Chromatographia最新文献

The conjugation technology is used for the manufacturing of polysaccharide conjugate vaccines against pathogenic organisms like Streptococcus pneumoniae, Haemophilus influenzae type b, etc. Conjugation using CDAP chemistry is widely utilized because of high yield and short reaction time. The cyano group of CDAP is attached to hydroxyl group of polysaccharides, resulting in activated polysaccharide with generation of process-related residual impurity, dimethyl aminopyridine (DMAP) which should be < 1.5 µg/day as per regulatory guidelines. In the present study, we reported the SEC-HPLC coupled with UV detector for quantitative estimation of residual DMAP content in pneumococcal polysaccharide–protein conjugates. The method was validated as per the ICH guidelines using representative pneumococcal conjugate of serotype 1, 6B and 23F. The method specificity was confirmed using placebo buffers and the identity of DMAP was ensured using orthogonal GC–MS method. The method showed linearity between 0.1 and 15 µg/mL with linear regression of > 0.99. Detection and quantitation limits of 0.03 µg/mL and 0.1 µg/mL were observed respectively. The overall % RSD of DMAP content was found to be between 0.94 and 1.91% suggesting precision of the method. Accuracy was performed by spiking known amount of DMAP at six different concentration levels and % recovery of 87 to 104% was noted. The validated SEC-HPLC–UV method was successfully applied for various stages of pneumococcal polysaccharide conjugate vaccines including crude, in-process and final purified samples. This has suggested that the SEC-HPLC–UV method can be used as routine process analytical tool to monitor residual DMAP.

Asymmetric flow field-flow fractionation (AF4) separates constituents based on particle size and is emerging as a powerful tool for obtaining high-resolution information on the size of nanoparticles in liquid media. However, the size measurement inaccuracy has been reported for traditional dynamic light scattering (DLS) detectors when coupled to AF4 systems for the reason of additional component caused by uniform translation motions. In this paper, we developed a flowing DLS for AF4 study based on our sinc model reported previously. We further investigated the reliability of the size measurement provided by sinc model. The experiments were performed with suspensions of mono-dispersed polystyrene microspheres with a nominal diameter of 201 nm, 400 nm, 596 nm, and 799 nm at a range of different detector flow rates. The results obtained demonstrate that sinc model not only can measure the particle size but also flow velocity accurately. We believe our model can provide particle size information by coupling AF4 to DLS more accurately and pave the way for the complex AF4 system study, such as the electrical asymmetric flow field-flow fractionation in the flowing dispersion.

N-glycosylation heterogeneity has a significant impact on the clinical safety, efficacy, pharmacokinetics (PK), and pharmacodynamics of monoclonal antibody (mAb) drugs, thus becoming a critical quality attribute (CQA) of great concern. In the registration quality standards for bevacizumab produced by different manufacturers, either lacking supervision of N-glycosylation or the detection methods and criteria vary greatly among manufacturers, leading to compromised comparability of results. Besides, the existing quality control system pays insufficient attention to the correlation between glyco-relevant physicochemical properties and glycosylated modification. In this study, a standardized hydrophilic interaction liquid chromatography-fluorescence detection (HILIC-FLD) N-glycan profiling method was developed, which had been proven to be more time-saving and easier to operate and was used for the parallel analysis of N-glycan profiles in bevacizumab products provided by 8 different manufacturers. We also paid attention to the glycosylation-related CQAs including charge heterogeneity, isoelectric point, non-glycosylated heavy chain (NGHC) and aggregates. Results revealed obvious difference in N-glycosylation, NGHC and aggregates among manufacturers. Among all the mAbs, the abundance of major glycoforms showed similar order yet the percentage of the same glycoform varies greatly. In summary, the standardized method could offer a reliable, efficient and practical approach for the parallel analysis and comparison of glycosylation and related CQA heterogeneity among manufacturers, providing a reference for more effective quality control of mAb drugs.

To comprehensively investigate the flavor characteristics of Shanyao yogurt, headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC–MS) and headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS) techniques were employed to analyze volatile flavor compounds(VFCs) in Shanyao yogurts fermented with Lactobacillus rhamnosus and Lactobacillus plantarum. The findings of the two techniques revealed variations in both components and concentrations of VFCs in the same sample. HS-SPME-GC-MS exhibited excellent performance in detecting acidic substances, while HS-GC-IMS demonstrated advantages in analyzing ester substances. Only 4 compounds were identified by both methods in Shanyao yogurt fermented by L. plantarum; whereas 7 compounds were identified by both methods in Shanyao yogurt fermented by L. rhamnosus. This research underscored the benefits of employing diverse analytical techniques for studying yogurt samples, as integrating multiple approaches provided a more comprehensive understanding of VFCs in Shanyao yogurt, thereby offering robust support for investigating VFCs in similar products.

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The application of artificial intelligence (AI) and machine learning (ML) is rapidly expanding and has begun to make a significant impact on polymer development and characterization. This perspective article explores the current state of AI in this field and highlights areas where its potential remains underutilized. While the optimization of polymer synthesis to achieve desired properties and the classification of polymer types are well-established, opportunities for AI integration in detailed characterization, analytical method development, and data processing remain largely untapped. Greater automation of the analytical laboratory, whether through dedicated algorithms or AI-driven solutions, will enable analytical chemists to focus more on addressing research questions and interpreting results, rather than on method development and routine measurements.

The simultaneous quantification bulk active pharmaceutical components and combined dosage forms is vital for ensuring quality control and therapeutic efficacy. This study addresses the need for a reliable analytical method for the simultaneous quantification of lobeglitazone sulphate (LBZ) and glimepiride (GPR), two drugs often used in the administration of type 2 diabetes. A review of existing literature reveals the absence of a quality by design (QbD)-assisted reverse phase high-performance liquid chromatography (RP-HPLC) method for this specific combination. Using a QbD approach, a robust RP-HPLC method was developed and optimized employing a central composite design (CCD). The Agilent Infinity 1270 Series HPLC system, equipped with a Zorbax SB 618 column (5 μm, 46 × 150 mm), was used. Method parameters were fine-tuned to achieve optimal resolution (7.6), tailing (1.8), and retention times of 5.6 min for LBZ and 8.6 min for GPR. The optimized mobile phase consisted of ACN:KH₂PO₄ buffer (pH 3.5, 50:50 v/v), a flow rate of 1 mL/min, and a detection wavelength of 227 nm. The developed method was validated as per current regulatory guidelines, demonstrating precision, accuracy, and sensitivity. These results underscore the effectiveness of a QbD framework in method development, ensuring reproducibility and robustness. This study highlights the broader potential of QbD-assisted analytical techniques in pharmaceutical research, paving the way for more efficient drug quality assessments and improved therapeutic outcomes across diverse drug combinations.

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N-Nitrosamines are raised as having health consequences by the European Medical Agencies ICH M7(R2) guidance on Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk. It has been suggested that nitrosamines have carcinogenic potential, and efforts should be made to control them at acceptable and safe levels in drug substances (DS). The detection of nitrosamine drug substance related impurities (NDSRIs) historically has encountered considerable challenges in establishing compound specific acceptable intake (AI) limits. Often, a default low AI limit of 18 ng/day was assigned to the NDSRIs due to the lack of their toxicology data, prior to the introduction of the current Carcinogenic Potency Categorization Approach (CPCA). Developing analytical methods to quantify NDSRIs posed a challenge due to an extremely low Quantitation Limit (QL), not exceeding 10% of the AI limit based on a conservative Threshold of Toxicological Concern (TTC) approach. This work presents the method development strategy for two complex diastereomeric NDSRIs in miglustat, a recently commercialized rare disease drug. A predictive chemistry for NDSRI formation and organic synthesis earlier was followed by in silico assessments of potential mutagenicity and carcinogenicity. This paper describes the development of a highly sensitive analytical method with a QL of 6.9 ppb for the combined NDSRIs, in alignment with the regulatory recommendation at the time of this work. The validated method was employed for confirmatory testing of miglustat batches for the combined NDSRIs. The results were below QL, and no routine NDSRI testing was required for future drug product (DP) batch release. This novel approach may offer valuable insights into the numerous molecules in development that will now require rigorous nitrosamine assessment.

This study introduces a rapid LC–MS/MS method designed to simultaneously quantify 14 water-soluble vitamins and their metabolites in human serum. The protein precipitation with metaphosphoric acid and stable isotope dilution techniques are utilized in this method. An Agilent XDB C18 column and dynamic ionization mode switching are adopted, enabling the completion of a single sample analysis within 5 min. The method has undergone rigorous validation. The lower limits of quantification (LLOQs) range from 0.3 to 400 ng/mL, with recovery rates between 80 and 120%. Intra-day and inter-day relative standard deviations (RSDs) are below 15%. The matrix effects are controlled within ± 15%, and the analyte carryover is less than 20% of the LLOQ. The stability tests have demonstrated that samples remain stable at room temperature (25 °C) for 10 h, at − 20 °C for 7 days, and can withstand one freeze–thaw cycle. Extracted samples are stable in the autosampler (4 °C) for 24 h. The method was applied to serum samples from 150 healthy individuals. Non-parametric statistical analysis was carried out to establish clinical reference intervals, which provides valuable data for the clinical evaluation of vitamin and metabolite levels in serum.

A novel epitope-imprinted polymer with specific recognition of trace tumor marker matrix metalloproteinase-9 (MMP-9) was synthesized and successfully applied for the enrichment, separation, and detection of the human serum samples. Based on catalytic domain characteristics containing zinc ion binding sites of MMP-9, an MMP-9 C-terminal nonapeptide was selected as the antigenic determinant template, while zinc acrylate and choline chloride-methacrylic acid were chosen as functional monomers. After optimizing the preparation conditions through single factor and mixed orthogonal experiments, molecularly imprinted polymers (MIPs) with a maximum adsorption capacity of 3.59 mg/g and an imprinting factor of 2.50 were obtained. MIPs showed good repeatability and reproducibility, and better selectivity for similar proteins than NIPs, with a 99.97% adsorption rate for 5 µg/mL MMP-9, which coincided with the results from sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of human serum samples spiked with nonapeptide was 90.04%, with a relative standard deviation (RSD) of 2.3% (n = 5), and the recovery of human serum samples spiked with MMP-9 protein was 75.05%, with an RSD of 0.88% (n = 5). The developed method had a linear range of 1–10 ng/mL for MMP-9, with a detection limit of 0.41 ng/mL. The synthesized MIPs were convenient, cost-effective and consistent. The results showed that they had the potential to be used as novel adsorption and enrichment materials. Moreover, they could offer valuable references for the further preparation of highly specific MMP-9 adsorbents.