A solid-liquid microextraction approach from a spot in an adsorbent layer of a chromatographic plate.

A new approach to extracting substances from a spot on a chromatographic plate for subsequent liquid chromatography-mass spectrometry analysis is described. This method involves extraction in a solid phase (an adsorbent layer of a chromatographic plate) - a liquid system using a simple device. For a single extraction of six selected coccidiostats from the adsorbent layer on the chromatographic plate with silica gel, 50 µL of methanol was used for 5 min. The data from the extraction experiments and liquid chromatography-mass spectrometry measurements demonstrated a good correlation between the ratio of the peak areas of the coccidiostats to the internal standard and the concentration of the substances in the range of two orders of magnitude. The coefficients of determination for the mentioned correlations range from 0.962 to 0.999. Moreover, the repeatability and reproducibility, expressed as the percentage values of relative standard deviation, do not exceed 7.5 % for any of the coccidiostats.

Comparison of the Determination of Some Antihypertensive Drugs in Clinical Human Plasma Samples by Solvent Front Position Extraction and Precipitation Modes.

The determination of the selected antihypertensive drugs in human plasma samples with the novel solvent front position extraction (SFPE) technique is presented. The SFPE procedure combined with LC-MS/MS analysis was used for the first time to prepare a clinical sample containing the drugs mentioned above from different therapeutic groups. The effectiveness of our approach was compared with the precipitation method. The latter technique is usually used to prepare biological samples in routine laboratories. During the experiments, the substances of interest and the internal standard were separated from other matrix components using a prototype horizontal chamber for thin-layer chromatography/high-performance thin-layer chromatography (TLC/HPTLC) with a moving pipette powered by a 3D mechanism, which distributed the solvent on the adsorbent layer. Detection of the six antihypertensive drugs was performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. Results obtained by SFPE were very satisfactory (linearity R2 ≥ 0.981; %RSD ≤ 6%; LOD and LOQ were in the range of 0.06-9.78 ng/mL and 0.17-29.64 ng/mL, respectively). The recovery was in the range of 79.88-120.36%. Intra-day and inter-day precision had a percentage coefficient of variation (CV) in the range of 1.10-9.74%. The procedure is simple and highly effective. It includes the automation of TLC chromatogram development, which significantly reduced the number of manual operations performed, the time of sample preparation and solvent consumption.

Solvent Front Position Extraction and some conventional sample preparation techniques for the determination of coccidiostats in poultry feed by LC-MS/MS.

Solvent Front Position Extraction is a novel technique developed for effective sample preparation of biological samples containing coccidiostats prior to LC-MS/MS. In this study the technique was used for isolation and determination of seven coccidiostats, from both main groups being: ionophores and chemical coccidiostats. Its effectiveness was evaluated by comparing with other sample preparation procedures, used in European routine laboratories. Results obtained by Solvent Front Position Extraction were very satisfactory (linearity R2 ≥ 0.971, recovery 90.1-111.1%, RSDr: 8.7-16.6%, RSDR: 9.0-17.7%) and fulfilled requirements described in Commission Regulation (EU) 2021/808 of 22 March 2021, which showed great potential of the technique in sample preparation of coccidiostats in poultry feed.

Preparation of Antihypertensive Drugs in Biological Matrix with Solvent Front Position Extraction for LC-MS/MS Analysis.

Solvent front position extraction procedure was used to prepare biological samples containing selected antihypertensive drugs (ramipril, lercanidipine, indapamide, valsartan, hydrochlorothiazide, perindopril, and nebivolol). Substances separated from the biological matrix components (bovine serum albumin) were quantified by means of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Sample preparation process was performed with the use of a prototype horizontal chamber with a moving pipette driven by a 3D printer mechanism enabling a controlled eluent flow velocity. Application of this device was advantageous for simultaneous preparation of several samples for further quantitative analysis, with a synchronized reduction of manual operations and solvent consumption. Quantitative results obtained for the majority of the investigated antihypertensive drugs in a complex biological matrix were satisfactory. The values of the %RSD were around 5% for six of the seven substances (with the exception of indapamide). The method exhibits a suitable accuracy (the relative error percentage was below 10% for most drugs). The values of LOD and LOQ were in the range of 1.19 µg/L-8.53 µg/L and 3.61 µg/L-25.8 µg/L, respectively.

Optimization of Adsorbent Layer Type and Developing Solvent in Coccidiostats Sample Preparation with Procedure of Solvent Front Position Extraction.

Coccidiostats are drugs used against coccidiosis, a common disease among breeding animals. Their widespread application leads to the appearance of their residues in food, which is potentially harmful for human health and life. The European Union has established limits of concentrations of these drugs in premixtures and food. Nowadays, there are many methods for monitoring coccidiostats' presence in market products, but their frequent weakness is sample preparation. Solvent Front Position Extraction is a planar chromatography-based sample preparation method that allows for effective assay of samples with coccidiostats when coupled with LC-MS/MS. The purpose of this research was to find common conditions for the effective isolation of eight coccidiostats from biological sample components with both lower and higher retention than the substances of interest. The acquired results were used for effective isolation of monensin and salinomycin from the premixture samples and allowed for their quantitative determination. The application of a semi-automatic device for the development of chromatograms positively impacted the results, confirming the effectiveness of the method for determining coccidiostats in biological samples.

Comparison of the Retention and Separation Selectivity of Aromatic Hydrocarbons with Polar Groups in RP-HPLC Systems with Different Stationary Phases and Eluents.

In this manuscript, the retention of aromatic hydrocarbons with polar groups has been compared for systems with various nonpolar columns of the types from C3 to C18 and different mobile phases composed of methanol, acetonitrile, or tetrahydrofuran as modifiers. The selectivity separation of the solutes in systems with different adsorbents, when one eluent modifier is swapped by another, has been explained, taking into account molecular interactions of the solutes with components of the stationary phase region (i.e., extracted modifier depending on the chain length of the stationary phase).

Solvent front position extraction with semi-automatic device as a powerful sample preparation procedure to quantitatitation of tryptophan in human plasma.

In the paper the results of the tryptophan determination in human plasma samples prepared with the novel Solvent Front Position Extraction (SFPE) technique are presented. The SFPE procedure is used for preparation of real biological sample for the first time. The results obtained using SFPE are compared with those using the classical sample preparation procedure. Under the optimal conditions, tryptophan and its internal standard were separated from other plasma compounds (matrix) as a small common zone/spot on a chromatographic plate using semiautomatic device equipped with moving pipet, which distributed developing solvent on the adsorbent layer. Tryptophan and the internal standard were evenly distributed within the small common zone from that the both substances were extracted and the solution obtained was transferred to quantitation with LC-MS and MS techniques. The determination results are satisfactory, the percentage values of relative error and RSD relative standard deviation do not exceed 5%. The procedure is characterized by simplicity, high analysis throughput, very good sample purification and seems to be easy applicable to other biological samples with these advantages mentioned.

Retardation of some drugs in thin-layer chromatographic systems with impregnated silica gel plates with hen's egg white and bovine serum albumin.

The influence of impregnation the chromatographic plate adsorbent layer, silica, with hen's egg white albumin (OVA) or bovine serum albumin (BSA) on the retention of some popular medicines (paracetamol, aminophenazone, theophylline, caffeine, acetanilide, ciprofloxacin, tramadol, acetylsalicylic acid, acebutolol) is investigated. The effect of composition and buffer pH of the mobile phase on solute separation selectivity is also studied. The chromatographic systems with and without above mentioned albumins and their influence on investigated drug retention are compared. In general, it has been turned out that retention of tested medicines in systems with the sorbent impregnated with albumin significantly increase relative to those with non-impregnated.

Optimization of the procedure of solvent front position extraction for preparation of multi-component sample for instrumental analysis.

Solvent Front Position Extraction (SFPE) procedure has been recently introduced as a novel concept for multi-component sample preparation. According to the procedure, thin-layer chromatography (TLC) is used to separate the compounds of interest from matrix components, and to focus them into a common zone from which the compounds are extracted and transferred to apparatus for instrumental analysis. In the paper, we investigate different adsorbent types of the chromatographic plates and various mobile phases, including pH of their buffers, in respect of optimization conditions of the SFPE procedure. The research was carried out using a test sample containing 9 compounds characterised by different chemical properties, hence the conclusions from the obtained results can be applied to other multi-component samples. Under the optimal conditions, all target compounds are separated from other compounds (matrix), and evenly distributed along a narrow strip, which is advantageous for their quantitation. The determination results are good, the percentage values of relative error and relative standard deviation do not exceed 6%.

Solvent Front Position Extraction with Semi-Automatic Device as a Powerful Sample Preparation Procedure Prior to Quantitative Instrumental Analysis.

The new prototype device is applied to the Solvent Front Position Extraction (SFPE) sample preparation procedure. The mobile phase is deposited onto the chromatographic plate adsorbent layer by the pipette, which is moved, according to programmed movement path, by a 3D printer mechanism. The application of the prototype device to SFPE procedure leads to the increased repeatability of the results and significant reduction of the analysis time in comparison to the classical procedure of chromatogram development. Additionally, the new equipment allows use procedures that are not possible to run using the classic chromatogram development. In this paper, the results of manual and semi-automatic sample preparation with SFPE are compared and the possible application of this prototype device is discussed.

Thin-layer chromatogram development with a moving pipette delivering the mobile phase onto the surface of the adsorbent layer.

The new approach to the development of thin-layer chromatograms is presented. For the first time we show flexible mobile phase dosage onto the surface of the adsorbent layer by moving pipette combined with precise syringe pumps. The pipette is driven into movement by computer controlled 3D machine (modified 3D printer mechanism). Delivery of the mobile phase to the adsorbent layer is equal to or lower than that of conventional development. Therefore chromatograms can be developed with optimal mobile phase velocity, adjusted to its absorption rate by the adsorbent layer. Under such conditions there is no excess of eluent on the surface of the adsorbent layer so higher performance of the chromatographic system can be obtained. Moreover chromatograms can be developed with constant linear mobile phase velocity and therefore the relationships the plate height vs. mobile phase linear velocity obtained with planar chromatography driven by capillary forces are investigated and reported. In addition the contribution of starting spot variance in total peak variance and the influence of narrowing of starting spots on performance of the chromatographic system have been studied. The results confirm a very significant starting spot variance contribution to total peak variance and consequently considerable influence of starting spot size on plate height of the separation system, when chromatogram is developed on a short distance. In the paper the advantages and disadvantages of the prototype device and its possible application are discussed.

Influence of carboxylic ion-pairing reagents on retention of peptides in thin-layer chromatography systems with C18 silica-based adsorbents.

One of the main problems related to chromatography of peptides concerns adverse interactions of their strong basic groups with free silanol groups of the silica based stationary phase. Influence of type and concentration of ion-pairing regents on peptide retention in reversed-phase high-performance liquid chromatography (RP-HPLC) systems has been discussed before. Here we present influence of these mobile phase additives on retention of some peptide standards in high-performance thin-layer chromatography (HPTLC) systems with C18 silica-based adsorbents. We prove, that due to different characteristic of adsorbents used in both techniques (RP HPLC and HPTLC), influence of ion-pairing reagents on retention of basic and/or amphoteric compounds also may be quite different. C18 silica-based HPTLC adsorbents provide more complex mechanism of retention and should be rather considered as mixed-mode adsorbents.

Comparison of the Retention of Aliphatic Hydrocarbons with Polar Groups in RP-HPLC Systems with Different Modifiers of the Binary Eluent.

The retention of aliphatic hydrocarbons with polar groups has been compared in respect to the separation selectivity changes in reversed-phase high-performance liquid chromatography with C18 stationary phase type and binary water eluent composed of methanol, acetonitrile, or tetrahydrofuran as modifiers. The changes in separation selectivity when one modifier is replaced by another in the eluent is explained, taking into consideration molecular interactions of the solutes with components of the stationary phase region, i.e., extracted modifier, and ordering of the stationary phase by the modifier.

Pressurized planar electrochromatography, high-performance thin-layer chromatography and high-performance liquid chromatography--comparison of performance.

Kinetic performance, measured by plate height, of High-Performance Thin-Layer Chromatography (HPTLC), High-Performance Liquid Chromatography (HPLC) and Pressurized Planar Electrochromatography (PPEC) was compared for the systems with adsorbent of the HPTLC RP18W plate from Merck as the stationary phase and the mobile phase composed of acetonitrile and buffer solution. The HPLC column was packed with the adsorbent, which was scrapped from the chromatographic plate mentioned. An additional HPLC column was also packed with adsorbent of 5 microm particle diameter, C18 type silica based (LiChrosorb RP-18 from Merck). The dependence of plate height of both HPLC and PPEC separating systems on flow velocity of the mobile phase and on migration distance of the mobile phase in TLC system was presented applying test solute (prednisolone succinate). The highest performance, amongst systems investigated, was obtained for the PPEC system. The separation efficiency of the systems investigated in the paper was additionally confirmed by the separation of test component mixture composed of six hormones.