Reversed-phase Liquid Chromatographic Separation Research Articles

Solvent selectivity and strength in reversed-phase liquid chromatography separation of peptides

A set of tryptic peptides was analyzed in reversed-phase liquid chromatography using gradient elution with acetonitrile, methanol, or isopropanol. We used these retention data as training sets to develop retention prediction models of peptides for the three organic eluents used. The coefficients of determination, R2, between predicted and observed data were approximately 0.95 for all systems. Retention coefficient values of twenty amino acids calculated from a model were utilized to investigate differences in separation selectivity between acetonitrile, methanol, or isopropanol eluents. The experimentally observed difference in separation selectivity appears to be a complex interplay of multiple amino acids, each contributing to a different degree to overall peptide retention. While retention contribution of hydrophilic amino acids was higher in methanol than acetonitrile, peptides containing aromatic amino acids (tyrosine, phenylalanine, tryptophan) exhibit relatively lower retention in methanol compared to acetonitrile. The differences between acetonitrile and isopropanol eluents were less pronounced. We also compared the relative elution strength of the three organic eluents for peptides. The relationship between the elution strength of two solvents is not linear, rather it was best fitted by a cubic polynomial function. Three solvents can be arranged in the order of increasing elution power as methanol<acetonitrile<isopropanol. The equations for relative solvent strength conversion were proposed.

A Highly Sensitive Targeted Mass Spectrometric Assay for Quantification of AGR2 Protein in Human Urine and Serum

Anterior gradient 2 (AGR2) is a secreted, cancer-associated protein in many types of epithelial cancer cells. We developed a highly sensitive targeted mass spectrometric assay for quantification of AGR2 in urine and serum. Digested peptides from clinical samples were processed by PRISM (high pressure and high resolution separations coupled with intelligent selection and multiplexing), which incorporates high pH reversed-phase liquid chromatography (LC) separations to fractionate and select target fractions for follow-on LC-selected reaction monitoring (LC-SRM) analyses. The PRISM-SRM assay for AGR2 showed a reproducibility of <10% CV and limit of quantification (LOQ) values of ∼130 pg/mL in serum and ∼10 pg per 100 μg of total protein mass in urine, respectively. A good correlation (R(2) = 0.91) was observed for the measurable AGR2 concentrations in urine between SRM and enzyme-linked immunosorbent assay (ELISA). On the basis of an initial cohort of 37 subjects, urinary AGR2/PSA concentration ratios showed a significant difference (P = 0.026) between noncancer and cancer. Large clinical cohort studies are needed for the validation of AGR2 as a useful diagnostic biomarker for prostate cancer. Our work validated the approach of identifying candidate secreted protein biomarkers through genomics and measurement by targeted proteomics, especially for proteins where no immunoassays are available.

The Use of Ammonium Formate as a Mobile-Phase Modifier for LC-MS/MS Analysis of Tryptic Digests

A major challenge facing current mass spectrometry (MS)-based proteomics research is the large concentration range displayed in biological systems, which far exceeds the dynamic range of commonly available mass spectrometers. One approach to overcome this limitation is to improve online reversed-phase liquid chromatography (RP-LC) separation methodologies. LC mobile-phase modifiers are used to improve peak shape and increase sample load tolerance. Trifluoroacetic acid (TFA) is a commonly used mobile-phase modifier, as it produces peptide separations that are far superior to other additives. However, TFA leads to signal suppression when incorporated with electrospray ionization (ESI), and thus, other modifiers, such as formic acid (FA), are used for LC-MS applications. FA exhibits significantly less signal suppression, but is not as effective of a modifier as TFA. An alternative mobile-phase modifier is the combination of FA and ammonium formate (AF), which has been shown to improve peptide separations. The ESI-MS compatibility of this modifier has not been investigated, particularly for proteomic applications. This work compares the separation metrics of mobile phases modified with FA and FA/AF and explores the use of FA/AF for the LC-MS analysis of tryptic digests. Standard tryptic-digest peptides were used for comparative analysis of peak capacity and sample load tolerance. The compatibility of FA/AF in proteomic applications was examined with the analysis of soluble proteins from canine prostate carcinoma tissue. Overall, the use of FA/AF improved online RP-LC separations and led to significant increases in peptide identifications with improved protein sequence coverage.

Fuzzy clustering evaluation of the discrimination power of UV–Vis and (±) ESI-MS detection system in individual or coupled RPLC for characterization of Ginkgo Biloba standardized extracts

Discrimination power evaluation of UV-Vis and (±) electrospray ionization/mass spectrometric techniques, (ESI-MS) individually considered or coupled as detectors to reversed phase liquid chromatography (RPLC) in the characterization of Ginkgo Biloba standardized extracts, is used in herbal medicines and/or dietary supplements with the help of Fuzzy hierarchical clustering (FHC). Seventeen batches of Ginkgo Biloba commercially available standardized extracts from seven manufacturers were measured during experiments. All extracts were within the criteria of the official monograph dedicated to dried refined and quantified Ginkgo extracts, in the European Pharmacopoeia. UV-Vis and (±) ESI-MS spectra of the bulk standardized extracts in methanol were acquired. Additionally, an RPLC separation based on a simple gradient elution profile was applied to the standardized extracts. Detection was made through monitoring UV absorption at 220 nm wavelength or the total ion current (TIC) produced through (±) ESI-MS analysis. FHC was applied to raw, centered and scaled data sets, for evaluating the discrimination power of the method with respect to the origins of the extracts and to the batch to batch variability. The discrimination power increases with the increase of the intrinsic selectivity of the spectral technique being used: UV-Vis<MS(-)<MS(+), but it is strongly sensitive to chemometric transformation of data. Comparison with cluster analysis (CA) and principal components analysis (PCA) indicates that the FHC algorithm produces better classification. However, PCA and CA may be successfully applied to discriminate between the manufacturing sources of the standardized extracts, and at some extent, to monitor the inter-batch variability. Although the chromatographic dimension sensibly contributes to the discrimination power, spectral MS data may be used as the lone powerful holistic alternative in characterization of standardized Ginkgo Biloba extracts.

Effect of mobile phase on resolution of the isomers and homologues of tocopherols on a triacontyl stationary phase

Reversed-phase liquid chromatographic (RPLC) separation of isomers and homologues of similar polarity is challenging. Tocopherol isomers and homologues are one such example. α, β, γ, and δ-tocopherols have been successfully separated by RPLC on triacontyl (C30) stationary phase. System suitability was tested by using four mobile phases, and observed chromatographic separations of β and γ-tocopherols were compared. Comparison indicated that methanol-tert-butyl methyl ether (TBME) 95:5 (v/v) at a flow rate of 0.75 mL min(-1) was the best mobile phase. Detection systems were also evaluated on the basis of limit of quantification; it was concluded that fluorescence detection was best. The method was validated by analysis of two homologues and two isomers of tocopherol in sesame, maize, and soybean samples. MS coupled with an ESI interface in negative-ion mode [M - H](-) was used for identification of individual components. It was concluded that addition of TBME to methanol was required to enhance the separation of β and γ-tocopherols, although methanol alone provided similar results. The applicability of the method to cereal, pulse, and oilseed samples was confirmed. The reproducibility of the procedure was good, with relative standard deviations in the range 1.7-3.9%. Recovery of tocopherols added to sesame samples ranged from 91 to 99%.

An Improved Liquid Chromatography-Tandem Mass Spectrometric Method to Quantify Formoterol in Human Urine

Formoterol is a new threshold substance in the prohibited list 2012 according to World Anti-Doping Agency. Extracted by ethyl acetate using formoterol-D6 as internal standard, formoterol underwent a constant flow rate gradient elution separation in reversed-phase liquid chromatography. Subsequently, mass spectrometry analysis was conducted by tandem mass spectrometry in the multiple reaction monitoring mode. According to the proposed method, a calibration curve was constructed in the range of 0.2-500 ng/mL (r(2) = 1) with a limit of quantification 0.2 ng/mL. The mean extracted recovery assessed at three different concentrations (1, 30 and 100 ng/mL) was more than 80%. The method was validated by the analysis of three quality control samples from World Association of Anti-Doping Scientists. In conclusion, the developed and validated method was sensitive, accurate and precise for the quantification of formoterol in human urine for doping control purposes.

A new approach to the preparation of large surface area poly(styrene-co-divinylbenzene) monoliths via knitting of loose chains using external crosslinkers and application of these monolithic columns for separation of small molecules

Poly(styrene-divinylbenzene) monoliths have been hypercrosslinked using a Fe3+ catalyzed Friedel–Crafts reaction involving three external crosslinkers (1) 4,4′-bis(chloromethyl)-1,1′-biphenyl, (2) α,α′-dichloro-p-xylene, and (3) formaldehyde dimethyl acetal. The first crosslinker was found to produce monoliths with the best chromatographic performance. The effects of a number of variables affecting the hypercrosslinking reaction were studied in detail. These variables included: (i) the temperature and time used for polymerization of the precursor monoliths, (ii) the amount and loading procedure of the external crosslinker and catalyst, and, (iii) the temperature and reaction time of the Friedel–Crafts alkylation. Polymers with extremely large surface areas reaching up to 900 m2/g were obtained using a precursor monolith polymerized for only 2.5 h, and hypercrosslinked with 4,4’-bis(chloromethyl)-1,1′-biphenyl. The increased performance of monoliths modified using this new procedure is comparable to the performance obtained with monolithic polymers containing chloromethylstyrene. However, the preparation of the poly(styrene-divinylbenzene) precursor is simpler than that of conterparts containing chloromethylstyrene. The hypercrosslinked monoliths in capillary columns were tested in an isocratic reversed phase liquid chromatography separation using a mixture comprised of acetone and six alkylbenzenes. Column efficiencies for retained analytes exceeded 70,000 plates/m.

Semi-automated liquid chromatography–mass spectrometric imaging platform for enhanced detection and improved data analysis of complex peptides

A semi-automated analytical platform featuring the coupling of monolithic reversed-phase liquid chromatography (RPLC) to matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI MSI) has been developed and evaluated. This is the first time that LC separation is readily coupled to MS imaging detection for the analysis of complex peptide mixtures both qualitatively and quantitatively. Methacrylate-based monolithic column with C12 functional groups is fabricated for fast RPLC separation. The LC flow and matrix flow are collected on a commercially available MALDI plate which is mechanically controlled and analyzed with MALDI MSI subsequently. Both tryptic peptides digested from bovine serum albumin (BSA) and endogenous neuropeptides extracted from the blue crab Callinectes sapidus are analyzed with this novel LC–MSI platform. Compared with regular offline LC fractionation coupled with MALDI MS detection, LC–MSI exhibits significantly increased MS signal intensity due to retaining of temporal resolution from separation dimension via continuous sampling, which results in increased number of peptides detected and accurate quantitation. In addition, imaging signals enable improved data analysis based on either mass-to-charge ratio or retention time, which is extremely beneficial for the analysis of complex analytes. These findings have demonstrated the potential of employing LC–MSI platform for enhanced proteomics and peptidomics studies.

Multi-residue method for determination of selected neonicotinoid insecticides in honey using optimized dispersive liquid–liquid microextraction combined with liquid chromatography-tandem mass spectrometry

The objective of this study was to develop analytical method based on optimized dispersive liquid–liquid microextraction (DLLME) as a pretreatment procedure combined with reversed phase liquid chromatographic separation on C18 column and isocratic elution for simultaneous MS/MS determination of selected neonicotinoid insecticides in honey. The LC–MS/MS parameters were optimized to unequivocally provide good chromatographic separation, low detection (LOD, 0.5–1.0μgkg−1) and quantification (LOQ, 1.5–2.5μgkg−1) limits for acetamiprid, clothianidin, thiamethoxam, imidacloprid, dinotefuran, thiacloprid and nitenpyram in honey samples. Using different types (chloroform, dichloromethane) and volumes of extraction (0.5–3.0mL) and dispersive (acetonitrile; 0.0–1.0mL) solvent and by mathematical modeling it was possible to establish the optimal sample preparation procedure. Matrix-matched calibration and blank honey sample spiked in the concentration range of LOQ-100.0μgkg−1 were used to compensate the matrix effect and to fulfill the requirements of SANCO/12495/2011 for the accuracy (R 74.3–113.9%) and precision (expressed in terms of repeatability (RSD 2.74–11.8%) and within-laboratory reproducibility (RSDs 6.64–16.2%)) of the proposed method. The rapid (retention times 1.5–9.9min), sensitive and low solvent consumption procedure described in this work provides reliable, simultaneous, and quantitative method applicable for the routine laboratory analysis of seven neonicotinoid residues in real honey samples.

Analysis of a Ricin Biomarker, Ricinine, in 989 Individual Human Urine Samples

Ricinine (3-cyano-4-methoxy-N-methyl-2-pyridone) is a urinary biomarker that can be measured to confirm human exposure to castor bean products such as ricin. Because many consumer products contain castor oil, another castor bean product, ricinine may be detectable in the general population. The following study characterized urinary ricinine concentrations from 989 individuals who were presumed to be unexposed to ricin. An automated diagnostic method was utilized to simplify the analysis of this large sample set. Sample preparation included a 96-well polystyrene divinylbenzene high throughput extraction and preconcentration step. Purified samples were analyzed by an efficient dual column, reversed-phase liquid chromatography separation and (13)C-isotope dilution tandem mass spectrometry. In this convenience sample set, only 1.2% of the urine specimens had detectable amounts of ricinine, randomly distributed between 0.186 and 4.15 ng/mL.

Application of hydrophilic interaction chromatography in phytochemical analysis

Summary Hydrophilic interaction chromatography (HILIC) is a liquid chromatography technique when a polar stationary phases — silica or polar bonded phases and aqueous mobile phase containing amount of a less polar solvent (often acetonitrile) is applied. HILIC is important for the separation of highly polar substances including biologically active compounds, such as drugs, amino acids, peptides, proteins, nucleosides, neurotransmitters, etc. HILIC is also the appropriate method for analysis of plant extract polar components such as carbohydrates, amino acids, peptides, phenolic acids, flavonoids and some alkaloids. Plant extracts are usually multicomponent mixtures of compounds of wide polarity range, which often cannot be analyzed by use of a single separation and detection method because of the high chemical diversity. Good results are obtained by use of coupled method, e.g., reversed-phase liquid chromatography (RP-LC) and HILIC mode separation. The elution order in HILIC is usually opposite to that in RP separations. This orthogonality determines one of the advantages of HILIC — the possibility of creating multidimensional separations. An important feature of HILIC is the improved sensitivity with electrospray mass spectrometry. This is significant for the analysis of components existing in small concentration in multicomponent mixtures. The high acetonitrile content also gives additional advantage of HILIC — faster separations due to the lower viscosity of HILIC eluents compared to standard RP ones. The presented review deals with the optimization of HILIC separations and application of the method for analysis of plant extracts components.

An Ion Mobility Assisted Data Independent LC-MS Strategy for the Analysis of Complex Biological Samples

The use of gas-phase ion mobility separation in LC-MS based bottom-up proteomic experiments is described. A data-independent acquisition approach was employed in combination with one and two dimensional reversed phase liquid chromatography separations. The ion mobility component in the acquisition schema affords additional acquisition specificity and improved peptide resolution in terms of system peak capacity. The additional acquisition specificity was mainly achieved by means of the combined retention and drift time association of product to precursor ions and chemical noise reduction. Experimental data from replicate experiments were utilized to determine the rate of increase for the number of detected ions and deconvoluted accurate mass – retention time pairs and protein and peptide identifications as a measure of peak capacity by varying mass spectral resolution, consideration of multidimensional separations and the incorporation of ion mobility into the analytical schema. The presented data reveals a high degree of reproducibility at the ion detection, peptide and protein level, when analyzed under identical conditions, with a concurrent average increase in the number of identified peptides and proteins of 160% in the instance of mobility assisted acquisitions. Two-dimensional separations independently improved the peptide and protein identification rates by another 260% and 320%, respectively. Collectively, both separation techniques, reversed phase chromatography and ion mobility, increased the peak capacity and number of peptides and proteins significantly. The applicability of the technique and instrument platform for qualitative and quantitative proteomics studies will be demonstrated.

Development of liquid microjunction extraction strategy for improving protein identification from tissue sections

MALDI Mass Spectrometry Imaging has shown important potential for molecular classification and pathology marker discovery. Protein markers identification is therefore of prime importance. Direct structural analysis from tissue sections has shown limitations for protein identification because of the high degree of complexity of tissues. Only proteins of major abundance are identified this way. On the contrary, conventional proteomics approaches clearly allow for reliable identification of complex protein extracts but do not provide fine correlation with protein location in their original context. Here is presented an approach to obtain identification of proteins of various abundances while keeping their localization within the section. On-tissue trypsin digestion followed by micro-extraction using a liquid micro-junction interface is an efficient strategy to extract tryptic peptides and further identify the associated proteins off tissues. It was shown that conventional Reverse Phase Liquid Chromatography separation on the extracted material followed by MS/MS analysis on a HR FTMS instrument enabled the identification of 1500 proteins on average with high confidence from an area of about 650μm in diameter, which corresponds to an estimated number of 1900 cells in average. The approach can be easily integrated in the MALDI MSI workflow and should provide interesting insights for clinical applications.

Reversed-phase high performance liquid chromatography separation of 2-(fluorophenyl)-5-methylbenzoxazole and 2-(chlorophenyl)-5-methylbenzoxazole positional isomers

The analytical method of 2-( fluorophenyl)-5-methylbenzoxazole ortho-, meta-, para-positional isomers and 2-( chlorophenyl) -5-methylbenzoxazole ortho-, meta-, para-positional isomers was developed with a commercial common liquid chromatography column. The separation was performed on an Inertsil ODS-SP C18 column (250 mm x 4.6 mm, 5 microm) at 40 degrees C with a linear gradient elution by mobile phases of acetonitrile (A) (from 60% to 80% within 15 min) and water (B) at a flow rate of 1.5 mL/min. The samples were detected by a diode array detector at 310 nm. Good linearities were obtained in the range of 2 - 200 mg/L for the six isomers. The limits of detection (LODs) (S/N = 3) of 2- (fluorophenyl) -5-methylbenzoxazole ortho-, meta-, para-isomers and 2-( chlorophenyl)-5-methylbenzoxazole ortho-, meta-, para-isomers were 0.0307, 0.0293, 0.0315, 0.0226, 0.0237, 0.0226 mg/L, respectively. The method provides not only a rapid detection method for the preparation of the isomers through hydrocarbon activation coupling reaction between the 5-methylbenzoxazole and fluorobenzene or chlorobenzene but also a reference for the separation and detection of 2-arylbenzoxazole isomers.

Urinary hydrophilic and hydrophobic metabolic profiling based on liquid chromatography‐mass spectrometry methods: Differential metabolite discovery specific to ovarian cancer

Discovery of novel metabolite biomarker(s) for improved ovarian cancer diagnosis is of great importance. In this paper, the differences of urinary hydrophilic and hydrophobic metabolic profiling between healthy women, benign ovarian tumor, and ovarian cancer patients were studied by metabolomics strategy. Metabolites in urine were analyzed on hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) coupled to MS. Data from HILIC or RPLC, positive or negative ion detection mode were found to be complementary. Data were filtered by orthogonal signal correction (OSC) method, and the three groups were discriminated by partial least squares discrimination analysis (PLS-DA) models. By combining the four datasets, maximum information can be collected, and a PLS-DA model was built after OSC filtering. The model based on combined dataset is superior to the ones based on the separate dataset, and important metabolites were screened based on the combined dataset model. Five metabolites were found to be specific to ovarian cancer and ten metabolites were considered commonly related to ovarian cancer and benign ovarian tumor. Combination of RPLC and HILIC separation, as well as positive and negative ion detection in metabolomic studies show advantages in collecting various metabolites information that helps us better understand the metabolic event.

Antibody-free, targeted mass-spectrometric approach for quantification of proteins at low picogram per milliliter levels in human plasma/serum

Sensitive detection of low-abundance proteins in complex biological samples has typically been achieved by immunoassays that use antibodies specific to target proteins; however, de novo development of antibodies is associated with high costs, long development lead times, and high failure rates. To address these challenges, we developed an antibody-free strategy that involves PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing) for sensitive selected reaction monitoring (SRM)-based targeted protein quantification. The strategy capitalizes on high-resolution reversed-phase liquid chromatographic separations for analyte enrichment, intelligent selection of target fractions via on-line SRM monitoring of internal standards, and fraction multiplexing before nano-liquid chromatography-SRM quantification. Application of this strategy to human plasma/serum demonstrated accurate and reproducible quantification of proteins at concentrations in the 50-100 pg/mL range, which represents a major advance in the sensitivity of targeted protein quantification without the need for specific-affinity reagents. Application to a set of clinical serum samples illustrated an excellent correlation between the results obtained from the PRISM-SRM assay and those from clinical immunoassay for the prostate-specific antigen level.

Chiral separations in reversed-phase liquid chromatography: Evaluation of several polysaccharide-based chiral stationary phases for a separation strategy update

By application of the reversed-phase generic screening conditions of a separation strategy defined in Matthijs et al. (2004) [18], the chiral discrimination abilities of six recently commercialized polysaccharide-based columns, Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, Lux Cellulose-4, Lux Amylose-2 and Sepapak-5, and of three classic ones, Chiralpak AD-RH, Chiralcel OD-RH and Chiralcel OJ-RH, were evaluated using a set of 58 compounds. Two mobile phases, an acidic and a basic, were sequentially applied on the columns. Using both mobile phases, a column set of Chiralcel OD-RH (or Lux Cellulose-1), Lux Cellulose-3 and Lux Amylose-2 gave the maximal number of cumulative separations, i.e. 51/58 (or 50), of which 35 (or 32) had baseline resolutions. Therefore, this set of systems was selected to update the screening step of the existing separation strategy. The selected columns were subsequently used to evaluate the applicability of the initial optimization steps, part of the existing RPLC strategy, using 66 different optimization cases. The existing optimization steps increased both the number of separations and of baseline separations by eight and by 18, respectively, relative to the screening results. Introduction of some modifications to the existing steps added eight more separations and three more baseline separations. In addition, a new optimization step for late eluting compounds was proposed and implemented. Based on these results, an updated chiral separation strategy in RPLC was defined.

Rapid reversed-phase liquid chromatography separation of cyclolinopeptides with monolithic and microparticulate columns

Three monolithic C18-bonded silica gel columns i.e. Chromolith® SpeedROD (CSR), Chromolith® Performance (CP), and Chromolith® High Resolution (CHR), MerckKGaA Darmstadt, Germany and two particle-based columns i.e. ZORBAX Eclipse XDB-C18 (ZEX), Agilent and POROS R1/20 (POR), Applied Biosystems were compared for their performance in separating a mixture of flaxseed cyclolinopeptides (CLs). Gradient mobile phases of acetonitrile and water were optimized for each column. The performance of CHR column in profiling CL standards, measured as the resolution of individual CL, selectivity, and peak asymmetry exceeded the performance of traditional particle-packed columns and the other monolithic columns. The profiling of CLs in aqueous methanolic flaxseed extract was optimized for high-throughput analysis. A total analysis time of 1.5min at a flow rate of 3.0mLmin−1 was achieved on a CSR column. Injection of over 2000 methanol extracts of flaxseed on a CSR column had no impact on backpressure or resolution of a standard CL mixture.

Effects of Chain Grafting Positions and Surface Coverage on Conformations of Model Reversed-Phase Liquid Chromatography Stationary Phases

Chemical separations in reversed-phase liquid chromatography (RPLC) are made possible by the detailed molecular-level interactions that take place between analyte molecules and the interface between the stationary and mobile phases. Changes in operational conditions lead to different retention times, suggesting that the structures of the stationary phase and the stationary/mobile phase interface are altered. However, the details of such alterations, and their relationship to separation performance, are not well understood. In this study, the conformations of model RPLC stationary phases were investigated with all-atom molecular dynamics simulations to elucidate the detailed structural response of the stationary phase to different conditions. The model system consists of a quartz surface functionalized with dimethyloctadecylsilane immersed in H2O. Pressurization of the mobile phase did not affect the structure of the stationary phase, in agreement with previous studies. The choice of grafting positions on ...

Recent theoretical and practical applications of micellar liquid chromatography (MLC) in pharmaceutical and biomedical analysis

Abstract Micellar liquid chromatography (MLC) is an analytical technique belonging to the wide range of reversed-phase liquid chromatographic (RP-LC) separation techniques. MLC with the use of surfactant solutions above its critical micellar concentration (CMC) and the addition of organic modifiers is currently an important analytical tool with still growing theoretical considerations and practical applications in pharmaceutical analysis of drugs and other biologically active compounds. The use of MLC as an alternative, relatively much faster in comparison to conventional chromatographic separation techniques has several advantages, especially as being suitable for screening pharmaceutical analysis. The analytical data received from MLC analysis are considered a useful source of information to predict passive drug absorption, drug transport and other pharmacokinetics and physicochemical measures of pharmaceutical substances. In the review several MLC assays for determination of drugs and other active compounds in biological samples were compared and critically discussed. The presented overview provides information on recent applications and achievements connected with the practical use of MLC. The review covers fields of interest related to theory and mechanism of MLC separation, direct applications of MLC in pharmaceutical analysis, including optimization and efficiency of separation with the use of modification of stationary phase and mobile phase compositions as well as the determination of physicochemical characteristics of drugs by MLC.