Hydroxypropyl methyl cellulose (HPMC) is a type of cellulose derivative with properties that render it useful in e.g. food, cosmetics, and pharmaceutical industry. The substitution degree and composition of the β-glucose subunits of HPMC affect its physical and functional properties, but HPMC characterization is challenging due to its high structural heterogeneity, including many isomers. In this study, comprehensive two-dimensional liquid chromatography-mass spectrometry was used to examine substituted glucose monomers originating from complete acid hydrolysis of HPMC. Resolution between the different monomers was achieved using a C18 and cyano column in the first and second LC dimension, respectively. The data analysis process was structured to obtain fingerprints of the monomers of interest. The results revealed that isomers of the respective monomers could be selectively separated based on the position of substituents. The examination of two industrial HPMC products revealed differences in overall monomer composition. While both products contained monomers with a similar degree of substitution, they exhibited distinct regioselectivity.

Lignans constitute a large group of phenolic plant secondary metabolites possessing high bioactivity. Their accurate determination in plant extracts with a complex chemical composition is challenging and requires advanced separation techniques. In the present study, a new approach to the determination of lignans in coniferous knotwood extracts as the promising industrial-scale source of such compounds based on comprehensive two-dimensional liquid chromatography separation and UV spectrophotometric detection is proposed. First and second-dimension column screening showed that the best results can be obtained using a combination of non-polar and polar hydroxy group embedded octadecyl stationary phases with moderate (~40%) \"orthogonality\". The optimization of LC × LC separation conditions allowed for the development of a new method for the quantification of the five lignans (secoisolariciresinol, matairesinol, pinoresinol, 7-hydroxymatairesinol, and nortrachelogenin) in knotwood extracts with limits of quantification in the range of 0.27-0.95 mg L-1 and a linear concentration range covering at least two orders of magnitude. Testing the developed method on coniferous (larch, fir, spruce, and pine) knotwood extracts demonstrated the high selectivity of the analysis and the advantages of LC × LC in the separation and accurate quantification of the compounds co-eluting in one-dimensional HPLC.

A unique and effective comprehensive two-dimensional liquid chromatography system was established and applied for the analysis of bioactive components in honeysuckle. Under the optimal conditions, Eclipse Plus C18 (2.1 × 100 mm, 3.5 μm, Agilent) and SB-C18 (4.6 × 50 mm, 1.8 μm, Agilent) columns were chosen for the first dimension (1D) and the second dimension (2D) separation. The optimal flow rates of 1D and 2D were 0.12 mL/min and 2.0 mL/min, respectively. Additionally, the proportion of organic solution was optimized to enhance orthogonality and integrated shift, and full gradient elution mode was adopted to improve chromatographic resolution. Furthermore, a total of 57 compounds were identified by molecular weight, retention time and collision cross-section value obtained from ion mobility mass spectrometry. Based on the data obtained from the principal component analysis, partial least squares discriminant analysis, and hierarchical cluster analysis, the categories of honeysuckle in different regions were significantly different. Moreover, the half maximal inhibitory concentration values of most samples were between 0.37 and 1.55 mg/mL, and most samples were potent α-glucosidase inhibitors, which is better for the evaluation of the quality of drugs from two aspects of substance content and activity.

Due to the growing trend of organic food, there is still concern over the use of chemicals and pesticides in agriculture. In recent years, several procedures have been validated for the control of pesticides in food. In the present research, a comprehensive two-dimensional liquid chromatography coupled with tandem mass spectrometry is proposed for the first time for a multi-class analysis of 112 pesticides in corn-based products. Notably, a \"reduced\" QuEChERS-based method as extraction and clean-up procedure prior to the analysis, was successfully employed. Limits of quantification values were lower than the ones fixed by the European legislation; intra-day and inter-day precision were lower than 12.9% and 15.1%, respectively (at the 500 μg/kg concentration levels). Over 70% of the analytes provided recoveries between 70% and 120% range (at 50, 500 and 1000 µg/kg concentration levels) with standard deviation values below 20%. In addition, matrix effect values were in the range between 13% to 161%. The method was applied to the analysis of real samples, and three pesticides were detected at trace levels in both samples. The findings of this work pave the way for the treatment of complex matrices such as corn products.

Comprehensive two-dimensional liquid chromatography (LC×LC) can provide enhanced resolving power and higher peak capacities for the separation of complex samples. The transfer of fractions of too high eluotropic strength from the first dimension, however, can lead to peak broadening. This process is related to the column dimensions, the flow rates, mobile phase compositions, and stationary phase compatibility. Temperature-responsive LC (TRLC) uses a smart polymer coupled to silica (poly(N-isopropylacrylamide), pNIPAAm), that exhibits a change in polarity upon modest variations in column temperature. Retention is thus modulated by temperature and not by organic solvents, allowing for the use of purely aqueous mobile phases. As these aqueous mobile phases depict a very low eluotropic strength on a reversed-phase column, it facilitates band refocusing at the second-dimension column head in TRLC×RPLC. One of the remaining obstacles of TRLC is the long analysis time. In this research, the potential of this column combination in terms of analyte refocusing will be exploited. First, it is shown that upwards flow gradients can be implemented in the first dimension of TRLC×RPLC. As the flow in the second dimension is maintained at a constant level, a first-dimension flow gradient does not lead to impaired sensitivity and has no negative effects on the resulting peak size. Then, the novel combination of a downwards temperature gradient with an upwards flow gradient will be introduced to speed up the analyses further. Analysis time was, depending on the method used, reduced by 36-54%, as demonstrated by the analysis of mixtures of food additives, phenolic compounds, and small molecule pharmaceuticals mimicking impurity analysis at a 0.05% level.

Casein hydrolysates are important additives to foods for elderly and sports nutrition. However, due to the enzymatic generation of so-called bitter peptides, their application is limited. Therefore, the procedure needs to be optimized in order to restrict their occurrence. For this, extensive sensory evaluations are necessary. By combining two separation techniques using comprehensive two-dimensional liquid chromatography, we present a novel method for estimating the bitter taste of hydrolysate samples on the basis of their elution pattern. Using a size exclusion column in the first and a reversed phase column in the second dimension allows for a detailed sample evaluation regarding peptide size and relative hydrophobicity. The results obtained for different casein hydrolysates were correlated with the sensory evaluation. We found that hydrolysates with increasing bitterness contain a higher amount of peptides of high hydrophobicity and a molecular weight less than 6.5 kDa.

In this work, a comprehensive two-dimensional liquid chromatography system, comprised of a ZIC-HILIC and C18 columns in the first and second dimensions, respectively, was tuned and employed for attaining high resolution profiles of the polyphenolic pattern in seven commercial berry juices. The developed HILIC × RP-LC method was validated in terms of linearity range, correlation coefficients, limit of detection, limit of quantification, precision (intra- and inter-day), and recovery. A total of 104 polyphenolic compounds belonging to different chemical classes (hydroxybenzoic and cinnamic acid derivatives, flavone glycosides, flavonols, flavonol glycosides, dihydroflavonols, and anthocyanin glycosides) have been characterized and quantified in the juices investigated. Despite the constituents being similar, a notable quantitative variation among the analyzed berry species was observed. Elderberry contained the highest amount of polyphenols (918 ± 1.10 mg 100 mL-1), followed by chokeberry (516 ± 0.08 mg 100 mL-1). On the other hand, raspberry contained the lowest amount (104 ± 1.21 mg 100 mL-1). Further, total phenolic, flavonoid, and anthocyanin contents were determined spectrophotometrically, yielding consistent results. The free-radical scavenging activity (DPPH test) and reducing power of the juices, expressed as IC50 (μL mL-1) and mg ASE mL-1, varied from 2.79 ± 0.03 (honeyberry) to 31.66 ± 0.02 (blueberry) and from 1.71 ± 0.01 (blueberry) to 8.89 ± 0.12 (chokeberry), respectively. Such a ZIC-HILIC × C18 platform based on focusing modulation, never employed so far for berry juices, showed a remarkable separation capability with high values of corrected peak capacity (up to 1372) and orthogonality (Ao up to 0.80), thus providing a great applicability to be advantageously employed for other complex food samples.

Understanding the relation between chemical characteristics and properties of synthetic polymers is one of the challenges faced by analytical chemists in industry. This is a complex task, as polymers are not synthesized as single molecule, but are populations of chemically similar compounds with distributions over several properties. The latter include, for example, molecular weight, nature of end-groups (functionality), and chemical composition. In this paper, comprehensive two-dimensional liquid chromatography was used to determine the combined functionality-type and molecular-weight distributions of hydroxy‑functionalized propoxylates. Propoxylates derived from different initiators (one up to eight terminal hydroxyl groups) were separated in the first dimension using a gradient normal-phase LC separation (NPLC). In the second dimension ultra-high pressure size-exclusion chromatography separation (UHPSEC), further speciating distributions based on molecular size. The developed NPLC × SEC method with evaporative light-scattering detection can be used for the fast screening (< 30 min) of mutually dependent functionality-type and molecular-weight distributions of unknown propoxylates.

Comprehensive two-dimensional liquid chromatography is a well-established method for the unraveling of very complex real-world samples. With regard to food and natural products such a technique turned out to be a very promising approach due to its high resolving power and improved identification capability, especially in combination with mass spectrometry. In this context, polyphenols comprise a particular complex class of bioactive compounds, due to their nature and content in commonly consumed foodstuffs, making their analysis challenging. The present contribution shows an overview of the two commonly employed approaches used for polyphenol analysis, viz. RP-LC × RP-LC and HILIC × RP-LC. Furthermore, the latest implementations as well as limitations and future perspectives are critically reported.

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.