Graphene oxide reinforced ionic liquid-functionalized adsorbent for solid-phase extraction of phenolic acids

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072
Author(s): Xiudan Hou, Xiaofeng Lu, Sheng Tang, Licheng Wang, Yong Guo
An environmental friendly sorbent of polymeric ionic liquids modified graphene oxide-grafted silica (PILs@GO@Sil) was synthesized for solid-phase extraction (SPE) of phenolic acids. The sorbent was prepared via a chemical layer-to-layer fabrication including amidation reaction, surface radical chain-transfer polymerization and in situ anion exchange. After modification with PILs, the silica surface had higher positive potential so that it would exhibit stronger electrostatic interaction for acidic compounds compared with GO@Sil. The adsorption performance of phenolic acids was investigated through the theoretical calculation and static, kinetic state adsorption experiments. Under the optimized conditions, wide linear ranges were obtained with correlation coefficients ranging from 0.9912 to 0.9998, and limits of detection were in the range of 0.20–0.50μgL−1. Compared with other reported methods, the proposed PILs@GO@Sil-SPE-HPLC showed higher extraction efficiency. Finally, the black wolfberry yogurt and urine were analyzed as real samples and good recoveries spiked with standard solution were obtained.

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072

Author(s): Xiudan Hou, Xiaofeng Lu, Sheng Tang, Licheng Wang, Yong Guo

An environmental friendly sorbent of polymeric ionic liquids modified graphene oxide-grafted silica (PILs@GO@Sil) was synthesized for solid-phase extraction (SPE) of phenolic acids. The sorbent was prepared via a chemical layer-to-layer fabrication including amidation reaction, surface radical chain-transfer polymerization and in situ anion exchange. After modification with PILs, the silica surface had higher positive potential so that it would exhibit stronger electrostatic interaction for acidic compounds compared with GO@Sil. The adsorption performance of phenolic acids was investigated through the theoretical calculation and static, kinetic state adsorption experiments. Under the optimized conditions, wide linear ranges were obtained with correlation coefficients ranging from 0.9912 to 0.9998, and limits of detection were in the range of 0.20–0.50 μgL−1. Compared with other reported methods, the proposed PILs@GO@Sil-SPE-HPLC showed higher extraction efficiency. Finally, the black wolfberry yogurt and urine were analyzed as real samples and good recoveries spiked with standard solution were obtained.





An UHPLC–MS/MS method for simultaneous quantification of human amyloid beta peptides Aβ1-38, Aβ1-40 and Aβ1-42 in cerebrospinal fluid using micro-elution solid phase extraction

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070
Author(s): Ping-ping Lin, Wei-li Chen, Fei Yuan, Lei Sheng, Yu-jia Wu, Wei-wei Zhang, Guo-qing Li, Hong-rong Xu, Xue-ning Li
Amyloid beta (Aβ) peptides in cerebrospinal fluid are extensively estimated for identification of Alzheimer’s disease (AD) as diagnostic biomarkers. Unfortunately, their pervasive application is hampered by interference from Aβ propensity of self-aggregation, nonspecifically bind to surfaces and matrix proteins, and by lack of quantitive standardization. Here we report on an alternative Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) method for simultaneous measurement of human amyloid beta peptides Aβ1-38, Aβ1-40 and Aβ1-42 in cerebrospinal fluid (CSF) using micro-elution solid phase extraction (SPE). Samples were pre-processing by the mixed-mode micro-elution solid phase extraction and quantification was performed in the positive ion multiple reaction monitoring (MRM) mode using electrospray ionization. The stable-isotope labeled Aβ peptides 15N51- Aβ1-38, 15N53- Aβ1-40 and 15N55- Aβ1-42 peptides were used as internal standards. And the artificial cerebrospinal fluid (ACSF) containing 5% rat plasma was used as a surrogate matrix for calibration curves. The quality control (QC) samples at 0.25, 2 and 15ng/mL were prepared. A “linear” regression (1/x2 weighting): y=ax+b was used to fit the calibration curves over the concentration range of 0.1–20ng/mL for all three peptides. Coefficient of variation (CV) of intra-batch and inter-batch assays were all less than 6.44% for Aβ1-38, 6.75% for Aβ1-40 and 10.74% for Aβ1-42. The precision values for all QC samples of three analytes met the acceptance criteria. Extract recoveries of Aβ1-38, Aβ1-40 and Aβ1-42 were all greater than 70.78%, both in low and high QC samples. The stability assessments showed that QC samples at both low and high levels could be stable for at least 24h at 4°C, 4h at room temperature and through three freeze-thaw cycles without sacrificing accuracy or precision. And no significant carryover effect was observed. This validated UHPLC/MS/MS method was successfully applied to the quantitation of Aβ peptides in real human CSF samples. Our work may provide a reference method for simultaneous quantitation of human Aβ1-38, Aβ1-40 and Aβ1-42 from CSF.

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070

Author(s): Ping-ping Lin, Wei-li Chen, Fei Yuan, Lei Sheng, Yu-jia Wu, Wei-wei Zhang, Guo-qing Li, Hong-rong Xu, Xue-ning Li

Amyloid beta (Aβ) peptides in cerebrospinal fluid are extensively estimated for identification of Alzheimer’s disease (AD) as diagnostic biomarkers. Unfortunately, their pervasive application is hampered by interference from Aβ propensity of self-aggregation, nonspecifically bind to surfaces and matrix proteins, and by lack of quantitive standardization. Here we report on an alternative Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) method for simultaneous measurement of human amyloid beta peptides Aβ1-38, Aβ1-40 and Aβ1-42 in cerebrospinal fluid (CSF) using micro-elution solid phase extraction (SPE). Samples were pre-processing by the mixed-mode micro-elution solid phase extraction and quantification was performed in the positive ion multiple reaction monitoring (MRM) mode using electrospray ionization. The stable-isotope labeled Aβ peptides 15N51- Aβ1-38, 15N53- Aβ1-40 and 15N55- Aβ1-42 peptides were used as internal standards. And the artificial cerebrospinal fluid (ACSF) containing 5% rat plasma was used as a surrogate matrix for calibration curves. The quality control (QC) samples at 0.25, 2 and 15ng/mL were prepared. A “linear” regression (1/x2 weighting): y=ax+b was used to fit the calibration curves over the concentration range of 0.1–20ng/mL for all three peptides. Coefficient of variation (CV) of intra-batch and inter-batch assays were all less than 6.44% for Aβ1-38, 6.75% for Aβ1-40 and 10.74% for Aβ1-42. The precision values for all QC samples of three analytes met the acceptance criteria. Extract recoveries of Aβ1-38, Aβ1-40 and Aβ1-42 were all greater than 70.78%, both in low and high QC samples. The stability assessments showed that QC samples at both low and high levels could be stable for at least 24h at 4°C, 4h at room temperature and through three freeze-thaw cycles without sacrificing accuracy or precision. And no significant carryover effect was observed. This validated UHPLC/MS/MS method was successfully applied to the quantitation of Aβ peptides in real human CSF samples. Our work may provide a reference method for simultaneous quantitation of human Aβ1-38, Aβ1-40 and Aβ1-42 from CSF.





First investigations for the characterization of glucosamine-6-phosphate synthase by capillary electrophoresis

Publication date: 1 January 2018 Source:Journal of Chromatography B, Volume 1072 Author(s): Miriam Beneito-Cambra, Pierre Gareil, Bernard Badet, Marie-Ange Badet-Denisot, Nathalie Delaunay The enzyme glucosamine-6-phosphate s…

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072

Author(s): Miriam Beneito-Cambra, Pierre Gareil, Bernard Badet, Marie-Ange Badet-Denisot, Nathalie Delaunay

The enzyme glucosamine-6-phosphate synthase (GlmS) is an important point of metabolic control in biosynthesis of amino sugar-containing macromolecules and is therefore a potential target in order to design antibacterial and antifungal drugs. It has two oligomerization states, which are the active dimer and the inactive hexamer. For the first time, the potential of CE to separate and quantify the two forms was studied. After incubating GlmS with the d-glucosamine 6-phosphate (GlcN6P) inhibitor, an electrolyte based on sodium phosphate at pH 7.2 and an ionic strength of 100mM plus GlcN6P (either 2 or 20mM) allowed the hexamer-dimer separation. However, the displacement of the dimer/hexamer equilibrium during the analysis time prevented any improvement of the resolution when varying the effective separation length or the temperature of the analysis. Therefore, the use of a short-end CE method allowed the decrease in the analysis time to about 1min. Some parameters such as the temperature and the time of incubation and the ratio of the inhibitor and enzyme concentrations were studied. Then, it was also possible to test, very rapidly and with a very small amount, some molecules having an inhibition potential for the GlmS enzyme (arabinose-5-phosphate oxime, 2-amino-2-deoxy-d-glucitol 6-phosphate, and glucose-6-phosphate).





Comparative study on the interaction between 3 CYP2C9 allelic isoforms and benzbromarone by using LC–MS/MS method

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070
Author(s): Lingli He, Chuan Li, Xuyuan Liu, Qingqing Yang, Haizhi Zhang, Weiren Xu, Luyong Zhang, Changxiao Liu
Benzbromarone is a uricosuric drug metabolized predominantly by cytochrome P450 2C9 from in vitro findings. Human CYP2C9 exhibits extensive genetic polymorphism and numbers of clinic studies have demonstrated that CYP2C9 genetic polymorphism has a significant influence on the pharmacokinetics of benzbromarone. But in vitro study on the interaction between CYP2C9 allelic isoforms and benzbromarone was rare. Here, an LC–MS/MS method was established and validated to determine the concentration of benzbromarone in different CYP2C9 enzyme incubation systems for the drug-enzyme interaction study. By selecting appropriate internal standard and optimizing separation system, including mobile phase, sample solvent and gradient elution condition, this LC–MS/MS method was developed with fine linearity (r2≥0.996), good reproducibility (RSD≤6.6%), high stability (92.37–114.67%), efficient recovery (91.23–109.82%) and acceptable matrix effect (110.54–115.31%). Based on this method, the interaction between 3 CYP2C9 allelic isoforms and benzbromarone was researched by kinetics parameters (Km, Vmax, Clint). As a result, CYP2C9*1 displayed the highest metabolic activity towards benzbromarone, CYP2C9*2 showed a little lower catalytic activity than CYP2C9*1 (relative clearance/*1=85.86%), CYP2C9*3 showed the lowest catalytic activity (relative clearance/*1=21.57%). The result illustrated that various CYP2C9 allelic isoforms showed different enzymatic activities towards benzbromarone, which could offer effective consultation for personalized administration in clinic.

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070

Author(s): Lingli He, Chuan Li, Xuyuan Liu, Qingqing Yang, Haizhi Zhang, Weiren Xu, Luyong Zhang, Changxiao Liu

Benzbromarone is a uricosuric drug metabolized predominantly by cytochrome P450 2C9 from in vitro findings. Human CYP2C9 exhibits extensive genetic polymorphism and numbers of clinic studies have demonstrated that CYP2C9 genetic polymorphism has a significant influence on the pharmacokinetics of benzbromarone. But in vitro study on the interaction between CYP2C9 allelic isoforms and benzbromarone was rare. Here, an LC–MS/MS method was established and validated to determine the concentration of benzbromarone in different CYP2C9 enzyme incubation systems for the drug-enzyme interaction study. By selecting appropriate internal standard and optimizing separation system, including mobile phase, sample solvent and gradient elution condition, this LC–MS/MS method was developed with fine linearity (r2 0.996), good reproducibility (RSD6.6%), high stability (92.37–114.67%), efficient recovery (91.23–109.82%) and acceptable matrix effect (110.54–115.31%). Based on this method, the interaction between 3 CYP2C9 allelic isoforms and benzbromarone was researched by kinetics parameters (Km, Vmax, Clint). As a result, CYP2C9*1 displayed the highest metabolic activity towards benzbromarone, CYP2C9*2 showed a little lower catalytic activity than CYP2C9*1 (relative clearance/*1=85.86%), CYP2C9*3 showed the lowest catalytic activity (relative clearance/*1=21.57%). The result illustrated that various CYP2C9 allelic isoforms showed different enzymatic activities towards benzbromarone, which could offer effective consultation for personalized administration in clinic.





Determination of isoquercitrin in rat plasma by high performance liquid chromatography coupled with a novel synergistic cloud point extraction

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072
Author(s): Jun Zhou, Jiang Bing Sun, Qiao Feng Wang
A novel improved preconcentration method known as synergistic cloud point extraction was established for isoquercitrin preconcentration and determination in rat plasma prior to its determination by high performance liquid chromatography. Synergistic cloud point extraction greatly simplified isoquercitrin extraction and detection. This method was accomplished at room temperature (about 22°C) in 1min with the nonionic surfactant Tergitol TMN-6 as the extractant, n-octanol as cloud point revulsant and synergic reagent. Parameters that affect the synergistic cloud point extraction processes, such as the concentrations of Tergitol TMN-6, volume of n-octanol, sample pH, salt content and extraction time were investigated and optimized. Under the optimum conditions, the calibration curve for the analyte was linear in the range from 5 to 500ngmL−1 with the correlation coefficients greater than 0.9996. Meanwhile, limit of detection (S/N=3) was less than 1.6ngmL−1 and limit of quantification (S/N=10) was less than 5ngmL−1. It demonstrated that the method can be successfully applied to the pharmacokinetic investigation of isoquercitrin.

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072

Author(s): Jun Zhou, Jiang Bing Sun, Qiao Feng Wang

A novel improved preconcentration method known as synergistic cloud point extraction was established for isoquercitrin preconcentration and determination in rat plasma prior to its determination by high performance liquid chromatography. Synergistic cloud point extraction greatly simplified isoquercitrin extraction and detection. This method was accomplished at room temperature (about 22°C) in 1min with the nonionic surfactant Tergitol TMN-6 as the extractant, n-octanol as cloud point revulsant and synergic reagent. Parameters that affect the synergistic cloud point extraction processes, such as the concentrations of Tergitol TMN-6, volume of n-octanol, sample pH, salt content and extraction time were investigated and optimized. Under the optimum conditions, the calibration curve for the analyte was linear in the range from 5 to 500ngmL−1 with the correlation coefficients greater than 0.9996. Meanwhile, limit of detection (S/N=3) was less than 1.6ngmL−1 and limit of quantification (S/N=10) was less than 5ngmL−1. It demonstrated that the method can be successfully applied to the pharmacokinetic investigation of isoquercitrin.





Improving the selectivity and sensitivity for quantifying 8-α-hydroxy-mutilin in rabbit tissues by using basic mobile phases and negative ionization

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072
Author(s): Aimin Tan, Guifen Gu, Xuan Gui, Yanxin Wu, Gordon Bolger, Albert Licollari, John C. Fanaras
Previously reported LC–MS methods for quantifying 8-α-hydroxy-mutilin (a marker residue of tiamulin) in tissues all used a pseudo MRM transition (from protonated molecular ion to protonated molecular ion, m/z 337→337) due to difficulties in finding a product ion, leading to suboptimal selectivity and sensitivity for detection. By using electrospray negative ionization in a basic medium, we, for the first time, found a highly selective and sensitive true MRM transition for 8-α-hydroxy-mutilin, m/z 335→179. With this newly found MRM transition and the use of pleuromutilin as the internal standard, a very sensitive, selective, and robust LC–MS/MS method has been developed and validated for quantifying 8-α-hydroxy-mutilin in rabbit tissues (muscle, liver, kidney, and fat). In comparison with the previously published methods, the selectivity and sensitivity were significantly improved. For the concentration range validated (0.2–10ppm or 0.2–10μg/g), the within-run and between-run accuracies (% bias) ranged from −5.0 to 3.1 and −4.9 to 3.0, respectively. The% CV ranged from 2.2 to 6.6 and 4.7 to 8.3 for within-run and between-run precisions, respectively. The validated method was successfully used to support two GLP tissue residue depletion studies in rabbits.

Publication date: 1 January 2018
Source:Journal of Chromatography B, Volume 1072

Author(s): Aimin Tan, Guifen Gu, Xuan Gui, Yanxin Wu, Gordon Bolger, Albert Licollari, John C. Fanaras

Previously reported LC–MS methods for quantifying 8-α-hydroxy-mutilin (a marker residue of tiamulin) in tissues all used a pseudo MRM transition (from protonated molecular ion to protonated molecular ion, m/z 337337) due to difficulties in finding a product ion, leading to suboptimal selectivity and sensitivity for detection. By using electrospray negative ionization in a basic medium, we, for the first time, found a highly selective and sensitive true MRM transition for 8-α-hydroxy-mutilin, m/z 335179. With this newly found MRM transition and the use of pleuromutilin as the internal standard, a very sensitive, selective, and robust LC–MS/MS method has been developed and validated for quantifying 8-α-hydroxy-mutilin in rabbit tissues (muscle, liver, kidney, and fat). In comparison with the previously published methods, the selectivity and sensitivity were significantly improved. For the concentration range validated (0.2–10ppm or 0.2–10μg/g), the within-run and between-run accuracies (% bias) ranged from −5.0 to 3.1 and −4.9 to 3.0, respectively. The% CV ranged from 2.2 to 6.6 and 4.7 to 8.3 for within-run and between-run precisions, respectively. The validated method was successfully used to support two GLP tissue residue depletion studies in rabbits.





Effective phospholipid removal from plasma samples by solid phase extraction with the use of copper (II) modified silica gel cartridges

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070
Author(s): Jolanta Flieger, Małgorzata Tatarczak-Michalewska, Anna Kowalska, Anna Madejska, Tomasz Śniegocki, Anna Sroka-Bartnicka, Monika Szymańska-Chargot
The new sorbent for solid phase extraction (SPE), based on silica gel modified with a copper (II), was obtained and its application for phospholipids removal from the human plasma was tested. SPE column conditioning requirements, the volume of the plasma, the composition of the elution solvent were all established. The efficacy of the removal of phospholipids was compared for different methods such as standard protein precipitation or HybridSPE Phospholipid Ultra and HybridSPE-PPT. The sample clean-up was verified by mass spectrometry (MS) and by monitoring of chromatograms in the region between 190nm and 400nm. The Fourier Transform Infrared Spectroscopy FT-IR and confocal Raman microscopy were used to evaluating the silica gel modifications and to show the structure of lipids confined in the silica pores.

Graphical abstract

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Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070

Author(s): Jolanta Flieger, Małgorzata Tatarczak-Michalewska, Anna Kowalska, Anna Madejska, Tomasz Śniegocki, Anna Sroka-Bartnicka, Monika Szymańska-Chargot

The new sorbent for solid phase extraction (SPE), based on silica gel modified with a copper (II), was obtained and its application for phospholipids removal from the human plasma was tested. SPE column conditioning requirements, the volume of the plasma, the composition of the elution solvent were all established. The efficacy of the removal of phospholipids was compared for different methods such as standard protein precipitation or HybridSPE Phospholipid Ultra and HybridSPE-PPT. The sample clean-up was verified by mass spectrometry (MS) and by monitoring of chromatograms in the region between 190nm and 400nm. The Fourier Transform Infrared Spectroscopy FT-IR and confocal Raman microscopy were used to evaluating the silica gel modifications and to show the structure of lipids confined in the silica pores.

Graphical abstract

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Enrichment of chelidonine from Chelidonium majus L. using macroporous resin and its antifungal activity

Publication date: 1 December 2017 Source:Journal of Chromatography B, Volume 1070 Author(s): Jialiang Pan, Yang Yang, Rui Zhang, Hanwen Yao, Kangkang Ge, Manyin Zhang, Ling Ma Chelidonium majus L. (greater celandine) has…

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070

Author(s): Jialiang Pan, Yang Yang, Rui Zhang, Hanwen Yao, Kangkang Ge, Manyin Zhang, Ling Ma

Chelidonium majus L. (greater celandine) has been used as an herbal medicine for several centuries. This study investigated an efficient method to purify chelidonine from the extract of C. majus L. using macroporous adsorption resins and evaluated the antifungal activity of chelidonine against Botryosphaeria dothidea as a model strain. Static adsorption and desorption tests revealed that D101 was the optimal resin for chelidonine purification. Pseudo-second-order kinetics model and Freundlich equation model were the most suitable for evaluating the endothermic and spontaneous adsorption processes of chelidonine on D101. Dynamic adsorption and desorption tests on D101 columns showed that the concentration of chelidonine increased 14.16-fold, from 2.67% to 37.81%, with the recovery yield of 80.77%. The antifungal activity of enriched chelidonine products was studied with B. dothidea. The results showed that the EC50 of crude extracts, enriched chelidonine products, and chelidonine standard against B. dothidea were 3.24mg/mL, 0.43mg/mL, and 0.77mg/mL, respectively. The result of antifungal activity test showed that chelidonine had the potential to be a useful antifungal agent. Moreover, the enrichment method of chelidonine was highly efficient, low cost, and harmless to the environment for industrial applications.





“One-pot” ethyl chloroformate derivatization and liquid-liquid extraction of reduced glutathione in erythrocyte and its quantitative GC–MS analysis

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070
Author(s): Alessandra Manca, Eugenio Alladio, Paola Massarenti, M. Paola Puccinelli, Antonella De Francesco, Erika Del Grosso, Giulio Mengozzi, Marco Pazzi, Marco Vincenti
A simple “one-pot” derivatization and liquid–liquid extraction (LLE) procedure was developed for GC–MS analysis of reduced glutathione (GSH) analysis in erythrocytes. The metabolite was extracted by 5% (w/v) TCA, the supernatant treated with ECF and ethanol-pyridine media, the derivative separated and detected by gas chromatography-mass spectrometry using a short non-polar capillary GC column at a high column-head pressure. Total analysis time was 11min. The process was optimized by a Design of Experiment. The method was validated showing a good linearity over the 25.4–813.4μM concentration range, providing satisfactory results in terms of intra-day and inter-day precision as well as an optimal accuracy. The new method was evaluated in a pilot study involving patients with severe protein malnutrition. Comparison of this group with a group of healthy subjects revealed significantly lower GSH concentrations in erythrocytes in the former, thus proving that the described GC–MS method could be employed for fast and simple GSH analysis in clinical studies.

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070

Author(s): Alessandra Manca, Eugenio Alladio, Paola Massarenti, M. Paola Puccinelli, Antonella De Francesco, Erika Del Grosso, Giulio Mengozzi, Marco Pazzi, Marco Vincenti

A simple “one-pot” derivatization and liquid–liquid extraction (LLE) procedure was developed for GC–MS analysis of reduced glutathione (GSH) analysis in erythrocytes. The metabolite was extracted by 5% (w/v) TCA, the supernatant treated with ECF and ethanol-pyridine media, the derivative separated and detected by gas chromatography-mass spectrometry using a short non-polar capillary GC column at a high column-head pressure. Total analysis time was 11min. The process was optimized by a Design of Experiment. The method was validated showing a good linearity over the 25.4–813.4μM concentration range, providing satisfactory results in terms of intra-day and inter-day precision as well as an optimal accuracy. The new method was evaluated in a pilot study involving patients with severe protein malnutrition. Comparison of this group with a group of healthy subjects revealed significantly lower GSH concentrations in erythrocytes in the former, thus proving that the described GC–MS method could be employed for fast and simple GSH analysis in clinical studies.





Determination of the authenticity of plastron-derived functional foods based on amino acid profiles analysed by MEKC

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070
Author(s): Lin-Qiu Li, Joewel T. Baibado, Qing Shen, Hon-Yeung Cheung
Plastron is a nutritive and superior functional food. Due to its limited supply yet enormous demands, some functional foods supposed to contain plastron may be forged with other substitutes. This paper reports a novel and simple method for determination of the authenticity of plastron-derived functional foods based on comparison of the amino acid (AA) profiles of plastron and its possible substitutes. By applying micellar electrokinetic chromatography (MEKC), 18 common AAs along with another 2 special AAs — hydroxyproline (Hyp) and hydroxylysine (Hyl) were detected in all plastron samples. Since chicken, egg, fish, milk, pork, nail and hair lacked of Hyp and Hyl, plastron could be easily distinguished. For those containing collagen, a statistical analysis technique — principal component analysis (PCA) was adopted and plastron was successfully distinguished. When applied the proposed method to authenticate turtle shell glue in the market, fake products were commonly found.

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070

Author(s): Lin-Qiu Li, Joewel T. Baibado, Qing Shen, Hon-Yeung Cheung

Plastron is a nutritive and superior functional food. Due to its limited supply yet enormous demands, some functional foods supposed to contain plastron may be forged with other substitutes. This paper reports a novel and simple method for determination of the authenticity of plastron-derived functional foods based on comparison of the amino acid (AA) profiles of plastron and its possible substitutes. By applying micellar electrokinetic chromatography (MEKC), 18 common AAs along with another 2 special AAs — hydroxyproline (Hyp) and hydroxylysine (Hyl) were detected in all plastron samples. Since chicken, egg, fish, milk, pork, nail and hair lacked of Hyp and Hyl, plastron could be easily distinguished. For those containing collagen, a statistical analysis technique — principal component analysis (PCA) was adopted and plastron was successfully distinguished. When applied the proposed method to authenticate turtle shell glue in the market, fake products were commonly found.





Determination of the R(−) and S(+)-enantiomers of vigabatrin in human plasma by ultra-high-performance liquid chromatography and tandem mass-spectrometry

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070
Author(s): Paul Duhamel, Marwa Ounissi, Thomas Le Saux, Hugues Bienayme, Catherine Chiron, Vincent Jullien
An analytical method was developed for the quantification in plasma of the R and S enantiomers of vigabatrin (VGB), a drug used for the treatment of some refractory pediatric epileptic syndromes. After adding 50μL of the internal standard, which consisted of a 15mg/L solution of deuterated racemic VGB, and 100μL of water to 100μL of plasma samples, a protein precipitation was performed by adding 600μL of methanol. The supernatant was evaporated to dryness under a stream of nitrogen and the dry residue was reconstituted with 500μL of water. Then, 100μL of 0.01M o-phthaldialdehyde and 0.01M N-acetyl-l-cysteine in borate buffer (0.1M, pH=9.5) were added for pre-column derivatization of the enantiomers as diastereomeric isoindoles. One microliter of the resulting mixture was injected in the chromatographic system. The chromatographic separation was performed in gradient elution mode at a flow rate of 400μL/min using a phenomenex EVO C-18 column with a mobile phase composed of 5mM ammonium acetate and a methanol:acetonitrile (63:37v/v) mixture. Detection was performed by mass spectrometry in selected reaction monitoring mode using heated electrospray ionization in positive mode as the ion source. Intra- and inter-day precision and accuracy were lower than 15% over the calibration range (0.2–50mg/L for each enantiomer) and the method was successfully used to assess plasma concentrations of VGB in epileptic children.

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070

Author(s): Paul Duhamel, Marwa Ounissi, Thomas Le Saux, Hugues Bienayme, Catherine Chiron, Vincent Jullien

An analytical method was developed for the quantification in plasma of the R and S enantiomers of vigabatrin (VGB), a drug used for the treatment of some refractory pediatric epileptic syndromes. After adding 50μL of the internal standard, which consisted of a 15mg/L solution of deuterated racemic VGB, and 100μL of water to 100μL of plasma samples, a protein precipitation was performed by adding 600μL of methanol. The supernatant was evaporated to dryness under a stream of nitrogen and the dry residue was reconstituted with 500μL of water. Then, 100μL of 0.01M o-phthaldialdehyde and 0.01M N-acetyl-l-cysteine in borate buffer (0.1M, pH=9.5) were added for pre-column derivatization of the enantiomers as diastereomeric isoindoles. One microliter of the resulting mixture was injected in the chromatographic system. The chromatographic separation was performed in gradient elution mode at a flow rate of 400μL/min using a phenomenex EVO C-18 column with a mobile phase composed of 5mM ammonium acetate and a methanol:acetonitrile (63:37v/v) mixture. Detection was performed by mass spectrometry in selected reaction monitoring mode using heated electrospray ionization in positive mode as the ion source. Intra- and inter-day precision and accuracy were lower than 15% over the calibration range (0.2–50mg/L for each enantiomer) and the method was successfully used to assess plasma concentrations of VGB in epileptic children.





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: 1 December 2017
Source:Journal of Chromatography B, Volume 1070
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: 1 December 2017
Source:Journal of Chromatography B, Volume 1070

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: 1 December 2017
Source:Journal of Chromatography B, Volume 1070
Author(s): Hong Pan, Linyan Huang, Yi Li, Xumei Zhou, Yuanfu Lu, Fuguo Shi
Arecoline, a predominant alkaloid in areca nut, shows several pharmacological and toxicological effects. In present study, a sensitive and accurate liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed and fully validated for quantification of arecoline in rat plasma. Importantly, our group found that arecoline was highly unstable in rat plasma samples, which brought challenges to accurately quantify in vivo. The hydrolysis of ester moiety of arecoline by carboxylesterases was responsible for its instability, and arecoline was hydrolyzed to arecaidine. The degradation of arecoline was completely inhibited using 5% formic acid as a stabilizer, which was immediately added to freshly collected rat plasma samples. EDTA was adopted as the anticoagulant to also reduce the degradation during blood collection and plasma separation owing to its anti-esterase effect. The plasma sample was separated by a C18 analytical column with a mobile phase of 0.1% formic acid and methanol (95:5v/v) at an isocratic flow rate of 300μL/min. The analyte was monitored on a tandem mass spectrometer using the multiple reaction monitoring scan in a positive electrospray ionization mode. The method exhibited high sensitivity and a good linearity rang of 1–1000ng/mL. The developed analytical method was employed in a pilot pharmacokinetic study of arecoline in rats. Arecoline was rapidly eliminated within 45min in rats after oral treatment of 150mg/kg arecoline.

Publication date: 1 December 2017
Source:Journal of Chromatography B, Volume 1070

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

Arecoline, a predominant alkaloid in areca nut, shows several pharmacological and toxicological effects. In present study, a sensitive and accurate liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed and fully validated for quantification of arecoline in rat plasma. Importantly, our group found that arecoline was highly unstable in rat plasma samples, which brought challenges to accurately quantify in vivo. The hydrolysis of ester moiety of arecoline by carboxylesterases was responsible for its instability, and arecoline was hydrolyzed to arecaidine. The degradation of arecoline was completely inhibited using 5% formic acid as a stabilizer, which was immediately added to freshly collected rat plasma samples. EDTA was adopted as the anticoagulant to also reduce the degradation during blood collection and plasma separation owing to its anti-esterase effect. The plasma sample was separated by a C18 analytical column with a mobile phase of 0.1% formic acid and methanol (95:5v/v) at an isocratic flow rate of 300μL/min. The analyte was monitored on a tandem mass spectrometer using the multiple reaction monitoring scan in a positive electrospray ionization mode. The method exhibited high sensitivity and a good linearity rang of 1–1000ng/mL. The developed analytical method was employed in a pilot pharmacokinetic study of arecoline in rats. Arecoline was rapidly eliminated within 45min in rats after oral treatment of 150mg/kg arecoline.





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.