Advances and Challenges in the Analysis of Boswellic Acids by Separation Methods.

Boswellia resin is an exudate from the cut bark of Boswellia trees. The main constituents of pharmacological interest are boswellic acids (pentacyclic triterpenoids), namely α-boswellic acid, ß-boswellic acid, 3-O-acetyl-α-boswellic acid, 3-O-acetyl-ß-boswellic acid, 11-keto-ß-boswellic acid, and 3-O-acetyl-11-keto-ß-boswellic acid. Nowadays, dietary supplements with Boswellia serrata extract are used in the treatment of inflammatory joint diseases. Additionally, the constituents of Boswellia resin have shown potential for the treatment of other chronic inflammatory diseases and various types of cancer. Separation methods including ultra/high-performance liquid chromatography, gas chromatography, thin layer chromatography, supercritical fluid chromatography, and capillary electrochromatography coupled with UV or MS detection have been used for the determination of boswellic acids in various matrices (mostly plant material and biological samples). This review aims to provide a comprehensive summary of these separation methods, offering a critical discussion of their strengths and limitations in the analysis of boswellic acids. The knowledge of various separation methods plays a pivotal role in the quality control of herbal dietary supplements and the monitoring of the metabolism and pharmacokinetics of their constituents. The approaches based on metabolomics and network pharmacology represent new ways of fingerprinting secondary metabolites in Boswellia resin increasing the comprehensiveness of the output of these methods resulting in safer dietary supplements.

Coupling of chiral and achiral stationary phases in supercritical fluid chromatography: Evaluating and improving retention prediction.

Isomers and stereoisomers are always challenging to separate. Column coupling may provide improved chromatographic selectivity, necessary for the separation of the compounds with similar chemical and structural properties. The relatively low viscosity of supercritical fluids, used as mobile phases allows for the coupling of several columns in series in supercritical fluid chromatography (SFC), without exceeding the pressure limits of the system. The aim of this study is to propose reliable prediction of the retention behaviour of analytes on a coupled column system, based on a limited number of initial analyses. The chiral compounds atenolol, ephedrine, propranolol, mianserin, labetalol and nadolol, besides the diastereomers quinine and quinidine, and the structural isomers of aminophenol and aminocresol were used as model analytes. The retention behaviour of the analytes was determined on the individual chiral columns Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, Lux Cellulose-4, Lux Amylose-2 and the achiral columns Luna NH2, Luna Silica, Synergi RP and FluoroSep RP. The mobile phase was composed of CO2 mixed with 20% (v/v) MeOH, which contained 0.1% (v/v) trifluoroacetic acid and 0.1% (v/v) isopropylamine. The retention factors of the analytes on coupled stationary phases were predicted, and subsequently compared to the experimentally obtained ones. Relative deviations of predicted and experimental retention factors were in range from 0.00% to 51.91%. Flow rate and back pressure of the screening conditions were adjusted to improve prediction precision on four column combinations, with varying success rates. The average relative deviations of retention factors were reduced to 2.84% - 6.59% by adjusting flow rate, and to 2.30% - 8.57% by adjusting back pressure. The most successful approach, flow rate adjustment, was then applied to select a column combination providing improved resolution of the structurally similar components of silymarin extract.

Development of a capillary electrophoresis method for the separation of flavonolignans in silymarin complex.

CE method for the baseline separation of structurally similar flavonolignans silybin A, silybin B, isosilybin A, isosilybin B, silychristin, silydianin, and their precursor taxifolin in silymarin complex has been developed and validated. The optimized background electrolyte was 100 mmol/L boric acid (pH 9.0) containing 5 mmol/L heptakis(2,3,6-tri-O-methyl)-ß-CD and 10% (v/v) of methanol. The separation was carried out in an 80.5/72 cm (50 µm id) fused silica capillary at +25 kV with UV detection at 200 nm. Genistein (10 µg/mL) was used as internal standard. The resolution between the diastereomers of silybin and isosilybin was 1.73 and 2.59, respectively. The method was validated for each analyte in a concentration range of 2.5-50 µg/mL. The calibration curves were rectilinear with correlation coefficients ≥0.9972. The method was applied to determine flavonolignans in two dietary supplements containing Silybum marianum extract. The accuracy was evaluated by comparing the results of the CE analyses of the dietary supplements with those of the reference United States Pharmacopeial HPLC method. The unpaired t-test did not show a statistically significant difference between the results of both the proposed CE and the reference method (p > 0.05, n = 3).

In-line molecularly imprinted polymer solid phase extraction-capillary electrophoresis coupled with tandem mass spectrometry for the determination of patulin in apple-based food.

We present a simple and sensitive method for the determination of patulin at µg·kg-1 level in apple-based products. Our method relies on the application of an in-line molecularly imprinted polymer solid-phase extraction microcartridge in capillary electrophoresis coupled with mass spectrometry. Capillary zone electrophoresis method has been developed and parameters affecting the in-line process have been carefully optimized. Validation parameters were assessed for patulin, giving LOQ of 1 µg·kg-1 and linearity range 1-100 µg·kg-1 with R2 ≥ 0.997. The LOQ was below the maximum content of patulin requested by the European Union in this type of products. The precision of the peak area and the migration time were less than 14.9 and 1.6%, respectively. Patulin has been analyzed in the presence of 5-hydroxymethylfurfural, which is the main interference in this kind of matrix. The method was applied to assay patulin content in various apple-based products.

Stationary-phase optimized selectivity in supercritical fluid chromatography using a customized Phase OPtimized Liquid Chromatography kit: comparison of different prediction approaches.

The use of stationary-phase optimized selectivity in liquid chromatography (SOS-LC) was shown to be successful for HPLC to analyze complex mixtures using a Phase OPtimized Liquid Chromatography (POPLC) kit. This commercial kit contains five stationary-phase types of varying lengths, which can be coupled to offer an improved separation of compounds. Recently, stationary-phase optimized selectivity supercritical fluid chromatography (SOS-SFC) has been introduced, transferring the methodology to SFC. In this study, the applicability of a customized POPLC expert kit for isocratic SFC runs was explored. Five stationary-phase chemistries were selected as potentially most suitable for achiral separations of polar compounds: aminopropyl (amino), cyanopropyl (CN), diol, ethylpyridine (EP), and silica. The retention factors (k) on the individual stationary phases were used for the prediction of the best stationary-phase combination, based on the POPLC algorithm (via the included software). As an alternative, the best column combination was predicted using multiple linear regression (MLR) models on the results obtained from a simplex centroid mixture design with only three stationary-phase types (amino, silica, and EP). A third approach applied the isocratic POPLC algorithm on the same three stationary-phase data. The proposed combinations were assembled and tested. The predicted and experimental retention factors were compared. The predictions based on the POPLC algorithm provided a stationary phase showing a complete separation of the mixture. The stationary phase suggested by the MLR models, on the other hand, showed co-elution of two compounds, due to an unexpected experimental retention shift. Overall, the customized POPLC kit showed good potential to be applied in SFC. Graphical abstract.

Development of micellar electrokinetic chromatography method for the determination of three defined impurities in indomethacin.

A micellar electrokinetic chromatography method for the determination of indomethacin impurities (4-chlorobenzoic acid, 5-methoxy-2-methyl-3-indoleacetic acid, and 3,4-dichloroindomethacin) has been developed. A 64.5/56-cm fused silica 50 µm id capillary with extended light path (150 µm id) detection window was used. Internal standard was 1-naphthylacetic acid. The analytes were separated at 30 kV with DAD detection at 224 nm. A central composite face-centered design was applied for the optimization of the separation conditions. The effect of SDS concentration, content of methanol, concentration of phosphate buffer, and pH of the buffer were studied at three levels. The optimized background electrolyte was 20 mmol/L phosphate buffer (pH 7.57) containing 58 mmol/L SDS and 0% MeOH. Sufficient resolution of all compounds with Rs ≥ 3.5 was achieved within 10 min. The method was validated for a range of 1.25-80 µg/mL of each impurity corresponding to 0.05-3.2% relative to the concentration of indomethacin (2.5 mg/mL). The calibration curves were rectilinear with correlation coefficients r2 exceeding 0.9994. The lower limit of quantification was 0.05% or 1.25 µg/mL that complies with the reporting limits regarding the ICH Q3A guideline. The method was applied to purity assay of indomethacin in both bulk drug and gel.