ABSTRACT
As a treasure resource of novel drug lead compounds, how to rapidly and high-efficiently screen and isolate active components from natural products is critical. Thanks to its high resolution, high automation and flexible integration, online two-dimensional liquid chromatography has great potential for screening active ingredients from complex matrices by integrating a highly specific bio-recognition process into a two-dimensional liquid chromatography system before, on or after the column separation. This review comprehensively summarized recent developments, applications and shortcomings of online two-dimensional liquid chromatography for natural product screening from different integration modes, including pre-column, on-column and post-column screening methods.
ABSTRACT
Cordyceps sinensis(C.sinensis)is a widely used and highly valuable traditional Chinese medicine.Several dipeptides have been detected in C.sinensis,but current scientific knowledge of its chemical makeup remains limited.In this study,an improved approach that integrates offline two-dimensional liquid chromatography(2D LC)separation,precursor ion list,library screening,and diagnostic ion filtering was established to systematically screen and characterize dipeptides in C.sinensis.Offline 2D LC integrating hydrophilic interaction LC and reverse phase separations was established to eliminate interference and identify the target dipeptides.A library containing the potential 400 dipeptides was created,and a precursor ion list with all theoretical precursor ions was adopted to trigger the MS/MS scan with high sensitivity.To identify dipeptides,the type and connection sequence of amino acids were determined according to the product ions.Ile and Leu residues were differentiated for the first time according to the characteristic ion at m/z 69.07.Ultimately,170 dipeptides were identified or tentatively characterized from C.sinensis,and most are reported for the first time in this species herein.In addition,the identified dipeptides were also applied for discrimination among the three Cordyceps species,and 11 markers were identified.The obtained results provide a deeper understanding of the chemical basis of C.sinensis.
ABSTRACT
Research on polymer impurities has always been important in the quality control of cephalosporins. Research on polymers in cephalosporins that lack active amino groups on the C-7 side chain has not been reported. Therefore, our study used cefazolin sodium, which is widely used in the clinic, as an example. The polymer in cefazolin sodium and its product "cefazolin sodium pentahydrate for injection" was analyzed by column switching liquid chromatography-high resolution mass spectrometry. Two polymer impurity peaks were detected and the possible structures of these polymers were suggested. Through two-dimensional liquid chromatography, the chromatographic peaks following Sephadex gel chromatography and high-performance gel chromatography were compared to those obtained by reverse high-performance liquid chromatography (HPLC) for cefazolin sodium as reported in the Chinese Pharmacopoeia. The HPLC method proves more suitable for polymer detection than Sephadex gel chromatography and high-performance gel chromatography. The method of polymer detection for cefazolin sodium was established using the method of related substances HPLC as described in the Chinese Pharmacopoeia.
ABSTRACT
Two dimensional liquid chromatography (2D-LC) has become an important way to achieve the separation of complex samples due to higher peak capacity, higher selectivity and better separation ability compared with one-dimensional liquid chromatography(1D-LC). 2D-LC has been developing rapidly in many fields, such as medicine, food, metabolites in vivo or in vitro and so on. This review illustrates various separation modes of 2D-LC and its applications and developments, with emphases on the research of quality control of drugs.
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Objective: To develop a new three-dimensional fingerprinting method and its assessing Methods: based on comprehensive two-dimensional liquid chromatography using Niuhuang Shangqing Pills (NSP) as an example. The developed method could offer new method for the quality control of NSP. Methods: In first dimension, the separation was achieved with an Acquity UPLC HSS CYANO column (100 mm × 2.1 mm, 1.8 μm), methanol-0.1% formic acid in water were used as mobile phases, flow rates were 0.1 mL/min. In second dimension, the separation was achieved with a Kinetex Phenyl-Hexyl column (50 mm × 3 mm, 2.6 μm), acetonitrile-0.1% formic acid in water were used as mobile phases, flow rates were 1.5 mL/min, detection wavelength was set at 254 nm, and acquiring frequency was at 12.5 Hz. Column temperature for each dimension was 40 ℃ and volume of loop linking the two dimensions was 100 μL. Three similarity-calculating Methods:, Euclidean Distance, Cosine, and Correlation Coefficient, were employed to assess the similarities among the 21 samples on the market using medians with arithmetic means of peak volumes of the common peaks as control fingerprints. Results: The three-dimensional fingerprints of 21 batches of NSP samples on the market were developed; Eighteen common peaks were assigned and five of them were identified, which were geniposide (1), pulegone (8), baicalin (9), imperatorin (15), and wogonin (16). Conclusion: A three-dimensional fingerprinting method and its assessing Methods: based on comprehensive two-dimensional liquid chromatography using NSP as an example were successfully developed for the first time, suggesting that it is a feasible method for developing fingerprints for Chinese materia medica. This work improves and supplements the traditional liquid-chromatography fingerprints.
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A two-dimensional liquid chromatography method was developed for the analysis of rice leaves proteomics based on the coupling of hydrophilic interaction liquid chromatography-reversed-phase liquid chromatography with online tandem mass spectrometry. The influence of pH value of chromatographic mobile phase on the orthogonality of the hydrophilic interaction-reversed-phase two-dimensional liquid chromatography was evaluated by the changes of standard peptide retention. The results indicated that the better orthogonality (R2=0.34113) was achieved from the system with hydrophilic interaction columns(pH 9.3) in the first and C18columns(pH 3.3) in the second LC dimension. Coupled with multiple fraction concatenation strategy,the orthogonality of two-dimensional liquid chromatography was further evaluated in the analysis of complex rice leaf proteins. The results showed that more than 50% of the total peptides were identified less than two times, and the peptides obtained from first-dimension were well distributed across the elution window,indicating that the method showed significant orthogonality in the identification of complex rice leaf proteins. Based on the proteome discoverer software,207345 peptides belonged to 2930 protein clusters were identified.
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By using a double loop technique based on a set of mechanical transmission components and a flow path system,a double function of injection and fraction collection was realized. On the basis of double loop technique,a novel type of online/offline interface for two-dimensional liquid chromatography was designed to construct an efficient separation system coupling two different separation modes with a higher peak capacity, and the functions of the interface were evaluated. By means of connecting the interface to an HPLC system, the multiple functions of injection, fraction collection and injection again were fulfilled for preparation and purity analysis of 4 kinds of aromatic compounds. As for combination of 2 sets of HPLC system,5 kinds of protein samples were separated preliminarily by strong cation exchange chromatography, and the components difficult to separate were collected and injected into reversed phase chromatographic system for further separation. Furthermore, the interface was applied to coupling two chromatographic systems in both strong cation exchange mode and microcolumn reversed-phase mode for the two-dimensional separation of bovine serum albumin enzymatic digest. When 1 mAU was set as the integral threshold,a total number of 292 peaks were identified. With the help of the online/offline interface, the preparation of microscale samples, fine separation of hardly separated samples and two-dimensional separation of complex samples were achieved flexibly. The result indicated that the system was a potent tool for the construction of two-dimensional chromatographic system and separation research.
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OBJECTIVE: To establish a detection method based on a novel type of two-dimensional liquid chromatographic system (2D-LC-UV) for determination of paraquat in poisoned patients' urine, and assess the methodology and characteristics of the two-dimensional chromatography. METHODS: 2D-LC-UV contains 1st and 2nd liquid chromatography and FLC transferor. Urine sample of 100 μL was directly injected without pretreatments, and paraquat was on-line concentrated and primarily separated on the 1st Aston SX1 column, then trapped on Aston SX column and separated by Aston SX1 column. The mobile phase of the 1st liquid chromatography, trapping column and 2nd liquid chromatography were acetonitrile-2 mol·L-1 ammonium acetate binary solution(1:10, V:V, 1.0 mL·min-1), pure water and 2.5 mol·L-1 ammonium acetate solution-methanol-acetonitrile ternary system (5:3:1, V:V:V, 1.0 mL·min-1), respectively. The column temperature was maitained at 40℃, and the UV adsorption wave length was set at 285 nm. RESULTS: The chromatographic system was capable of processing 500 μL urine. Paraquat was transferred and separated completely in the two-dimensional system with linear calibration curve over the concentration range of 20-20 000 ng·mL-1 (r=0.999 9). The urine matrixes from different samples had little interference effect. The chromatographic balance time was less than 15 min, and the calibration curve was stable for more than 90 d. CONCLUSION: Because of its accuracy, stability, automation, and independence of specialized technical personnel, the established chromatographic method is ideal for quick test of paraquat in poisoned patients, providing scientific basis for clinical treatment of patients with paraquat poisoning.
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As one kind of lipid excipients, triacylglycerols (TAGs) are being more and more widely used in pharmaceutics with the development of advanced drug delivery systems. As their characteristics are mainly determined by their structures and compositions, strict quality control ought to be performed on TAGs, in order to guide their use in pharmaceutics. However, because of their complex compositions, no qualtity standard is set for TAGs in Ch.P currently. In this paper, we reviewed the methods for the separation and analysis of TAGs, includingthin-layer chromatog raphy, solid-phase extraction, gas chromotography, liquid chromatography, two-dimensional liquid chromatography, and ultraperformance convergence chromatography, and summarized their advantages and disadvantages. The application scope of each method was also overviewed in this review. Besides that we also pointed outthat according to our own study the newly emerged ultraperformance convergence chromatography would be one of the best methods to analyze TAGs.
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OBJECTIVE: To establish a method for determination of diclofenac sodium in human plasma under high-fat meal condition using two-dimensional liquid chromatography (2D-HPLC) coupled with trapping column, and to evaluate the bioequi valence and the bioavailability of diclofenac sodium sustain-released tablets in healthy volunteers. METHODS: Under fed state, eighteen healthy male volunteers were divided into two groups by an open, randomized two period crossover design with a single dose of diclofenac sodium sustain-released tablets. The plasma concentrations were determined by 2D-HPLC method, and calculated pharmacokinetic parameters and bioavailability. RESULTS: The main pharmacokinetic parameters of test and reference preparations after a single dose were: AUC0-16 was (2 591.6 ± 705.8) and (2 588.8 ± 772.0) ng · h · mL-1; AUC1→∞ was (2 896.4 ±839.7) and (2 700.3 ± 806.1) ng · h · mL-1; tmax was (4.6 ± 0.7) and (4.4 ± 0.9)h; ρmax was(1 332.8 ± 912.5) and (1 325.7 ± 706.3) ng · mL-1, respectively. The relative bioavailability of test preparation in single dose was (102.4 ± 15.1)%. CONCLUSION: The 2D-HPLC method coupled with trapping column is a simple, rapid and specific for determination of diclofenac sodium in human plasma. The results of statistical analysis indicate that the two preparations are bioequivalent in healthy volunteers with a single dose under high-fat fed condition.
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A method of on-line solid phase extraction ( SPE )-two dimensional liquid chromatography electrospary-tandem mass spectrometric method was established for the determination of Teicoplanin concentrations in human cerebrospinal fluid. Cerebrospinal fluid samples were treated by the on-line SPE treatment, and analyzed by LC-MS/MS. The chromatographic separation was performed on a Shiseido CAPCALL-PAK C18 column with gradient elution by using 25 mmol/L ammonium acetate ( pH 6. 0 )-acetonitrile as mobile phases, and the flow rate of 1 mL/min. Detection was carried out under the selected reaction monitoring ( SRM) in positive ionization mode with scopolamine hydrobromide as internal standard. Matrix-matched calibration curves with good correlation coefficients (R2=0. 9993, n=6) were obtained in the concentration range of 25-5000 μg/L. The average recoveries varied from 100. 8% to 109. 9%. The intra-and inter-day precisions were less than 6%. The method is proved to be rapid, sensitive, accurate, and suitable to determine Teicoplanin concentrations in human cerebrospinal fluid.
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An on-line two dimensional liquid chromatographic (2D-LC) method was established by using an ultimate dual gradient liquid chromatography, three chromatographic columns and valve-switching technology to detect 2460A in trichoderma hazianum fermentation liquor. MF C8 column (10 mm×4. 6 mm, 5. 0 μm) was used as purification column and MG C18 column (20 mm×4. 6 mm, 5. 0 μm) was used as enriching column. Methanol and water were used as mobile phase with a gradient elution at a flow rate of 2. 0 mL/min. The sample was separated on the Thermo Hypersil GOLD C18 column (250 mm×4. 6 mm, 5. 0μm) maintained at 40 ℃ using methanol and water. The flow rate was 1. 0 mL/min and 1. 0 mL sample was injected into the 2D-LC system. The detection wavelength was 424 nm. The whole analytical time was less than 60 min. The standard curve was linear over the 2460A concentration range of 0. 0025-10. 0 mg/L(r=0. 9981, n=8). The limit of detection was calculated to be 1 . 2μg/L ( S/N=3 ) and the limit of quantification was calculated to be 2 . 5 μg/L ( S/N=10 ) . The average recoveries varied from 88 . 0% to 104 . 4%.
ABSTRACT
Qualitative and quantitative analyses of biological samples containing drugs, toxicants and endogenous substances play an important role in the researches of life sciences, as well as in new drug discovery and development. Biological samples are characterized by complex matrix, multiple endogenous interferences, significantly lower concentrations of measured analytes compared to endogenous components and small sampling volume. Consequently, it often requires bioanalysis methods with superior specificity, high sensitivity and good reproducibility. The two-dimensional liquid chromatography (2D-LC) technique, which allows for high peak capacity, significant reduced matrix effect and carryover of complex matrix samples and automated sample pre-treatment and analysis, has been the powerful solution to the separation and analysis of biological sample and widely applied to environment, food and pharmaceutical analysis. On the basis of introduction of the principle and equipments of 2D-LC, the application of this technique in the pharmacokinetics, toxicological and biological study was reviewed.