Li Guang, Li jing, Zhenxing Zou, Liu Bo, Weiping Li, Ding Yang, Song Xuri, Xiaolan Fang, Hu Daoqi
The elimination of residual host cell DNA (HCD) and proteins (HCPs) is a pivotal step in the purification process for biological products such as monoclonal antibodies, recombinant proteins, vaccines, gene therapy vectors, and cell‐based therapies. During the preparation of recombinant staphylokinase (r‐SAK), a potential therapeutic protein for thrombotic disorders expressed in Escherichia coli cells, an efficient chromatography purification process, incorporating anion exchange, cation exchange, and gel filtration techniques, was developed to effectively eliminate HCPs and residual DNA. This multistep chromatography approach yielded r‐SAK with a residual HCD concentration below 1 ng/mL, a residual HCP concentration below 0.01%, and purity exceeding 98%. Comparative analysis revealed that modified cellulose‐based matrix resins exhibited superior efficiency compared to dextran and agarose matrix resins for eliminating residual HCPs and HCD under identical conditions. Based on the different properties of the matrix, deductions were made regarding the reasons for the differentiation in separation efficiency. The physical strength of the cellulose‐based matrix ensures the structural stability of macroporous resin and can guarantee efficient separation under conditions of high flow and heavy load. This study suggests that maintaining the structural stability of macropores in bioseparation materials is crucial for improving the efficiency of separating biological products.
{"title":"Removal of Residual DNA and Host Cell Proteins for the Purification of Recombinant Staphylokinase Expressed in Escherichia coli","authors":"Li Guang, Li jing, Zhenxing Zou, Liu Bo, Weiping Li, Ding Yang, Song Xuri, Xiaolan Fang, Hu Daoqi","doi":"10.1002/sscp.202300214","DOIUrl":"https://doi.org/10.1002/sscp.202300214","url":null,"abstract":"The elimination of residual host cell DNA (HCD) and proteins (HCPs) is a pivotal step in the purification process for biological products such as monoclonal antibodies, recombinant proteins, vaccines, gene therapy vectors, and cell‐based therapies. During the preparation of recombinant staphylokinase (r‐SAK), a potential therapeutic protein for thrombotic disorders expressed in Escherichia coli cells, an efficient chromatography purification process, incorporating anion exchange, cation exchange, and gel filtration techniques, was developed to effectively eliminate HCPs and residual DNA. This multistep chromatography approach yielded r‐SAK with a residual HCD concentration below 1 ng/mL, a residual HCP concentration below 0.01%, and purity exceeding 98%. Comparative analysis revealed that modified cellulose‐based matrix resins exhibited superior efficiency compared to dextran and agarose matrix resins for eliminating residual HCPs and HCD under identical conditions. Based on the different properties of the matrix, deductions were made regarding the reasons for the differentiation in separation efficiency. The physical strength of the cellulose‐based matrix ensures the structural stability of macroporous resin and can guarantee efficient separation under conditions of high flow and heavy load. This study suggests that maintaining the structural stability of macropores in bioseparation materials is crucial for improving the efficiency of separating biological products.","PeriodicalId":21639,"journal":{"name":"SEPARATION SCIENCE PLUS","volume":null,"pages":null},"PeriodicalIF":1.3,"publicationDate":"2024-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141797304","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
This research presents the development and validation of chromatographic method for analyzing sorafenib and its pharmacopeial impurities, with a focus on stability studies and degradation product (DP) characterization. Initial method optimization involved exploring various column and buffer combinations, ultimately achieving optimal separation and peak symmetry using an ODS‐AQ YMC (150 mm) column with 0.6 mL/min gradient flow of 10 mM ammonium formate buffer adjusted to pH 3.4 with formic acid as solvent A, and ethanol as solvent B as mobile phase and 246 nm wavelength. Method exhibits calibration curve linear in 50–300 µg/mL for sorafenib and 0.050–0.30 µg/mL for impurities with a detection limit of 0.015 µg/mL for impurities. A structural elucidation of DPs was performed using LC–MS/MS, providing valuable insights into their molecular compositions, and was characterized as 4‐[4‐(carboxyamino)phenoxy]pyridine‐2‐carboxylic acid (DP 1) and 4‐(4‐aminophenoxy)pyridine‐2‐carboxamide (DP 2). Using AGREE and GAPI metrics, evaluation highlighted method sustainability through ethanol–water solvents and shorter column to reduce energy consumption. Toxicity assessments revealed differences in environmental impact and toxicological profiles of DPs, emphasizing importance of managing safety considerations for sorafenib and its DPs. This research offers novel insights into sorafenib analysis by addressing pharmacopeial impurities, characterizing DPs, and evaluating method sustainability and safety.
{"title":"Green Analytical Approach for HPLC Method Development for Quantification of Sorafenib and Its Pharmacopeia Impurities: LC–MS/MS Characterization and Toxicity Prediction of Stress Degradation Products","authors":"Rajesh Varma Bhupatiraju, Pavani Peddi, Subhashini . Edla, Kandula Rekha, Bikshal Babu Kasimala","doi":"10.1002/sscp.202400106","DOIUrl":"https://doi.org/10.1002/sscp.202400106","url":null,"abstract":"This research presents the development and validation of chromatographic method for analyzing sorafenib and its pharmacopeial impurities, with a focus on stability studies and degradation product (DP) characterization. Initial method optimization involved exploring various column and buffer combinations, ultimately achieving optimal separation and peak symmetry using an ODS‐AQ YMC (150 mm) column with 0.6 mL/min gradient flow of 10 mM ammonium formate buffer adjusted to pH 3.4 with formic acid as solvent A, and ethanol as solvent B as mobile phase and 246 nm wavelength. Method exhibits calibration curve linear in 50–300 µg/mL for sorafenib and 0.050–0.30 µg/mL for impurities with a detection limit of 0.015 µg/mL for impurities. A structural elucidation of DPs was performed using LC–MS/MS, providing valuable insights into their molecular compositions, and was characterized as 4‐[4‐(carboxyamino)phenoxy]pyridine‐2‐carboxylic acid (DP 1) and 4‐(4‐aminophenoxy)pyridine‐2‐carboxamide (DP 2). Using AGREE and GAPI metrics, evaluation highlighted method sustainability through ethanol–water solvents and shorter column to reduce energy consumption. Toxicity assessments revealed differences in environmental impact and toxicological profiles of DPs, emphasizing importance of managing safety considerations for sorafenib and its DPs. This research offers novel insights into sorafenib analysis by addressing pharmacopeial impurities, characterizing DPs, and evaluating method sustainability and safety.","PeriodicalId":21639,"journal":{"name":"SEPARATION SCIENCE PLUS","volume":null,"pages":null},"PeriodicalIF":1.3,"publicationDate":"2024-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141798291","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Identifying and quantifying stabilizers in recycled plastics is crucial for utilizing recycled plastics in high‐value‐added applications while avoiding inhibitory effects between additives. However, identifying and quantifying stabilizers composed of multiple chemical classes with different chemical structures presents significant challenges. In this study, the time‐dependent extraction of 17 stabilizers, including six phenolic antioxidants, two phosphite antioxidants, two sulfur antioxidants, two hindered amine light stabilizers (HALS), and five ultraviolet absorbers, from a model polypropylene (PP), was investigated to observe the extraction behavior of these stabilizers. Using ultrasonic extraction on cryogenically ground PP powder, the 17 stabilizers were extracted almost quantitatively within a short duration of 1 h. Utilizing the developed extraction method, stabilizers in 10 types of commercially available recycled PP were quantified by liquid chromatography–tandem mass spectrometry, and no significant matrix effects were observed. On average, nine types of stabilizers were detected per sample, highlighting the complexity of the stabilizer formulations in recycled PP. Sulfur antioxidants and HALS, which require caution due to their inhibitory effects, were found simultaneously in five samples, underscoring the importance of identifying and quantifying stabilizers in recycled PP.
{"title":"Quantification of Recycled Polypropylene Stabilizers for Quality Control by Liquid Chromatography–Tandem Mass Spectrometry Using Simple, Fast and Efficient Ultrasonic Extraction","authors":"Shotaro Ito, Asahiro Nagatani, Hirohmi Watanabe, Masaru Aoyagi","doi":"10.1002/sscp.202400130","DOIUrl":"https://doi.org/10.1002/sscp.202400130","url":null,"abstract":"Identifying and quantifying stabilizers in recycled plastics is crucial for utilizing recycled plastics in high‐value‐added applications while avoiding inhibitory effects between additives. However, identifying and quantifying stabilizers composed of multiple chemical classes with different chemical structures presents significant challenges. In this study, the time‐dependent extraction of 17 stabilizers, including six phenolic antioxidants, two phosphite antioxidants, two sulfur antioxidants, two hindered amine light stabilizers (HALS), and five ultraviolet absorbers, from a model polypropylene (PP), was investigated to observe the extraction behavior of these stabilizers. Using ultrasonic extraction on cryogenically ground PP powder, the 17 stabilizers were extracted almost quantitatively within a short duration of 1 h. Utilizing the developed extraction method, stabilizers in 10 types of commercially available recycled PP were quantified by liquid chromatography–tandem mass spectrometry, and no significant matrix effects were observed. On average, nine types of stabilizers were detected per sample, highlighting the complexity of the stabilizer formulations in recycled PP. Sulfur antioxidants and HALS, which require caution due to their inhibitory effects, were found simultaneously in five samples, underscoring the importance of identifying and quantifying stabilizers in recycled PP.","PeriodicalId":21639,"journal":{"name":"SEPARATION SCIENCE PLUS","volume":null,"pages":null},"PeriodicalIF":1.3,"publicationDate":"2024-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141813582","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Anika Lokker, Pierre‐Hugues Stefanuto, Dries Cnuts, V. Rots, J.-F. Focant
The characterization of Paleolithic adhesives holds great potential for understanding human behavior and its evolution. Gas chromatography–mass spectrometry (GC–MS) is the most accurate identification method; unfortunately, it is destructive and requires a large sample size. Hence, most Paleolithic adhesives are not characterized with GC–MS. Here, a new nondestructive identification method is introduced; dynamic headspace (DHS) with two‐dimensional GC coupled to a time‐of‐flight MS. The DHS extraction is optimized with an experimental design approach. Four parameters were selected, and the optimized values were as follows: incubation temperature: 50°C, incubation time: 20 min, purge volume: 450 mL, and purge flow: 22.5 mL min−1, pine resin was chosen as a proxy for Paleolithic adhesives. Subsequently, DHS was also tested on hide glue, which has less volatile than pine resin, and the universality of the extraction was tested. With untargeted techniques, a distinction between hide glue and pine resin could be made based on their chromatographic profiles. Lastly, DHS was tested against an existing HS‐solid‐phase microextraction method. DHS showed a higher response in the total area of the chemical groups of interest. Thus, DHS has a higher sensitivity for prehistoric adhesives than solid‐phase microextraction, which is desired for minimal samples.
{"title":"Optimization of Dynamic Headspace Sampling Conditions for the Identification of Paleolithic Adhesives","authors":"Anika Lokker, Pierre‐Hugues Stefanuto, Dries Cnuts, V. Rots, J.-F. Focant","doi":"10.1002/sscp.202400085","DOIUrl":"https://doi.org/10.1002/sscp.202400085","url":null,"abstract":"The characterization of Paleolithic adhesives holds great potential for understanding human behavior and its evolution. Gas chromatography–mass spectrometry (GC–MS) is the most accurate identification method; unfortunately, it is destructive and requires a large sample size. Hence, most Paleolithic adhesives are not characterized with GC–MS. Here, a new nondestructive identification method is introduced; dynamic headspace (DHS) with two‐dimensional GC coupled to a time‐of‐flight MS. The DHS extraction is optimized with an experimental design approach. Four parameters were selected, and the optimized values were as follows: incubation temperature: 50°C, incubation time: 20 min, purge volume: 450 mL, and purge flow: 22.5 mL min−1, pine resin was chosen as a proxy for Paleolithic adhesives. Subsequently, DHS was also tested on hide glue, which has less volatile than pine resin, and the universality of the extraction was tested. With untargeted techniques, a distinction between hide glue and pine resin could be made based on their chromatographic profiles. Lastly, DHS was tested against an existing HS‐solid‐phase microextraction method. DHS showed a higher response in the total area of the chemical groups of interest. Thus, DHS has a higher sensitivity for prehistoric adhesives than solid‐phase microextraction, which is desired for minimal samples.","PeriodicalId":21639,"journal":{"name":"SEPARATION SCIENCE PLUS","volume":null,"pages":null},"PeriodicalIF":1.3,"publicationDate":"2024-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141813658","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Xiaofei He, Mengxue Feng, Xiaofeng Xu, Hong Zhao, Xueqing Qian
Liquid chromatography–triple quadrupole mass spectrometry (LC–MS/MS) is becoming an increasingly essential analytical technique in many fields, such as food, environmental, biochemical, pharmaceutical, and clinical chemistry. The testing matrices, such as plasma and serum, cannot usually be sent directly to the LC–MS/MS system because of the large number of unrelated substances in addition to target analytes in samples, such as cells, tissues, and proteins. Protein precipitation (PPT) is the most utilized pretreatment method in clinical analysis because of its ease of use; its performance always involves the separation of the liquid phase from the solid phase, in which the most commonly used manipulation is centrifugation. However, there is an upper limit of throughput for centrifugation, and the cost and difficulty of integrating centrifugation into automatic equipment are usually high. To solve the drawbacks of the current PPT method, we developed a cartridge structure that can omit the centrifugation step in PPT, which, in turn, can facilitate the incorporation of the PPT method into an automatic clinical pretreatment procedure for LC–MS/MS. We used vitamin D analysis as a representative application in our endeavor to develop a centrifugation‐free PPT method that can be easily applied to a new automatic clinical pretreatment procedure.
{"title":"A Cartridge Structure Facilitates Clinical Mass Spectrometry Pretreatment and Can be Integrated Into a High‐Throughput Instrument","authors":"Xiaofei He, Mengxue Feng, Xiaofeng Xu, Hong Zhao, Xueqing Qian","doi":"10.1002/sscp.202400011","DOIUrl":"https://doi.org/10.1002/sscp.202400011","url":null,"abstract":"Liquid chromatography–triple quadrupole mass spectrometry (LC–MS/MS) is becoming an increasingly essential analytical technique in many fields, such as food, environmental, biochemical, pharmaceutical, and clinical chemistry. The testing matrices, such as plasma and serum, cannot usually be sent directly to the LC–MS/MS system because of the large number of unrelated substances in addition to target analytes in samples, such as cells, tissues, and proteins. Protein precipitation (PPT) is the most utilized pretreatment method in clinical analysis because of its ease of use; its performance always involves the separation of the liquid phase from the solid phase, in which the most commonly used manipulation is centrifugation. However, there is an upper limit of throughput for centrifugation, and the cost and difficulty of integrating centrifugation into automatic equipment are usually high. To solve the drawbacks of the current PPT method, we developed a cartridge structure that can omit the centrifugation step in PPT, which, in turn, can facilitate the incorporation of the PPT method into an automatic clinical pretreatment procedure for LC–MS/MS. We used vitamin D analysis as a representative application in our endeavor to develop a centrifugation‐free PPT method that can be easily applied to a new automatic clinical pretreatment procedure.","PeriodicalId":21639,"journal":{"name":"SEPARATION SCIENCE PLUS","volume":null,"pages":null},"PeriodicalIF":1.3,"publicationDate":"2024-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141812919","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
In traditional Chinese medicine research, seaweeds used for drug and health food development mainly refer to Sargassum pallidum (Turn.) C. Ag. (SP) and Sargassum fusiforme (Harv.) Setch. (SF). In the Chinese Pharmacopoeia, polysaccharides are the main quality control indicators. The total sugar content is determined by the anthrone–sulfuric acid method to evaluate the quality of seaweeds. However, this method cannot reflect the structural characteristics of seaweed polysaccharides and cannot distinguish among different seaweed varieties. Given this, to comprehensively evaluate the quality of different seaweeds, this study conducted a thorough analysis of the primary structure of polysaccharides in 11 batches of SF and 7 batches of SP. The neutral sugar content was determined by the anthrone–sulfuric acid colorimetric method, weight average molecular weight (Mw) by high‐performance size exclusion chromatography coupled with multi‐angle laser light scattering and refractive index detector method, sulfate content by ion chromatography and monosaccharide composition by ultrahigh‐performance liquid chromatography method, while also using professional software for statistical and similarity analysis. The results showed significant differences (p < 0.05) in neutral sugar content and Mw between S. fusiforme polysaccharides and S. pallidum polysaccharides, which can be used to distinguish SF and SP. The monosaccharide composition fingerprint was analyzed using chemometric methods, and it was found that fucose and glucose could serve as differential markers to distinguish SF and SP. This study further deepens the understanding of polysaccharides in seaweeds and more comprehensively evaluates the quality of different seaweeds.
在传统中药研究中，用于药物和保健食品开发的海藻主要是马尾藻（Sargassum pallidum (Turn.) C. Ag.）（SP）和马尾藻（Sargassum fusiforme (Harv.) Setch.(SF）。在《中国药典》中，多糖是主要的质量控制指标。采用蒽酮-硫酸法测定总糖含量，以评价海藻的质量。然而，这种方法无法反映海藻多糖的结构特征，也无法区分不同的海藻品种。有鉴于此，为全面评价不同海藻的质量，本研究对 11 批 SF 和 7 批 SP 的多糖一级结构进行了全面分析。采用蒽酮-硫酸比色法测定中性糖含量，采用高效尺寸排阻色谱-多角度激光光散射和折射率检测器法测定重量平均分子量（Mw），采用离子色谱法测定硫酸盐含量，采用超高效液相色谱法测定单糖组成，并使用专业软件进行统计和相似性分析。结果表明，扶桑多糖与苍白球多糖在中性糖含量和Mw上存在显著差异（p < 0.05），可用于区分SF和SP。利用化学计量学方法分析了单糖组成指纹，发现岩藻糖和葡萄糖可作为区分SF和SP的差异标记。该研究进一步加深了人们对海藻多糖的认识，更全面地评价了不同海藻的品质。
{"title":"Comparison of Polysaccharide Profiles of Different Seaweeds Based on Ion Chromatography and Ultrahigh‐Performance Liquid Chromatography","authors":"Jing Fan, Donglin Gu, Weiyi Xv, Tingting Zhou, Anzhen Chen, Jingguang Lu, Ying Wang, Hongyu Jin, Feng Wei, ShuangCheng Ma","doi":"10.1002/sscp.202400060","DOIUrl":"https://doi.org/10.1002/sscp.202400060","url":null,"abstract":"In traditional Chinese medicine research, seaweeds used for drug and health food development mainly refer to Sargassum pallidum (Turn.) C. Ag. (SP) and Sargassum fusiforme (Harv.) Setch. (SF). In the Chinese Pharmacopoeia, polysaccharides are the main quality control indicators. The total sugar content is determined by the anthrone–sulfuric acid method to evaluate the quality of seaweeds. However, this method cannot reflect the structural characteristics of seaweed polysaccharides and cannot distinguish among different seaweed varieties. Given this, to comprehensively evaluate the quality of different seaweeds, this study conducted a thorough analysis of the primary structure of polysaccharides in 11 batches of SF and 7 batches of SP. The neutral sugar content was determined by the anthrone–sulfuric acid colorimetric method, weight average molecular weight (Mw) by high‐performance size exclusion chromatography coupled with multi‐angle laser light scattering and refractive index detector method, sulfate content by ion chromatography and monosaccharide composition by ultrahigh‐performance liquid chromatography method, while also using professional software for statistical and similarity analysis. The results showed significant differences (p < 0.05) in neutral sugar content and Mw between S. fusiforme polysaccharides and S. pallidum polysaccharides, which can be used to distinguish SF and SP. The monosaccharide composition fingerprint was analyzed using chemometric methods, and it was found that fucose and glucose could serve as differential markers to distinguish SF and SP. This study further deepens the understanding of polysaccharides in seaweeds and more comprehensively evaluates the quality of different seaweeds.","PeriodicalId":21639,"journal":{"name":"SEPARATION SCIENCE PLUS","volume":null,"pages":null},"PeriodicalIF":1.3,"publicationDate":"2024-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141812957","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Currently, the therapeutic potential of complex biomolecules such as peptides has significantly enhanced the demand for analytical method development and validation for their quality control. Peptides are more complex to analyze than small molecules because of their larger size and unique physical–chemical properties, which create different challenges for analytical method development. Reversed‐phase high‐performance liquid chromatography (RP‐HPLC) with ultraviolet or mass spectrometry is the most widely used technique for analyzing peptides and related substances. In the present study, a decapeptide and its six related possible impurities were synthesized in‐house and identified. A stability‐indicating RP‐HPLC method has been developed for the estimation of the decapeptide and its related substances. The developed method was validated according to the International Conference on Harmonization guidelines for specificity, linearity, limit of detection, quantification, accuracy, precision, and robustness. The stability‐indicating capability of the proposed method was studied under the stress conditions of acid, base, oxidative, thermal, humidity, and photolytic degradation. The decapeptide peak and its degradant were found to be homogeneous and pure in the studied stress trials, with mass balance for all types of degradation exceeding 95%. It confirms the reliability and appropriateness of determining the integrity and purity of the decapeptide and its related substances.
{"title":"A Stability‐Indicating RP‐HPLC Method for the Simultaneous Analysis of a Novel Synthetic Decapeptide and Six Related Substances","authors":"Ramesh Pawar, Sunil Tivari, Divya Panchani, Jayanti Makasana","doi":"10.1002/sscp.202400015","DOIUrl":"https://doi.org/10.1002/sscp.202400015","url":null,"abstract":"Currently, the therapeutic potential of complex biomolecules such as peptides has significantly enhanced the demand for analytical method development and validation for their quality control. Peptides are more complex to analyze than small molecules because of their larger size and unique physical–chemical properties, which create different challenges for analytical method development. Reversed‐phase high‐performance liquid chromatography (RP‐HPLC) with ultraviolet or mass spectrometry is the most widely used technique for analyzing peptides and related substances. In the present study, a decapeptide and its six related possible impurities were synthesized in‐house and identified. A stability‐indicating RP‐HPLC method has been developed for the estimation of the decapeptide and its related substances. The developed method was validated according to the International Conference on Harmonization guidelines for specificity, linearity, limit of detection, quantification, accuracy, precision, and robustness. The stability‐indicating capability of the proposed method was studied under the stress conditions of acid, base, oxidative, thermal, humidity, and photolytic degradation. The decapeptide peak and its degradant were found to be homogeneous and pure in the studied stress trials, with mass balance for all types of degradation exceeding 95%. It confirms the reliability and appropriateness of determining the integrity and purity of the decapeptide and its related substances.","PeriodicalId":21639,"journal":{"name":"SEPARATION SCIENCE PLUS","volume":null,"pages":null},"PeriodicalIF":1.3,"publicationDate":"2024-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141823621","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
A new stability‐indicating RPHPLC method with short run time has been developed and validated for tofacitinib citrate and its related substances. The novel HPLC method integrates hydrophilic interaction liquid chromatography (HILIC) technology as the stationary phase and employs a mobile phase composed of phosphate buffer (pH 7.0) and acetonitrile (45:55, %v/v) at a flow rate of 0.5 mL/min under isocratic elution. Analytes were monitored via a UV detector at 210 nm and with the column oven temperature at 30°C for a 20‐min analysis. Precision (%RSD) for Impurity‐A and Impurity‐B and tofacitinib met specifications at 2.4%, 0.8%, and 0.0%, respectively. Accuracy ranged from 86% to 100% for impurities, with LOD at 0.03% and LOQ at 0.05%–0.06%. Correlation coefficients exceeded 0.999 for impurities and tofacitinib citrate. Solution stability was confirmed for 24 h at room temperature. The method range was extended from LOQ to ∼1.5% for impurities and LOQ to ∼150% for tofacitinib citrate. The method's stability was evaluated under acid and base hydrolysis, oxidative and water hydrolysis, and thermal and photolytic degradation. Introducing HILIC as the stationary phase for this work proved effective, eliminating the need of ion pair reagents, reducing analysis time, and ensuring consistent results.
{"title":"Stability‐Indicating HPLC Method Development and Validation for the Quantification of Tofacitinib Citrate and Its Related Substances Using Hydrophilic Liquid Interaction Chromatography","authors":"G. Sivaprasadu, Muralidhar Pamerla, Podilapu Atchutha Rao, Adapa Venkateswara Rao, Suresh Salakolusu, Harihara Padhy, Ravikumar Ganta","doi":"10.1002/sscp.202400048","DOIUrl":"https://doi.org/10.1002/sscp.202400048","url":null,"abstract":"A new stability‐indicating RPHPLC method with short run time has been developed and validated for tofacitinib citrate and its related substances. The novel HPLC method integrates hydrophilic interaction liquid chromatography (HILIC) technology as the stationary phase and employs a mobile phase composed of phosphate buffer (pH 7.0) and acetonitrile (45:55, %v/v) at a flow rate of 0.5 mL/min under isocratic elution. Analytes were monitored via a UV detector at 210 nm and with the column oven temperature at 30°C for a 20‐min analysis. Precision (%RSD) for Impurity‐A and Impurity‐B and tofacitinib met specifications at 2.4%, 0.8%, and 0.0%, respectively. Accuracy ranged from 86% to 100% for impurities, with LOD at 0.03% and LOQ at 0.05%–0.06%. Correlation coefficients exceeded 0.999 for impurities and tofacitinib citrate. Solution stability was confirmed for 24 h at room temperature. The method range was extended from LOQ to ∼1.5% for impurities and LOQ to ∼150% for tofacitinib citrate. The method's stability was evaluated under acid and base hydrolysis, oxidative and water hydrolysis, and thermal and photolytic degradation. Introducing HILIC as the stationary phase for this work proved effective, eliminating the need of ion pair reagents, reducing analysis time, and ensuring consistent results.","PeriodicalId":21639,"journal":{"name":"SEPARATION SCIENCE PLUS","volume":null,"pages":null},"PeriodicalIF":1.3,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141831843","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
An analytical method based on high‐performance liquid chromatography (HPLC) method was developed for the determination of β‐hydroxy‐β‐methylbutyrate (HMB) content in food raw materials. The quantitative detection was performed by HPLC on diode array detector mode under external standard method. The limits of quantification, calculated as 10 times the standard deviation, were 2.0 g/100 g. The average recoveries were in the range of 92.3%–101%, with relative standard deviations below 3.87%, measured at three concentration levels. This method was used to determine the purity of HMB in 10 commercially available food raw materials. The real sample analysis results showed that the content and measurement uncertainties of HMB were 77.1–81.8 g/100 g and 4.51–4.68 g/100 g respectively, which met the requirement of HMB content in the range of 77–82 g/100 g in the HMB announcement. There are no studies on the detection of HMB in food raw materials in the previous reports. The established method provided reference for the rapid quantification and purity analysis of β‐hydroxy‐β‐methylbutyrate in food raw materials.
{"title":"Determination of β‐Hydroxy‐β‐Methylbutyrate Content in Food Raw Materials by High‐Performance Liquid Chromatography","authors":"Wen‐hua Zhang, Cunxian Xi, Xionghai Yi, Ya-qin Zhang, Weiqi Bao, Li-juan Shao, Dun-ming Xu","doi":"10.1002/sscp.202400144","DOIUrl":"https://doi.org/10.1002/sscp.202400144","url":null,"abstract":"An analytical method based on high‐performance liquid chromatography (HPLC) method was developed for the determination of β‐hydroxy‐β‐methylbutyrate (HMB) content in food raw materials. The quantitative detection was performed by HPLC on diode array detector mode under external standard method. The limits of quantification, calculated as 10 times the standard deviation, were 2.0 g/100 g. The average recoveries were in the range of 92.3%–101%, with relative standard deviations below 3.87%, measured at three concentration levels. This method was used to determine the purity of HMB in 10 commercially available food raw materials. The real sample analysis results showed that the content and measurement uncertainties of HMB were 77.1–81.8 g/100 g and 4.51–4.68 g/100 g respectively, which met the requirement of HMB content in the range of 77–82 g/100 g in the HMB announcement. There are no studies on the detection of HMB in food raw materials in the previous reports. The established method provided reference for the rapid quantification and purity analysis of β‐hydroxy‐β‐methylbutyrate in food raw materials.","PeriodicalId":21639,"journal":{"name":"SEPARATION SCIENCE PLUS","volume":null,"pages":null},"PeriodicalIF":1.3,"publicationDate":"2024-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141831994","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Staphylococcus aureus Protein A was immobilized on an in‐house made neutravidin poly (glycidyl methacrylate‐co‐ethylene dimethacrylate) capillary column with a 25 µm internal diameter, a length of 30 mm and a mass loadability of 60 ng. The immobilized quantity of protein A on the organic monolith was very low, in the pico mole range (1.80 pmol). This was of significance importance when working with less available or expensive purified enzyme. This capillary column was integrated into a nano liquid chromatographic system and used for the fast determination without dilution of the doses of therapeutic monoclonal antibodies (mAbs) in standardized infusion bags prepared in advance in a pharmacy department. This chromatographic method was linear in the studied concentration range with good precision and accuracy. Heat stressed studies indicated that the protein A affinity capillary column was able to monitor degraded mAbs. As well, the high specificity of this column to capture immunoglobulin G2 in cell culture supernatant was visualized. As the mAbs are produced through genetic engineering of animal cells this last result demonstrated that this novel protein A column could be used in the feature for rapid screening of immunoglobulin G concentration in cell culture.
金黄色葡萄球菌蛋白 A 被固定在自制的中性聚（甲基丙烯酸缩水甘油酯-二甲基丙烯酸乙烯酯）毛细管色谱柱上，该色谱柱内径 25 微米，长度 30 毫米，质量负载能力为 60 纳克。有机整体柱上固定的蛋白质 A 数量非常少，仅为微摩尔范围（1.80 pmol）。这在使用较少或昂贵的纯化酶时具有重要意义。这种毛细管色谱柱被集成到纳米液相色谱系统中，用于快速测定药剂部门提前准备好的标准输液袋中的治疗性单克隆抗体（mAbs）剂量，无需稀释。该色谱法在所研究的浓度范围内呈线性关系，具有良好的精密度和准确度。热应激研究表明，蛋白 A 亲和毛细管色谱柱能够监测降解的 mAbs。此外，该色谱柱捕获细胞培养上清液中免疫球蛋白 G2 的特异性也很高。由于 mAbs 是通过动物细胞基因工程产生的，因此最后一项结果表明，这种新型蛋白 A 柱可用于快速筛选细胞培养物中的免疫球蛋白 G 浓度。
{"title":"Fast quantitative determination of monoclonal antibody in infusion bags using protein A nano liquid chromatography","authors":"C. André, Yves Claude Guillaume","doi":"10.1002/sscp.202400050","DOIUrl":"https://doi.org/10.1002/sscp.202400050","url":null,"abstract":"Staphylococcus aureus Protein A was immobilized on an in‐house made neutravidin poly (glycidyl methacrylate‐co‐ethylene dimethacrylate) capillary column with a 25 µm internal diameter, a length of 30 mm and a mass loadability of 60 ng. The immobilized quantity of protein A on the organic monolith was very low, in the pico mole range (1.80 pmol). This was of significance importance when working with less available or expensive purified enzyme. This capillary column was integrated into a nano liquid chromatographic system and used for the fast determination without dilution of the doses of therapeutic monoclonal antibodies (mAbs) in standardized infusion bags prepared in advance in a pharmacy department. This chromatographic method was linear in the studied concentration range with good precision and accuracy. Heat stressed studies indicated that the protein A affinity capillary column was able to monitor degraded mAbs. As well, the high specificity of this column to capture immunoglobulin G2 in cell culture supernatant was visualized. As the mAbs are produced through genetic engineering of animal cells this last result demonstrated that this novel protein A column could be used in the feature for rapid screening of immunoglobulin G concentration in cell culture.","PeriodicalId":21639,"journal":{"name":"SEPARATION SCIENCE PLUS","volume":null,"pages":null},"PeriodicalIF":1.1,"publicationDate":"2024-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141101977","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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