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1.
Talanta ; 236: 122836, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34635226

RESUMO

Bispecific antibodies (bsAbs) are considered as an important class of biopharmaceutical drugs, with about 160 products in clinical trials. From an analytical point of view, the correct chain-association is one of the most critical challenge to monitor during bsAbs development and production. In the present study, a full analytical workflow has been developed based on the use of various chromatographic modes: size exclusion chromatography (SEC), ion exchange chromatography (IEX), reversed phase liquid chromatography (RPLC), and hydrophilic interaction chromatography (HILIC), all combined with high resolution mass spectrometry (MS). This analytical strategy was applied to Hemlibra® (emicizumab), which is certainly the most successful commercial bsAb to date. Using this strategy, it was possible to monitor the presence of mispaired bsAb species and detect and identify additional post-translational modifications (PTMs).


Assuntos
Anticorpos Biespecíficos , Anticorpos Monoclonais , Cromatografia em Gel , Cromatografia de Fase Reversa , Espectrometria de Massas
2.
Talanta ; 236: 122889, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34635268

RESUMO

Phenolic compounds are an interesting class of natural products because of their proposed contribution to health benefits of foods and beverages and as a bio-source of organic (aromatic) building blocks. Phenolic extracts from natural products are often highly complex and contain compounds covering a broad range in molecular properties. While many 1D-LC and mass spectrometric approaches have been proposed for the analysis of phenolics, this complexity inevitably leads to challenging identification and purification. New insights into the composition of phenolic extracts can be obtained through online comprehensive two-dimensional liquid chromatography (LC × LC) coupled to photodiode array and mass spectrometric detection. However, several practical hurdles must be overcome to achieve high peak capacities and to obtain robust methods with this technique. In many LC × LC configurations, refocusing of analytes at the head of the 2D column is hindered by the high eluotropic strength of the solvent transferred from the 1D to the 2D, leading to peak breakthrough or broadening. LC × LC combinations whereby a purely aqueous mobile phase is used in the 1D and RPLC is used in the 2D are unaffected by these phenomena, leading to more robust methods. In this contribution, the combination of temperature-responsive liquid chromatography (TRLC) with RPLC is used for the first time for the analysis of phenolic extracts of natural origin to illustrate the potential of this alternative combination for natural product analyses. The possibilities of the combination are investigated through analysis of wine extracts by TRLC × RPLC-DAD and TRLC × RPLC-ESI-MS.


Assuntos
Cromatografia de Fase Reversa , Vinho , Cromatografia Líquida , Fenóis/análise , Temperatura
3.
J Pharm Biomed Anal ; 207: 114420, 2022 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-34662781

RESUMO

Retention behavior of two structural isomeric pentacyclic triterpenic acids, maslinic acid and corosolic acid, was investigated by reverse phase high performance liquid chromatography (HPLC) with hydroxypropyl-ß-cyclodextrin (HP-ß-CD) as mobile phase additive. Inclusion complexation of maslinic acid, corosolic acid with hydroxypropyl-ß-cyclodextrin was evaluated under different concentration of hydroxypropyl-ß-cyclodextrin. Apparent formation constant (Km) between methanol and hydroxypropyl-ß-cyclodextrin was determined to be 13.82 L mol-1 under 25 °C using UV-spectrophotometry. Two retention models were employed individually for evaluation of inclusion complexation between the two pentacyclic triterpenic acids and hydroxypropyl-ß-cyclodextrin. It was found that a higher apparent formation constant (Kf) for corosolic acid and hydroxypropyl-ß-cyclodextrin was obtained, 19115 L mol-1, indicating that a greater affinity of hydroxypropyl-ß-cyclodextrin with corosolic acid was produced compared with that of maslinic acid, 11775 L mol-1, in the selected mobile phase, and stoichiometric ratio for both of inclusion complex was found to be 1:1. Thermodynamic analysis showed that a negative standard enthalpy change (ΔH) and an entropy change (ΔS*) for analyte transfer were obtained, where ΔH of maslinic acid and corosolic acid was found to be -10.188 kJ mol-1 and -10.650 kJ mol-1, ΔS* of two compounds was -2.092 and -2.180, respectively, indicating that transfer of structural isomers from mobile phase to stationary phase was enthalpically driven. Meanwhile, positive values were obtained for standard enthalpy change and standard entropy change, 136 kJ mol-1 and 274 kJ mol-1 and 536 J mol-1 K-1and 1004 J mol-1 K-1, for inclusion complexation between maslinic acid, corosolic acid and hydroxypropyl-ß-cyclodextrin, while negative values were obtained for Gibbs free energy during formation of inclusion complex, -160 kJ mol-1 and -299 kJ mol-1, indicating a spontaneous inclusion reaction happened.


Assuntos
beta-Ciclodextrinas , 2-Hidroxipropil-beta-Ciclodextrina , Cromatografia Líquida , Cromatografia de Fase Reversa , Triterpenos Pentacíclicos , Termodinâmica
4.
Zhongguo Zhong Yao Za Zhi ; 46(19): 5020-5026, 2021 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-34738397

RESUMO

The chemical constituents in the ethyl acetate extract of Corydalis tomentella was isolated and purified with normal and reversed phase silica gel column chromatography, Sephadex LH-20, MCI, and semi-preparative HPLC. The compound structures were identified based on spectroscopic experiments and reported papers. Finally, eighteen compounds(1-18) were obtained from C. tomentella, including 17 alkaloids and 1 terpenoid. Among them, compound 1(tomentellaine A) was a novel alkaloid. Compounds 2-5, 7-14, and 16-18 were isolated from this plant for the first time.


Assuntos
Alcaloides , Corydalis , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Extratos Vegetais
5.
Anal Chim Acta ; 1184: 339023, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34625255

RESUMO

Total triacylglycerol (TAG) level is a key clinical marker of metabolic and cardiovascular diseases. However, the roles of individual TAGs have not been thoroughly explored in part due to their extreme structural complexity. We present a targeted mass spectrometry-based method combining multiple reaction monitoring (MRM) and multiple stage mass spectrometry (MS3) for the comprehensive qualitative and semiquantitative profiling of TAGs. This method referred as TriP-MS3 - triacylglycerol profiling using MS3 - screens for more than 6,700 TAG species in a fully automated fashion. TriP-MS3 demonstrated excellent reproducibility (median interday CV âˆ¼ 0.15) and linearity (median R2 = 0.978) and detected 285 individual TAG species in human plasma. The semiquantitative accuracy of the method was validated by comparison with a state-of-the-art reverse phase liquid chromatography (RPLC)-MS (R2 = 0.83), which is the most commonly used approach for TAGs profiling. Finally, we demonstrate the utility and the versatility of the method by characterizing the effects of a fatty acid desaturase inhibitor on TAG profiles in vitro and by profiling TAGs in Caenorhabditis elegans.


Assuntos
Cromatografia de Fase Reversa , Plasma , Humanos , Espectrometria de Massas , Reprodutibilidade dos Testes , Triglicerídeos
6.
Se Pu ; 39(11): 1230-1238, 2021 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-34677018

RESUMO

The n-octanol/water partition coefficient (log P) is an important parameter to characterize the overall hydrophobicity of organic compounds. Reversed-phase liquid chromatography (RPLC) has been recommended as an effective method for the indirect determination of log P by the Organization for Economic Cooperation and Development (OECD). Using RPLC, most studies focus on the determination of log P or the apparent n-octanol/water partition coefficient (log D) of neutral compounds and weakly ionized compounds. However, the experimental log P or log D values of strongly ionized compounds have rarely been reported. In our previous work, the experimental log D of strongly ionized compounds could be determined well by ion-pair reversed-phase liquid chromatography (IP-RPLC) on an octadecyl-poly(vinyl alcohol) column using the log D-log kw-IP model established by different types of model compounds. However, the universality of this strategy for different chromatographic columns has yet to be verified. In this study, the retention behavior of neutral compounds, phenolic acids, carboxylic acids, sulfonic acids, and some amphoteric compounds was systematically investigated on a silica-based C18 column (150 mm×4.6 mm, 5 µm) via ion-suppressed RPLC (IS-RPLC) and IP-RPLC, respectively. In the IS-RPLC mode, methanol and 20 mmol/L ammonium dihydrogen phosphate buffer (pH 7.0) were used as the mobile phase to perform isocratic elution at different methanol ratios. The log kw values of the test compounds were obtained using the linear solvent strength (LSS) model. Neutral compounds, weakly ionized phenolic acids and benzene carboxylic acids were then used as model compounds to establish the log D-log kw-IS model. The quantitative structure-retention relationship (QSRR) model, including structure-related descriptors like the charge (ne) and Abraham solvation parameters (A and B), exhibited much better correlation than the unary linear regression model between log D and log kw-IS. The log D7.0 (log D under pH 7.0) values of 19 ionized compounds were then determined by the model; the determined compounds were used as model compounds and validation compounds in IP-RPLC. In the IP-RPLC mode, besides methanol and ammonium dihydrogen phosphate buffer, the mobile phase also contained tetrabutylammonium bromide, as an ion-pair reagent. The retention behaviors of all tested compounds conformed well with the LSS model even under IP-RPLC, with a log k-φ linear correlation coefficient (R 2) greater than 0.99. The log D-log kw-IP model was then established using 62 compounds as a mixed model set, including neutral, weakly ionized, and strongly ionized compounds. Similarly, by introducing ne, A, and B, the log D-log kw-IP model showed good linearity, with R 2 greater than 0.94. Comparing the log D-log kw-IP model established on the silica-based C18 column in this work with that established on a poly(vinyl alcohol)-based C18 column in our previous work, ne, A, and B contributed more to the model in this work, indicating there was a greater secondary effect on the silica-based column. To confirm the reliability of the log D-log kw-IP model, three different types of acidic compounds were used as validation compounds. The predicted log D of the three ionic compounds was very similar to that determined by the shake-flask method (SFM)/slow stirring method (SSM) or IS-RPLC method in this work, confirming the reliability of the model. Based on the above results, the log D7.0 values of eight strongly ionized compounds were predicted by IP-RPLC. The findings suggested that IP-RPLC is a promising method to predict the experimental log D of strongly ionized compounds, and that the conventional silica-based C18 column offers more flexible options in log D determination.


Assuntos
Cromatografia de Fase Reversa , Água , 1-Octanol , Cromatografia Líquida de Alta Pressão , Reprodutibilidade dos Testes , Dióxido de Silício
7.
J Phys Chem B ; 125(40): 11320-11336, 2021 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-34610741

RESUMO

The interfacial phenomena behind analyte separation in a reversed-phase liquid chromatography column take place nearly exclusively inside the silica mesopores. Their cylindrical geometry can be expected to shape the properties of the chromatographic interface with consequences for the analyte density distribution and diffusivity. To investigate this topic through molecular dynamics simulations, we introduce a cylindrical pore inside a slit pore configuration, where the inner curved and outer planar silica surface bear the same bonded phase. The present model replicates an average-sized (9 nm) mesopore in an endcapped C18 column equilibrated with a mobile phase of 70/30 (v/v) water/acetonitrile. Simulations performed for ethylbenzene and acetophenone show that the surface curvature shifts the bonded phase and analyte density toward the pore center, decreases the solvent density in the bonded-phase region, increases the acetonitrile excess in the interfacial region, and considerably enhances the surface diffusivity of both analytes. Overall, the cylindrical pore provides a more hydrophobic environment than the slit pore. Ethylbenzene density is decidedly increased in the cylindrical pore, whereas acetophenone density is nearly equally distributed between the cylindrical and slit pore. The cylindrical pore geometry thus sharpens the discrimination between the apolar and moderately polar analytes while enhancing the mass transport of both.


Assuntos
Cromatografia de Fase Reversa , Água , Interações Hidrofóbicas e Hidrofílicas , Dióxido de Silício , Solventes
8.
Molecules ; 26(20)2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34684754

RESUMO

High-molecular-weight glutenin subunits (HMW-GS) account for only 10% of total wheat storage proteins, but play an important role in the processing quality of wheat flour. Therefore, identifying HMW-GS alleles associated with good end-use quality provides important information for wheat breeders. To rapidly, accurately and reproducibly identify HMW-GS, we established an optimized reversed-phase ultra-performance liquid chromatography (RP-UPLC) method. Separation parameters were optimized using an ACQUITY UPLC Protein BEH C4 column and stepwise ACN gradient, and the separation patterns and retention times (RTs) of 22 subunits were comparatively analyzed in 16 standard wheat cultivars. All HMW-GS proteins were well separated within about 5.5 min, and all analyses were complete within 12 min. We distinguished the 16 subunits based on RT, although three subunits in 1Bx (1Bx7/1Bx7OE and 1Bx17) and three subunits in 1By (1By8*, 1By9 and 1By15) had overlapping RTs; these were differentiated by SDS-PAGE. To distinguish 1Bx7 and 1Bx7OE, which differ in protein abundance, RP-UPLC was combined with PCR analysis of DNA junction markers. The optimized method was successfully applied to determine HMW-GS alleles in a large collection of bread wheat germplasm (1787 lines). This protocol is an appropriate option for selecting lines harboring favorable HMW-GS alleles in wheat breeding.


Assuntos
Cromatografia de Fase Reversa/métodos , Glutens/genética , Triticum/genética , Alelos , Eletroforese em Gel de Poliacrilamida/métodos , Farinha , Expressão Gênica/genética , Regulação da Expressão Gênica de Plantas/genética , Frequência do Gene/genética , Glutens/análise , Peso Molecular , Melhoramento Vegetal , Subunidades Proteicas/química , Transcriptoma/genética , Triticum/química
9.
Zhongguo Zhong Yao Za Zhi ; 46(17): 4438-4445, 2021 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-34581048

RESUMO

The chemical constituents from the stems and leaves of Clausena excavata were isolated and purified by column chromatography with silica gel, ODS, Sephadex LH-20 and RP-HPLC. The chemical structures of the isolated compounds were identified on the basis of physicochemical properties, spectroscopic analysis, as well as the comparisons with the data reported in literature. Nineteen compounds were isolated from the 90% ethanol extract of the stems and leaves of C. excavata, which were identified as methyl orsellinate(1), syringaresinol(2), lenisin A(3), scopoletin(4), osthenol(5), N-benzoyltyrarnine methyl ether(6), N-p-coumaroyltyramine(7), aurantiamide acetate(8), 1H-indole-3-carboxaldehyde(9), furostifoline(10), clausenalansine E(11), 3-formylcarbazole(12), clausine L(13), clausine E(14), methyl carbazole-3-carboxylate(15), glycosinin(16), murrayafoline A(17), clausine H(18) and 2,7-dihydroxy-3-formyl-1-(3'-methyl-2'-butenyl)carbazole(19). Among these isolated compounds, compounds 1-11 were isolated from C. excavata for the first time, and compounds 1, 2 and 10 were isolated from the genus Clausena for the first time. In addition, this study evaluated the anti-rheumatoid arthritis activities of compounds 1-19 by measuring their anti-proliferative effects on synoviocytes in vitro according to MTS method. Compounds 10-19 displayed remarkable anti-rheumatoid arthritis activities, which exhibited the inhibitory effects on the proliferation of MH7 A synovial fibroblast cells with the IC_(50) values ranging from(27.63±0.18) to(235.67±2.16) µmol·L~(-1).


Assuntos
Clausena , Sinoviócitos , Proliferação de Células , Cromatografia de Fase Reversa , Folhas de Planta
10.
Se Pu ; 39(9): 1021-1029, 2021 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-34486842

RESUMO

Ion-pair reversed-phase liquid chromatography (IP-RPLC) enhances separation by adding ion-pair reagents to the mobile phase, thereby improving the retention of oppositely charged solutes. IP-RPLC is primarily used for the separation and analysis of strongly ionized compounds. In IP-RPLC, researchers often focus more on the influence of the counter-ion type and concentration, buffer salt concentration and pH, and column temperature, on the retention behavior of solutes. However, the effects of the buffer salt type and non-counter ions in ion-pair reagents on the retention behavior of solutes have rarely been investigated. Accordingly, in this work, the effects of buffer salt types and non-counter ions on the retention behavior of strongly ionized compounds were investigated by IP-RPLC using 14 sulfonic acid compounds as model compounds. Experiments were performed using a silica-based C18 column with methanol as the organic modifier. In the first type of experiment, tetrabutylammonium bromide was kept unchanged as the ion-pair reagent in the mobile phase, and ammonium dihydrogen phosphate, ammonium chloride, and ammonium acetate were used as buffer salts, respectively. The retention factor (k) was obtained at different methanol ratios, and linear solvent strength (LSS) models were established to determine the log kw (logarithm of retention factors of solutes when 100% aqueous phases were used as the mobile phase) and S (intercept of the LSS model) values of each solute. All solutes exhibited the highest log kw with the ammonium chloride buffer system; most compounds also exhibited the highest S values with this system, except for 1,5-naphthalenedisulfonic acid, 4-methylbenzenesulfonic acid, 5-amino-2-nanphthalenesulfonic acid, and 4-hydroxybenzenesulfonic acid. However, the chromatographic hydrophobic indices (CHIs, log kw/S) of the solutes with different buffer salts were approximately equal. In the second type of experiment, ammonium dihydrogen phosphate was kept unchanged as the buffer salt in the mobile phase, and tetrabutylammonium bromide, tetrabutylammonium dihydrogen phosphate, tetrabutylammonium hydrogen sulfate, tetrabutylammonium nitrate, and tetrabutylammonium acetate were used as ion-pair reagents, respectively. Almost all solutes exhibited the highest S with the tetrabutylammonium acetate system, indicating that weakly ionized anions (such as acetate ions) in ion-pair reagents will improve the S values of sulfonic acid compounds. Interestingly, the CHIs of the solutes were almost the same for solutes with different non-counter ions. These results suggest that both, the buffer salt types and non-counter ions, influence the log kw and S values of sulfonic acid compounds. Comparison of the retention behavior of solutes with different mobile phases suggested ion-pair mechanisms as well as dynamic ion-exchange mechanisms plays role in the IP-RPLC retention of sulfonic acid compounds. In addition, with all the experimental mobile phases, the apparent n-octanol/water partition coefficient (log D) presented a good linear correlation with log kw, S, and CHI, respectively, by the introduction of structure-related descriptors such as charge (ne), Abraham solvation parameters (A and B), and the polar surface area (PSA). Considering the differences in the log kw and S values obtained with different buffer salts and non-counter ions, the CHIs were relatively stable; therefore, the CHI is more suitable for establishing a quantitative structure-retention relationship (QSRR) model in IP-RPLC, compared to log kw and S.


Assuntos
Cromatografia de Fase Reversa , Cromatografia Líquida de Alta Pressão , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Íons
11.
J Pharm Biomed Anal ; 206: 114358, 2021 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-34534866

RESUMO

The chromatographic and lipophilicity characters of seven cephalosporins of different four classes (cephradine, cefaclor, cefprozil, cefixime, cefotaxime, ceftazidime and cefepime) were examined by salting out thin-layer chromatography (SOTLC). SOTLC using ammonium sulfate salt was employed to predict the lipophilicity of the proposed drugs via their retention behavior. The calculated RM0 values showed liner relationship with the molar concentration of ammonium sulfate in mobile phase in the range of 0.5-2.5 mol/L. Additionally, quantitative structure retention relationship (QSRR) was generated to figure out the relationship between the calculated chromatographic parameters (RM0 and C0) and log P of the studied cephalosporins. Good correlations were found between the chromatographically obtained retention parameters (RM0 and C0) and some molecular descriptors of the examined drugs. Furthermore, an efficient QSAR model was carried out using the calculated chromatographic parameters (RM0 and C0) and log P of the studied cephalosporins to predict minimum inhibitory concentration (MIC) and blood brain barrier (BBB) penetration of the examined drugs. The study was extended to separate and quantify the selected antibiotics in their pure forms and pharmaceutical formulations. Normal phase thin layer chromatographic (NP-TLC) method using a usable developing system of acetone: methanol: water: ammonium hydroxide: glacial acetic acid (90: 10: 18: 3: 2, by volume) was successfully applied to resolve the studied cephalosporins. Linearity was achieved in the range of 0.2-3 µg/mL for most of the studied antibiotics. The developed SOTLC method can be considered as a good start alternative to reversed phase thin layer chromatography (RP-TLC) for prediction of the lipophilic properties of examined cephalosporins. Moreover, the proposed NP-TLC densitometric method can be easily applied for quality control analysis of the chosen drugs and other structurally related components.


Assuntos
Cefalosporinas , Cromatografia de Fase Reversa , Cefepima , Cromatografia em Camada Delgada , Testes de Sensibilidade Microbiana
12.
Molecules ; 26(17)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34500554

RESUMO

Untargeted (NMR) and targeted (RP-HPLC-PDA-ESI-MSn, RP-HPLC-FD) analytical methodologies were used to determine the bioactive components of 19 tea samples, characterized by different production processes (common tea and GABA tea), degrees of fermentation (green and oolong teas), and harvesting season (autumn and spring). The combination of NMR data and a multivariate statistical approach led to a statistical model able to discriminate between GABA and non-GABA teas and green and oolong teas. Targeted analyses showed that green and GABA green teas had similar polyphenol and caffeine contents, but the GABA level was higher in GABA green teas than in regular green tea samples. GABA oolong teas showed lower contents of polyphenols, caffeine, and amino acids, and a higher content of GABA, in comparison with non-GABA oolong teas. In conclusion, the results of this study suggest that the healthy properties of teas, especially GABA teas, have to be evaluated via comprehensive metabolic profiling rather than only the GABA content.


Assuntos
Camellia sinensis/química , Chá/química , Aminoácidos/química , Cafeína/química , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Extratos Vegetais/química , Folhas de Planta/química , Polifenóis/química
13.
Anal Chim Acta ; 1179: 338838, 2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34535262

RESUMO

Chemical cross-linking in combination with mass spectrometry (XL-MS) has emerged as a useful method for structural elucidation of proteins and protein complexes. Due to the low stoichiometry of cross-linked peptides, a specific enrichment method is always necessary prior to LC-MS/MS analysis, especially for complex samples. Currently, strong cation exchange chromatography (SCX), size exclusion chromatography (SEC), and affinity tag-based enrichment are among the widely used enrichment strategies. Herein, we present a two-dimensional strategy combining basic pH reversed-phase liquid chromatography (bRPLC) fractionation and tip-based SCX (SCX-Tip) enrichment, termed ReST, for the characterization of cross-linked peptides. We revealed the unbiased separation effects of the bRPLC in the cross-linked peptide fractionation. We optimized the enrichment conditions of SCX-Tip using well-designed cross-linked peptides. Taking advantage of the high resolution of bRPLC separation and the high enrichment efficiency of SCX-Tip, we were able to identify 43.6% more cross-linked peptides than the conventional SCX approach. The presented ReST is a simple and efficient approach for proteome-scale protein-protein interaction studies.


Assuntos
Cromatografia de Fase Reversa , Proteômica , Cátions , Cromatografia por Troca Iônica , Cromatografia Líquida , Concentração de Íons de Hidrogênio , Peptídeos , Espectrometria de Massas em Tandem
14.
Anal Chem ; 93(37): 12565-12573, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34491041

RESUMO

In comparison to proteomics, the application of two-dimensional liquid chromatography (2D LC) in the field of metabolomics is still premature. One reason might be the elevated chemical complexity and the associated challenge of selecting proper separation conditions in each dimension. As orthogonality of dimensions is a major issue, the present study aimed for the identification of successful stationary phase combinations. To determine the degree of orthogonality, first, six different metrics, namely, Pearson's correlation coefficient (1 - |R|), the nearest-neighbor distances (H̅NND), the "asterisk equations" (AO), and surface coverage by bins (SCG), convex hulls (SCCH), and α-convex hulls (SCαH), were critically assessed by 15 artificial 2D data sets, and a systematic parameter optimization of α-convex hulls was conducted. SGG, SCαH with α = 0.1, and H̅NND generated valid results with sensitivity toward space utilization and data distribution and, therefore, were applied to pairs of experimental retention time sets obtained for >350 metabolites, selected to represent the chemical space of human urine. Normalized retention data were obtained for 23 chromatographic setups, comprising reversed-phase (RP), hydrophilic interaction liquid chromatography (HILIC), and mixed-mode separation systems with an ion exchange (IEX) contribution. As expected, no single LC setting provided separation of all considered analytes, but while conventional RP×HILIC combinations appeared rather complementary than orthogonal, the incorporation of IEX properties into the RP dimension substantially increased the 2D potential. Eventually, one of the most promising column combinations was implemented for an offline 2D LC time-of-flight mass spectrometry analysis of a lyophilized urine sample. Targeted screening resulted in a total of 164 detected metabolites and confirmed the outstanding coverage of the 2D retention space.


Assuntos
Cromatografia de Fase Reversa , Metabolômica , Cromatografia Líquida , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas
15.
J Chromatogr A ; 1654: 462449, 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34399143

RESUMO

In both biologics quality control experiments and protein post-translational modification studies, the analytical system used is not supposed to bring any artefactual modifications which could impair the results. In this work, we investigated oxidation of methionine-containing peptides during reversed-phase (RP) chromatographic separation. We first used a synthetic methionine-containing peptide to evaluate this artefactual phenomenon and then considered more complex samples (i.e., plasma and HeLa protein digests). The methionine oxidation levels of the peptides were systematically assessed and compared for the long-term use of the analytical column, the sample trapping time, the gradient length, the sample load and the nature of the stationary phase (HSS T3 from Waters, YMC Triart C18 from YMC Europe GmbH and BEH130 C18 from Waters). In addition to the oxidation of methionine in solution, we observed on the HSS T3 and the BEH130 stationary phases an additional broad peak corresponding to an on-column oxidized species. Considering the HSS T3 phase, our results highlight that the on-column oxidation level significantly increases with the age of the analytical column and the gradient length and reaches 56 % when a 1-year-old column set is used with a 180 min-long LC method. These levels go to 0 % and 18 % for the YMC Triart C18 and the BEH130 C18 phases respectively. Interestingly, the on-column oxidation proportion decreases as the injected sample load increases suggesting the presence of a discrete number of oxidation sites within the stationary phase of the analytical column. Those findings observed in different laboratories using distinct set of columns, albeit to varying degrees, strengthen the need for a standard of methionine-containing peptide that could be used as a quality control to appraise the status of the liquid chromatographic columns.


Assuntos
Cromatografia de Fase Reversa , Metionina , Peptídeos , Cromatografia de Fase Reversa/instrumentação , Cromatografia de Fase Reversa/normas , Metionina/metabolismo , Oxirredução , Peptídeos/metabolismo , Controle de Qualidade
16.
J Proteome Res ; 20(9): 4589-4597, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34384028

RESUMO

Metaproteomics is a powerful analytical approach that can assess the functional capabilities deployed by microbial communities in both environmental and biomedical microbiome settings. Yet, the mass spectra resulting from these mixed biological communities are challenging to obtain due to the high number of low intensity peak features. The use of multiple dimensions of chromatographic separation prior to mass spectrometry analyses has been applied to proteomics previously but can require increased sampling handling and instrument time. Here, we demonstrate an automated online comprehensive active modulation two-dimensional liquid chromatography method for metaproteome sample analysis. A high pH PLRP-S column was used in the first dimension followed by low pH separation in the second dimension using dual modulating C18 traps and a C18 column. This method increased the number of unique peptides found in ocean metaproteome samples by more than 50% when compared to a one-dimension separation while using the same amount of sample and instrument time.


Assuntos
Cromatografia de Fase Reversa , Microbiota , Espectrometria de Massas , Peptídeos , Proteômica
17.
J Chromatogr A ; 1653: 462421, 2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34343783

RESUMO

We present a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of sodium lauryl sulfate and sodium laureth sulfate homologues in the range of alkyl chain length C12-C16 with 0-5 ethoxy groups. The method is based on scanning the precursor ions fragmenting to m/z 80 and 97 (Precursor Ion Scanning mode), which makes it specific for species with easily cleavable sulfate groups. By monitoring fragmentation of thus discovered quasi-molecular ions we were able to unequivocally identify all sulfate species present in complex mixtures of alkyl and alkyl-ether sulfates with molecular weight ranging from 200 to 600 m/z. Because of the intrinsic sulfate-sensitivity, the presented method can be also applied to non-sodium salts of alkyl- and alkyl-ether sulfates (e.g. ammonium, mono- or triethanolamine, etc.), which are often used by cosmetic manufacturers to justify the misleading SLS- and SLES-free claims (where SLS and SLES refer to sodium lauryl sulfate and sodium laureth sulfate, respectively). The use of reversed phase liquid chromatography (RPLC) column with C4 instead of C18 shortened significantly the overall analysis time and allowed us to use a semiquantitative method (based on single standard for Quantitative Analysis of Multi-component System, QAMS) to determine several SLS and SLES homologues in one run with the limit of quantification (LOQ) = 0.4 µg/mL and of detection (LOD) in the range 0.12-0.97 µg/mL. The method was successfully applied to 17 commercially available cosmetic/household products allowing verification of their manufacturers' declarations.


Assuntos
Ácidos Alcanossulfônicos , Cromatografia Líquida , Tensoativos , Espectrometria de Massas em Tandem , Cromatografia de Fase Reversa , Íons
18.
J Chromatogr A ; 1653: 462429, 2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34371364

RESUMO

Many materials used in a wide range of fields consist of polymers that feature great structural complexity. One particularly suitable technique for characterising these complex polymers, that often feature correlated distributions in e.g. microstructure, chemical composition, or molecular weight, is comprehensive two-dimensional liquid chromatography (LC × LC). For example, using a combination of reversed-phase LC and size-exclusion chromatography (RPLC × SEC). Efficient and sensitive LC × LC often requires focusing of the analytes between the two stages. For the analysis of large-molecule analytes, such as synthetic polymers, thermal modulation (or cold trapping) may be feasible. This approach is studied for the analysis of a styrene/butadiene "star" block copolymer. Trapping efficiency is evaluated qualitatively by monitoring the effluent of the trap with an evaporative light-scattering detector and quantitatively by determining the recovery of polystyrene standards from RPLC × SEC experiments. The recovery was dependant on the molecular weight and the temperatures of the first-dimension column and of the trap, and ranged from 46% for a molecular weight of 2.78 kDa to 86% (or up to 94.5% using an optimized set-up) for a molecular weight of 29.15 kDa, all at a first-dimension-column temperature of 80 °C and a trap temperature of 5 °C. Additionally a strategy to reduce the pressure pulse from the modulation has been developed, bringing it down from several tens of bars to only a few bar.


Assuntos
Cromatografia de Fase Reversa , Polímeros , Cromatografia em Gel , Calefação , Peso Molecular
19.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445404

RESUMO

Chronic UV radiation causes oxidative stress and inflammation of skin and blood cells. Therefore, in this study, we assessed the effects of cannabidiol (CBD), a natural phytocannabinoid with antioxidant and anti-inflammatory properties, on the phospholipid (PL) and ceramide (CER) profiles in the plasma of nude rats irradiated with UVA/UVB and treated topically with CBD. The results obtained showed that UVA/UVB radiation increased the levels of phosphatidylcholines, lysophospholipids, and eicosanoids (PGE2, TxB2), while downregulation of sphingomyelins led to an increase in CER[NS] and CER[NDS]. Topical application of CBD to the skin of control rats significantly upregulated plasma ether-linked phosphatidylethanolamines (PEo) and ceramides. However, CBD administered to rats irradiated with UVA/UVB promoted further upregulation of CER and PEo and led to significant downregulation of lysophospholipids. This was accompanied by the anti-inflammatory effect of CBD, manifested by a reduction in the levels of proinflammatory PGE2 and TxB2 and a dramatic increase in the level of anti-inflammatory LPXA4. It can therefore be suggested that topical application of CBD to the skin of rats exposed to UVA/UVB radiation prevents changes in plasma phospholipid profile resulting in a reduction of inflammation by reducing the level of LPE and LPC species and increasing antioxidant capacity due to upregulation of PEo species.


Assuntos
Canabidiol/administração & dosagem , Ceramidas/sangue , Eicosanoides/sangue , Fosfolipídeos/sangue , Raios Ultravioleta/efeitos adversos , Administração Tópica , Animais , Canabidiol/farmacologia , Ceramidas/efeitos da radiação , Cromatografia de Fase Reversa , Eicosanoides/efeitos da radiação , Masculino , Fosfolipídeos/efeitos da radiação , Ratos , Ratos Nus , Espectrometria de Massas em Tandem
20.
Anal Chem ; 93(35): 12090-12095, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34431663

RESUMO

Ultraperformance liquid chromatography (UPLC) and ion mobility (IM) spectrometry were interfaced with mass spectrometry (MS) and tandem mass spectrometry (MS/MS) to characterize a complex nonionic surfactant mixture. The surfactant was composed of a glycerol core, functionalized with poly(ethylene oxide) units (PEOn) that were partially esterified by caprylic and/or capric acid. Reversed-phase UPLC classified the blend based on polarity into four groups of eluates, corresponding to compounds with zero, one, two, or three fatty acid residues. Additional separation within each eluate group was achieved according to the length of the fatty acid chains. Coeluting molecules of similar polarity were dispersed in the gas phase by their collision cross section in the IM dimension. Performed in series, UPLC and IM allowed for the separation and detection of several isomeric and isobaric blend constituents, thereby enabling their isolation for conclusive MS/MS analysis to confirm or elucidate their primary structures and architectures (overall four-dimensional, 4D, characterization).


Assuntos
Cromatografia de Fase Reversa , Espectrometria de Massas em Tandem , Cromatografia Líquida , Espectrometria de Mobilidade Iônica , Tensoativos
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