2D Separation Research Articles

Charge variant analysis of protein-based biopharmaceuticals using two-dimensional liquid chromatography hyphenated to mass spectrometry

The profile of charge variants represents an important critical quality attribute of protein-based biopharmaceuticals, in particular of monoclonal antibodies, and must therefore becontrolled. In this work, 2D-LC methods for charge variant analysis were developed using a strong cation-exchange chromatography (SCX) as first dimension (1D) separation. Non-porous SCX (3 µm) particle columns and different mobile phases were evaluated using a test mixture of some standard proteins of different size and pI (comprising myoglobin, bovine serum albumin, cytochrome c, lysozyme and β-lactoglobulin) and two monoclonal IgG1 antibodies (NIST mAb and Secukinumab). The most promising 1D eluent for SCX was a salt-mediated pH-gradient system using a ternary mobile phase system with 2-(N-morpholino)ethanesulfonic acid, 1,3-diamino-2-propanol and sodium chloride. For the second dimension (2D), a desalting reversed-phase liquid chromatography (RP-LC) was chosen to enable the hyphenation of the charge variant separation with mass spectrometric (MS) detection. While for intact mAbs the 2D just served for desalting without additional selectivity, the 2D contributed some orthogonal selectivity for the mAb fragment separation. Various core-shell and monolithic columns were tested and variables such as gradient time and flow rate systematically optimized. Unexpectedly, a C4 400 Å column (3.4 µm diameter with 0.2 µm porous shell) provided higher peak capacities compared to the same 1000 Å column (2.7 µm diameter with 0.5 µm porous shell). A thinner shell appeared to be more advantageous than wider pores under high flow regime. An ultra-fast RP-LC method with a run time of one minute was developed using trifluoroacetic acid which was later replaced by formic acid as additive for better MS compatibility. The successful hyphenation of the two orthogonal separation modes, SCX and RP-LC, could be demonstrated in the multiple heart-cutting and the full comprehensive mode. MS analysis using a high-resolution quadrupole time-of-flight instrument enabled to identify different glycoforms and some major charge variants of the antibody at the intact protein level as well as on the subunit level (Fc/2, Lc, Fd’) in a middle-up approach by 2D-LC-ESI-MS analysis.

Validation of Extended Statistical Overlap Theory for Application to Small Second Dimension Peak Capacity

Approximate equations for the numbers of singlet peaks and total peaks in two-dimensional (2D) separations of randomly distributed component zones predict values of zero as the second-dimension peak capacity 2nc goes to zero. This is the result of deriving the equations for an unbounded 2D space. More rigorous equations are needed to assess how large 2nc must be before the error in the approximate equations is acceptably small. An exact equation for the number of singlet peaks in a 2D separation with a bounded second dimension is derived in Online Resource 1. The equation accounts for the reduced likelihood of overlap of zones represented as circles or ellipses near the second-dimension boundaries and is the result of the lengthy derivation of different expressions of probability, with the appropriate expression depending on the value of 2nc. In this paper, the equation is used to modify the prediction of the Roach equation for the number of total peaks in the 2D separation. Computer simulations of the clustering of circles confirm the equation for singlet peaks over a wide range of first-dimension saturations α1D (0.05 ≤ α1D ≤ 5) and 2nc values (0.1 ≤ 2nc ≤ 15). The saturation α1D, equal to the ratio of the number of components requiring separation to the first-dimension peak capacity available for separation, is a metric of chromatographic crowdedness. Over the same α1D range, the equation for total peaks slightly underestimates the simulations when 2nc < 2.5 but agrees with them at larger 2nc. It is shown that the extended theory differs most from the approximate one when α1D is large and 2nc is small.

Comprehensive two-dimensional liquid chromatography as a biomimetic screening platform for pharmacokinetic profiling of compound libraries in early drug development

A comprehensive two-dimensional liquid chromatography-based biomimetic platform (LCxLC) has been developed and validated for drug diffusion studies. Human serum albumin (HSA) and immobilized artificial membrane (IAM) were thereby used in the first (1D) and second (2D) separation dimension, respectively. While the former was meant to emulate the blood, the latter was instead intended to mimic the intestinal mucosa epithelium. Therefore, the experimental conditions, i.e. pH, temperature and buffer composition, were modulated to reflect faithfully in vivo conditions. 30 compounds, whose effective intestinal permeability (Peff) assayed in situ on humans by a validated technique was known from the literature, were used as model drugs.A good and orthogonal separation was achieved for the whole dataset, although for a better distribution of the most polar compounds in the elution window a segmented gradient elution program had to be employed. Interestingly, the passively uptaken compounds having the most favourable Peff populated a specific area of the 2D plots, implying that the affinity for HSA and IAM has to lie in specific ranges in order for a compound to be satisfactorily absorbed from the intestinal lumen.Although these results should be regarded as preliminary, this work paves an entirely new and unprecedented way to profile pharmaceutically relevant compounds for their in vivo absorption and distribution potential.

Identification of proteins associated with development of metastasis from cutaneous squamous cell carcinomas (cSCCs) via proteomic analysis of primary cSCCs.

Cutaneous squamous cell carcinoma (cSCC) is one of the most common cancers capable of metastasizing. Proteomic analysis of cSCCs can provide insight into the biological processes responsible for metastasis, as well as future therapeutic targets and prognostic biomarkers. To identify proteins associated with development of metastasis in cSCC. A proteomic-based approach was employed on 105 completely excised, primary cSCCs, comprising 52 that had metastasized (P-M) and 53 that had not metastasized at 5years post-surgery (P-NM). Formalin-fixed, paraffin-embedded cSCCs were microdissected and subjected to proteomic profiling after one-dimensional (1D), and separately two-dimensional (2D), liquid chromatography fractionation. A discovery set of 24 P-Ms and 24 P-NMs showed 144 significantly differentially expressed proteins, including 33 proteins identified via both 1D and 2D separation, between P-Ms and P-NMs. Several differentially expressed proteins were also associated with survival in SCCs of other organs. The findings were verified by multiple reaction monitoring on six peptides from two proteins, annexin A5 (ANXA5) and dolichyl-diphosphooligosaccharide-protein glycosyltransferase noncatalytic subunit (DDOST), in the discovery group and validated on a separate cohort (n=57). Increased expression of ANXA5 and DDOST was associated with reduced time to metastasis in cSCC and decreased survival in cervical and oropharyngeal cancer. A prediction model using ANXA5 and DDOST had an area under the curve of 0·93 (confidence interval 0·83-1·00), an accuracy of 91·2% and higher sensitivity and specificity than cSCC staging systems currently in clinical use. This study highlights that increased expression of two proteins, ANXA5 and DDOST, is significantly associated with poorer clinical outcomes in cSCC.

A systematic investigation of comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry with dynamic pressure gradient modulation for high peak capacity separations

Dynamic pressure gradient modulation (DPGM) in full modulation mode is optimized for comprehensive two-dimensional (2D) gas chromatography (GC × GC) with time-of-fight mass spectrometry (TOFMS) detection to obtain high peak capacity separations and demonstrate broad applicability for complex samples. A pulse valve introduces an auxiliary carrier gas flow at a T-union connecting the first dimension (1D) column to the second dimension (2D) column. At a sufficiently high auxiliary pressure (Paux) the 1D flow is temporarily stopped. Then, during each modulation period (PM) the valve is turned off briefly, a period termed the pulse width (pw), allowing the 1D effluent to essentially be reinjected onto the 2D column for the modulated separations. Modifications to the modulator assembly are provided to improve performance. Method optimization is demonstrated for a 116-component test mixture by tuning the Paux and the pw. For a PM = 2 s and 1F of 0.10 ml/min, the optimal pw and initial Paux selected were 200 ms and 330.9 kPa (33 psig), respectively. The 30 min separation of the test mixture provided a 1D peak capacity of 1nc = 330 and a 2D peak capacity of 2nc = 15, hence an ideal 2D peak capacity nc,2D = 1nc × 2nc = 4950. Likewise, the 2D peak capacity corrected for undersampling of the 1D separation was 4500 and corrected for both undersampling and sampling variation via statistical overlap theory was 4090. These results provide a 2-fold improvement in peak capacity relative to the previous DPGM study in full modulation mode for GC × GC-TOFMS. The optimized conditions were applied for a variety of applications: diesel fuel, derivatized cow serum, solid phase microextraction (SPME) of coffee headspace, and SPME of river water headspace. Additionally, the fraction of 2D separation space utilized (fcoverage), as defined by the minimum convex hull method, ranged from 0.60 to 0.85. We observed that any fcoverage correction to 2D peak capacity is highly sample dependent, since all samples, except for the diesel sample, were run with the same separation conditions, and yet the fcoverage ranged from 0.60 to 0.80.

Multidimensional gas chromatographic‒Mass spectrometric method for separation and identification of triacylglycerols in olive oil.

A 'heart-cut' multidimensional gas chromatography‒mass spectrometry (H/C MDGC‒MS) method for separation and identification of triacylglycerols (TAGs) in extra virgin olive oil was developed. A GC configuration, comprising a non-polar first dimension (1D) column (15 m length) and a mid-polarity second dimension (2D) column (9 m length), was employed. Standard TAGs were used to test and demonstrate the H/C MDGC method, for identification of TAG components and to validate the method. Various chromatographic conditions such as column flow and temperature program were evaluated. The 1D separation resulted in overlap of some standard TAG peaks. These overlapped 1D regions of the standard TAGs were H/C to 2D for further separation and resulted in clearly distinguished individual TAG component peaks. The 1D separation of olive oil TAGs displayed three major peaks and four minor peaks. The application of the H/C MDGC method to olive oil TAGs resulted in the separation of each sampled 1D region into two or more TAG peaks. TAG components in olive oil resolved on the 2D column were identified based on characteristic mass fragment ions such as [M-RCO2]+, [RCO+128]+, [RCO+74]+ and RCO+ and comparison of their mass spectra with that of the standard TAGs. Sixteen olive oil TAGs were identified by MS after 2D separation. The repeatability of the H/C method was evaluated in terms of retention time shift and area response in the 2D and found to be <0.02% and <8% RSD respectively.

Comprehensive Two-Dimensional Gas Chromatography with Mass Spectrometry: Toward a Super-Resolved Separation Technique

A data interpretation and processing approach for improved compound identification and data presentation in comprehensive two-dimensional gas chromatography (GC×GC) is described. A footprint peak of a compound in 2D space can be represented by a centroid or peak apex, similar to the data-reduced histogram spectra used in mass spectrometry. The workflow was demonstrated on data from GC×GC-TOFMS. Peaks in a modulated chromatogram were initially detected by conventional chromatographic integration, followed by a curve-fitting approach, which interpolated high-precision, absolute retention times for all modulated peaks. First dimension retention time (1tR) was obtained by using an exponentially modified Gaussian (EMG) fitting model for near-Gaussian distributed subpeaks, polynomial fitting for highly asymmetrical peaks, and parabolic fitting for under-sampled peaks, which allows determination of a precise 1tR, considering the dwell-time arising from modulation and 2tR. Area summation of the modulated peaks belonging to the same compound was then performed to yield the total peak area. Each compound in the GC×GC-MS result was then represented by its position at the intersecting coordinates, (1tR, 2tR), in the 2D separation plane, having a height of the same magnitude as the total component summed area. This results in a novel and uncluttered GC×GC output convention based on the scripted total ion chromatogram (TIC) data with precise 1tR, 2tR, and area. Comparison between the contour plots from the scripted and conventional TIC revealed improved data presentation, accompanied by an apparent enhanced resolution. The described approach was applied to the identification of 177 aroma compounds from peaches as indicators of fruit quality.

The Morphology–Density Relationship in 1 &lt; z &lt; 2 Clusters

Abstract The morphology–density relationship states that dense cosmic environments such as galaxy clusters have an overabundance of quiescent elliptical galaxies, but it is unclear at which redshift this relationship is first established. We study the morphology of four clusters with 1.2 &lt; z &lt; 1.8 using Hubble Space Telescope imaging and the morphology computation code statmorph. By comparing the median morphology of cluster galaxies to CANDELS field galaxies using Monte Carlo analysis, we find that two out of four clusters (at z = 1.19 and 1.75) have an established morphology–density relationship with more than 3σ significance. Approximately 50% of the galaxies in these clusters are bulge-dominated, compared to ∼30% in the field, and they are significantly more compact. This result is more significant for low-mass galaxies with , showing that low-mass galaxies are affected the most in clusters. We also find an intriguing system of two z ≈ 1.45 clusters at a unusually small 2D separation of 3′ and 3D separation of ≈73 Mpc that exhibit no morphology–density relationship but have enhanced merger signatures. We conclude that the environmental mechanism responsible for the morphology–density relationship is (1) already active as early as z = 1.75; (2) forms compact, bulge-dominated galaxies; and (3) affects primarily low-mass galaxies. However, there is a significant degree of intracluster variance that may depend on the larger cosmological environment in which the cluster is embedded.

A selective comprehensive reversed-phase×reversed-phase 2D-liquid chromatography approach with multiple complementary detectors as advanced generic method for the quality control of synthetic and therapeutic peptides.

There is a huge, still increasing market for synthetic and therapeutic peptides. Their quality control is commonly based on a generic reversed-phase liquid chromatography (RPLC) method with C18 stationary phase and acetonitrile gradient with 0.1% trifluoroacetic acid in the mobile phase. It performs exceptionally well for a wide variety of impurities, yet structurally closely related impurities with similar sequences, not resolved in preparative RPLC, may easily coelute in the corresponding QC run as well. To address this problem an advanced generic 2D-LC impurity profiling method was developed in this work. It employs a selective comprehensive (high resolution sampling) RP×RP 2D-LC separation using a 100×2.1mm ID column with the common acidic generic gradient in the first dimension, while RPLC under basic pH on a short 30×3mm ID column is used in the second dimension. Recording data with a UV detector at 215nm after 1D separation provides the common generic 1D chromatogram. However, after the 2D separation a flow splitter enabled recording of the signals of complementary detectors comprising a diode array detector (DAD) in-line with a charged aerosol detector (CAD) and a quadrupole-time-of-flight (QTOF) mass spectrometer (MS) with an electrospray ionization (ESI) source. Generic conditions of this 2D-LC method have been established through optimization of 2D stationary and mobile phase considering different pH values and buffer concentrations. The orthogonal separation principle has been documented by a number of therapeutic peptides including Exenatide, Octreotide, Cyclosporine A and Oxytocin as well as some other proprietary synthetic peptides. The information density can be further enhanced by using the QTOF-MS detector by data independent acquisition with SWATH. Through this sequential window acquisition of all theoretical fragment ion mass spectra it became possible to collect MS/MS data comprehensively in the high-resolution sampling window, thus enabling the extraction of 2D-EICs from fragment ions and the generation of 2D-contour plots of all product ions. Using Oxytocin as an example for an important therapeutic peptide, the ability of this advanced generic sRP-UV×RP-DAD-CAD-ESI-QTOF-MS/MS method with SWATH for peptide quality control is discussed.

Engineering solutions for flow control in microfluidic devices for spatial multi-dimensional liquid chromatography

Spatial multi-dimensional liquid chromatography can provide unprecedent resolving power by utilizing subsequent orthogonal separation mechanisms, while the analysis time is minimized thanks to parallel development of the analyzed fractions in each dimension. In this study, different engineering solutions have been realized aiming at achieving flow confinement and control during subsequent 1D and 2D chromatographic developments in a microfluidic device for spatial two-dimensional separations. First, the flow distributor design was optimized and physical barriers, i.e., reducing the cross section of microchannels as well as locally integrated monolithic substrates in chip segments, were assessed as a means of flow confinement. Furthermore, an on-chip active-valving, high-pressure approach was successfully developed. The flow in first-dimension separation is confined within a channel situated in a rotating axis containing through-holes. These are either closed during the 1st development or opened when aligned with the 2D flow distributor and the 2D channels after rotation of the axis by 90°, allowing for sample transfer and executing the subsequent 2D analysis. The on-chip active-valving concept has been successfully demonstrated in combination with stationary-phase analyte focusing prior to the 2D development using a microfluidic spatial 2D-LC device containing polymer monolithic stationary phases locally synthesized in the parallel 2D separation channels. Finally, this workflow was successfully employed to separate a mixture of dyes applying gradient RPLC during the 2D development.

Microextraction assisted multiple heart-cutting and comprehensive two-dimensional liquid chromatography hyphenated to Q-TOF/MS for the determination of multiclass compounds from Dendrobium species

Chromatographic separation and determination of closely related classes are one of the most difficult tasks in the analysis of medicinal plants. In addition, two-dimensional ultra-high performance liquid chromatography (2DLC) has enormous potential and become a significant tool for enhancing peak capacity, selectivity and sensitivity. Workflows based on multiple heart-cutting (MHC) and comprehensive 2DLC (LC × LC) hyphenated to quadrupole time-of-flight mass spectrometry (Q-TOF/MS) were described that allow to identify and separate the analytes in Dendrobium species. Three Dendrobium genus were Dendrobium officinale Kimura et Migo, Dendrobium nobile Lindl and Dendrobium chrysotoxum Lindl. In this study, excellent selectivity, resolution, and peak shape were obtained through combining reversed-phase SB C18 column (2.1 × 100 mm, 1.8 μm) in the first dimension with 3.0 × 50 mm Poroshell 120 Bonus column (1.8 μm particle size) in the second dimension (2D), together with the application of synchronized mode in both dimensions. The matrix solid phase dispersion method was applied to extract Dendrobium species and 31 compounds were characterized and separated within 40 min. In terms of 2D flow rate and separation, LC × LC mode was faster than MHC mode. However, too high 2D flow rate would exacerbate the damage to the column in the LC × LC mode. MHC mode was used to genernally separate and analyze specific parts of the sample, otherwise it would take too much time. In short, MHC and LC × LC coupled with Q-TOF/MS method was a powerful technique and shown to be applicable for the analysis and determination of trace compounds in Dendrobium genus extracts and could be easily expanded to other complex mixtures.

Uncertainty in the separation properties of functionalized porous graphenes

High energy means (e.g. electron/ion beam bombardment) are often used to punch holes in graphene sheets, making them into useful two-dimensional (2D) separation materials. After pore saturation, the best controlled pores may have the same geometry and chemical composition but still of various configurations. Using first-principles calculations, we found that even energy-degenerate pores often have very different separation properties. More importantly, this uncertainty can be even greater than the difference in the separation properties among pores passivated with different functional groups, thus defeating the purpose of pore functionalization. Single-layered porous graphene sheets were modeled with pores saturated by representative atoms and chemical groups of H-, N-, F- and OH-. The pore configuration-dependent separation property was characterized by passing molecules of various sizes (from methane to much bigger dibranched alkanes). Pore configurations within 0.1 eV energy difference often exhibit different penetration barriers for the same type of passing molecules. The difference in barriers ranges from 0.2 eV to 0.6 eV. Our study highlights the uncertainty in the separation properties of porous 2D membranes prepared by high energetic irradiations and the following functionalization. To have well-defined separation properties, position-specific pore passivation methods need to be invoked.

Simultaneous determination of loading capacity and selectivity in preparative off-line two-dimensional separation: An application for purification of corilagin from Pomegranate flower extracts

This work describes the development of capacity orthogonal chromatography (COC), a new technique for simultaneously determining the loading capacity and orthogonality during the construction of two-dimensional (2D) separations. Three steps were required for the construction of a COC based on the correlation between the selectivity factor (α) and both orthogonality and loading capacity. (1) α values of the impurities-target compound were used to normalize the retention of the impurities around the target compound. (2) α values were input into four quadrants of a coordinate system to identify correlations between orthogonality and loading capacity. α values of the impurities must be greater in the first dimension than the second dimension, with iterated analyses performed until an αmax is obtained for the two purification methods. (3) Touch-peak separation using the first-dimensional αmax was performed and the target compound was collected. The co-eluted impurities are further separated in the second dimension. To test the efficiency of this technique, a COC using two methods on a standard C18 column was developed to purify corilagin from pomegranate flower extract. Despite its low abundance, 288 mg of corilagin was obtained by COC and further purified by LH-20 gel chromatography to obtain the compound with an 80.0% recovery and 98.4% purity. Compared to COC, the purity of corilagin independently obtained using the same purification methods and identical loading capacity was poor (60.1% and 61.6%). These results indicate that COC is a useful tool for extending loading capacity in the development of preparative 2D separations.

Determination of the Metabolite Content of Brassica juncea Cultivars Using Comprehensive Two-Dimensional Liquid Chromatography Coupled with a Photodiode Array and Mass Spectrometry Detection

Plant-based foods are characterized by significant amounts of bioactive molecules with desirable health benefits beyond basic nutrition. The Brassicaceae (Cruciferae) family consists of 350 genera; among them, Brassica is the most important one, which includes some crops and species of great worldwide economic importance. In this work, the metabolite content of three different cultivars of Brassica juncea, namely ISCI Top, “Broad-leaf,” and ISCI 99, was determined using comprehensive two-dimensional liquid chromatography coupled with a photodiode array and mass spectrometry detection. The analyses were carried out under reversed-phase conditions in both dimensions, using a combination of a 250-mm microbore cyano column and a 50-mm RP-Amide column in the first and second dimension (2D), respectively. A multi (three-step) segmented-in-fraction gradient for the 2D separation was advantageously investigated here for the first time, leading to the identification of 37 metabolites. In terms of resolving power, orthogonality values ranged from 62% to 69%, whereas the corrected peak capacity values were the highest for B. juncea ISCI Top (639), followed by B. juncea “Broad-leaf” (502). Regarding quantification, B. juncea cv. “Broad-leaf” presented the highest flavonoid content (1962.61 mg/kg) followed by B. juncea cv. ISCI Top (1002.03 mg/kg) and B. juncea cv. ISCI 99 (211.37 mg/kg).

Polypropylene capillary-channeled polymer fiber column as the second dimension in a comprehensive two-dimensional RP × RP analysis of a mixture of intact proteins.

Online, comprehensive two-dimensional liquid chromatography (2D-LC) has become an attractive option for the analysis of complex samples of relevance in various fields (e.g., environmental, food, biology, and polymer sciences). In general, the second-dimension (2D) separation plays a more crucial role than the first (1D) in the total performance of LC × LC systems. Speed and efficiency of the 2D separation are of primary importance. These challenges are amplified in the case of protein separations where mass transfer limitations effect elution kinetics. The research presented here leverages the developments of capillary-channeled polymer (C-CP) fiber stationary phases directed at high-throughput and rapid protein separations, evaluating them as 2D material in comprehensive reversed-phase × reversed-phase (RP × RP) 2D-LC versus conventional, packed-bed columns as 2D. The ability to operate fiber columns at high linear velocities (> 75mms-1 at < 1000psi) without sacrifice in resolution is the first step towards potential 2D implementation, alleviating the need for ultrahigh-pressure pumping (i.e., UHPLC) in that stage. What must be realized are fast elution gradients and rapid re-equilibration to minimize cycling times (modulation periods). Different flow rates and gradient programs were evaluated. Rapid elution allows the use of shorter shift-gradient windows. At the optimized gradient recycling time on this instrument, 36s, there is no significant degradation in peak capacity. Finally, a graphic comparison of the separation efficiency between seven commercial reversed-phase columns and the polypropylene C-CP fiber column is presented for a synthetic, eleven-protein mixture using the recommended operating conditions for each of the 2D columns. The kinetic advantages of the fiber columns are clearly demonstrated.

Review of Three-Dimensional Liquid Chromatography Platforms for Bottom-Up Proteomics.

Proteomics is a large-scale study of proteins, aiming at the description and characterization of all expressed proteins in biological systems. The expressed proteins are typically highly complex and large in abundance range. To fulfill high accuracy and sensitivity of proteome analysis, the hybrid platforms of multidimensional (MD) separations and mass spectrometry have provided the most powerful solution. Multidimensional separations provide enhanced peak capacity and reduce sample complexity, which enables mass spectrometry to analyze more proteins with high sensitivity. Although two-dimensional (2D) separations have been widely used since the early period of proteomics, three-dimensional (3D) separation was barely used by low reproducibility of separation, increased analysis time in mass spectrometry. With developments of novel microscale techniques such as nano-UPLC and improvements of mass spectrometry, the 3D separation becomes a reliable and practical selection. This review summarizes existing offline and online 3D-LC platforms developed for proteomics and their applications. In detail, setups and implementation of those systems as well as their advances are outlined. The performance of those platforms is also discussed and compared with the state-of-the-art 2D-LC. In addition, we provide some perspectives on the future developments and applications of 3D-LC in proteomics.

Determination of Risperidone and 9-Hydroxyrisperidone in Human Serum by Heart-Cutting Isocratic Two-Dimensional Liquid Chromatography

This study reports a novel heart-cutting two-dimensional high performance liquid chromatography (2D-LC) method with ultraviolet detection for the determination of risperidone and 9-hydroxyrisperidone in human serum. In this 2D system, a strong cation-exchange column was applied for the first dimensional (1D) separation to isolate the targets from the complex serum matrices, and a middle column was used to transfer the targets from the first dimension to the second dimension (2D) through a six-port valve. A reverse-phase C18 column was selected in the second dimension for further separation. An assistant pump was optimized to prevent the peak dispersion and the loss of resolution. This method has the advantages of high sensitivity by large injection volume, simple one-step sample preparation and short analysis time. A linear range between 5 and 146 ng/mL for risperidone and 7 and 200 ng/mL for 9-hydroxyrisperidone was achieved. All of the results showed that the coupling of ion-exchange with reversed-phase liquid chromatography had powerful separation capabilities for risperidone and 9-hydroxyrisperidone in human serum. The method exhibited good repeatability and recoveries and may be used as a reliable approach for therapeutic drug monitoring in clinical analysis.

Separation of twelve posaconazole related stereoisomers by multiple heart-cutting chiral–chiral two-dimensional liquid chromatography

Posaconazole represents a triazole antifungal agent which is used to treat various fungal infections. It contains four chiral centers leading to 16 stereoisomers. With the convergent synthesis route, only 11 related stereoisomeric impurities may potentially exist in the active pharmaceutical ingredient (API). It is a challenge to separate all the stereoisomers in one run. To address this problem, a multiple heart-cutting chiral–chiral two-dimensional liquid chromatography (2D-LC) method was developed. The multiple heart-cutting 2D-LC separation was implemented on 2D-LC system with three chiral columns with immobilized polysaccharide chiral stationary phases, namely Chiralpak IB, IC and IF3. In the system, the column Chiralpak IB was used as the 1D separation column and IC and IF3 columns were used parallelly for the 2D separation. The twelve stereoisomers were all well separated in one run by the multiple heart-cutting chiral–chiral 2D-LC system.

Chemometric decomposition of comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry data employing partial modulation in the negative pulse mode

Partial modulation in the negative pulse mode was optimized for comprehensive two-dimensional gas chromatography (GC × GC) coupled to time-of-flight mass spectrometry (TOFMS). With partial modulation in the negative pulse mode, a flow of auxiliary carrier gas is applied through a pulse valve to a T-junction joining the first (1D) and second (2D) dimension columns for nearly all of the modulation period (PM). This results in dilution of the 1D eluate, followed by briefly turning off the pulse valve for a specified pulse width (pw), effectively “injecting” undiluted 1D eluate onto the 2D column. The raw data has the appearance of 2D separations superimposed on top of the 1D separation. While high peak capacity GC × GC data are produced, there are challenges that needed to be addressed regarding the use of chemometrics for analyte decomposition, identification, and quantification. Herein, these data analysis challenges are addressed using multivariate curve resolution - alternating least squares (MCR-ALS). An isothermal separation of a 15-component mixture of similar compounds is obtained in 20 s using a PM = 250 ms and a pw = 6 ms. Various peak overlap situations were purposely produced to facilitate the chemometric method demonstration. The MCR-ALS chromatographic loadings (peak profiles) were used to determine retention times (tR) and width-at-base (Wb) in both separation dimensions. MCR-ALS readily decomposed 1D × 2D regions for analytes with severe 1D overlap if they were fully resolved on 2D. Decomposition was more challenging for analytes severely overlapped on both GC × GC dimensions when their spectra were similar, though ultimately all 15 compounds were successfully decomposed. Additionally, the 2D concentration peak profiles obtained via MCR-ALS are demonstrated to be reliable for identification and quantifiable. The predicted versus prepared concentration values are in good agreement for two representative analytes, with percent deviation values of −5.6% (±2.2%) for 1-hexene, and 1.8% (±3.4%) for 2-pentanone.

Deep Intact Proteoform Characterization in Human Cell Lysate Using High-pH and Low-pH Reversed-Phase Liquid Chromatography.

Post-translational modifications (PTMs) play critical roles in biological processes and have significant effects on the structures and dynamics of proteins. Top-down proteomics methods were developed for and applied to the study of intact proteins and their PTMs in human samples. However, the large dynamic range and complexity of human samples makes the study of human proteins challenging. To address these challenges, we developed a 2D pH RP/RPLC-MS/MS technique that fuses high-resolution separation and intact protein characterization to study the human proteins in HeLa cell lysate. Our results provide a deep coverage of soluble proteins in human cancer cells. Compared to 225 proteoforms from 124 proteins identified when 1D separation was used, 2778 proteoforms from 628 proteins were detected and characterized using our 2D separation method. Many proteoforms with critically functional PTMs including phosphorylation were characterized. Additionally, we present the first detection of intact human GcvH proteoforms with rare modifications such as octanoylation and lipoylation. Overall, the increase in the number of proteoforms identified using 2DLC separation is largely due to the reduction in sample complexity through improved separation resolution, which enables the detection of low-abundance PTM-modified proteoforms. We demonstrate here that 2D pH RP/RPLC is an effective technique to analyze complex protein samples using top-down proteomics.