Validation of a multiplexed and targeted lipidomics assay for accurate quantification of lipidomes.

A major challenge of lipidomics is to determine and quantify the precise content of complex lipidomes to the exact lipid molecular species. Often, multiple methods are needed to achieve sufficient lipidomic coverage to make these determinations. Multiplexed targeted assays offer a practical alternative to enable quantitative lipidomics amenable to quality control standards within a scalable platform. Herein, we developed a multiplexed normal phase liquid chromatography-hydrophilic interaction chromatography multiple reaction monitoring method that quantifies lipid molecular species across over 20 lipid classes spanning wide polarities in a single 20-min run. Analytical challenges such as in-source fragmentation, isomer separations, and concentration dynamics were addressed to ensure confidence in selectivity, quantification, and reproducibility. Utilizing multiple MS/MS product ions per lipid species not only improved the confidence of lipid identification but also enabled the determination of relative abundances of positional isomers in samples. Lipid class-based calibration curves were applied to interpolate lipid concentrations and guide sample dilution. Analytical validation was performed following FDA Bioanalytical Method Validation Guidance for Industry. We report repeatable and robust quantitation of 900 lipid species measured in NIST-SRM-1950 plasma, with over 700 lipids achieving inter-assay variability below 25%. To demonstrate proof of concept for biomarker discovery, we analyzed plasma from mice treated with a glucosylceramide synthase inhibitor, benzoxazole 1. We observed expected reductions in glucosylceramide levels in treated animals but, more notably, identified novel lipid biomarker candidates from the plasma lipidome. These data highlight the utility of this qualified lipidomic platform for enabling biological discovery.

A chiral porous organic polymer COP-1 used as stationary phase for HPLC enantioseparation under normal-phase and reversed-phase conditions.

A spherical chiral porous organic polymer (POPs) COP-1 is synthesized by the Friedel-Crafts alkylation reaction of Boc-3-(4-biphenyl)-L-alanine (BBLA) and 4,4'-bis(chloromethyl)-1,1'-biphenyl (BCMBP), which was used as a novel chiral stationary phase (CSPs) for mixed-mode high-performance liquid chromatography (HPLC) enantioseparation. The racemic compounds were resolved in normal-phase liquid chromatography (NPLC) using n-hexane/isopropanol as mobile phase and reversed-phase liquid chromatography (RPLC) using methanol/water as mobile phase. The COP-1-packed column exhibited excellent separation performance toward various racemic compounds including alcohols, amines, ketones, esters, epoxy compounds, organic acids, and amino acids in NPLC and RPLC modes. The effects of analyte mass and column temperature on the separation efficiency of racemic compounds were investigated. In addition, the chiral resolution ability of the COP-1-packed column not only can be complementary in RPLC/NPLC modes but also exhibit a good chiral recognition complementarity with Chiralpak AD-H column and chiral porous organic cage (POC) NC1-R column. The relative standard deviations (RSD) (n = 5) of the retention time, resolution value, and peak area by repeated separation of 1-(4-chiorophenyl)ethanol are all below 3.0%. The COP-1 column shows high column efficiency (e.g., 17,320 plates m-1 for 1-(4-chlorophenyl)ethanol on COP-1 column in NPLC), high enantioselectivity, and good reproducibility toward various racemates. This work demonstrates that chiral POPs microspheres are promising chiral materials for HPLC enantioseparation.

Normal-phase liquid chromatography retention behavior of polycyclic aromatic sulfur heterocycles and alkyl-substituted polycyclic aromatic sulfur heterocycle isomers on an aminopropyl stationary phase.

Retention indices for 67 polycyclic aromatic sulfur heterocycles (PASHs) and 80 alkyl-substituted PASHs were determined using normal-phase liquid chromatography (NPLC) on an aminopropyl (NH2) stationary phase. The retention behavior of PASH on the NH2 phase is correlated with the number of aromatic carbon atoms and two structural characteristics have a significant influence on their retention: non-planarity (thickness, T) and the position of the sulfur atom in the bay-region of the structure. Correlations between solute retention on the NH2 phase and T of PASHs were investigated for three cata-condensed (cata-) PASH isomer groups: (a) 13 four-ring molecular mass (MM) 234 Da cata-PASHs, (b) 20 five-ring MM 284 Da cata-PASHs, and (c) 12 six-ring MM 334 Da cata-PASHs. Correlation coefficients ranged from r = -0.49 (MM 234 Da) to r = -0.65 (MM 334 Da), which were significantly lower than structurally similar PAH isomer groups (r = -0.70 to r = -0.99). The NPLC retention behavior of the PASHs are compared to similar results for PAHs.

Qualitative characterization of three combustion-related standard reference materials for polycyclic aromatic sulfur heterocycles and their alkyl-substituted derivatives via normal-phase liquid chromatography and gas chromatography/mass spectrometry.

The research described here provides the most comprehensive qualitative characterization of three combustion-related standard reference materials (SRMs) for polycyclic aromatic sulfur heterocycles (PASHs) and some alkyl-substituted (alkyl-) derivatives to date: SRM 1597a (coal tar), SRM 1991 (coal tar/petroleum extract), and SRM 1975 (diesel particulate extract). An analytical approach based on gas chromatography/mass spectrometry (GC/MS) is presented for the determination of three-, four-, and five-ring PASH isomers and three- and four-ring alkyl-PASHs in the three SRM samples. The benefit of using a normal-phase liquid chromatography (NPLC) fractionation procedure prior to GC/MS analysis was demonstrated for multiple isomeric PASH groups. Using a semi-preparative aminopropyl (NH2) LC column, the three combustion-related samples were fractionated based on the number of aromatic carbon atoms. The NPLC-GC/MS method presented here allowed for the following identification breakdown: SRM 1597a - 35 PASHs and 59 alkyl-PASHs; SRM 1991-31 PASHs and 58 alkyl-PASHs; and SRM 1975-13 PASHs and 25 alkyl-PASHs. These identifications were based on NPLC retention data, the GC retention times of reference standards, and the predominant molecular ion peak in the mass spectrum. Prior to this study, only 11, 1, and 0 PASHs/alkyl-PASHs had been identified in SRM 1597a, SRM 1991, and SRM 1975, respectively. Graphical abstract NPLC-GC/MS analysis for the three- and four-ring parent PASH isomers in SRM 1597a.

Normal-phase liquid chromatography retention behavior of polycyclic aromatic hydrocarbon and their methyl-substituted derivatives on an aminopropyl stationary phase.

Retention indices for 124 polycyclic aromatic hydrocarbons (PAHs) and 62 methyl-substituted (Me-) PAHs were determined using normal-phase liquid chromatography (NPLC) on a aminopropyl (NH2) stationary phase. PAH retention behavior on the NH2 phase is correlated to the total number of aromatic carbons in the PAH structure. Within an isomer group, non-planar isomers generally elute earlier than planar isomers. MePAHs generally elute slightly later but in the same region as the parent PAHs. Correlations between PAH retention behavior on the NH2 phase and PAH thickness (T) values were investigated to determine the influence of non-planarity for isomeric PAHs with four to seven aromatic rings. Correlation coefficients ranged from r = 0.19 (five-ring peri-condensed molecular mass (MM) 252 Da) to r = -0.99 (five-ring cata-condensed MM 278 Da). In the case of the smaller PAHs (MM ≤ 252 Da), most of the PAHs had a planar structure and provided a low correlation. In the case of larger PAHs (MM ≥ 278 Da), nonplanarity had a significant influence on the retention behavior and good correlation between retention and T was obtained for the MM 278 Da, MM 302 Da, MM 328 Da, and MM 378 Da isomer sets. Graphical abstract NPLC separation of the three-, four-, five-, and six-ring PAH isomers with different number of aromatic carbon atoms and degrees of non-planarity (Thickness, T). The inserted figure plots the number of aromatic carbon atoms vs. the log I value for the 124 parent PAHs.

Qualitative characterization of SRM 1597a coal tar for polycyclic aromatic hydrocarbons and methyl-substituted derivatives via normal-phase liquid chromatography and gas chromatography/mass spectrometry.

A normal-phase liquid chromatography (NPLC) fractionation procedure was developed for the characterization of a complex mixture of polycyclic aromatic hydrocarbons (PAHs) from a coal tar sample (Standard Reference Material (SRM) 1597a). Using a semi-preparative aminopropyl (NH2) LC column, the coal tar sample was separated using NPLC based on the number of aromatic carbons; a total of 14 NPLC fractions were collected. SRM 1597a was analyzed before and after NPLC fractionation by using gas chromatography/mass spectrometry (GC/MS) with a 50% phenyl stationary phase. The NPLC-GC/MS method presented in this study allowed for the identification of 72 PAHs and 56 MePAHs. These identifications were based on the NPLC retention times for authentic reference standards, GC retention times for authentic reference standards, and the predominant molecular ion peak in the mass spectrum. Most noteworthy was the determination of dibenzo[a,l]pyrene, which could not be measured directly by GC/MS because of low concentration and co-elution with dibenzo[j,l]fluoranthene. The NPLC-GC/MS procedure also allowed for the tentative identification of 74 PAHs and 117 MePAHs based on the molecular ion peak only. This study represents the most comprehensive qualitative characterization of SRM 1597a to date. Graphical abstract NPLC-GC/MS analysis for the six-ring MM 302 Da PAH isomers in SRM 1597a.

Identification and quantification of six-ring C26H16 cata-condensed polycyclic aromatic hydrocarbons in a complex mixture of polycyclic aromatic hydrocarbons from coal tar.

We applied a combination of normal-phase liquid chromatography (NPLC) with ultraviolet-visible spectroscopy and gas chromatography with mass spectrometry (GC/MS) for the fractionation, identification, and quantification of six ring C26H16 cata-condensed polycyclic aromatic hydrocarbons, PAHs, in the Standard Reference Material 1597a, Complex Mixture of PAHs from Coal Tar. For the characterization analysis, we calculated the GC retention indices of 17 C26H16 PAH authentic reference standards using the Rxi-PAH and DB-5 GC columns. Then, we used NPLC with ultraviolet-visible spectroscopy to isolate the fractions containing the C26H16 PAHs, and subsequently, we used GC/MS to establish the identity and quantity of the C26H16 PAHs using authentic reference standards. Following this procedure, 12 C26H16 cata-condensed PAHs benzo[c]pentaphene, dibenzo[f,k]tetraphene, benzo[h]pentaphene, dibenzo[a,l]tetracene, dibenzo[c,k]tetraphene, naphtho[2,3-c]tetraphene, dibenzo[a,c]tetracene, benzo[b]picene, dibenzo[a,j]tetracene, naphtho[2,1-a]tetracene, dibenzo[c,p]chrysene, and dibenzo[a,f]tetraphene were identified and quantified for the first time, and benzo[c]picene was quantified for the first time in an environmental combustion sample.

Chiral separability of boron cluster species studied by screening approaches utilizing polysaccharide-based chiral stationary phases.

In this study, the screening steps of chiral separation strategies with polysaccharide-based chiral stationary phases were applied on boron cluster compounds in normal-phase liquid chromatography (NPLC) and polar organic solvents chromatography (POSC). Since the screening steps were initially developed to analyze organic compounds, their applicability for boron clusters was investigated. Overall, the screening steps in NPLC were applicable for the separation of zwitterions, while for anions mostly no elution was observed. A hypothesis for the latter behavior is precipitation of anions in the nonpolar mobile phases. Ten out of 11 compounds could be partially or baseline separated on the NPLC screening systems. In POSC, all zwitterions were separated on at least one of the screening systems, with an overall lower retention as in NPLC. Anions were detected but not separated in the majority of the experiments. Also their retention on the chiral stationary phases was very limited. This study showed that the chiral discrimination potential of chemically modified polysaccharides is meaningful for chiral separations of structurally chiral boron cluster species, but needs further systematic research, in which recognition mechanisms should be further explored. In addition, some unusual peaks also indicated that conditions with a high separation efficiency must first be searched for some of the tested systems.

Lipid Readjustment in Yarrowia lipolytica Odd-Chain Fatty Acids Producing Strains.

Yarrowia lipolytica is a promising oleaginous yeast for producing unusual lipids, such as odd-chain fatty acids (OCFA). Their diverse applications and low natural production make OCFA particularly interesting. In recent studies, inhibiting the catabolic pathway of precursor, boosting precursor pools, and optimizing substrate combination greatly improved the production of OCFA in Y. lipolytica. We explored the lipid readjustment of OCFA in engineered Y. lipolytica strains. NPLC-Corona-CAD® evidenced a time-dependent overproduction of free fatty acids, diglycerides, and phosphatidylcholine (PC) in obese LP compared to obese L. Phosphatidylethanolamine (PE) and phosphatidylinositol, largely overproduced in obese LP at 72 h compared to obese L, vanished at 216 h. The fatty acyls (FAs) composition of glycero- and glycerophospholipids was determined by NPLC-APPI+-HRMS from in-source generated monoacylglycerol-like fragment ions. C18:1 and C17:1 were predominant acylglycerols in obese L and obese LP, respectively. Phosphatidic acid, PE, and PC exhibited similar FAs composition but differed in their molecular species distributions. Cardiolipin (CL) is known to contain mostly C18:2 FAs corresponding to the composition in obese L, 50% of C18:2, and 35% of C18:1. In obese LP, both FAs dropped to drop to 20%, and C17:1 were predominant, reaching 55%. We hypothesize that CL-modified composition in obese LPs may alter mitochondrial function and limit lipid production.

Determination of total and unbound ribociclib in human plasma and brain tumor tissues using liquid chromatography coupled with tandem mass spectrometry.

Ribociclib is a selective, orally bioavailable inhibitor of cyclin-dependent kinase (CDK) 4/6, which has therapeutic potential for a variety of cancer types. This study was to develop and validate a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for determining total and unbound concentrations of ribociclib in human plasma and brain tumor tissue samples. Plasma and tumor homogenate samples were extracted using protein precipitation with acetonitrile. Unbound fraction in plasma or tumor homogenate was determined by equilibrium dialysis method. Chromatographic separation was achieved based on aqueous normal-phase chromatography mechanism on a Waters XBridge™ Amide column under isocratic elution with acetonitrile-ammonium formate (10 mM, pH 3) (75:25, v/v) at a flow rate of 0.8 mL/min. Ribociclib and the internal standard ([13C6]ribociclib) were monitored at the mass transitions m/z, 435.3 > 367.2 and 441.3 > 373.2, respectively, using positive electrospray ionization mode. The lower limit of quantitation (LLOQ) was 0.5 nM of ribociclib in plasma. Linear calibration curve was established at the concentration range of 0.5-1000 nM in plasma. Intra- and inter-day precision and accuracy were within the generally accepted criteria for bioanalytical method. The developed method was successfully applied to determine the plasma pharmacokinetics and central nervous system penetration of ribociclib in patients with malignant primary brain cancer.

Linear solvation energy relationship (LSER) characterization of the normal phase retention mechanism on hypercrosslinked polystyrenes.

Linear solvation energy relationships (LSERs) were applied to retention on hypercrosslinked polystyrene on silica (HC-Tol) to elucidate the type and relative importance of molecular interactions between model solutes and the HC-Tol stationary phase. Classical amino phase and another hypercrosslinked phase (5-HGN) were used as reference columns. On both the HC-Tol and amino, polar interactions predominate and contribute to retention. Solute volume V has no impact on retention on the amino column, while V has a slightly negative influence on retention for the HC-Tol column. The differences in coefficient v between the amino and the HC-Tol columns might explain why the HC-Tol is capable of group-type separations. 5-HGN phase has smaller a and b values compared to HC-Tol, which means that 5-HGN is not as basic or acidic in terms of hydrogen bonds as is HC-Tol. This suggests that the hydrogen bonding character of the HC-Tol phase arises from its silica substrate.

Liposomes as potential masking agents in sport doping. Part 1: analysis of phospholipids and sphingomyelins in drugs and biological fluids by aqueous normal-phase liquid chromatography-tandem mass spectrometry.

In the present work, aqueous normal-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), in different acquisition modes, was employed for the direct analysis and profiling of nine phospholipid classes (phosphatidic acids, phosphatidylserines, phosphatidylethanolamines, lysophosphatidylethanolamines, phosphatidylglycerols, phosphatidylinositols, phosphatidylcholines, lysophosphatidylcholines, and sphingomyelins) in biological and pharmaceutical matrices. After chromatographic separation by a diol column, detection and elucidation of phospholipid and sphingomyelin classes and molecular species were performed by different scan acquisition modes. For screening analysis, molecular ions [M + H]+ were detected in positive precursor ion scan of m/z 184 for the classes of phosphatidylcholines, lyso-phosphatidylcholines and sphingomyelins; while phosphatidylethanolamines and lyso-phosphatidylethanolamines were detected monitoring neutral loss scan of 141 Da; and phosphatidylserines detected using neutral loss scan of 184 Da. Molecular ions [M-H]- were instead acquired in negative precursor ion scan of m/z 153 for the classes of phosphatidic acids and phosphatidylglycerols; and of m/z 241 for the phosphatidylinositols. For the identification of the single molecular species, product ion scan mass spectra of the [M + HCOO]- ions for phosphatidylcholines and [M + H]+ ions for the other phospholipids considered were determined for each class and compared with the fragmentation pattern of model phospholipid reference standard. By this approach, nearly 100 phospholipids and sphingomyelins were detected and identified. The optimized method was then used to characterize the phospholipid and sphingomyelin profiles in human plasma and urine samples and in two phospholipid-based pharmaceutical formulations, proving that it also allows to discriminate compounds of endogenous origin from those resulting from the intake of pharmaceutical products containing phospholipidic liposomes. Copyright © 2016 John Wiley & Sons, Ltd.

Optimization of normal phase chromatographic conditions for lipid analysis and comparison of associated detection techniques.

One important challenge in lipid class analysis is to develop a method suitable or, at least adaptable, for a vast diversity of samples. In the current study, an improved normal-phase liquid chromatography (NPLC) method allowed analyzing the lipid classes present in mammalian, vegetable as well as microorganism (yeast and bacteria) lipid samples. The method effectively separated 30 lipid classes or subclasses with a special focus on medium polarity lipids. The separation was carried out with bare silica stationary phase and was coupled to evaporative light scattering detection (ELSD), charged aerosol detection (Corona-CAD®) and mass spectrometry. Solutions are provided to circumvent technical issues (such as pumping solvents of low viscosity, solvent purity, rinsing step). The influence of mobile phase composition and addition of ionic modifiers on the chromatographic behavior of particular lipid classes is documented. A comparison between ELSD and Corona-CAD® confirmed the interest of this later detector for samples with a wide range of concentration of different lipids. Three common atmospheric pressure ionization interfaces were used for coupling the NPLC separation to a LTQ Velos Pro® mass spectrometer. The comparison of the chromatographic profiles showed that atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) are both suitable to detect the different lipid classes whereas APPI allows a better sensitivity for lipids at low-concentration.

Synthesis and performance of chiral ferrocene modified silica gel for mixed-mode chromatography.

A brush-type chiral stationary phase, N-ferrocenyl benzoyl-(1S, 2R)-1, 2-diphenyl ethanol-bonded on the silica gel (NFcBEs) for high performance liquid chromatography (HPLC), was prepared using γ-glycidoxypropyltrimethoxysilane as coupling reagent. The structure of this novel material was characterized by infrared spectroscopy, elemental analysis and thermogravimetric analysis. Mechanism involved in the chromatographic separation is the multi-interaction including hydrophobic, π-π, hydrogen-bonding, π-charge transfer, dipole-dipole and acid-base equilibrium interactions. Based on these interactions, successful separation could be achieved among polycyclic aromatic hydrocarbons, mono-substituted benzenes, aromatic amines, quinolines, nucleosides, phenols and 5-nitroimidazoles drugs in reversed phase liquid chromatography (RPLC). Good resolutions for substituted amine isomers were also obtained with NFcBEs. Racemates of amino acids and drug carvedilol mixtures were well separated on NFcBEs in the normal phase liquid chromatography (NPLC) mode. Such stationary phase with characteristics of multi-interaction mechanism and mixed-mode separation is potential for the analysis of complex samples. The retention behaviors of R- and S-carvedilol on NFcBEs column were investigated with the assistance of quantum chemistry calculation using the density functional theory (DFT) B3LYP method.

Comprehensive two-dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography for analysis of toad skin.

An analytical two-dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography (2D NPLC × RPLC) system was constructed with a newly developed thermal evaporation assisted adsorption (TEAA) interface. This novel TEAA interface with heating temperature above solvent boiling point allowed fast removal of organic NPLC solvent and successfully solved the solvent incompatibility problem between NPLC and RPLC. The system achieved rapid on-line solvent exchange between the two dimensions within a short modulation time of 190 s and was applied in the analysis of an extract from the skin of Bufo bufo gargarizans. This is the first time to realize the on-line comprehensive analysis of a moderate polar natural product by coupling NPLC with reversed phase ultra-high performance liquid chromatography (UHPLC). To be highlighted, with the TEAA interface, the 2D NPLC × RPLC system provided excellent resolution and orthogonality (75.2%), when compared with that of 2D RPLC × RPLC.

Linear solvation energy relationships in normal phase chromatography based on gradient separations.

Coupling the modified Soczewiñski model and one gradient run, a gradient method was developed to build a linear solvation energy relationship (LSER) for normal phase chromatography. The gradient method was tested on dinitroanilinopropyl (DNAP) and silica columns with hexane/dichloromethane (DCM) mobile phases. LSER models built based on the gradient separation agree with those derived from a series of isocratic separations. Both models have similar LSER coefficients and comparable goodness of fit, but the LSER model based on gradient separation required fewer trial and error experiments.

Investigation of the temperature dependence of water adsorption on silica-based stationary phases in hydrophilic interaction liquid chromatography.

In the present work, the adsorption of water was investigated in aqueous normal-phase liquid chromatography on Cogent Silica C and Cogent Phenyl hydride stationary phases at different temperatures by frontal analysis - using coulometric Karl Fischer titration - to compare the temperature dependence of adsorption of water from aqueous acetonitrile. The Cogent Silica-C and Cogent Phenyl Hydride columns have a silicon hydride surface (silica hydride) with less than 2% free silanol group; therefore, they do not have a strong association with water. The adsorption behavior of water on the mentioned stationary phases was modeled by Langmuir isotherm. The preferentially adsorbed water was expressed in terms of a hypothetical monomolecular water layer equivalent in the inner pores. The uptake of water slightly depends on the temperature. The adsorbed water may fill four to eight percent of the pore volume over the studied temperature range, which approximately corresponds to the equivalent of 0.24-0.68 water layer coverage of the adsorbent surface. The phenyl hydride stationary phase shows decreased water uptake in comparison to the Silica C stationary phase.

Alternative solvents for improving the greenness of normal phase liquid chromatography of lipid classes.

An evaluation of solvents alternative to n-heptane (d-limonene and hexamethyldisiloxane) and chloroform (cyclopentyl methyl ether, 2-methyltetrahydrofuran and isopentyl acetate) was developed for lipid classes separation of non-polar cholesteryl ester to highly polar phospholipids by high-performance liquid chromatography on bare silica stationary phase and evaporative light-scattering detection. Screening of alternative solvents was used to estimate their compatibility with liquid chromatography and evaporative light-scattering detection and to evaluate their chromatographic selectivity. This work shows that n-heptane can be advantageously replaced by hexamethyldisiloxane. An increase of non-polar lipids retention is observed with hexamethyldisiloxane as weak solvent. Chloroform, which is largely used for lipid analysis, might be replaced efficaciously by cyclopentyl methyl ether, 2-methyltetrahydrofuran or isopentyl acetate. Aside from offering a different selectivity, the gradients composed by one or both alternative solvents gave efficient and comparable or even better separations than those obtained with conventional solvents.

Separation and characterization of bufadienolides in toad skin using two-dimensional normal-phase liquid chromatography×reversed-phase liquid chromatography coupled with mass spectrometry.

Bufadienolides possess various bioactivities especially antitumor. Due to the high structural diversity, the separation of bufadienolides often suffers from coelution problem on conventional RP columns. In this work, an off-line two-dimensional normal-phase liquid chromatography×reversed-phase liquid chromatography (2D-NPLC×RPLC) method was developed to separate and characterize bufadienolides in toad skin. Several RP and NP columns were evaluated with five reference bufadienlides. The XUnion C18 and XAmide columns exhibited superior chromatographic performances for bufadienlide separation, and were selected in RPLC and NPLC, respectively. RPLC was used in the second-dimension for the good compatibility with MS, while NPLC was adopted in the first-dimension. The orthogonality of the 2D-NPLC×RPLC system was investigated by the geometric approach using fifteen bufadienolide mixtures. The result was 49.6%, demonstrating reasonable orthogonality of this 2D-LC system. By combining the 2D-LC system with MS, 64 bufadienlides including 33 minor ones and 11 pairs of isomers in toad skin were identified. This off-line 2D-NPLC×RPLC allowed to solve the coelution problem of bufadienlides in one-dimension RPLC, and thus facilitated the identification significantly.

On-line comprehensive two-dimensional normal-phase liquid chromatography×reversed-phase liquid chromatography for preparative isolation of toad venom.

An on-line comprehensive preparative two-dimensional normal-phase liquid chromatography×reversed-phase liquid chromatography (2D NPLC×RPLC) system was constructed with a newly developed vacuum evaporation assisted adsorption (VEAA) interface, allowing fast removal of NPLC solvent in the vacuum condition and successfully solving the solvent incompatibility problem between NPLC and RPLC. The system achieved on-line solvent exchange within the two dimensions and its performance was illustrated by gram-scale isolation of crude extract from the venom of Bufo bufo gargarizans. Within separation time of ∼20h, 19 compounds were obtained with high purity in a single run. With the VEAA interface, the 2D system exhibited apparent advantages in separation efficiency and automation compared with conventional methods, indicating its promising application in the routine separation process for complicated natural products.