正相模式/反相模式的二维液相色谱系统的构建与应用

以4.6mm×50 mm i. d.的Hypersil SiO2正相色谱柱为第一维,4.6mm×250 mm i. d.的Kromasil C18反相色谱柱为第二维,通过升高第二维色谱温度的方法增加两维流动相间互溶性的方法构建了定量环-阀切换接口的二维液相色谱系统(NPLC×RPLC)。根据有机溶剂的特征,在第一维正相色谱流动相中加入二氧六环;第二维反相色谱流动相中加入异丙醇,在改善流动相兼容性的同时,有效调整分离选择性。采用此系统对正天丸样品进行分离分析,达到1120的峰容量。     

Online trace enrichment to determine pyrethroids in river water by HPLC with column switching and photochemical induced fluorescence detection

The potential of online trace enrichment on a highly apolar short column in LC was evaluated for the determination of pyrethroids in river water. Twelve millilitres of water samples, modified with 8 mL ACN (ACN/water 40:60, v/v), were passed through 50 x 4.6 mm ID first separation column packed with 5 microm Hypersil Elite C18. Pesticides were preconcentrated in this column while the matrix background was eluted to waste. Separation of pesticides was performed on a 3.5 microm symmetric C18 column (250 x 4.6 mm ID) with an ACN step gradient as mobile phase and fluorescence detection was used after postcolumn derivatization by using UV light. The use of photochemically induced fluorescence for detection improved sensitivity and selectivity. Quantification limits ranged from 0.05 to 0.1 microg/L and pesticide recoveries at two concentration levels (0.1 and 0.5 microg/L) were between 93.1 and 118.6%, with RSD between 2.5 and 7.5% (n = 3) in river water samples. No matrix effect was detected.     

High throughput screening of active pharmaceutical ingredients by UPLC

Ultra performance LC (UPLC) was evaluated as an efficient screening approach to facilitate method development for drug candidates. Three stationary phases were screened: C-18, phenyl, and Shield RP 18 with column dimensions of 150 mm x 2.1 mm, 1.7 microm, which should theoretically generate 35,000 plates or 175% of the typical column plate count of a conventional 250 mm x 4.6 mm, 5 microm particle column. Thirteen different active pharmaceutical ingredients (APIs) were screened using this column set with a standardized mobile-phase gradient. The UPLC method selectivity results were compared to those obtained for these compounds via methods developed through laborious trial and error screening experiments using numerous conventional HPLC mobile and stationary phases. Peak capacity was compared for columns packed with 5 microm particles and columns packed with 1.7 microm particles. The impurities screened by UPLC were confirmed by LC/MS. The results demonstrate that simple, high efficiency UPLC gradients are a feasible and productive alternative to more conventional multiparametric chromatographic screening approaches for many compounds in the early stages of drug development.     

Comprehensive two-dimensional liquid chromatography applying two parallel columns in the second dimension

The design of a new interface for comprehensive two-dimensional liquid chromatography (LC x LC) is described. To the conventionally used LC x LC system with the loop-type interface consisting of a two-position/ten-port switching valve equipped with two loops, an extra two-position/ten-port switching valve, a detector, a pump and a second column placed in parallel with the column in the second dimension, are added. The features of the interface are that the separation space in the second dimension is significantly enlarged and that the number of fractions transferred from the first to the second dimension can be increased, reducing the risk to lose resolution of the primary dimension. The potential of the system in NPLC x 2RPLC is illustrated with the analysis of a standard mixture and a lemon oil extract. For the lemon oil analysis, the effective peak capacity was increased from 437 using a conventional interface to 1095 with the new interface. RPLC x 2RPLC in combination with reduced modulation times was applied to the analysis of steroids and to the detection of impurities at the 0.05% relative concentration level in a sulfonamide drug sample.     

Simultaneous determination of tretinoin and clindamycin phosphate and their degradation products in topical formulations by reverse phase HPLC

A new HPLC method based on reverse phase separation and photodiode-array detection has been developed for the simultaneous determination of tretinoin and clindamycin phosphate, and their degradation products in topical formulations. The method has been shown to be stability indicating, accurate, and precise for two different formulation vehicles. Separation was achieved on a reverse phase C18 column (Lichrospher, RP18, 5 microm, 25 cm x 4.6 mm ID, Phenomenex, USA) using a simple gradient with aqueous-acetonitrile and aqueous-methanol mobile phases. The method recovery averaged 100.3% for tretinoin and 99.6% for clindamycin phosphate at a concentration range between 80% and 120% of the label claim. The method can be applied to assess the stability of tretinoin and clindamycin phosphate in pharmaceutical formulations containing tretinoin and clindamycin phosphate individually or in combination as active drugs.     

Advantages of ultra performance liquid chromatography over high-performance liquid chromatography: comparison of different analytical approaches during analysis of diclofenac gel

Miniaturization embracing instrumentation, column particle size, and column dimensions is one of the major current trends in separation techniques. This leads to shortening of analysis time and great savings in solvent consumption. Ultra performance liquid chromatography (UPLC) is one of the new developments in liquid chromatography. An ultra-high pressure system allows using of small particle-packed columns with small diameter, which has a positive effect on both system efficiency and analysis time. An analytical method for determination of the active substance diclofenac, the degradation product 1-(2,6-dichlorphenyl)-2-indolinone, and the preservatives methylparaben and propylparaben was used for testing and comparing LC systems. Various octadecylsilica-based analytical columns were examined. Acquity UPLC BEH C18 (2.1 x 50 mm, 1.7 microm) and (2.1 x 100 mm, 1.7 microm) were tested for UPLC. The following analytical columns were used in a test for HPLC: Purospher RP 18e (125 x 4.0 mm, 5 microm), Zorbax Eclipse XDB C18 (75 x 4.6 mm, 3.5 microm), Zorbax Eclipse SB C18 (50 x 4.6 mm, 1.8 microm), as was a monolithic column (Chromolith Performance RP-18e (100 x 4.6 mm). Results of a System Suitability Test (SST) were calculated and compared for each chromatographic peak. System efficiency and analysis duration were compared with regard to solvent consumption and system maintenance     

Construction of a new interface for comprehensive supercritical fluid chromatography x reversed phase liquid chromatography (SFC x RPLC)

The design of a new interface to hyphen high efficiency supercritical fluid chromatography (SFC) and fast RPLC in a comprehensive configuration is described. SFC x RPLC is a viable alternative to normal phase (NP) LC x RPLC and is characterized by high orthogonality. Compared to NPLC x RPLC an additional advantage is the expansion of supercritical carbon dioxide (CO(2)) when exposed to atmospheric pressure leading to fractions consisting of solvents that are miscible with the second dimension RPLC mobile phase. The interface consists of a two-position/ten-port switching valve equipped with two packed octadecyl silica (C(18)) loops for effective trapping and focusing of the analytes after elution from the SFC dimension. The addition of a water make-up flow to the SFC effluent prior to entering the loops is of fundamental importance to efficiently focus the solutes on the C(18) material and to reduce interferences of expanded CO(2) gas on the second dimension separation. The features of the system are illustrated with the analysis of a lemon oil sample.     

Evaluation of an International Pharmacopoeia method for the analysis of nelfinavir mesilate by liquid chromatography

A gradient LC method for the determination of related substances in nelfinavir mesilate (NFVM) has been recently published in the International Pharmacopoeia. The method uses a base deactivated reversed phase C18 column (25 cm x 4.6 mm I.D.), 5 microm kept at a temperature of 35 degrees C. The mobile phases consist of acetonitrile, methanol, phosphate buffer pH 3.4 and water. The flow rate is 1.0 ml/min. UV detection is performed at 225 nm. A system suitability test (SST) is described to govern the quality of the separation. The separation towards NFVM components was investigated on 18 C18 columns and correlation was made with the column classification system developed in our laboratory. The method was evaluated using a Hypersil BDS C18 column (25 cm x 4.6 mm I.D.), 5 microm. A two level fractional factorial design was applied to examine the robustness of the method. The method shows good selectivity, precision, linearity and sensitivity. Seven commercial samples were examined using this method.     

High-efficiency liquid chromatography on conventional columns and instrumentation by using temperature as a variable I. Experiments with 25 cm x 4.6 mm I.D., 5 microm ODS columns

High plate numbers were obtained in conventional LC by coupling columns and by using temperature to reduce the viscosity of the mobile phase. At 80 degrees C up to eight columns of 25 cm x 4.6 mm I.D. packed with 5 microm ODS particles could be coupled generating 180,000 effective plates while the pressure drop was only 350bar. For routine work, a set of four columns is preferred. The analysis times on one column operated at 30 degrees C and 1 mL/min flow rate and on four columns at 80 degrees C and 2 mL/min flow rate are the same in isoeluotropic conditions while the resolution is doubled. Multicolumn systems were successfully applied in isocratic and gradient mode for the analysis of pharmaceutical and environmental samples.     

Elevated temperature-extended column length conventional liquid chromatography to increase peak capacity for the analysis of tryptic digests

High efficiency separations (200 000 plates) were obtained on conventional LC equipment by coupling 8 x 25 cm x 2.1 (or 4.6) mm id x 5 microm d(p) ODS columns (total length 2 m) and operation at 60 degrees C using a dedicated LC oven. The peak capacity in this 1-D set-up was 900 for the separation of human serum tryptic peptides analyzed after depletion of six highly abundant proteins. The chromatographic performance of an elevated temperature-extended column length conventional LC is highlighted.     

Cellulose dimethylphenylcarbamate-bonded carbon-clad zirconia for chiral separation in high performance liquid chromatography.

Porous zirconia particles are very robust material and have received considerable attention as a stationary phase support for HPLC. We prepared cellulose dimethylphenylcarbamate-bonded carbon-clad zirconia (CDMPCCZ) as a chiral stationary phase (CSP) for separation of enantiomers of a set of 14 racemic compounds in normal phase (NP) and reversed-phase (RP) liquid chromatography. Retention and enantioselectivity on CDMPCCZ were compared to those on CDMPC-coated zirconia (CDMPCZ) to see how the change in immobilization method of the chiral selector affects the retention and chiral selectivity. In NPLC, retention was longer and the number of resolved racemates was smaller on CDMPCCZ than on CDMPCZ. However, chiral selectivity factors for some resolved racemates were better on CDMPCCZ than on CDMPCZ. The longer retention on CDMPCCZ is likely due to strong, non-chiral discriminating interactions with the carbon layer on CDMPCZ. In RPLC only two racemates were resolved on CDMPCCZ, but retention times were shorter than, and resolutions were comparable to, those in NPLC, indicating a potential for improving chromatographic performance of the CDMPCCZ column in RPLC with optimized column preparation and separation conditions.     

A vacuum assisted dynamic evaporation interface for two-dimensional normal phase/reverse phase liquid chromatography

A vacuum assisted dynamic solvent evaporation interface for coupling of two-dimensional normal phase/reverse phase liquid chromatography was developed and evaluated. A normal-phase liquid chromatographic (NPLC) column of a 250 mm × 4.6 mm I.D. 5 μm CN phase was used as the first dimension, and a reversed-phase liquid chromatographic (RPLC) column of 250 mm × 4.6 mm I.D. 5 μm C₁₈ phase was used as the second dimension. The eluent from the first dimension flowed into a fraction loop, and the solvent in the eluent was dynamically evaporated and removed by vacuum as it was entering the fraction loop of the interface. The non-evaporable analytes was retained and enriched in about 5–25 μL solution within the loop. Up to 1 mL/min of mobile phase from the first dimension can be evaporated and removed dynamically by the interface. The mobile phase from the second dimension then entered the loop, and dissolved the concentrated analytes retained inside the loop, and carried them onto the second dimension column for further separation. The operation conditions of the two dimensions were independent from each other, and both dimensions were operated at their optimal chromatographic conditions. We evaluated the interface by controlling the loop temperature in a water bath at normal temperature, and investigated the sample losses by using standard samples with different boiling points. It was found that the sample loss due to evaporation in the interface was negligible for non-volatile samples or for components with boiling point above 340 °C. The interface realizes fast solvent removal of mL volume of fraction and concentration of the fraction into tenth of μL volume, and injection of the concentrated fraction on the secondary column. The chromatographic performance of the two-dimensional LC system was enhanced without compromise of separation efficiency and selectivity on each dimension.     

Comparative performance of ion exchange and ion-paired reversed phase high performance liquid chromatography for the determination of nucleotides in biological samples.

We have compared anion exchange chromatography on APS-Hypersil (4.6 x 100 mm) eluted with a phosphate gradient with reversed phase chromatography on ODS-Hypersil (4.6 x 100 mm) in the presence of either tetrabutylammonium (TBA) or triethylammonium (TEA) ions with a methanol gradient. The systems have been compared both for ease of operation and for their resolving power with standard mixtures and acid extracts of both normal red cells (RBC) and ischaemic tissues. The two chromatographic modes exhibited similar separating efficiencies for standard mixtures of nucleotides but retention times were most stable using reversed phase liquid chromatography (RPLC) with TEA. Anion exchange columns slowly lost ion exchange capacity but selectivity was unchanged. RPLC in the presence of TBA gave reproducibile capacity factors only when operated isocratically due to irreversible changes to the silica surface. For RBCs the RPLC with TEA and anion exchange systems resolved 17 and 15 peaks, respectively, and for the ischaemic samples 22 and 14 peaks, respectively. However, nucleosides and bases were also resolved by the ODS column causing chromatographic crowding and uncertain peak identification.     

Dual-purpose sample trap for on-line strong cation-exchange chromatography/reversed-phase liquid chromatography/tandem mass spectrometry for shotgun proteomics. Application to the human Jurkat T-cell proteome

A dual-purpose sample-trapping column is introduced for the capacity enhancement of proteome analysis in on-line two-dimensional nanoflow liquid chromatography (strong cation-exchange chromatography followed by reversed-phase liquid chromatography) and tandem mass spectrometry. A home-made dual trap is prepared by sequentially packing C18 reversed-phase (RP) particles and SCX resin in a silica capillary tubing (1.5 cm x 200 microm I.D. for SCX, 0.7 cm x 200 microm for RP) ended with a home-made frit and is connected to a nanoflow column having a pulled tip treated with an end frit. Without having a separate fraction collection and concentration process, digested peptide mixtures were loaded directly in the SCX part of the dual trap, and the SCX separation of peptides was performed with a salt step elution initiated by injecting only 8 microL of NH4HCO3 solution from the autosampler to the dual trap. The fractionated peptides at each salt step were directly transferred to the RP trap packed right next to the SCX part for desalting, and a nanoflow LC-MS-MS run was followed. During the sample loading-SCX fractionation-desalting, flow direction was set to bypass the analytical column to prevent contamination. The entire 2D-LC separation and MS-MS analysis were automated. Evaluation of the technique was made with an injection of 15 microg peptide mixtures from human Jurkat T-cell proteome, and the total seven salt step cycles followed by each RPLC run resulted in an identification of 681 proteins.     

A comprehensive two-dimensional normal-phase × reversed-phase liquid chromatography based on the modification of mobile phases

A comprehensive orthogonal two-dimensional liquid chromatography (2D-LC) based on the modification of mobile phases was developed with a sample loop–valve interface. To improve the compatibility of mobile phases and analysis speed, some special solvents were chosen as the mobile phases, and the column temperature was elevated to decrease the viscosity of mobile phases of reversed-phase liquid chromatography (RPLC). Based on this principle, the first dimension was normal-phase liquid chromatography (NPLC) with a SiO₂ column, and the second dimension was reversed-phase liquid chromatography containing two tandem C18 columns. 1,4-Dioxane was used in the NPLC mobile phase, and isopropyl alcohol was employed in the RPLC mobile phase. Moreover, the elevated column temperature enabled the reduction of the backpressure and using tandem C18 columns to improve the resolving power in RPLC. The new comprehensive 2D-LC system and applied strategy offered a novel idea for construction of 2D-LC system. A traditional Chinese medicine, Zhengtian pill, was used as the test sample to evaluate the constructed 2D-LC system. 876 peaks were detected, and the peak capacity reached 1740.     

Selective preconcentration of chemical warfare agent degradation products using a zirconia preconcentration column

Zirconia (ZrO(2)) has strong Lewis acid sites which have an affinity for the strongly electronegative phosphonate group of organo-phosphates. To investigate whether this affinity can be used for selective preconcentration, the retention of methyl, ethyl, and propylphosphonic acid (MPA, EPA and PPA) and inorganic anion matrix components on ZrO(2) was investigated. Only organo-phosphates and sulfate exhibited retention on zirconia. After preconcentration, the retained species were eluted from ZrO(2) by 0.75 mM Na(2)CO(3), and separated by a Dionex Ionpac AS11 anion-exchange column (250 mm x 2 mm I.D.) and a Hypercarb RPLC column (50 mm x 4.6 mm I.D., 3 microm) in series followed by suppressed conductivity detection. Calibration curve showed a linear response for MPA, EPA and PPA in the range of 0.01 microM to 1 microM (R(2)>0.9989). Detection limits after preconcentration of a 10 mL sample were 0.16, 0.19 and 0.16 microg/L for MPA, EPA, and PPA, respectively.     

Liquid chromatographic method for analysis of saponins in Maesa balansae extract active against leishmaniasis

A liquid chromatographic method was developed for the separation of six related triterpenoid saponins in Maesa balansae extracts with different purity, active against leishmaniasis. As stationary phase a Hypersil BDS C18 column (3 microm), 100 x 4.6 mm was used. The mobile phase was a mixture of methanol, acetonitrile, 5% (m/v) ammonium acetate, pH 6.5 and water. A linear gradient was developed for the analysis of crude extracts. An isocratic method was developed to analyze purified samples that mainly contained saponins 3 and 4, the most active saponins. The isocratic LC method was optimized and the robustness was evaluated with an experimental design. The method showed good selectivity, repeatability, linearity and sensitivity.     

Tandem column for the simultaneous determination of arginine, ibuprofen and related impurities by liquid chromatography

Ibuprofen arginate is a rapidly absorbed salt designed to promote more rapid onset of analgesia than commercially available forms of ibuprofen. Ibuprofen and arginine have very different polarities and this becomes in a chromatographic problem, further complicated with the determination of related compounds, which is necessary in stability assays of the pharmaceutical forms. The common solution is the employment of two separate methods, but this is time consuming. A LC method has been developed to determinate both compounds and related impurities in one run. Ibuprofen, arginine and three ibuprofen related impurities (B, E and J) have been baseline separated with isocratic conditions at pH 3.0 and run time under 20 min by employing a tandem combination of two different stationary phases: first a ZORBAX SB-C18 column from Agilent (250 mm x 4.6 mm and 5 microm) and downstream a SUPELCOSIL LC-NH2 column from Supelco (150 mm x 4.6 mm and 3 microm). The octadecyldiisobutylsilane column provides the separation of ibuprofen and its impurities by a hydrophobic mechanism, whereas aminopropyl column offers selective retention of arginine by dipolar interaction mechanism. Method has been successfully validated following ICH guidelines and it has been demonstrated to be reliable for arginine, ibuprofen and related impurities determination in sachets of two different dosages as pharmaceutical forms. Moreover, stress test has proved the selectivity of the method for degradation products, such as those that can emerge throughout long-term stability assays.     

Fluoxetine and norfluoxetine analysis by direct injection of human plasma in a column switching liquid chromatographic system

A column switching LC method is presented for the analysis of fluoxetine (FLU) and norfluoxetine (NFLU) by direct injection of human plasma using a lab-made restricted access media (RAM) column. A RAM-BSA-octadecyl silica (C-18) column (40 mm x 4.6 mm, 10 microm) is evaluated in both backflush and foreflush elution modes and coupled with a C-18 lab-made (50 mm x 4.6 mm, 3 microm) analytical column in order to perform online sample preparation. Direct injection of 100 microL of plasma samples is possible with the developed approach. In addition, reduction of sample handling is obtained when compared with traditional liquid-liquid extraction (LLE) and SPE. The total analysis time is around 20 min. A LOQ of 15 ng/mL is achieved in a concentration range of 15-500 ng/mL, allowing the therapeutic drug monitoring of clinical samples. The precision values achieved are lower than 15% for all the evaluated points with adequate recovery and accuracy. Furthermore, no matrix interferences are found in the analysis and the proposed method shows to be an adequate alternative for analysis of FLU in plasma.