[Impurity profile analysis of amphotericin B using on-line two-dimensional high performance liquid chromatography-quadrupole time-of-flight mass spectrometry].

Amphotericin B (AmB) is a polyene-macrolide antimicrobial drug with a broad antibacterial spectrum and remarkable efficacy against deep fungal infections. It binds to ergosterol on the fungal cell membrane and alters its permeability, thereby destroying the membrane. AmB is a multicomponent antimicrobial medication that contains a wide range of impurities, rendering quality analysis extremely difficult. In the current Chinese Pharmacopoeia (Edition 2020) and European Pharmacopoeia (EP10.3), high performance liquid chromatography (HPLC) is applied to examine related substances in AmB. However, this technique presents a number of issues. For instance, the mobile phases used in the HPLC method described in both references contain nonvolatile inorganic salts, which cannot be coupled with a mass spectrometry (MS) detector. In addition, because the mobile phases used have a low pH, the component/impurities of AmB drug can easily be degraded or interconverted during the analytical process, leading to reduced analytical accuracy. Therefore, the accuracy and sensitivity of this method must be improved. In this study, a method based on on-line two-dimensional high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (2D HPLC-Q TOF/MS) was developed to analyze the impurity profile of AmB in accordance with the Chinese Pharmacopoeia (Edition 2020) and European Pharmacopoeia (EP10.3). The method combines on-line dilution and a multiple-capture HPLC system to achieve the efficient separation of AmB component/impurities. It also resolves the issue of poor solvent compatibility in 2D HPLC, increases the analytical flux, enhances the automation capability, reduces the mutual conversion of AmB and its impurities during the analytical process, and increases the detection sensitivity of the method. MS was also used to determine the structural inference of unstable components and impurities. An XBridge Shield C18 column (250 mm×4.6 mm, 3 µm) was used for first-dimensional-liquid chromatography with gradient elution using methanol-acetonitrile-4.2 g/L citric acid monohydrate solution (10∶30∶60, v/v/v, pH 4.7) as mobile phase A and methanol-acetonitrile-4.2 g/L citric acid monohydrate solution (12∶68∶20, v/v/v, pH 3.9) as mobile phase B. An Xtimate C8 column (10 mm×2.1 mm, 5 µm) was used as the trap column, and trapping and desalting were performed using 10 mmol/L ammonium formate aqueous solution containing 0.1% formic acid-acetonitrile (95∶5, v/v). An Xtimate C8 column (250 mm×2.1 mm, 5 µm) was used for second-dimensional-liquid chromatography with gradient elution using 10 mmol/L ammonium formate aqueous solution containing 0.1% formic acid-acetonitrile (95∶5, v/v) and 10 mmol/L ammonium formate aqueous solution containing 0.1% formic acid-acetonitrile (5∶95, v/v) as mobile phases. The data were collected in positive-ion mode. In this study, the structures of six impurities in amphotericin B were inferred, according to the fragmentation, the MS and MS2 spectra of each impurity. The developed method can be used to quickly and sensitively analyze the impurity profile of AmB. Furthermore, the research results on impurity profiles can be applied to guide improvements in AmB production.

Development and Validation of LC-MS/MS Method for the Determination of 1-Methyl-4-Nitrosopiperazine (MNP) in Multicomponent Products with Rifampicin-Analytical Challenges and Degradation Studies.

Rifampicin is an essential medicine for treating and preventing tuberculosis (TB). TB is a life-threatening infectious disease and its prevention and treatment are public health imperatives. In the time of a global crisis of nitrosamine contamination of medicinal products, patient safety and a reduction in the number of drug recalls at the same time are crucial. In this work, the LC-MS/MS method was developed for the determination of the 1-methyl-4-nitrosospiperazine (MNP), a genotoxic nitrosamine impurity in various products containing rifampicin at a 5.0 ppm limit level according to Food and Drug Administration (FDA). Extraction with neutralization was necessary due to the matrix and solvent effect associated with the complexity of the rifampicin product. The developed method was validated in accordance with regulatory guidelines. Specificity, accuracy, precision, limit of detection, and limit of quantification parameters were evaluated. The recovery of the MNP was 100.38 ± 3.24% and the intermediate precision was 2.52%. The contamination of MNP in Rifampicin originates in the manufacturing process of the drug. Furthermore, the results of the forced degradation experiments show that the formation of MNP is possible by two mechanisms: through degradation of rifampicin and the oxidation of 1-amino-4-methyl-piperazine. This article points out that it is necessary to monitor and describe degradation products and the mechanism of degradation of potentially affected active pharmaceutical ingredient (API) with respect to the formation of nitrosamines during stress testing, as it was done in the following work for rifampicin in multicomponent products.

Changes in methamphetamine impurity profiles induced by tert-butoxycarbonylation.

Analysis of impurities in methamphetamine (MA) can be used to characterize MA seizures, investigate the relationship among MA seizures, and provide information on their synthetic routes. Recently, chemically derivatized MA, such as tert-butoxycarbonyl (t-Boc) MA, has been seized and attracted attention because routine forensic analysis methods may fail to correctly identify them. Chemical derivatization is a simple method for protection and deprotection of a compound, and protection of MA using t-Boc can be used to mask the MA. Although t-Boc derivatization might alter the impurity profile of MA, the actual changes in the impurity profile have not been investigated. In this study, changes in the MA impurity profile with tert-butoxycarbonylation were explored. MA and some typical impurities were derivatized using di-tert-butyl dicarbonate and water. Analysis of the impurities in five MA samples by gas chromatography showed that peaks both appeared and disappeared for the deprotected MA compared with the original MA. However, typical impurities important for characterizing MA seizures were conserved after derivatization and deprotection. Most of the new peaks were speculated to be contaminants introduced during derivatization and deprotection. A peak giving a mass spectrum similar to that of t-Boc MA was detected in the chromatograms of t-Boc MA and deprotected MA. Although the origin of this peak was not determined, it might be a marker for the MA involving tert-butoxycarbonylation. These results indicate that tert-butoxycarbonylation can alter the MA impurity profile; therefore, care is needed when interpreting results for derivatized MA.

Study on the impurity profile and influencing factors of photodegradation in non-aqueous ofloxacin ear drops using liquid chromatography combined with ion trap/time-of-flight mass spectrometry.

Ofloxacin ear drops contain a large proportion of organic solvents, which have a great effect on the photodegradation of ofloxacin. The photodegradation impurities of ofloxacin in aqueous solution has been studied, however, the photodegradation of ofloxacin in non-aqueous solution with a high proportion of organic solvents has not been reported. In this article, the impurity profile in non-aqueous ofloxacin ear drops was studied for further improvement of official monograph in pharmacopoeia and quality control of drug. The liquid chromatography combined with ion trap/time-of-flight mass spectrometry was applied to separate and characterize the structures of the impurities in non-aqueous ofloxacin ear drops. Mass fragmentation pattern of ofloxacin and its impurities were studied. The structures of seventeen impurities in ofloxacin ear drops were elucidated based on the high-resolution MSn data in positive ion modes, and ten of them were unknown impurities. The results showed that the impurity profile of non-aqueous ofloxacin solution was significantly different from that of aqueous ofloxacin solution. The effects of packaging materials and excipients on the photodegradation of ofloxacin ear drops were also investigated. The results of correlation analysis showed that the packaging materials with low light transmittance could reduce the light degradation, and ethanol of excipients could significantly decrease the light stability of ofloxacin ear drops. This study revealed the impurity profile and key factors affecting the photodegradation of non-aqueous ofloxacin ear drops, and guided enterprises to improve drug prescription and packaging materials to ensure the safety of drug use by the public.

Azobisisobutyronitrile loaded on mesoporous silica particles: A new stressor for solid-state oxidative forced degradation studies.

A new approach for testing drug sensitivity to autooxidative degradation in the solid state is demonstrated in this work. A novel solid-state form of stressing agent for autooxidation has been proposed, based on azobisisobutyronitrile loaded into mesoporous silica carrier particles. The new solid-state form of the stressing agent was applied in degradation studies of two active pharmaceutical ingredients: bisoprolol and abiraterone acetate. The effectiveness and predictivity of the method were evaluated by comparing impurity profiles with those obtained by traditional stability testing of commercial tablets containing the investigated APIs. The results obtained by the new solid-state stressor were also compared with those obtained by an existing method for testing peroxide oxidative degradation in the solid state using a complex of polyvinylpyrrolidone with hydrogen peroxide. It was found that the new silica particle-based stressor was able to effectively predict which impurities could be formed by autooxidation in tablets and that this new approach is complementary to methods for testing peroxide oxidative degradation known from the literature.

Quality control and consistency evaluation of ceftriaxone sodium for injection.

Ceftriaxone sodium for injection is an antibiotic used clinically. Here, we developed a strategy for evaluating the consistency of ceftriaxone sodium for injection. Comparison of the quality of the generic and original raw materials, and analysis of the production process revealed that the quality of the ceftriaxone sodium raw material is the most important factor affecting the quality of preparation, while the ceftriaxone sodium crystallization process is the key factor affecting the quality of raw materials. The solution clarity of the formulation, another key aspect, was addressed by controlling the leachable components found in the rubber closures used in the packaging. The time to achieve therapeutic efficacy of the preparation could be preliminarily evaluated by evaluating the rate of salt formation and the protein binding rate. Finally, the results of the tests (including water, pH, impurity profile and solution clarity) and assay were compared with the original preparation. On this basis, the critical quality attributes (CQAs) that reflect the quality of the product could be determined and a strategy for evaluating ceftriaxone sodium for injection was developed.

Effect of mobile phase pH on liquid chromatography retention of mepartricin related compounds and impurities as support to the structural investigation by liquid chromatography-mass spectrometry.

Mepartricin is a semisynthetic polyene macrolide with antifungal and anti-protozoal activities, and it is widely used for the treatment of benign prostatic hyperplasia. Mepartricin is produced by synthetic methyl esterification of the more toxic partricin, and its activity is due to a complex of related compounds. Among them, the main ones are mepartricin B and mepartricin A which are characterized by the presence of a primary and a secondary amine group, respectively. In this work a previously reported HPLC-UV method was properly modified to make it MS-compatible. The selected conditions entail the use of a C18 reverse phase column, and a mobile phase composed by ammonium formate and acetonitrile, with the addition of heptafluorobutyric acid as modifier. The developed method was applied to the characterization of a mepartricin reference standard and a mepartricin experimental batch. All the UV responding peaks, 30 for the standard and 21 for the experimental batch, were successfully detected by MS, allowing to define their m/z values and acquire their fragmentation spectra. For the structural elucidation of isobaric species and, in particular, the identification of toxic partricin-related impurities, the presence of differently ionisable chemical groups was considered, as partricins contain free caboxy-groups, while mepartricins represent their estherified counterparts. A deep study of the effect of mobile phase pH on the chromatographic retention of partricin and mepartricin related compounds was performed in the pH range 2.5-6.5. This study allowed to successfully cluster all the detected species and asses, in the considered batch, the absence of other partricin-related impurities in addition to partricin B and partricin A.

Assay of tirofiban and identification of oxidative impurity in aqueous injection by using UPLC-PDA/QDa detectors.

The basic objective of this study is to propose a short, reliable, mass compatible ultra-performance liquid chromatography (UPLC) method to confirm the identity of impurities and to estimate the assay and purity of Tirofiban simultaneously in aqueous injection (5mg/100mL bag). Aqueous formulations are susceptible to oxidation, hence the possible oxidative degradation impurities of Tirofiban were studied in this experiment by using UPLC coupled with photodiode array/Quadrupole Dalton Analyzer (PDA/QDa) detectors. The required separations were achieved in the column: ACQUITY HSS T3 (100×2.1) mm, 1.7µm, operated at 30°C by using 0.02% Triethyl amine (TEA) in water, pH 2.8 with formic acid as solution-A and 0.1% formic acid in 9:1 acetonitrile, water as solution-B. Binary gradient flow is delivered at the rate of 0.5mL/min and the detection of impurities specifically carried out at 227nm using empower3 software. RP-UPLC/PDA with QDa detector was used for the experiment. The method was linear and accurate from the concentrations: 0.04 to 0.38µg/mL for impurity-A and 0.04 to 75µg/mL for Tirofiban. The major unknown degradation impurity generated during the oxidative degradation has been identified as N-oxide derivative (Impurity-B) [(M+H)+ 455.1] by using QDa detector operated in an electro spray positive ion mode by applying a voltage of 0.8kV. This method was further validated as per ICH Q2 (R2) guidelines. Hence, the proposed method is said to be a fast, sensitive and comprehensive technique, which could give a clear idea about the assay and impurity profile of Tirofiban injection.

Impurity profiling of Compound Amino Acid Injection (6AA) using ion-pair high performance liquid chromatography coupled with corona-charged aerosol detection and high resolution mass spectrometry.

The complex industrial production process of amino acids (AAs) leads to the existence of a certain amount of impurities in Compound Amino Acid Injection (6AA). It is difficult to obtain its comprehensive and systematic impurity profile using conventional ultraviolet (UV) detectors due to lack of a suitable chromophore in the structures of AAs and their impurities. In our study, a universal ion-pair high performance liquid chromatography (HPLC) method combined with high resolution mass spectrometer (HRMS) and charged aerosol detection (CAD) was developed to identify and determine the content of impurities in Compound Amino Acid Injection (6AA), respectively. After optimizing the content of trifluoroacetic acid (TFA) and heptafluorobutyric acid (HFBA) in the mobile phase on a C18 AQ column, HPLC-CAD method was developed and nine unknown impurities were detected. These impurities were successfully identified using HPLC coupled with orbitrap mass spectrometry and confirmed with their reference substances. The CAD parameters setting was optimized to improve the sensitivity and linearity of the methods before the developed method was validated. The results of validation reflected that the limit of detection (LOD) was approximately 2 ng (corresponding to approximately 0.02 % of L-isoleucine in injection). Under the optimized power function value (PFV) of CAD, the linear range of each impurity was 1 ∼ 200 µg mL-1 (the linear range of one of the impurities with higher content was 2 ∼ 400 µg mL-1) with coefficients of determination (R2) greater than 0.998. The recovery rates for nine impurities were 93.37 % ∼ 110.23 %. This study made full use of the qualitative functions of HRMS and the versatility of CAD, revealing possible impurities in the 6AA injection, which could provide reference for the safety research of it.

Impurity Profiling and Identification of 2,6-Diisopropylphenol by HPLC Method and Raman Spectroscopy Method.

Identification of 2,6-diisopropylphenol by Raman spectroscopy using the TruScan Raman spectrum is validated by specificity test and robustness. HPLC assay method is developed to detect the 2,6-diisopropylphenol and its impurities A = determination of 2,6-diisopropylphenol; its main impurities related compound A = 2,6-diisopropylphenyl isopropyl ether, related compound B = 2,6-diisopropylquinone, and related compound C = 3,3'-5,5'-tetraisopropyl diphenol; and unknown impurities done by HPLC method. Related compound A and C determination has been developed by normal phase HPLC; good resolution peak shapes have resulted from the peak results. The limit of impurities A and C is reported to be 0.05% and 0.03%, and the limit of impurity B is also detected by using the same above procedure, but the column has been changed for the maximum wavelength detection and better elution from the peak results. The reported impurity level was 0.02%, the unknown impurity limit was 0.0149%, and the total impurity level of 2,6-diisopropylphenol was reported to be 0.06% which are in the threshold limit level. It specifies that the drug is safe and efficient without any toxicity.

Formulation and sterilization assessment of kanamycin sulfate injection based on impurity profiles / 药学学报

The compatibility of kanamycin with sodium citrate for the formulation of kanamycin sulfate injection was determined, including optimization of the amount of sodium citrate in the injection and the sterilization process. An HPLC coupled with an evaporative light scattering detector (ELSD) was used to measure the amount of sodium citrate and the impurity profiles. A validated post-column derivatization HPLC coupled with a fluorescence detector (FLD) was used to determine the correlation between specific impurities in a domestic factory and sodium citrate, and then the formulation was evaluated by HPLC coupled with mass detector (MS) characterization of degradation products. The results show that the amount of sodium citrate in kanamycin sulfate injection from a domestic factory is about 40 times higher than that of the Meiji formulation. Several specific impurities can be detected in solutions heated under simulated sterilization conditions (121 ℃), which were correlated with the amount of sodium citrate. Impurities were characterized by HPLC-MS/MS, and data showed that the identified impurities were interaction products of kanamycin and sodium citrate. These results indicate that greater attention should be directed at formula optimization in domestic factories, as it is crucial to the safety and efficacy of the preparations. Drug-excipient chemical compatibility should also be evaluated in the development of pharmaceutical dosages forms especially when the active pharmaceutical ingredients have a primary amine group.

Development of a method of analysis for profiling of the impurities in phenoxymethylpenicillin potassium based on the analytical quality by design concept combined with the degradation mechanism of penicillins.

Accurate analysis of all of the impurities present in a substance is critical for controlling the impurity profiles of drugs. Penicillins can easily yield a formidable array of degradation-related impurities (DRIs) with significantly different polarities and charge properties, which renders identifying each one a complicated matter. In this work, phenoxymethylpenicillin potassium (Pen V) was selected to find a way to quickly establish a robust analysis method for the impurity profiling of penicillin. Based on the analytical quality by design (AQbD) concept and the degradation mechanism of the drug, structures of all of the DRIs were first proposed. Then Pen V and its detected DRIs were separated and identified by liquid chromatography-tandem mass spectrometry method (LC-MS). Characteristic fragment ions and mass fragmentation process of Pen V and its detected DRIs were summarized. In addition, a quantitative structure-retention relationship (QSRR) model was constructed to predict the retention times of undetected impurities and to evaluate whether the chromatographic system can separate them. Finally, a stability-indicating high-performance liquid chromatography (HPLC) method was developed that can separate all of the DRIs of Pen V.

Rapid Characterization of Impurity Profiles in Vancomycin Raw Material by Column-Switching LC/MSn / 中国药学杂志

OBJECTIVE: To characterize the impurity structures in vancomycin raw material. METHODS: Impurities were eluted gradiently on a Chromasil 100-5 C18(4.6 mm×250 mm, 5 μm) column, with triethylamine buffer solution (pH 3.2)-acetonitrile-tetrahydrofuran (92∶7∶1, V/V/V) as mobile phase A, and triethylamine buffer solution (pH 3.2)-acetonitrile-tetrahydrofuran (70∶29∶1, V/V/V) as mobile phase B. Stress tests were performed on the raw material to specify those impurities. By application of on-line LC/MSn method, the impurities were analyzed in positive mode and their structures were characterized based on the degradation mechanism and mass fragmentation regularity of sugar-lipopeptides. RESULTS: Totally 14 impurities were characterized in the raw material, seven of which were reported for the first time. The relationships between the chemical structures and chromatographic behaviors of vancomycin, demethylvancomycin and methylated vancomycin with their two β-isomers were summarized. CONCLUSION: The structures of related impurities in vancomycin raw material can be rapidly identified by on-line LC/MSn method together with stress degradation experiments.

Quality control of gold nanoparticles as pharmaceutical ingredients.

Nanoparticles are being developed for a wide range of medical applications such as, controlled release, drug delivery systems or imagery, theranostics, implants…. For the moment, there is no legal definition of nanoparticles or nanomaterials for therapeutic use. The specific case of gold nanoparticles is not an exception: their current definition as nanoparticle material does not correspond to classic pharmaceutical ingredients as described in Pharmacopoeias. In this study, more than 30 different batches of citrate stabilized gold nanoparticles (AuNP) were synthesized and analyzed thanks to both classical approaches (UV-Vis spectrophotometry, dynamic light scattering coupled or not to electrophoresis …) and capillary zone electrophoresis (CZE) coupled to diode array detection to assess their purity and impurity profiles. These techniques led to the beginning of defined specifications, a key step for the use of gold nanoparticles as pharmaceutical ingredients. CZE was demonstrated suitable to evaluate a batch-to-batch quality control, to monitor the purification processes and to follow the stability of 18 different batches for 20 days. Finally, commercially available AuNP samples were tested and the results compared to the provided certificates of analysis.

Translation of nanomedicines from lab to industrial scale synthesis: The case of squalene-adenosine nanoparticles.

A large variety of nanoparticle-based delivery systems have become increasingly important for diagnostic and/or therapeutic applications. Yet, the numerous physical and chemical parameters that influence both the biological and colloidal properties of nanoparticles remain poorly understood. This complicates the ability to reliably produce and deliver well-defined nanocarriers which often leads to inconsistencies, conflicts in the published literature and, ultimately, poor translation to the clinics. A critical issue lies in the challenge of scaling-up nanomaterial synthesis and formulation from the lab to industrial scale while maintaining control over their diverse properties. Studying these phenomena early on in the development of a therapeutic agent often requires partnerships between the public and private sectors which are hard to establish. In this study, through the particular case of squalene-adenosine nanoparticles, we reported on the challenges encountered in the process of scaling-up nanomedicines synthesis. Here, squalene (the carrier) was functionalized and conjugated to adenosine (the active drug moiety) at an industrial scale in order to obtain large quantities of biocompatible and biodegradable nanoparticles. After assessing nanoparticle batch-to-batch consistency, we demonstrated that the presence of squalene analogs resulting from industrial scale-up may influence several features such as size, surface charge, protein adsorption, cytotoxicity and crystal structure. These analogs were isolated, characterized by multiple stage mass spectrometry, and their influence on nanoparticle properties further evaluated. We showed that slight variations in the chemical profile of the nanocarrier's constitutive material can have a tremendous impact on the reproducibility of nanoparticle properties. In a context where several generics of approved nanoformulated drugs are set to enter the market in the coming years, characterizing and solving these issues is an important step in the pharmaceutical development of nanomedicines.

Exploring the Critical Factors Limiting Polyaniline Biocompatibility.

Today, the application of polyaniline in biomedicine is widely discussed. However, information about impurities released from polyaniline and about the cytotoxicity of its precursors aniline, aniline hydrochloride, and ammonium persulfate are scarce. Therefore, cytotoxicity thresholds for the individual precursors and their combinations were determined (MTT assay) and the type of cell death caused by exposition to the precursors was identified using flow-cytometry. Tests on fibroblasts revealed higher cytotoxicity of ammonium persulfate than aniline hydrochloride. Thanks to the synergic effect, both monomers in combination enhanced their cytotoxicities compared with individual substances. Thereafter, cytotoxicity of polyaniline doped with different acids (sulfuric, nitric, phosphoric, hydrochloric, and methanesulfonic) was determined and correlated with impurities present in respective sample (HPLC). The lowest cytotoxicity showed polyaniline doped with phosphoric acid (followed by sulfuric, methanesulfonic, and nitric acid). Cytotoxicity of polyaniline was mainly attributed to the presence of residual ammonium persulfate and low-molecular-weight polar substances. This is crucial information with respect to the purification of polyaniline and production of its cytocompatible form.

Chitosan oligosaccharide (COS): An overview.

The frequently studied polysaccharide, chitosan oligosaccharide/chitooligosaccharide (COS) is the major degradation product of chitosan/chitin via chemical hydrolysis or enzymatic degradation involving deacetylation and depolymerization processes. Innumerable studies have revealed in the recent decade that COS has various promising biomedical implications in the past analysis, current developments and potential applications in a biomedical, pharmaceutical and agricultural sector. Innovations into COS derivatization has broadened its application in cosmeceutical and nutraceutical productions as well as in water treatment and environmental safety. In relation to its parent biomaterials and other available polysaccharides, COS has low molecular weight (Mw), higher degree of deacetylation (DD), higher degree of polymerization (DP), less viscous and complete water solubility, which endowed it with significant biological properties like antimicrobial, antioxidant, anti-inflammatory and antihypertensive, as well as drug/DNA delivery ability. In addition, it is also revealed to exhibit antidiabetic, anti-obesity, anti-HIV-1, anti-Alzheimer's disease, hypocholesterolemic, calcium absorption and hemostatic effects. Furthermore, COS is shown to have higher cellular transduction and completely absorbable via intestinal epithelium due to its cationic sphere exposed on the more exposed shorter N-glucosamine (N-Glc) units. This paper narrates the recent developments in COS biomedical applications while paying considerable attention to its physicochemical properties and its chemical composition. Its pharmacokinetic aspects are also briefly discussed while highlighting potential overdose or lethal dosing. In addition, due to its multiple NGlc unit composition and vulnerability to degradation, its safety is given significant attention. Finally, a suggestion is made for extensive study on COS anti-HIV effects with well-refined batches.

A two-step process for capture and purification of human basic fibroblast growth factor from E. coli homogenate: Yield versus endotoxin clearance.

A two-step purification process for human basic fibroblast growth factor (FGF-2) from clarified E. coli homogenate has been developed in which the impurity level after the second step is below the limit of quantification. Endotoxin content is cleared to 0.02 EU/µg FGF-2 and the overall yield is 67%. The performance of the cation exchanger Carboxymethyl-Sepharose Fast Flow (CM-SFF) was compared to the affinity resin Heparin-SFF regarding the impurity profile and product quality in the elution peak. The CM-SFF eluate was further purified using hydrophobic interaction resin Toyopearl-Hexyl-650C. The relative amounts of target product, host cell proteins (HCPs), dsDNA, endotoxin, monomer content, and high molecular weight impurities differed along the elution peak depending on the applied method. The bioactive monomer (>99%) was obtained with a yield of 48% for CM-SFF and 68% for Heparin-SFF. A half-load reduction in CM-SFF increased the yield up to 67% without deterioration of the impurity content. Assuming a dose of 400 µg FGF-2, endotoxin was reduced to 188 EU/dose, dsDNA <10 ng/dose, and HCP <2 ppm/dose using the cation exchanger. In the pooled eluate fractions, dsDNA was removed 4-fold (291 ng/mL) and endotoxin 14-fold (0.47 EU/µg FGF-2) more efficiently by CM-SFF than by affinity chromatography. In contrast, HCP clearance was 3-fold (13 ppm) more efficient with Heparin-SFF than CM-SFF. In contrast to process monitoring by UV280nm or SDS-PAGE, this characterization is the basis for a Process Analytical Technology attempt when correlated with online monitored signals, as it enables knowledge-based pooling according to defined quality criteria.

Current situation and the trend in impurity profiling of chemical drugs / 药学学报

Impurity profiling is one of the most important activities in both assuring drug safety and improving the quality of domestic drugs. Since the basic strategy of impurity profile control was put forward in 2010, a mature control procedure for impurity profile in drugs has been formed in China after nearly ten years of continuous efforts. The progress in impurity profiling before 2010 and from 2010 to 2015 have been reviewed. Since 2015, the concepts, analytical techniques and the application of these techniques in this field have developed rapidly. As a result, the progress in impurity profiling of chemical drugs since 2015 was reviewed in this paper. And the views on future development of impurity profiling in drugs were also put forward.

Development of a reversed phase liquid chromatographic method for analysis of pyridoxal-5'-phosphate and its impurities.

Since no proper method is available in literature for the analysis of pyridoxal-5'-phosphate, a reversed phase liquid chromatographic method was developed and validated for specificity, sensitivity, linearity, precision and accuracy. Nine potential related substances and forced degradation products could be successfully separated from the main peak. The separation was achieved on a Polaris C18 column (250 × 4.6 mm i.d., 5 µm) using a mobile phase consisting of 20 mM ammonium formate in 0.65% formic acid - acetonitrile (98.8:1.2, v/v). Isocratic elution was performed at a flow rate of 1.0 mL/min and the analytes were detected by UV at 240 nm. The volatile mobile phase allowed also direct coupling to an ion-trap mass spectrometer with a positive electrospray ionization source to characterize unknown peaks in the chromatogram. The method can be used for quality control purposes as required by regulatory authorities to ensure the product's safety and efficacy.