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Due to the complex manufacturing process of therapeutic monoclonal antibodies, it is hardly possible to produce an identical copy of the original product (originator). Consequently, follow-on products (biosimilars) must demonstrate their efficacy being similar to the originator in terms of structure and function. During this process, a variety of analytical methods are required for this purpose. This study focuses on three particularly relevant analytical techniques: high-resolution mass spectrometry, fragment crystallisable (Fc) affinity chromatography, and two-dimensional peptide mapping. Each analytical method proved able to identify specific differences between originator and biosimilar. High-resolution mass spectrometry was used to characterize the glycan pattern. It was shown that a trastuzumab biosimilar did not have the G0:G0F sugar modification identified in the originator. The application of affinity chromatography to rituximab showed that originator and biosimilar interacted differently with the immobilized Fc receptor. Furthermore, 2D-HPLC peptide mapping demonstrated the influence of orthogonality of separation dimensions, leading to differentiation of a rituximab originator and biosimilar.
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Antineoplásicos Imunológicos , Medicamentos Biossimilares , Anticorpos Monoclonais/química , Medicamentos Biossimilares/química , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , RituximabRESUMO
Complete characterization and quantification of monoclonal antibodies often rely on enzymatic digestion with trypsin. In order to accelerate and automate this frequently performed sample preparation step, immobilized enzyme reactors (IMER) compatible with standard HPLC systems were used. This allows an automated online approach in all analytical laboratories. We were able to demonstrate that the required digestion time for the model monoclonal antibody rituximab could be reduced to 20 min. Nevertheless, a previous denaturation of the protein is required, which also needs 20 min. Recoveries were determined at various concentrations and were 100% ± 1% at 100 ng on column, 96% ± 7% at 250 ng on column and 98% ± 2% at 450 ng on column. Despite these good recoveries, complete digestion was not achieved, resulting in a poorer limit of quantification. This is 50 ng on column under optimized IMER conditions, whereas an offline digest on the same system achieved 0.3 ng on column. Furthermore, our work revealed that TRIS buffers, when used with an IMER system, led to alteration of the peptides and induced modifications in the peptides. Therefore, the addition of TRIS should be avoided when working at elevated temperatures of about 60 °C. Nevertheless, our results have shown that the recovery is not significantly influenced whether TRIS is used or not (recovery: 96 ± 7% with TRIS vs. 100 ± 9% without TRIS).
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Anticorpos Monoclonais/análise , Reatores Biológicos , Enzimas Imobilizadas/química , Anticorpos Monoclonais/química , Automação , Desnaturação Proteica , Rituximab/análise , Rituximab/química , Tripsina/químicaRESUMO
The plant hormone abscisic acid (ABA) regulates many processes, including response to drought, seed dormancy and abscission of leaves and fruits. For maintenance of ABA homeostasis, catabolism of ABA by 8'-hydroxylation and subsequent cyclisation to phaseic acid (PA) is crucial. However, detection of ABA 8'-hydroxylation activity is tedious. We present a simple and rapid method for detection of ABA 8'-hydroxylase activity by cloning cDNAs of interest and expressing the respective protein in yeast. Upon addition of ABA, PA is formed and subsequently quantified in the yeast cell culture supernatant by heart cutting 2D-HPLC or GC-MS.
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Sistema Enzimático do Citocromo P-450/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Proteínas de Plantas/metabolismo , Plantas/enzimologia , Saccharomyces cerevisiae/enzimologia , CiclizaçãoRESUMO
INTRODUCTION: The root of Angelica dahurica is a traditional Chinese medicine that used for the treatment of headache, toothache, abscess, furunculosis and acne. Coumarins were the major bioactive constituents of A. dahurica, hence it is worthwhile developing a method to simultaneously characterise them, especially those in trace amounts. OBJECTIVE: To develop an efficient method for the simultaneous characterisation of coumarins in A. dahurica. METHODS: A method using off-line two-dimensional high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (off-line 2D-HPLC-ESI/MS(n) ) was developed. RESULTS: In total 50 coumarins, including 32 linear furanocoumarins, 16 bifuranocoumarins and two non-furanocoumarins, were identified from the roots of A. dahurica. The possible MS fragmentations of these coumarins are also proposed. CONCLUSION: The method described here allows rapid and convenient identification of the coumarins in A. dahurica, and may be applied to other herbal medicines containing linear furanocoumarins.
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Angelica/química , Cromatografia Líquida de Alta Pressão/métodos , Cumarínicos/isolamento & purificação , Medicamentos de Ervas Chinesas/química , Furocumarinas/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , Cumarínicos/química , Medicamentos de Ervas Chinesas/isolamento & purificação , Furocumarinas/química , Estrutura Molecular , Raízes de Plantas/química , Plantas Medicinais , Espectrofotometria Ultravioleta/métodosRESUMO
AICAR (5-amino-4-imidazolecarboxyamide ribonucleoside), as a metabolic modulator, is classified in the S4 category by the World Anti-Doping Agency (WADA). Carbon Isotope Ratio Mass Spectrometry (CIR) is the mainstream method for distinguishing the endogenous and exogenous sources of AICAR in urine due to the significant individual difference in the concentration. The purpose of this study is to establish a gas chromatography combustion Isotope Ratio Mass Spectrometry (GC/C/IRMS) method for AICAR based on efficient two-dimensional liquid chromatography (2D-HPLC) separation. METHOD: In this study, an automated 2D-HPLC separation technique was used to separate and purify AICAR and endogenous reference substances in urine samples. Then, AICAR was derivatized with 3-TMS as the main derivative product, while the endogenous reference compounds remained in their original form. Subsequently, the developed GC/C/IRMS method was utilized for the detection of the target and reference substances. Followed, we evaluated the applicability of this method using urine samples from two Asian males administered a low dose of AICAR (3 grams). RESULTS: The advantages of this study include: 1) reduced sample pretreatment time: the established 2D-HPLC separation method can separate the target and endogenous reference substances in one step; 2) low interference: the isotope chromatograms have low background interference, and the separation of endogenous reference substances is purer; 3) more accurate result calculations: this method only requires derivatization and result correction for AICAR, with the endogenous reference substances measured in their original form, reducing biases from corrections of multiple substances. The detection method performed well, with a concentriton limit of 2500 ng/mL, meeting the needs of routine detection concentrations. The CIR results from volunteer samples indicated that samples collected within 16 hours post-administration exceeded the threshold set in the literature. CONCLUSION: This study successfully established a 2D-HPLC-GC/IRMS method that integrates CIR as the most stable indicator for distinguishing the internal and external sources of AICAR. After administering a low dose of AICAR to the Asian population, exogenous drug characteristics were manifested within 16 hours. This observation, when compared to the 40-hour detection window cited in the literature, suggests that the length of the detection window is positively correlated with the dosage of the test drug.
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Glycans mediate various biological processes through carbohydrate-protein interactions, and glycan microarrays have become indispensable tools for understanding these mechanisms. However, advances in functional glycomics are hindered by the absence of convenient and universal methods for obtaining natural glycan libraries with diverse structures from glycoconjugates. To address this challenge, we have developed an integrative approach that enables one-pot release and simultaneously capture, separation, structural characterization, and functional analysis of N/O-glycans. Using this approach, glycoconjugates are incubated with a pyrazolone-type heterobifunctional tag-ANPMP to obtain glycan-2ANPMP conjugates, which are then converted to glycan-AEPMP conjugates. We prepared a tagged glycan library from porcine gastric mucin, soy protein, human milk oligosaccharides, etc. Following derivatization by N-acetylation and permethylation, glycans were subjected to detailed structural characterization by ESI-MSn analysis, which revealed >83 highly pure glycan-AEPMPs containing various natural glycan epitopes. A shotgun microarray is constructed to study the fine details of glycan-bindings by proteins and antisera.
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Proteínas , Pirazolonas , Animais , Humanos , Suínos , Glicoconjugados , Polissacarídeos/química , Glicômica/métodosRESUMO
Oxylipins are important low abundant signaling molecules in living organisms. In platelets they play a primary role in platelet activation and aggregation in the course of thrombotic events. In vivo, they are enzymatically synthesized by cyclooxygenases, lipoxygenases, or cytochrome P450 isoenzmes, resulting in diverse polyunsaturated fatty acid (FA) metabolites including hydroxy-, epoxy-, oxo-FAs, and endoperoxides with pro-thrombotic or anti-thrombotic effects. In a recent study, it was reported that hemin induces platelet death which was accompanied by enhanced reactive oxygen species (ROS) production (measured by flow cytometry) and lipid peroxidation (as determined by proxy using flow cytometry with BODIPY-C11 as sensor). Lipidomic studies further indicated significant changes of the platelet lipidome upon ex vivo hemin treatment, amongst others oxylipins were increased. The effect could be (at least partly) reversed by riociguat/diethylamine NONOate diethylammonium salt (DEA/NO) which modulates the soluble guanylate cyclase(sGC)-cGMP-cGMP-dependent protein kinase I(cGKI) signaling axis. In the original work, oxylipins were measured by a non-enantioselective UHPLC-tandem-MS assay which may not give the full picture whether oxylipin elevation is due to ROS or by enzymatic processes. We present here the study of the stereochemical disposition of hemin-induced platelet lipidome alterations using Chiralpak IA-U column with amylose tris(3,5-dimethylphenylcarbamate) chiral selector immobilized on 1.6⯵m silica particles. It was found that the major platelet oxylipins 12-HETE, 12-HEPE and 14-HDoHE (from 12-LOX) and 12-HHT (from COX-1) were present in S-configuration indicating their enzymatic formation. On the other hand, both R and S enantiomers of 9- and 13-HODE, 11- and 15-HETE were detected, possibly due to enzyme promiscuity rather than non-specific oxidation (by ROS or autoxidation), as confirmed by multi-loop based two-dimensional LC-MS using selective comprehensive mode with achiral RPLC in the 1st dimension and chiral LC in the 2nd using a multiple heart-cutting interface. For 12-HETrE, a peak at the retention time of the R-enantiomer was ruled out as isobaric interference by 2D-LC-MS. In particular, arachidonic acid derivates 12(S)-HHT, 11(R)-HETE and 15(S)-HETE were found to be sensitive to hemin and cGMP modulation.
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Plaquetas , GMP Cíclico , Hemina , Oxilipinas , Espectrometria de Massas em Tandem , Oxilipinas/farmacologia , Oxilipinas/química , Espectrometria de Massas em Tandem/métodos , Estereoisomerismo , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , GMP Cíclico/metabolismo , Humanos , Hemina/metabolismo , Hemina/química , Cromatografia Líquida/métodos , Espécies Reativas de Oxigênio/metabolismo , Lipidômica/métodos , Peroxidação de Lipídeos/efeitos dos fármacosRESUMO
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 (10â¶30â¶60, v/v/v, pH 4.7) as mobile phase A and methanol-acetonitrile-4.2 g/L citric acid monohydrate solution (12â¶68â¶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 (95â¶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 (95â¶5, v/v) and 10 mmol/L ammonium formate aqueous solution containing 0.1% formic acid-acetonitrile (5â¶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.
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Anfotericina B , Contaminação de Medicamentos , Espectrometria de Massas , Cromatografia Líquida de Alta Pressão/métodos , Anfotericina B/análise , Anfotericina B/química , Espectrometria de Massas/métodosRESUMO
Two multidimensional HPLC separations of an Australian red wine are presented, >70% of the available separation space was used. A porous graphitic carbon (PGC) stationary phase was used as the first dimension in both separations with both RP core-shell and hydrophilic interaction chromatography fully porous columns used separately in the second dimension. To overcome peak analysis problems caused by signal noise and low detection limits, the data were pre-processed with penalised least-squares smoothing. The PGC × RP combination separated 85 peaks with a spreading angle of 71° and the PGC × hydrophilic interaction chromatography separated 207 peaks with a spreading angle of 80°. Both 2D-HPLC steps were completed in 76 min using a comprehensive stop-and-go approach. A smoothing step was added to peak-picking processes and was able to greatly reduce the number of false peaks present due to noise in the chromatograms. The required thresholds were not able to ignore the noise because of the small magnitude of the peaks; 1874 peaks were located in the non-smoothed PGC × RP separation that reduced to 227 peaks after smoothing was included.
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Algoritmos , Cromatografia Líquida de Alta Pressão/métodos , Vinho/análise , Carbono/químicaRESUMO
Gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) can be used to detect the synthetic forms of endogenous anabolic androgenic steroids (EAAS) by comparing the 13C/12C ratios of the endogenous reference compound to that of the target compound. Isolation and enrichment of the target compound from urinary matrices is an essential prerequisite for the GC/C/IRMS confirmation procedure in doping control analysis. Boldenone (Bo) is a natural anabolic androgenic steroid (AAS) and a derivative of testosterone. The GC/C/IRMS confirmation procedure for Bo and its main metabolite 5ß-androst-1-en-17ß-ol-3-one (BoM) is extremely complicated due to the low concentrations and the enormously complex matrices in urine. The present study demonstrated a sample purification procedure for GC/C/IRMS by using online 2D-HPLC to purify Bo and BoM in urine samples. Bo and BoM with concentrations as low as 2 ng/mL were isolated and enriched with superior purity and selectivity. The validity of the method was verified with the technical document issued by the world anti-doping agency. The online 2D-HPLC purification procedure featured high selectivity for the analytes and no isotopic fractionation in the collection process. The present method can be used as a routine method allowing doping control laboratories to perform Boldenone confirmation.
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Esteróides Androgênicos Anabolizantes , Testosterona , Cromatografia Gasosa-Espectrometria de Massas , IsótoposRESUMO
Oligonucleotides are commonly analysed using one dimensional chromatography (1D-LC) to resolve and characterise manufacturing impurities, structural isomers and (in respect to emerging oligonucleotide therapeutics) drug substance and drug product. Due to low selectivity and co-elution of closely related oligonucleotides using 1D-LC, analyte resolution is challenged. This leads to the requirement for improved analytical methods. Multidimensional chromatography has demonstrated utility in a range of applications as it increases peak capacity using orthogonal separations, however there are limited studies demonstrating the 2D-LC analysis of closely related oligonucleotides. In this study we optimised OGN size and sequence based separations using a variety of 1D-LC methods and coupled these orthogonal modes of chromatography within a 2D-LC workflow. Theoretical 2D-LC workflows were evaluated for optimal orthogonality using the minimum convex hull metric. The most orthogonal workflow identified in this study was ion-pair reversed phase using tributylammonium acetate (IP-RP-TBuAA) coupled with strong anion exchange in conjunction with sodium perchlorate (SAX-NaClO4) at high mobile phase pH. We developed a heart-cut (IP-RP-TBuAA)-(SAX-NaClO4) 2D-LC method for analysis of closely related size and sequence variant OGNs and OGN manufacturing impurities. The 2D-LC method resulted in an increased orthogonality and a reduction in co-elution (or close elution). Application of a UV based reference mapping strategy in conjunction with the 2D-LC method demonstrated a reduction in analytical complexity by reducing the reliance on mass based detection methods.
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Cromatografia de Fase Reversa , Oligonucleotídeos , Oligonucleotídeos/análise , Cromatografia Líquida/métodos , Cromatografia de Fase Reversa/métodos , Cromatografia por Troca Iônica/métodos , ÂnionsRESUMO
d-Amino acids, rare enantiomers of amino acids, have been identified as biomarkers and therapeutic options for COVID-19. Methods for monitoring recovery are necessary for managing COVID-19. On the other hand, the presence of SARS-CoV2 virus in the blood is associated with worse outcomes. We investigated the potential of d-amino acids for assessing recovery from severe COVID-19. In patients with severe COVID-19 requiring artificial ventilation, the blood levels of d-amino acids, including d-alanine, d-proline, d-serine, and d-asparagine, which were lower than the normal range before treatment, quickly and transiently increased and surpassed the upper limit of the normal range. This increase preceded the recovery of respiratory function, as indicated by ventilation weaning. The increase in blood d-amino acid levels was associated with the disappearance of the virus in the blood, but not with inflammatory manifestations or blood cytokine levels. d-Amino acids are sensitive biomarkers that reflect the recovery of the clinical course and blood viral load. Dynamic changes in blood d-amino acid levels are key indicators of clinical course.
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Glycomics analysis has been undermined by the lack of structurally defined individual glycans as model compounds. However, it is challenging to prepare individual glycans from natural resources, mainly due to separation difficulties caused by highly diverse structure, complicated mixture form and chromophore-free property of naturally-existing glycans. In this study, we report a simple, universal and low-cost glycan separation strategy, glycoselection, which allows preparation of individual reducing glycans from their mixtures through reversible chromogenic derivatization by hydrazide chemistry in combination with two-dimensional high-performance liquid chromatography (2D-HPLC). Investigations on reaction conditions using lactose and maltodextrin as model glycans showed the feasibility of reversible hydrazide labeling and one-pot hydrazone conversion under mild conditions, the good stability of hydrazone-form derivatives of glycans in solution and the difference among seven selected hydrazine-carrying chromogenic reagents in product yields during glycan labeling and post-column detagging. The 2D-HPLC separation conditions were established on maltodextrin, from which fourteen highly-purified individual reducing oligo-glucans were ultimately obtained. Using this strategy, we also successfully prepared and identified eleven individual neutral reducing N-glycans from chicken ovalbumin and thirteen individual neutral reducing oligosaccharides and eight individual sialylated ones from human milk, demonstrating its good applicability to different types of reducing glycans as well as biological samples. Given the compatibility of individual reducing glycans with almost all of glycan derivatization protocols and analytical techniques of glycans and the potential of the method for larger scale application, this work provides a universal approach to compound-specific analysis of natural glycans and has great significance for glycomics studies.
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Glicômica , Hidrazonas , Humanos , Cromatografia Líquida de Alta Pressão , Glicômica/métodos , Polissacarídeos/química , HidrazinasRESUMO
BACKGROUND: Endogenous molecules that provide an unbiased and a precise evaluation of kidney function are still necessary. We explored the potential of clearance of d-serine, a rare enantiomer of serine and a biomarker of kidney function, as a measure of glomerular filtration rate (GFR). METHODS: This was a cross-sectional observational study of 200 living kidney transplant donors and recipients enrolled between July 2019 and December 2020 in a single Japanese center, for whom GFR was measured by clearance of inulin (C-in). Clearance of d-serine (C-dSer) was calculated based on blood and urine levels of d-serine, as measured by two-dimensional high-performance liquid chromatography. Analytical performance was assessed by calculating biases. Utilizing data from 129 participants, we developed equations for C-in based on C-dSer and C-cre using a linear regression model, and the performance was validated in 68 participants. FINDINGS: The means of C-in and C-dSer were 66.7 and 55.7 mL/min/1.73 m2 of body surface area, respectively, in the entire cohort. C-dSer underestimated C-in with a proportional bias of 22.0% (95% confidence interval, 14.2-29.8%) and a constant bias of -1.24 (-5.78-3.31), whereas the proportional bias was minor to that of C-cre (34.6% [31.1-38.2%] and 2.47 (-1.18-6.13) for proportional and constant bias, respectively). Combination of C-dSer and C-cre measured C-in with an equation of 0.391 × C-dSer + 0.418 × C-cre + 3.852, which reduced the proportional bias (6.5% [-0.2-13.1%] and -4.30 [-8.87-0.28] for proportional and constant bias, respectively). In the validation dataset, this equation performed well with median absolute residual of 3.5 [2.3-4.8], and high ratio of agreement (ratios of 30% and 15% different from C-in [P30 and P15] of 98.5 [91.4-100] and 89.7 [80.0-95.2], respectively). INTERPRETATION: The smaller proportional bias compared to that of C-cre is an advantage of C-dSer as a measure of C-in. Combinational measurement of d-serine and creatinine, two endogenous molecules, has the potential to serve as a measure of GFR with precision and minor biases and can support important clinical decisions. FUNDING: Japan Society for the Promotion of Science (JSPS, grant number 17H04188), Japan Agency of Medical Research and Development (AMED, JP20gm5010001), Osaka Kidney Bank (OKF19-0010), Shiseido Co., Ltd and KAGAMI Inc.
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19-Norandrosterone (19-NA) is the main metabolite of nandrolone and/or its precursors, which can be found naturally in human urine in trace amount. Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) confirmation procedure can be used to identify a potential exogenous origin of 19-NA in urine sample. Sample purification for GC-C-IRMS analysis is crucial to the whole confirmation procedure because the concentration of 19-NA in the urine to be tested is very low. Online two-dimensional high-performance liquid chromatography (2D-HPLC) clean-up procedure with high separation capacity is used to isolate and enrich 19-NA as a sample pretreatment process. Linearity, lowest detectable concentration, uncertainty, and selectivity of the method are validated according to the World Anti-doping Agency's (WADA) requirement. Isotope fractionation effect was not observed during the 2D-HPLC purification process. The validated method provides a high efficient and convenient confirmation procedure to determine the origin of 19-NA.
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Cromatografia Líquida de Alta Pressão/métodos , Estranos/urina , Cromatografia Gasosa-Espectrometria de Massas/métodos , Cromatografia Líquida de Alta Pressão/instrumentação , Desenho de Equipamento , Estranos/isolamento & purificação , Humanos , Limite de Detecção , Detecção do Abuso de Substâncias/métodosRESUMO
d-Amino acids, enantiomers of l-amino acids, are increasingly recognized as physiologically active molecules as well as potential biomarkers for diseases. d-Amino acid oxidase (DAO) catalyzes the oxidative deamination of d-amino acids and is present in a wide variety of organisms from yeasts to humans. Previous studies indicated that LEA rats lacked DAO activity, and levels of d-Ser and d-Ala were markedly increased in their tissues, suggesting a mutated locus responsible for the lack of Dao activity (ldao) existed in the LEA genome. Sequence analysis identified deletion breakpoints located in intron 4-5 of the Dao gene and intron 1-2 of the Svop gene, resulting in a 54.1-kb deletion which encompassed exons 5-12 of the Dao gene and exons 2-16 of the Svop gene. We developed a novel congenic rat strain, F344-Daoldao, harboring the Daoldao mutation from LEA rats delivered onto the F344 genetic background. Compared to the parental F344 strain, in F344-Daoldao rats d-Ala was markedly increased in both cerebrum and cerebellum, while d-Ser content was increased in cerebellum but not cerebrum. d-Ala, d-Ser, d-Pro and d-Leu levels were also elevated in F344-Daoldao plasma. F344-Daoldao rats represent a novel model system that will aid in elucidating the physiological functions of d-amino acids in vivo. (203 words).
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D-Aminoácido Oxidase/genética , D-Aminoácido Oxidase/metabolismo , Mutação , Aminoácidos/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Rim , Masculino , Ratos , Ratos Endogâmicos F344 , Análise de Sequência de DNA , TranscriptomaRESUMO
Flos Lonicerae Japonicae(Jinyinhua) possesses clearing heat and detoxification activity, and has been used as a traditional Chinese medicine to treat influenza for many years. Due to the complex chemical composition and diverse content of Jinyinhua, especially the many trace ingredients, the effective components are unknown. In this study, an improved two-dimensional high performance liquid chromatography-ultrafiltration combined with electrospray ionization-time-of-flight/mass spectroscopy (ESI-TOF/MS) approach was designed and used for the enrichment, screening and characterization of minor neuraminidase inhibitors in Jinyinhua. In the first dimension, semi-prep-HPLC was employed for the preliminary separation of different polarity components and enrichment of low content components from Jinyinhua extract. In the second dimension, hydrophilic interaction liquid chromatography and reverse phase-HPLC were used to separate the different polar fractions, respectively. The fractions then underwent ultrafiltration and ESI-TOF/MS for the comprehensive screening and characterization of potential neuraminidase inhibitors. As a result, a total of 44 compounds were found to have neuraminidase inhibitory activity, and 22 of these compounds were preliminarily identified by accurate molecular weight and UV absorption data compared with standards and references. The activity of 16 of these compounds was verified by the neuraminidase inhibition assay. This study provides support for the rapid screening of minor neuraminidase inhibitors from complex natural medicines.
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Inibidores Enzimáticos/química , Neuraminidase/antagonistas & inibidores , Extratos Vegetais/química , Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas/química , Lonicera/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Ultrafiltração/métodosRESUMO
To study the structure of ß-glucans, we developed a separation method and molecular library of ß-glucan oligosaccharides. The oligosaccharides were prepared by partial acid hydrolysis from laminarin, which is a ß-glucan of Laminaria digitata. They were labeled with the 2-aminopyridine fluorophore and separated to homogeneity by size-fractionation and reversed phase high-performance liquid chromatography (HPLC). Branching structures of all isomeric oligosaccharides from trimers to pentamers were determined, and a two-dimensional (2D)-HPLC map of the ß-glucan oligosaccharides was made based on the data. Next, structural analysis of the longer ß-glucan oligosaccharide was performed using the 2D-HPLC map. A branched decamer oligosaccharide was isolated from the ß-glucan and cleaved to smaller oligosaccharides by partial acid hydrolysis. The structure of the longer oligosaccharide was successfully elucidated from the fragment structures determined by the 2D-HPLC map. The molecular library and the 2D-HPLC map described in this study will be useful for the structural analysis of ß-glucans.
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Monoclonal antibodies are mainly produced by mammalian cell culture, which due to its complexity, results in a wide range of product variants/isoforms. With the growing implementation of Quality by Design (QbD) and Process Analytical Technology (PAT) in drug manufacturing, monitoring and controlling quality attributes within a predefined range during manufacturing may provide added consistency to product quality. To implement these concepts, more robust analytical tools could reduce the time needed for monitoring quality attributes during upstream processing. The formation of protein aggregates is one such quality attribute that can lead to safety and efficacy issues in the final drug product. Described in this study is a fully automated two-dimensional high performance liquid chromatography (2D-HPLC) method for characterizing protein aggregation of crude in-process bioreactor samples. It combines protein A purification and separation by size exclusion into a single analytical module that has the potential to be employed at-line within a bioprocessing system. This method utilizes a novel in-line fraction collection device allowing for the collection of up to twelve fractions from a single sample or peak which facilitates the subsequent linked analysis of multiple protein peaks of interest in one chromatography module.
Assuntos
Anticorpos Monoclonais , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Cromatografia em Gel/instrumentação , Cromatografia em Gel/métodos , Desenho de Equipamento , Agregados Proteicos , Proteína Estafilocócica A/análise , Proteína Estafilocócica A/química , Proteína Estafilocócica A/isolamento & purificaçãoRESUMO
In-silico optimisation of a two-dimensional high performance liquid chromatography (2D-HPLC) separation protocol has been developed for the interrogation of methamphetamine samples including model, real world seizure, and laboratory synthesised samples. The protocol used Drylab(®) software to rapidly identify the optimum separation conditions from a library of chromatography columns. The optimum separation space was provided by the Phenomonex Kinetex PFP column (first dimension) and an Agilent Poroshell 120 EC-C18 column (second dimension). To facilitate a rapid 2D-HPLC analysis the particle packed C18 column was replaced with a Phenomenex Onyx Monolithic C18 withought sacrificing separation performance. The Drylab(®) optimised and experimental separations matched very closely, highlighting the robust nature of HPLC simulations. The chemical information gained from an intermediate methamphetamine sample was significant and complimented that generated from a pure seizure sample. The influence of the two-dimensional separation on the analytical figures of merit was also investigated. The limits of detection for key analytes in the second dimension determined for methamphetamine (4.59×10(-4)M), pseudoephedrine (4.03×10(-4)M), caffeine (5.16×10(-4)M), aspirin (9.32×10(-4)M), paracetamol (5.93×10(-4)M) and procaine (2.02×10(-3)M).