaofei Liu, Jia’an Qin, Meihua Yang, Hongzheng Zhao, Yong Wang, Weiying Guo, Zhijie Ma, Weijun Kong

A simple and rapid gas chromatography-flame photometric detection (GC-FPD) method was developed for the determination of 12 organophosphorus pesticides (OPPs) in Salvia miltiorrhizae by using ultrasonication assisted one-step extraction (USAE) without any clean-up steps. Some crucial parameters such as type of extraction solvent and temperature programming were optimized to improve the method performance for trace analysis. Any clean-up steps were negligent as no interferences were detected in the GC-FPD chromatograms for sensitive detection. Under the optimized conditions, limits of detection (LODs) and quantitation (LOQs) for all pesticides were in the range of 0.001–0.002mg/kg and 0.004–0.01mg/kg, respectively, which were all below the regulatory maximum residue limits suggested. RSDs for method precision (intra- and inter-day variations) were lower than 6.8% in approval with international regulations. Average recovery rates for all pesticides at three fortification levels (0.5, 1.0 and 5.0mg/kg) were in the range of 71.2–101.0% with relative standard deviations (RSDs) <13%. The developed method was evaluated for its feasibility in the simultaneous pre-concentration and determination of 12 OPPs in 32 batches of real S. miltiorrhizae samples. Only one pesticide (dimethoate) out of the 12 targets was simultaneously detected in four samples at concentrations of 0.016–0.02mg/kg. Dichlorvos and omethoate were found in the same sample from Sichuan province at 0.004 and 0.027mg/kg, respectively. Malathion and momocrotophos were determined in the other two samples at 0.014 and 0.028mg/kg, respectively. All the positive samples were confirmed by LC–MS/MS. The simple, reliable and rapid USAE-GC-FPD method with many advantages over traditional techniques would be preferred for trace analysis of multiple pesticides in more complex matrices.





The rapid determination of bromadiolone in liver and blood plasma by in-injector pyrolysis gas chromatography – ion trap tandem mass spectrometry

Publication date: Available online 14 October 2017
Source:Journal of Chromatography B
Author(s): Veronika Doubková, Petr Maršálek, Vladimír Večerek
The unintentional poisoning of off-target animals by bromadiolone, a second generation anticoagulant rodenticide, is an undesirable outcome requiring sensitive analytical methods. In this study, a rapid and sensitive method for the determination of bromadiolone in liver and blood plasma by means of gas chromatography coupled with tandem mass spectrometry without need for derivatization was developed. The method is based on the in-injector pyrolysis of bromadiolone and subsequent gas chromatography coupled with ion trap tandem mass spectrometry with electron ionization. Sample preparation includes extraction with methanol, evaporation under nitrogen stream, and dissolution in toluene. The pyrolysis of bromadiolone was carried out in an injector at 390°C. Chromatographic separation of the pyrolytical fragment of bromadiolone was achieved using a VF–5ms column with helium as the mobile phase. Tandem in-time mass spectrometry of the separated pyrolytical fragment of bromadiolone was carried out using an ion trap mass spectrometer after electron ionization. Recovery ranged from 94 to 98%. The method showed good linearity up to 1000μgkg−1 for liver and 1000μgL−1 for plasma. The limit of detection was 0.38μgkg−1 for liver and 0.26μgL−1 for plasma. The developed method was used successfully in several animal poisoning cases.

Publication date: Available online 14 October 2017
Source:Journal of Chromatography B

Author(s): Veronika Doubková, Petr Maršálek, Vladimír Večerek

The unintentional poisoning of off-target animals by bromadiolone, a second generation anticoagulant rodenticide, is an undesirable outcome requiring sensitive analytical methods. In this study, a rapid and sensitive method for the determination of bromadiolone in liver and blood plasma by means of gas chromatography coupled with tandem mass spectrometry without need for derivatization was developed. The method is based on the in-injector pyrolysis of bromadiolone and subsequent gas chromatography coupled with ion trap tandem mass spectrometry with electron ionization. Sample preparation includes extraction with methanol, evaporation under nitrogen stream, and dissolution in toluene. The pyrolysis of bromadiolone was carried out in an injector at 390°C. Chromatographic separation of the pyrolytical fragment of bromadiolone was achieved using a VF–5ms column with helium as the mobile phase. Tandem in-time mass spectrometry of the separated pyrolytical fragment of bromadiolone was carried out using an ion trap mass spectrometer after electron ionization. Recovery ranged from 94 to 98%. The method showed good linearity up to 1000μgkg−1 for liver and 1000μgL−1 for plasma. The limit of detection was 0.38μgkg−1 for liver and 0.26μgL−1 for plasma. The developed method was used successfully in several animal poisoning cases.





Liquid chromatography-tandem mass spectrometric assay for determination of unstable arecoline in rat plasma and its application

Publication date: Available online 14 October 2017 Source:Journal of Chromatography B Author(s): Hong Pan, Linyan Huang, Yi Li, Xumei Zhou, Yuanfu Lu, Fuguo Shi

Publication date: Available online 14 October 2017
Source:Journal of Chromatography B

Author(s): Hong Pan, Linyan Huang, Yi Li, Xumei Zhou, Yuanfu Lu, Fuguo Shi







Stability indicating HPLC-DAD method for analysis of Ketorolac binary and ternary mixtures in eye drops: Quantitative analysis in rabbit aqueous humor

Publication date: Available online 14 October 2017
Source:Journal of Chromatography B
Author(s): Fawzy A. El Yazbi, Ekram M. Hassan, Essam F. Khamis, Marwa A.A. Ragab, Mohamed M.A. Hamdy
Ketorolac tromethamine (KTC) with phenylephrine hydrochloride (PHE) binary mixture (mixture 1) and their ternary mixture with chlorpheniramine maleate (CPM) (mixture 2) were analyzed using a validated HPLC-DAD method. The developed method was suitable for the in vitro as well as quantitative analysis of the targeted mixtures in rabbit aqueous humor. The analysis in dosage form (eye drops) was a stability indicating one at which drugs were separated from possible degradation products arising from different stress conditions (in vitro analysis). For analysis in aqueous humor, Guaifenesin (GUF) was used as internal standard and the method was validated according to FDA regulation for analysis in biological fluids. Agilent 5 HC-C18(2) 150×4.6mm was used as stationary phase with a gradient eluting solvent of 20mM phosphate buffer pH 4.6 containing 0.2% triethylamine and acetonitrile. The drugs were resolved with retention times of 2.41, 5.26, 7.92 and 9.64min for PHE, GUF, KTC and CPM, respectively. The method was sensitive and selective to analyze simultaneously the three drugs in presence of possible forced degradation products and dosage form excipients (in vitro analysis) and also with the internal standard, in presence of aqueous humor interferences (analysis in biological fluid), at a single wavelength (261nm). No extraction procedure was required for analysis in aqueous humor. The simplicity of the method emphasizes its capability to analyze the drugs in vivo (in rabbit aqueous humor) and in vitro (in pharmaceutical formulations).

Publication date: Available online 14 October 2017
Source:Journal of Chromatography B

Author(s): Fawzy A. El Yazbi, Ekram M. Hassan, Essam F. Khamis, Marwa A.A. Ragab, Mohamed M.A. Hamdy

Ketorolac tromethamine (KTC) with phenylephrine hydrochloride (PHE) binary mixture (mixture 1) and their ternary mixture with chlorpheniramine maleate (CPM) (mixture 2) were analyzed using a validated HPLC-DAD method. The developed method was suitable for the in vitro as well as quantitative analysis of the targeted mixtures in rabbit aqueous humor. The analysis in dosage form (eye drops) was a stability indicating one at which drugs were separated from possible degradation products arising from different stress conditions (in vitro analysis). For analysis in aqueous humor, Guaifenesin (GUF) was used as internal standard and the method was validated according to FDA regulation for analysis in biological fluids. Agilent 5 HC-C18(2) 150×4.6mm was used as stationary phase with a gradient eluting solvent of 20mM phosphate buffer pH 4.6 containing 0.2% triethylamine and acetonitrile. The drugs were resolved with retention times of 2.41, 5.26, 7.92 and 9.64min for PHE, GUF, KTC and CPM, respectively. The method was sensitive and selective to analyze simultaneously the three drugs in presence of possible forced degradation products and dosage form excipients (in vitro analysis) and also with the internal standard, in presence of aqueous humor interferences (analysis in biological fluid), at a single wavelength (261nm). No extraction procedure was required for analysis in aqueous humor. The simplicity of the method emphasizes its capability to analyze the drugs in vivo (in rabbit aqueous humor) and in vitro (in pharmaceutical formulations).





A high-throughput UHPLC method for the analysis of 5-nitroimidazole residues in milk BASED ON SALTing-out ASSISTED LIQUID-LIQUID EXTRACTION

Publication date: Available online 13 October 2017
Source:Journal of Chromatography B
Author(s): Maykel Hernández-Mesa, Laura Carbonell-Rozas, Carmen Cruces-Blanco, Ana M. García-Campaña
A novel salting-out assisted liquid-liquid extraction (SALLE) method has been developed for the extraction of eight 5-NDZ antibiotics from milk samples prior to their analysis by UHPLC. An exhaustive study of the parameters involved in the SALLE procedure has been carried out, optimizing the extraction solvent volume, the amount of the salt agent, and the centrifugation and vortex timing by an experimental design. After sample treatment, the obtained extract was reconstituted in the mobile phase (6:94 (v/v) acetonitrile/water containing 0.1% (v/v) of formic acid) and analyzed by the proposed UHPLC-UV method. Separation was accomplished in a C18 Zorbax Eclipse Plus (50mm×2.1mm, 1.8μm) column at 45°C within 8min. Gradient mode was considered using a mobile phase consisting of 0.1% (v/v) formic acid aqueous solution and acetonitrile containing 0.1% (v/v) formic acid at a flow rate of 0.45mL/min. Analytical signals were monitored at 320nm. Matrix-matched calibration curves showed satisfactory linearity (R2≥0.996). LODs, ranging from 2 to 4μg/L, were achieved and precision studies showed RSDs lower than 12.8% in terms of peak height. Additionally, recoveries higher than 62.8% were obtained for all studied compounds in milk samples.

Publication date: Available online 13 October 2017
Source:Journal of Chromatography B

Author(s): Maykel Hernández-Mesa, Laura Carbonell-Rozas, Carmen Cruces-Blanco, Ana M. García-Campaña

A novel salting-out assisted liquid-liquid extraction (SALLE) method has been developed for the extraction of eight 5-NDZ antibiotics from milk samples prior to their analysis by UHPLC. An exhaustive study of the parameters involved in the SALLE procedure has been carried out, optimizing the extraction solvent volume, the amount of the salt agent, and the centrifugation and vortex timing by an experimental design. After sample treatment, the obtained extract was reconstituted in the mobile phase (6:94 (v/v) acetonitrile/water containing 0.1% (v/v) of formic acid) and analyzed by the proposed UHPLC-UV method. Separation was accomplished in a C18 Zorbax Eclipse Plus (50mm×2.1mm, 1.8μm) column at 45°C within 8min. Gradient mode was considered using a mobile phase consisting of 0.1% (v/v) formic acid aqueous solution and acetonitrile containing 0.1% (v/v) formic acid at a flow rate of 0.45mL/min. Analytical signals were monitored at 320nm. Matrix-matched calibration curves showed satisfactory linearity (R2 0.996). LODs, ranging from 2 to 4μg/L, were achieved and precision studies showed RSDs lower than 12.8% in terms of peak height. Additionally, recoveries higher than 62.8% were obtained for all studied compounds in milk samples.





Different in vitro Cellular Responses to Tamoxifen Treatment in Polydimethylsiloxane-based Devices Compared to Normal Cell Culture

Publication date: Available online 12 October 2017
Source:Journal of Chromatography B
Author(s): Lingyu Wang, Linfen Yu, Samantha Grist, Karen C. Cheung, David D.Y. Chen
Cell culture systems based on polydimethylsiloxane (PDMS) microfluidic devices offer great flexibility because of their simple fabrication and adaptability. PDMS devices also make it straightforward to set up parallel experiments and can facilitate process automation, potentially speeding up the drug discovery process. However, cells grown in PDMS-based systems can develop in different ways to those grown with conventional culturing systems because of the differences in the containers’ surfaces. Despite the growing number of studies on microfluidic cell culture devices, the differences in cellular behavior in PDMS-based devices and normal cell culture systems are poorly characterized. In this work, we investigated the proliferation and autophagy of MCF7 cells cultured in uncoated and Parylene-C coated PDMS wells. Using a quantitative method combining solid phase extraction and liquid chromatography mass spectrometry we developed, we showed that Tamoxifen uptake into the surfaces of uncoated PDMS wells can change the drug’s effective concentration in the culture medium, affecting the results of Tamoxifen-induced autophagy and cytotoxicity assays. Such changes must be carefully analyzed before transferring in vitro experiments from a traditional culture environment to a PDMS-based microfluidic system. We also found that cells cultured in Parylene-C coated PDMS wells showed similar proliferation and drug response characteristics to cells cultured in standard polystyrene (PS) plates, indicating that Parylene-C deposition offers an easy way of limiting the uptake of small molecules into porous PDMS materials and significantly improves the performance of PDMS-based device for cell related research.

Publication date: Available online 12 October 2017
Source:Journal of Chromatography B

Author(s): Lingyu Wang, Linfen Yu, Samantha Grist, Karen C. Cheung, David D.Y. Chen

Cell culture systems based on polydimethylsiloxane (PDMS) microfluidic devices offer great flexibility because of their simple fabrication and adaptability. PDMS devices also make it straightforward to set up parallel experiments and can facilitate process automation, potentially speeding up the drug discovery process. However, cells grown in PDMS-based systems can develop in different ways to those grown with conventional culturing systems because of the differences in the containers’ surfaces. Despite the growing number of studies on microfluidic cell culture devices, the differences in cellular behavior in PDMS-based devices and normal cell culture systems are poorly characterized. In this work, we investigated the proliferation and autophagy of MCF7 cells cultured in uncoated and Parylene-C coated PDMS wells. Using a quantitative method combining solid phase extraction and liquid chromatography mass spectrometry we developed, we showed that Tamoxifen uptake into the surfaces of uncoated PDMS wells can change the drug’s effective concentration in the culture medium, affecting the results of Tamoxifen-induced autophagy and cytotoxicity assays. Such changes must be carefully analyzed before transferring in vitro experiments from a traditional culture environment to a PDMS-based microfluidic system. We also found that cells cultured in Parylene-C coated PDMS wells showed similar proliferation and drug response characteristics to cells cultured in standard polystyrene (PS) plates, indicating that Parylene-C deposition offers an easy way of limiting the uptake of small molecules into porous PDMS materials and significantly improves the performance of PDMS-based device for cell related research.





Identification and Quantification of Signal Peptide Variants in an IgG1 Monoclonal Antibody Produced in Mammalian Cell Lines

Publication date: Available online 12 October 2017 Source:Journal of Chromatography B Author(s): Yunping Huang, Jinmei Fu, Richard Ludwig, Li Tao, Jacob Bongers, Li Ma, Ming Yao, Mingshe Zhu, Tapan Das, Reb Russell …

Publication date: Available online 12 October 2017
Source:Journal of Chromatography B

Author(s): Yunping Huang, Jinmei Fu, Richard Ludwig, Li Tao, Jacob Bongers, Li Ma, Ming Yao, Mingshe Zhu, Tapan Das, Reb Russell

Sequence variants of a monoclonal antibody resulting from incomplete processing of signal peptide were identified and characterized using multiple mass spectrometry platforms and reverse phase chromatography. Detection and quantification of these variants by three LC/MS platforms were assessed. Quantification was also performed by mass spectrometric analysis of the subunits of the antibody generated by reduction and IdeS proteolysis. Peptide mapping with LC/MS/MS detection was used to quantify and confirm the identities of signal peptide sequence variants. Although quantification of the signal peptide variants thru mass spectrometry approaches is system dependent, our data revealed the results are close to the values determined by chromatographic separation with UV detection. Each of the methods have proven effective in demonstrating the consistency of signal peptide variants levels across the manufacture history of the antibody.





Biophysical analysis of BMV virions purified using a novel method

Publication date: Available online 12 October 2017
Source:Journal of Chromatography B
Author(s): Aleksander Strugała, Monika Kręcisz, Jakub Dalibor Rybka, Anna Urbanowicz, Kamil Szpotkowski, Paulina Bierwagen, Marek Figlerowicz, Maciej Kozak, Christoph Böttcher, Michał Giersig
Brome mosaic virus (BMV) has been successfully loaded with different types of nanoparticles. However, studies concerning its application as a nanoparticle carrier demand high-purity virions in large amounts. Existing BMV purification protocols rely on multiple differential ultracentrifugation runs of the initially purified viral preparation. Herein, we describe an alternative method for BMV purification based on ion-exchange chromatography and size-exclusion chromatography (SEC) yielding 0.2mg of virus from 1g of plant tissue. Our method is of similar efficiency to previously described protocols and can easily be scaled up. The method results in high-quality BMV preparations as confirmed by biophysical analyses, including cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), static light scattering (SLS), and circular dichroism (CD) measurements and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy. Our results revealed that purified BMV capsids are stable and monodisperse and can be used for further downstream applications. In this work, we also characterize secondary structure and size fluctuations of the BMV virion at different pH values.

Publication date: Available online 12 October 2017
Source:Journal of Chromatography B

Author(s): Aleksander Strugała, Monika Kręcisz, Jakub Dalibor Rybka, Anna Urbanowicz, Kamil Szpotkowski, Paulina Bierwagen, Marek Figlerowicz, Maciej Kozak, Christoph Böttcher, Michał Giersig

Brome mosaic virus (BMV) has been successfully loaded with different types of nanoparticles. However, studies concerning its application as a nanoparticle carrier demand high-purity virions in large amounts. Existing BMV purification protocols rely on multiple differential ultracentrifugation runs of the initially purified viral preparation. Herein, we describe an alternative method for BMV purification based on ion-exchange chromatography and size-exclusion chromatography (SEC) yielding 0.2mg of virus from 1g of plant tissue. Our method is of similar efficiency to previously described protocols and can easily be scaled up. The method results in high-quality BMV preparations as confirmed by biophysical analyses, including cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), static light scattering (SLS), and circular dichroism (CD) measurements and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy. Our results revealed that purified BMV capsids are stable and monodisperse and can be used for further downstream applications. In this work, we also characterize secondary structure and size fluctuations of the BMV virion at different pH values.





Determination of l-glutamic acid and γ–aminobutyric acid in mouse brain tissue utilizing GC–MS/MS

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069
Author(s): Christine A. Farthing, Don E. Farthing, Ronald E. Gress, Douglas H. Sweet
A rapid and selective method for the quantitation of neurotransmitters, l-Glutamic acid (GA) and γ–Aminobutyric acid (GABA), was developed and validated using gas chromatography-tandem mass spectrometry (GC–MS/MS). The novel method utilized a rapid online hot GC inlet gas phase sample derivatization and fast GC low thermal mass technology. The method calibration was linear from 0.5 to 100μg/mL, with limits of detections of 100ng/mL and 250ng/mL for GA and GABA, respectively. The method was used to investigate the effects of deletion of organic anion transporter 1 (Oat1) or Oat3 on murine CNS levels of GA and GABA at 3 and 18 mo of age, as compared to age matched wild-type (WT) animals. Whole brain concentrations of GA were comparable between WT, Oat1−/−, and Oat3−/− 18 mo at both 3 and 18 mo of age. Similarly, whole brain concentrations of GABA were not significantly altered in either knockout mouse strain at 3 or 18 mo of age, as compared to WT. These results indicate that the developed GC–MS/MS method provides sufficient sensitivity and selectivity for the quantitation of these neurotransmitters in mouse brain tissue. Furthermore, these results suggest that loss of Oat1 or Oat3 function in isolation does not result in significant alterations in brain tissue levels of GA or GABA.

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069

Author(s): Christine A. Farthing, Don E. Farthing, Ronald E. Gress, Douglas H. Sweet

A rapid and selective method for the quantitation of neurotransmitters, l-Glutamic acid (GA) and γ–Aminobutyric acid (GABA), was developed and validated using gas chromatography-tandem mass spectrometry (GC–MS/MS). The novel method utilized a rapid online hot GC inlet gas phase sample derivatization and fast GC low thermal mass technology. The method calibration was linear from 0.5 to 100μg/mL, with limits of detections of 100ng/mL and 250ng/mL for GA and GABA, respectively. The method was used to investigate the effects of deletion of organic anion transporter 1 (Oat1) or Oat3 on murine CNS levels of GA and GABA at 3 and 18 mo of age, as compared to age matched wild-type (WT) animals. Whole brain concentrations of GA were comparable between WT, Oat1−/−, and Oat3−/− 18 mo at both 3 and 18 mo of age. Similarly, whole brain concentrations of GABA were not significantly altered in either knockout mouse strain at 3 or 18 mo of age, as compared to WT. These results indicate that the developed GC–MS/MS method provides sufficient sensitivity and selectivity for the quantitation of these neurotransmitters in mouse brain tissue. Furthermore, these results suggest that loss of Oat1 or Oat3 function in isolation does not result in significant alterations in brain tissue levels of GA or GABA.





Broad-spectrum immunoaffinity cleanup for the determination of aflatoxins B1, B2, G1, G2, M1, M2 in Ophiocordyceps sinensis and its pharmaceutical preparations by ultra performance liquid chromatography tandem mass spectrometry

Publication date: Available online 10 October 2017
Source:Journal of Chromatography B
Author(s): Shujuan Sun, Jie Xie, Tao Peng, Bing Shao, Kui Zhu, Yuanze Sun, Kai Yao, Qiang Gu, Jing Zhang, Chunlin Fan, Ying Chen, Haiyang Jiang
An ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for the simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 (AFB1, AFB2, AFG1, AFG2, AFM1 and AFM2) in Ophiocordyceps sinensis and its pharmaceutical preparations. A rapid and reliable immunoaffinity column containing a broad-spectrum monoclonal antibody for six aflatoxins was used for sample cleanup. Under the optimized conditions, the home-made immunoaffinity column capacity were about 315, 319, 292, 102, 444 and 369ng/mL gel for AFB1, AFB2, AFG1, AFG2, AFM1 and AFM2, respectively. Recoveries for all tested aflatoxins ranged from 79.28% to 103.42% with relative standard deviation less than 8%. The limits of quantitation were in the range of 0.008–0.045μg/kg. Among 31 real samples analyzed, one sample was contaminated with AFB1, AFB2 and AFM1 at levels of 0.483, 0.068 and 0.104μg/kg, respectively. The established method is simple, accurate, and can be effectively used to determine the aflatoxins in Ophiocordyceps sinensis and its pharmaceutical preparations.

Publication date: Available online 10 October 2017
Source:Journal of Chromatography B

Author(s): Shujuan Sun, Jie Xie, Tao Peng, Bing Shao, Kui Zhu, Yuanze Sun, Kai Yao, Qiang Gu, Jing Zhang, Chunlin Fan, Ying Chen, Haiyang Jiang

An ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for the simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 (AFB1, AFB2, AFG1, AFG2, AFM1 and AFM2) in Ophiocordyceps sinensis and its pharmaceutical preparations. A rapid and reliable immunoaffinity column containing a broad-spectrum monoclonal antibody for six aflatoxins was used for sample cleanup. Under the optimized conditions, the home-made immunoaffinity column capacity were about 315, 319, 292, 102, 444 and 369ng/mL gel for AFB1, AFB2, AFG1, AFG2, AFM1 and AFM2, respectively. Recoveries for all tested aflatoxins ranged from 79.28% to 103.42% with relative standard deviation less than 8%. The limits of quantitation were in the range of 0.008–0.045μg/kg. Among 31 real samples analyzed, one sample was contaminated with AFB1, AFB2 and AFM1 at levels of 0.483, 0.068 and 0.104μg/kg, respectively. The established method is simple, accurate, and can be effectively used to determine the aflatoxins in Ophiocordyceps sinensis and its pharmaceutical preparations.





Acetonitrile extraction coupled with UHPLC–MS/MS for the accurate quantification of 17 heterocyclic aromatic amines in meat products

Publication date: Available online 9 October 2017
Source:Journal of Chromatography B
Author(s): Yan Yan, Shuang Zhang, Guan-jun Tao, Feng-hui You, Jie Chen, Mao-mao Zeng
This study proposed a simple and accurate acetonitrile extraction pretreatment method coupled with ultrahigh-performance liquid chromatography with tandem mass spectrometry for the simultaneous determination of 17 heterocyclic aromatic amines (HAAs) in meat products. With this new method, all 17 HAAs, including 11 polar and 6 nonpolarHAAs, were simultaneously extracted by acetonitrile and purified by one-step Oasis MCX cartridge purification. Compared with two different improved reference methods, the acetonitrile method could obtain higher recoveries (in the range of 42.5% to 99.0%) and better repeatability (lower than 12.2%). The limits of quantification were calculated between 0.028ngg−1and0.648ngg−1 with high correlation coefficients (r>0.9976) in wide linear ranges. The proposed acetonitrile method was successfully applied to the analysis of the HAAs levels in 10 commercial meat products with satisfactory recoveries.

Publication date: Available online 9 October 2017
Source:Journal of Chromatography B

Author(s): Yan Yan, Shuang Zhang, Guan-jun Tao, Feng-hui You, Jie Chen, Mao-mao Zeng

This study proposed a simple and accurate acetonitrile extraction pretreatment method coupled with ultrahigh-performance liquid chromatography with tandem mass spectrometry for the simultaneous determination of 17 heterocyclic aromatic amines (HAAs) in meat products. With this new method, all 17 HAAs, including 11 polar and 6 nonpolarHAAs, were simultaneously extracted by acetonitrile and purified by one-step Oasis MCX cartridge purification. Compared with two different improved reference methods, the acetonitrile method could obtain higher recoveries (in the range of 42.5% to 99.0%) and better repeatability (lower than 12.2%). The limits of quantification were calculated between 0.028ngg−1and0.648ngg−1 with high correlation coefficients (r> 0.9976) in wide linear ranges. The proposed acetonitrile method was successfully applied to the analysis of the HAAs levels in 10 commercial meat products with satisfactory recoveries.





Corrigendum to “Protocols for the analytical characterization of therapeutic monoclonal antibodies. I—Non-denaturing chromatographic techniques” [J. Chromatogr. B 1058 (2017) 73–84]

Publication date: Available online 10 October 2017
Source:Journal of Chromatography B
Author(s): Alexandre Goyon, Valentina D’Atri, Balazs Bobaly, Elsa Wagner-Rousset, Alain Beck, Szabolcs Fekete, Davy Guillarme

Publication date: Available online 10 October 2017
Source:Journal of Chromatography B

Author(s): Alexandre Goyon, Valentina D’Atri, Balazs Bobaly, Elsa Wagner-Rousset, Alain Beck, Szabolcs Fekete, Davy Guillarme







Immobilization of sugars in supermacroporous cryogels for the purification of lectins by affinity chromatography

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069
Author(s): Gabriel Ramos Ferreira Gonçalves, Olga Reinert Ramos Gandolfi, Leandro Soares Santos, Renata Cristina Ferreira Bonomo, Cristiane Martins Veloso, Lizzy Ayra Alcântara Veríssimo, Rafael da Costa Ilhéu Fontan
Lectins are glycoproteins that bind to carbohydrates or glycoconjugates by specific interactions. The specificity of lectins to various carbohydrates is a determinant factor in the choice of ligand for the chromatographic matrix when using chromatography as a lectin purification technique. In this work, the immobilization of three different aminated carbohydrates on the surface of macroporous polymeric cryogels was evaluated. Carbohydrates were immobilized on cryogel surfaces via the glutaraldehyde method to create spacer arms, reducing steric hindrance. The immobilized N-acetyl-d-glucosamine and N-acetyl-d-mannosamine concentrations contained approximately 130mg of carbohydrate/g dehydrated cryogel, while the N-acetyl-d-galactosamine contained 105mg of carbohydrate/g dehydrated cryogel. Scanning electron microscopy showed that the physical structure and porosity of the chromatographic columns were not affected by the immobilization process, maintaining an elevated hydration capacity and the macroporous structure of the cryogels. Adsorption of concanavalin A on cryogels functionalized with N-acetyl-d-glucosamine (cryo-d-GlcNAc) was tested, as well as its reuse capability. After 5 cycles of use, cryo-d-GlcNAc was shown to be stable, with an adsorptive capacity of around 50mg/g. Carbohydrate immobilization in polyacrylamide cryogels was satisfactory, with promise for applications in lectin purification processes.

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069

Author(s): Gabriel Ramos Ferreira Gonçalves, Olga Reinert Ramos Gandolfi, Leandro Soares Santos, Renata Cristina Ferreira Bonomo, Cristiane Martins Veloso, Lizzy Ayra Alcântara Veríssimo, Rafael da Costa Ilhéu Fontan

Lectins are glycoproteins that bind to carbohydrates or glycoconjugates by specific interactions. The specificity of lectins to various carbohydrates is a determinant factor in the choice of ligand for the chromatographic matrix when using chromatography as a lectin purification technique. In this work, the immobilization of three different aminated carbohydrates on the surface of macroporous polymeric cryogels was evaluated. Carbohydrates were immobilized on cryogel surfaces via the glutaraldehyde method to create spacer arms, reducing steric hindrance. The immobilized N-acetyl-d-glucosamine and N-acetyl-d-mannosamine concentrations contained approximately 130mg of carbohydrate/g dehydrated cryogel, while the N-acetyl-d-galactosamine contained 105mg of carbohydrate/g dehydrated cryogel. Scanning electron microscopy showed that the physical structure and porosity of the chromatographic columns were not affected by the immobilization process, maintaining an elevated hydration capacity and the macroporous structure of the cryogels. Adsorption of concanavalin A on cryogels functionalized with N-acetyl-d-glucosamine (cryo-d-GlcNAc) was tested, as well as its reuse capability. After 5 cycles of use, cryo-d-GlcNAc was shown to be stable, with an adsorptive capacity of around 50mg/g. Carbohydrate immobilization in polyacrylamide cryogels was satisfactory, with promise for applications in lectin purification processes.





Multiplex quantitative analysis of eicosanoid mediators in human plasma and serum: Possible introduction into clinical testing

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069
Author(s): Atsushi Yasumoto, Suzumi M. Tokuoka, Yoshihiro Kita, Takao Shimizu, Yutaka Yatomi
Eicosanoid mediators play important roles in maintaining the physiological and pathophysiological homeostasis in the body. Their measurements, however, are rarely performed in clinical practice. In the present study, we analyzed 30 varieties of eicosanoid mediators that were detectable in human plasma and serum collected from healthy donors, using liquid chromatography-tandem mass spectrometry from the viewpoint of the clinical application of the multiplex quantitation of eicosanoid mediators. Wider variety of eicosanoid mediators were detected in serum (27 out of 30) than in plasma (14 out of 30), since the serum was thought to contain lipid mediators released from activated platelets. Larger inter-individual variations were observed in the plasma and serum eicosanoid levels. On the other hand, the concentrations of eicosanoids were not affected by the platelet count but were affected by the concentration of arachidonic acid (AA) within the reference interval (17.4–40.5×1010/L). When serum samples from patients with hematological disorders were analyzed, the concentrations of AA were positively correlated with the platelet count. When the patients underwent ASA therapy, a marked decrease in the concentrations of thromboxane B2 (TXB2) and 12-hydroxyl-heptadecatrienoic acid (12-HHT) was observed. Considering the availability of serum samples in clinical settings, the serum analysis of eicosanoids may be clinically useful.

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069

Author(s): Atsushi Yasumoto, Suzumi M. Tokuoka, Yoshihiro Kita, Takao Shimizu, Yutaka Yatomi

Eicosanoid mediators play important roles in maintaining the physiological and pathophysiological homeostasis in the body. Their measurements, however, are rarely performed in clinical practice. In the present study, we analyzed 30 varieties of eicosanoid mediators that were detectable in human plasma and serum collected from healthy donors, using liquid chromatography-tandem mass spectrometry from the viewpoint of the clinical application of the multiplex quantitation of eicosanoid mediators. Wider variety of eicosanoid mediators were detected in serum (27 out of 30) than in plasma (14 out of 30), since the serum was thought to contain lipid mediators released from activated platelets. Larger inter-individual variations were observed in the plasma and serum eicosanoid levels. On the other hand, the concentrations of eicosanoids were not affected by the platelet count but were affected by the concentration of arachidonic acid (AA) within the reference interval (17.4–40.5×1010/L). When serum samples from patients with hematological disorders were analyzed, the concentrations of AA were positively correlated with the platelet count. When the patients underwent ASA therapy, a marked decrease in the concentrations of thromboxane B2 (TXB2) and 12-hydroxyl-heptadecatrienoic acid (12-HHT) was observed. Considering the availability of serum samples in clinical settings, the serum analysis of eicosanoids may be clinically useful.





Simultaneous determination of a novel c-Met/AXL dual-target small-molecule inhibitor BPI-9016M and its metabolites in human plasma by liquid chromatography-tandem mass spectrometry: Application in a pharmacokinetic study in Chinese advanced solid tumor patients

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069
Author(s): Xinge Cui, Xin Zheng, Ji Jiang, Fenlai Tan, Lieming Ding, Pei Hu
BPI-9016M is a novel dual-target small-molecule inhibitor targeting c-Met and AXL, which was developed by Betta Pharmaceuticals Co., Ltd (Hangzhou, China). It has great potential in the treatment of advanced cancer. A high throughput quantitation method, based on liquid chromatography-tandem mass spectrometry, was developed and validated for the simultaneous determination of BPI-9016M and its main metabolite, M1 and M2-2, in human plasma with a sample preparation method of precipitation of protein. Liquid chromatographic separation was performed with a gradient elution of formic acid–10mM ammonium acetate aqueous solution (1:1000, v/v) and acetonitrile at a flow rate of 0.4mL/min within 2.2min. A Waters ACQUITY UPLC BEH C18 column (1.7μm, 2.1×50mm) was chosen, of which the temperature was set to be 40°C. Mass spectrometric detection, which were achieved in positive mode, were performed by multiple reaction monitoring with SCIEX API 5500 Qtrap equipped with an ESI ion source. This method showed good linearity, accuracy and precision in the range of 0.4–200ng/mL for BPI-9016M and 0.8–800ng/mL for M1 and M2-2, with high recovery and slight matrix effect for all analytes. And under the conditions same as stability assessments in method validation, the three analytes stayed stable during the entire destiny of a clinical sample from the collection of whole blood to the analysis of plasma by this method. The validated method was successfully applied to a first-in-human, dose-escalation phase I clinical trial in Chinese advanced solid tumor patients for the pharmacokinetic research of BPI-9016M tablet after oral administration. The concentration-time curves of BPI-9016M, M1, M2-2 were detailly captured with good veracity. And according to the results of hemolysis assessment, plasma concentrations of analytes in hemolyzed plasma samples could be reported normally without label.

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069

Author(s): Xinge Cui, Xin Zheng, Ji Jiang, Fenlai Tan, Lieming Ding, Pei Hu

BPI-9016M is a novel dual-target small-molecule inhibitor targeting c-Met and AXL, which was developed by Betta Pharmaceuticals Co., Ltd (Hangzhou, China). It has great potential in the treatment of advanced cancer. A high throughput quantitation method, based on liquid chromatography-tandem mass spectrometry, was developed and validated for the simultaneous determination of BPI-9016M and its main metabolite, M1 and M2-2, in human plasma with a sample preparation method of precipitation of protein. Liquid chromatographic separation was performed with a gradient elution of formic acid–10mM ammonium acetate aqueous solution (1:1000, v/v) and acetonitrile at a flow rate of 0.4mL/min within 2.2min. A Waters ACQUITY UPLC BEH C18 column (1.7μm, 2.1×50mm) was chosen, of which the temperature was set to be 40°C. Mass spectrometric detection, which were achieved in positive mode, were performed by multiple reaction monitoring with SCIEX API 5500 Qtrap equipped with an ESI ion source. This method showed good linearity, accuracy and precision in the range of 0.4–200ng/mL for BPI-9016M and 0.8–800ng/mL for M1 and M2-2, with high recovery and slight matrix effect for all analytes. And under the conditions same as stability assessments in method validation, the three analytes stayed stable during the entire destiny of a clinical sample from the collection of whole blood to the analysis of plasma by this method. The validated method was successfully applied to a first-in-human, dose-escalation phase I clinical trial in Chinese advanced solid tumor patients for the pharmacokinetic research of BPI-9016M tablet after oral administration. The concentration-time curves of BPI-9016M, M1, M2-2 were detailly captured with good veracity. And according to the results of hemolysis assessment, plasma concentrations of analytes in hemolyzed plasma samples could be reported normally without label.





Application of novel ternary deep eutectic solvents as a functional monomer in molecularly imprinted polymers for purification of levofloxacin

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069
Author(s): Xiaoxia Li, Kyung Ho Row
A series of ecofriendly ternary deep eutectic solvents (DESs) with different molar ratios were prepared as candidate functional monomers. Three of the optimal ternary DESs as functional monomers were applied to the preparation of molecularly imprinted polymers (MIPs). After synthesis, the proposed polymers were characterized by elemental analysis (EA), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), Brunauer-Emmett-Teller surface area measurements (BET) and Fourier transform infrared spectroscopy (FT-IR). These MIPs based on ternary DESs with different molar ratios exhibited different absorption capacities of levofloxacin. A sample of levofloxacin (500ng) was dissolved in a millet extractive (10mL). All MIPs were used as SPE adsorbents to purify the extracts. According to characterization result, the ternary DES-3 (1:3:1.5) was joined in the synthetic process of MIP-1. The green ternary DES-3-based MIPs had the best selectivity recovery for levofloxacin (91.4%) from the millet extractive. The best selectivity of MIP-1 was attributed to the novel monomer (ternary DES) in the preparation of the materials. Overall, ternary DES-based MIPs have potential applications as media in many research areas.

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069

Author(s): Xiaoxia Li, Kyung Ho Row

A series of ecofriendly ternary deep eutectic solvents (DESs) with different molar ratios were prepared as candidate functional monomers. Three of the optimal ternary DESs as functional monomers were applied to the preparation of molecularly imprinted polymers (MIPs). After synthesis, the proposed polymers were characterized by elemental analysis (EA), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), Brunauer-Emmett-Teller surface area measurements (BET) and Fourier transform infrared spectroscopy (FT-IR). These MIPs based on ternary DESs with different molar ratios exhibited different absorption capacities of levofloxacin. A sample of levofloxacin (500ng) was dissolved in a millet extractive (10mL). All MIPs were used as SPE adsorbents to purify the extracts. According to characterization result, the ternary DES-3 (1:3:1.5) was joined in the synthetic process of MIP-1. The green ternary DES-3-based MIPs had the best selectivity recovery for levofloxacin (91.4%) from the millet extractive. The best selectivity of MIP-1 was attributed to the novel monomer (ternary DES) in the preparation of the materials. Overall, ternary DES-based MIPs have potential applications as media in many research areas.





A selective knockout method for discovery of minor active components from plant extracts: Feasibility and challenges as illustrated by an application to Salvia miltiorrhiza

Publication date: Available online 7 October 2017
Source:Journal of Chromatography B
Author(s): Xue-Yan Li, Lin Yang, Ping Li, Jun Chen
Natural products have been recognized to play an invaluable role in drug discovery. However, efficient discovery of minor active constituents from natural sources is challenging due to low abundance and complex matrices. In this study, we developed a selective knockout method to discover minor bioactive components from complex phytochemical mixtures, using a Chinese medicine as an example. Based on the chromatographic fingerprint, six major components in the ethyl acetate extract of Salvia miltiorrhiza (EASM) were selectively knocked out via high-resolution peak fraction (HRPF) approach. The remaining extract was automatically enriched and fractionated to generate a chemical library consisting of 62 minor components with contents less than 3‰. Simultaneously, a parallel mass-spectrometry (MS) analysis was performed to ensure purity and to characterize the structures of compounds in each fraction. Via an antioxidant response element (ARE)-driven luciferase reporter system, 33 minor components were screened out as nuclear factor erythroid 2-related factor 2 (Nrf2) activators and 30 components were identified. Here, the Nrf2 activation activities of 21 components have been reported for the first time. Different from the existing methods for discovery of active compounds from natural products, in the developed method of this manuscript, the major components are selectively removed, and the fractions of the minor components are prepared after several times of preparative HPLC enrichment by high-resolution peak fraction approach. It improves the prospective discovery of minor active components from complex medicinal herbs.

Publication date: Available online 7 October 2017
Source:Journal of Chromatography B

Author(s): Xue-Yan Li, Lin Yang, Ping Li, Jun Chen

Natural products have been recognized to play an invaluable role in drug discovery. However, efficient discovery of minor active constituents from natural sources is challenging due to low abundance and complex matrices. In this study, we developed a selective knockout method to discover minor bioactive components from complex phytochemical mixtures, using a Chinese medicine as an example. Based on the chromatographic fingerprint, six major components in the ethyl acetate extract of Salvia miltiorrhiza (EASM) were selectively knocked out via high-resolution peak fraction (HRPF) approach. The remaining extract was automatically enriched and fractionated to generate a chemical library consisting of 62 minor components with contents less than 3‰. Simultaneously, a parallel mass-spectrometry (MS) analysis was performed to ensure purity and to characterize the structures of compounds in each fraction. Via an antioxidant response element (ARE)-driven luciferase reporter system, 33 minor components were screened out as nuclear factor erythroid 2-related factor 2 (Nrf2) activators and 30 components were identified. Here, the Nrf2 activation activities of 21 components have been reported for the first time. Different from the existing methods for discovery of active compounds from natural products, in the developed method of this manuscript, the major components are selectively removed, and the fractions of the minor components are prepared after several times of preparative HPLC enrichment by high-resolution peak fraction approach. It improves the prospective discovery of minor active components from complex medicinal herbs.





One single standard substance for the simultaneous determination of 17 triterpenes in Ganoderma lingzhi and its related species using high-performance liquid chromatography

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069
Author(s): Wei Liu, Jingsong Zhang, Wei Han, Yanfang Liu, Jie Feng, Chuanhong Tang, Na Feng, Qingjiu Tang
Due to the difficulty and high cost for the preparation of triterpenes, one single standard for the simultaneous determination of multi-components (SSDMC) with high performance liquid chromatography (HPLC) is an advanced solution for multi-component analysis. Experiments were carried out to investigate the feasibility of SSDMC for the analysis of Ganoderma triterpenes, with external standard method (ESM) compared, and the samples of Ganoderma were classified by the content of Ganoderma triterpenes. The analysis was performed by using a Fortis Speed Core-C18 column (150mm×4.6mm I.D., 2.6μm) at gradient elution of 0.01% glacial acetic acid-water (V/V) and acetonitrile with diode array detection (252nm), at a flow rate of 1mL/min. The results showed that all calibration curves had good linearity (r2>0.9999) within test ranges. The LOD and LOQ were lower than 2.52ng and 6.43ng, respectively. The RSD for intra-day and inter-day of the seventeen analytes were less than 3.12% at three levels, and the recoveries were 91.4–103.0%. The contents of other 16 triterpenes were determined with ganoderic acid A by SSDMC, which showed that there were few differences compared with the results obtained by ESM. Moreover, the classification of 25 different species and strains of Ganoderma by using the content of triterpenes intuitively reflected the distinction among Ganoderma. In summary, the developed method could be readily utilized as a method of quality evaluation for Ganoderma triterpenes.

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069

Author(s): Wei Liu, Jingsong Zhang, Wei Han, Yanfang Liu, Jie Feng, Chuanhong Tang, Na Feng, Qingjiu Tang

Due to the difficulty and high cost for the preparation of triterpenes, one single standard for the simultaneous determination of multi-components (SSDMC) with high performance liquid chromatography (HPLC) is an advanced solution for multi-component analysis. Experiments were carried out to investigate the feasibility of SSDMC for the analysis of Ganoderma triterpenes, with external standard method (ESM) compared, and the samples of Ganoderma were classified by the content of Ganoderma triterpenes. The analysis was performed by using a Fortis Speed Core-C18 column (150mm×4.6mm I.D., 2.6μm) at gradient elution of 0.01% glacial acetic acid-water (V/V) and acetonitrile with diode array detection (252nm), at a flow rate of 1mL/min. The results showed that all calibration curves had good linearity (r2 >0.9999) within test ranges. The LOD and LOQ were lower than 2.52ng and 6.43ng, respectively. The RSD for intra-day and inter-day of the seventeen analytes were less than 3.12% at three levels, and the recoveries were 91.4–103.0%. The contents of other 16 triterpenes were determined with ganoderic acid A by SSDMC, which showed that there were few differences compared with the results obtained by ESM. Moreover, the classification of 25 different species and strains of Ganoderma by using the content of triterpenes intuitively reflected the distinction among Ganoderma. In summary, the developed method could be readily utilized as a method of quality evaluation for Ganoderma triterpenes.





Quantitative analysis of antithrombin III binding site in low molecular weight heparins by exhausetive heparinases digestion and capillary electrophoresis

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069
Author(s): Mingyu Zhang, Gong Li, Yi Zhang, Jingwu Kang
The antithrombin III (ATIII)-binding site, which contains a special 3-O-sulfated, N-sulfated glucosamine residue with or without 6-O-sulfation, is mainly responsible for the anticoagulant activity of heparin. Undergoing the chemical depolymerization process, the preservation of the ATIII-binding site in low molecular weight heparins (LMWHs) are varied leading to the fluctuation of the anticoagulant activity. Herein we report a capillary electrophoresis (CE) method in combination with heparinase digestion and affinity chromatography for the measurement of molar percentage of ATIII-binding site of LMWHs. After exhaustively digesting LMWHs with the mixture of heparinase I, II and III, almost all the resulting oligosaccharide building blocks, including the three 3-O-sulfated tetrasaccharides derived from the ATIII-binding site, were resolved by CE separation. The peak area of each building block permits quantification of the molar percentage of the ATIII-binding site. The peaks corresponding to the 3-O-sulfated tetrasaccharides were assigned based on the linear relationship between the electrophoretic mobilities of the oligosaccharides and their charge to mass ratios. The peak assignment was further confirmed by analysis of the high ATIII affinity fractions, which contains much high 3-O-sulfated tetrasaccharides. With the method, the molar percentage of the ATIII-binding site of enoxaparin from different batches and different manufactures were measured and compared. It was demonstrated that the CE method provides more precise data for assessing the anti-FXa activity than that of the biochemical assay method.

Publication date: 15 November 2017
Source:Journal of Chromatography B, Volumes 1068–1069

Author(s): Mingyu Zhang, Gong Li, Yi Zhang, Jingwu Kang

The antithrombin III (ATIII)-binding site, which contains a special 3-O-sulfated, N-sulfated glucosamine residue with or without 6-O-sulfation, is mainly responsible for the anticoagulant activity of heparin. Undergoing the chemical depolymerization process, the preservation of the ATIII-binding site in low molecular weight heparins (LMWHs) are varied leading to the fluctuation of the anticoagulant activity. Herein we report a capillary electrophoresis (CE) method in combination with heparinase digestion and affinity chromatography for the measurement of molar percentage of ATIII-binding site of LMWHs. After exhaustively digesting LMWHs with the mixture of heparinase I, II and III, almost all the resulting oligosaccharide building blocks, including the three 3-O-sulfated tetrasaccharides derived from the ATIII-binding site, were resolved by CE separation. The peak area of each building block permits quantification of the molar percentage of the ATIII-binding site. The peaks corresponding to the 3-O-sulfated tetrasaccharides were assigned based on the linear relationship between the electrophoretic mobilities of the oligosaccharides and their charge to mass ratios. The peak assignment was further confirmed by analysis of the high ATIII affinity fractions, which contains much high 3-O-sulfated tetrasaccharides. With the method, the molar percentage of the ATIII-binding site of enoxaparin from different batches and different manufactures were measured and compared. It was demonstrated that the CE method provides more precise data for assessing the anti-FXa activity than that of the biochemical assay method.





A HILIC-UHPLC-MS/MS untargeted urinary metabonomics combined with quantitative analysis of five polar biomarkers on osteoporosis rats after oral administration of Gushudan

Publication date: Available online 6 October 2017
Source:Journal of Chromatography B
Author(s): Xiao Wu, Yue Huang, Jinghan Sun, Yongqing Wen, Feng Qin, Longshan Zhao, Zhili Xiong
A HILIC-UHPLC-MS/MS untargeted urinary metabonomic method combined with quantitative analysis of five potential polar biomarkers in rat urine was developed and validated, to further understand the anti-osteoporosis effect of Gushudan(GSD) and its mechanism on prednisolone-induced osteoporosis(OP) rats in this study. The metabolites were separated and identified on Waters BEH HILIC (2.1mm×100mm, 1.7μm) column using the Waters ACQUITY™ ultra performance liquid chromatography system (Waters Corporation, Milford, USA) coupled with a Micromass Quattro Micro™ API mass spectrometer (Waters Corp, Milford, MA, USA). Principal component analysis (PCA) was used to identify potential biomarkers. Primary potential polar biomarkers including creatinine, taurine, betaine, hypoxanthine and cytosine, which were related to energy metabolism, lipid metabolism and amino acid metabolism, were found in the untargeted metabonomic research. Moreover, these targeted biomarkers were further separated and quantified in multiple-reaction monitoring (MRM) with positive ionization mode, using tinidazole as internal standard (I.S.). Good linearities (r>0.99) were obtained for all the analytes with the low limit of quantification from 1.00 to 12.8μg/mL. The relative standard deviation (RSD) of the intra-day and inter-day precisions were within 15.0% and the accuracy ranged from −14.3% to 13.5%. The recovery was more than 85.0%. And the validated method was successfully applied to investigate the urine samples of the control group, prednisolone-induced osteoporosis model group and Gushudan-treatment group in rats. Compared to the control group, the level of creatinine, taurine, betaine, hypoxanthine and cytosine in the model group revealed a significant decrease trend (p<0.05), while the Gushudan-treatment group showed no statistically differences by an independent sample t-test. This paper provided a better understanding of the therapeutic effect and mechanism of GSD on prednisolone-induced osteoporosis rats.

Publication date: Available online 6 October 2017
Source:Journal of Chromatography B

Author(s): Xiao Wu, Yue Huang, Jinghan Sun, Yongqing Wen, Feng Qin, Longshan Zhao, Zhili Xiong

A HILIC-UHPLC-MS/MS untargeted urinary metabonomic method combined with quantitative analysis of five potential polar biomarkers in rat urine was developed and validated, to further understand the anti-osteoporosis effect of Gushudan(GSD) and its mechanism on prednisolone-induced osteoporosis(OP) rats in this study. The metabolites were separated and identified on Waters BEH HILIC (2.1mm×100mm, 1.7μm) column using the Waters ACQUITY™ ultra performance liquid chromatography system (Waters Corporation, Milford, USA) coupled with a Micromass Quattro Micro™ API mass spectrometer (Waters Corp, Milford, MA, USA). Principal component analysis (PCA) was used to identify potential biomarkers. Primary potential polar biomarkers including creatinine, taurine, betaine, hypoxanthine and cytosine, which were related to energy metabolism, lipid metabolism and amino acid metabolism, were found in the untargeted metabonomic research. Moreover, these targeted biomarkers were further separated and quantified in multiple-reaction monitoring (MRM) with positive ionization mode, using tinidazole as internal standard (I.S.). Good linearities (r>0.99) were obtained for all the analytes with the low limit of quantification from 1.00 to 12.8μg/mL. The relative standard deviation (RSD) of the intra-day and inter-day precisions were within 15.0% and the accuracy ranged from −14.3% to 13.5%. The recovery was more than 85.0%. And the validated method was successfully applied to investigate the urine samples of the control group, prednisolone-induced osteoporosis model group and Gushudan-treatment group in rats. Compared to the control group, the level of creatinine, taurine, betaine, hypoxanthine and cytosine in the model group revealed a significant decrease trend (p<0.05), while the Gushudan-treatment group showed no statistically differences by an independent sample t-test. This paper provided a better understanding of the therapeutic effect and mechanism of GSD on prednisolone-induced osteoporosis rats.