In liquid chromatography differences between the pH of an injected sample and the pH of the mobile phase can have a significant impact on retention times, peak widths, and resolution.

Separations of complex peptide mixtures have been a common target application for two-dimensional liquid chromatography over the last few decades. These separations have most frequently been carried out at the capillary scale, with columns on the order of 75 mu m i.d. and flow rates on the order of 500 nL/min. Recently, however, several groups have worked to optimize comprehensive 2D-LC (LC x LC) separations of peptides at the analytical scale (i.e., 2 mm i.d. columns, and ca. 1 mL/min flow rates) and demonstrated peak capacities on the order of 5000 in analysis times of a few hours, using reversed-phase separations in both dimensions. In this paper we aim to advance the performance of such separations in two primary ways. First, we demonstrate that active solvent modulation (ASM) can be used to improve the 2D peak capacity by both enabling use of long and highly efficient first dimension (D-1) columns, and by mitigating the deleterious effects of injecting large fractions of D-1 effluent into the small columns that are required for fast and highly sensitive second dimension (D-2) separations. Taken together these two benefits enable the realization of a peak capacity of 10,000 in an analysis time of four hours. This comes at the cost of increased instrument complexity compared to 1D-LC separations, but the 2D-LC approach is unquestionably the most efficient way to improve upon the resolving power of existing 1D-LC. Second, we have systematically studied the compromise between the peak capacity of each D-2 separation and the operating pressure required to achieve that peak capacity. Understanding this compromise will be important to the development of LC x LC methods that both produce high peak capacities, and are sufficiently robust to operate for days at a time without significant losses in separation performance. Based on the results of this study we chose conditions for subsequent separations that required less than 400 bar operating pressure in the second dimension, but yielded a 2D peak capacity of about 3500 in 2 h. After 160 h of continuous operation of the LC x LC separation under these conditions (and about 20,000 injections into the D-2 column) the D-2 column had only lost about 18% of its initial isocratic efficiency. These results should motivate further development and implementation of such high performing and robust separations for the identification and quantification of peptides in a variety of application areas, including digests of therapeutic proteins such as monoclonal antibodies.

Monoclonal antibodies (mAb) and related molecules are being developed at a remarkable pace as new therapeutics for the treatment of diseases ranging from cancer to inflammatory disorders. However, characterization of these molecules at all stages of development and manufacturing presents tremendous challenges to existing analytical technologies because of their large size (ca. 150 kDa) and inherent heterogeneity resulting from complex glycosylation patterns and other post-translational modifications. Multidimensional liquid chromatography is emerging as a powerful platform technology that can be used to both improve analysis speed for these molecules by combining existing one-dimensional separations into a single method (e.g., Protein A affinity separation and size-exclusion chromatography) and increasing the resolving power of separations by moving from one dimension of separation to two. In the current study, we have demonstrated the ability to combine hydrophilic interaction (HILIC) and RP separations in an online comprehensive 2D separation coupled with high resolution MS detection (HILIC × RP-HRMS). We find that active solvent modulation (ASM) is critical for coupling these two separation modes, because it mitigates the otherwise serious negative impact of the acetonitrile-rich HILIC mobile phase on the second dimension RP separation. The chromatograms obtained from these HILIC × RP-HRMS separations of mAbs at the subunit level reveal the extent of glycosylation on the Fc/2 and Fd subunits in analysis times on the order of 2 h. In comparison to previous CEX × RP separations of the same molecules, we find that chromatograms from the HILIC × RP separations are richer and reveal separation of some glycoforms that coelute in the CEX × RP separations.

Comprehensive two-dimensional liquid chromatography (LC × LC) has been gaining popularity for the analysis of complex samples in a wide range of fields including metabolomics, environmental analysis, and food analysis. While LC × LC can provide greater chromatographic resolution than one-dimensional LC (1D-LC), overlapping peaks are often still present in separations of complex samples, a problem that can be alleviated by chemometric curve resolution techniques such as multivariate curve resolution-alternating least squares (MCR-ALS). MCR-ALS has also been previously shown to assist in the quantitative analysis of LC x LC data by isolating pure analyte signals from background signals which are often present at higher levels in LC x LC compared to 1D-LC. In this work we present the analysis of a dataset from the LC × LC analyses of parsley, parsnip and celery samples for the presence and concentrations of 14 furanocoumarins. Several MCR-ALS implementations are compared for the analysis of LC × LC data. These implementations include analyzing the LC x LC chromatogram alone, analyzing the one-dimensional chromatogram alone, as well as two hybrid approaches that make use of both the first and second dimension chromatograms. Furthermore, we compared manual integration of resolved chromatograms versus a simple summation approach, using the resolved chromatographic peaks in both cases. It is found that manual integration of the resolved LC × LC chromatograms provides the best quantification as measured by the consistency between replicate injections. If the summation approach is desired for automation, the choice of MCR-ALS implementation has a large effect on the precision of the analysis. Based on these results, the concentrations of the 14 furanocoumarins are determined in the three apiaceous vegetable types by analyzing the LC × LC chromatograms with MCR-ALS and manual integration for peak area determination. The concentrations of the analytes are found to vary greatly between samples, even within a single vegetable type.

As research, development, and manufacturing of biosimilar protein therapeutics proliferates, there is great interest in the continued development of a portfolio of complementary analytical methods that can be used to efficiently and effectively characterize biosimilar candidate materials relative to the respective reference (i.e., originator) molecule. Liquid phase separation techniques such as liquid chromatography and capillary electrophoresis are powerful tools that can provide both qualitative and quantitative information about similarities and differences between reference and biosimilar materials, especially when coupled with mass spectrometry. However, the inherent complexity of these protein materials challenges even the most modern one-dimensional (1D) separation methods. Two-dimensional (2D) separations present a number of potential advantages over 1D methods, including increased peak capacity, 2D peak patterns that can facilitate unknown identification, and improvement in the compatibility of some separation methods with mass spectrometry. In this study, we demonstrate the use of comprehensive 2D-LC separations involving cation-exchange (CEX) and reversed-phase (RP) separations in the first and second dimensions to compare 3 reference/biosimilar pairs of monoclonal antibodies (cetuximab, trastuzumab and infliximab) that cover a range of similarity/disimilarity in a middle-up approach. The second dimension RP separations are coupled to time-of-flight mass spectrometry, which enables direct identification of features in the chromatograms obtained from mAbs digested with the IdeS enzyme, or digestion with IdeS followed by reduction with dithiothreitol. As many as 23 chemically unique mAb fragments were detected in a single sample. Our results demonstrate that these rich datasets enable facile assesment of the degree of similarity between reference and biosimilar materials.

Two persistent impediments to wider adoption of two-dimensional liquid chromatography (2D-LC) are the perceptions that 2D methods are generally less sensitive than 1D ones, and that coupling of certain separation modes in a 2D system is difficult because of the negative impact of the effluent of the first separation on the second separation. In this work we address these problems in the specific case where reversed-phase separations are used in both dimensions of a 2D-LC system, but the pH is varied such that the ionization state of carboxylic acid analytes is different (i.e., neutral or negatively charged, in eluents buffered at pH 2 or 7) in the two columns. We first demonstrate that the effect of first dimension ((1)D) effluent on the performance of second dimension ((2)D) separation of ionogenic solutes is much more serious than it is for neutral compounds where the pH of the eluent does not play a role in retention. We have systematically varied the properties of the sample solution injected into the (2)D column (i.e., the (1)D effluent), as well as the (2)D eluent, with the goal of establishing guidelines for conditions that yield acceptable (2)D performance. We find that the organic solvent content of the (1)D effluent and (2)D eluent is not as important as the buffer concentrations in these two solutions, and that the greater the ratio of buffer concentration in the (1)D effluent relative to the (2)D eluent, the smaller the volume one can inject into the (2)D column before dramatic peak splitting occurs. We have then used the information from these simple experiments to guide both 1D experiments that mimic the (2)D separation, and actual 2D separations, to demonstrate that online adjustment of the properties of the (1)D effluent by dilution with a buffered solvent prior to injection into the (2)D column is a very effective solution to the pH mismatch problem. We find that when the buffer capacity of the diluent is high enough to effectively titrate the (1)D effluent such that its pH approaches that of the (2)D eluent, excellent (2)D peak shape is obtained for the carboxylic acid analytes, even when the volume of injected sample solution exceeds the (2)D column volume.

In this paper, we describe the findings of a study aimed at assessing the detection sensitivity of comprehensive two-dimensional high-performance liquid chromatography (LCxLC) separation of a degraded active pharmaceutical ingredient (API) with UV absorption as the detection technique. Specifically, we have examined the impact of the volume and solvent composition (referred to as “interface conditions”) of fractions of first-dimension column effluent transferred to the second dimension for further separation on the ability to resolve and detect low-abundance compounds. Historically, LCxLC has been perceived as being inferior to 1D-LC from the point of view of detection sensitivity. In this work, we demonstrate that LCxLC is sufficiently sensitive to be useful in the pharmaceutical context where in general impurities present at 0.05 % (relative to the API concentration) should be quantified. Moreover, we find that this level of sensitivity is only attained under certain conditions: dilution of the first column effluent with weak solvent (water in this case) prior to injection into the second-dimension column is very beneficial because it promotes focusing of the analyte band in the second column, thereby improving the detection sensitivity of the LCxLC system; and, quantitation limits are also a strong function of peak location in the second-dimension separation window, where baseline disturbances near the dead time of the second column can limit reliable detection of low-abundance compounds.

Various implementations of two-dimensional high-performance liquid chromatography are increasingly being developed and applied to the analysis of complex materials, including those encountered in the analysis of foods, beverages, and nutraceuticals. Previously, we introduced the concept of selective comprehensive two-dimensional liquid chromatography (sLC × LC) as a hybrid between the more conventional, but extreme opposite sampling modes of heartcutting (LC–LC) and fully comprehensive (LC × LC) 2D separation. The sLC × LC approach breaks the link between first dimension (1D) sampling time and second dimension (2D) analysis time that is faced in LC × LC and allows very rapid (as low as 1 s) sampling of highly efficient 1D separations, while at the same time allowing efficient 2D separations on the timescale of tens of seconds. In this paper, we improve upon our previous sLC × LC work by demonstrating the ability to perform the processes of 1D sampling and 2D separation in parallel. This significantly improves the flexibility of the technique and allows targeted analysis of analytes that elute close together in time in the 1D separation. To demonstrate the value of this added capability, we have developed a sLC × LC method using multi-wavelength ultraviolet absorbance detection for the quantitative analysis of six target furanocoumarin compounds in extracts of celery, parsley, and parsnips. We show that 2D separations of 1D effluent containing the target compounds of interest reveal the presence of unanticipated interferent peaks that would otherwise compromise the quantitative accuracy of the method. We also demonstrate the application of the chemometric method iterative key set factor analysis with alternating least-squares to sLC × LC to mathematically resolve target compounds that are only slightly separated chromatographically but not sufficiently resolved for accurate quantitation.