Isocratic Elution Mode Research Articles

Application of a HPLC-Q/TOF Method with Post-column Compensation for Separation and Identification of Polar Impurities in Cytidine Disodium Triphosphate for Injection

Background: Cytidine Disodium Triphosphate (CTP-2Na) for injection is mainly used for treating nervous system diseases. Currently, there are few studies focused on the separation and identification of polar impurities in CTP-2Na for injection, which is important for ensuring drug safety and efficacy. Objective: The study aimed to establish an HPLC-Q/TOF method for the separation and identification of polar impurities in CTP-2Na for injection. Methods: Chromatographic separation was achieved on a Waters Atlantis T3 column using 5 mM aqueous ammonium acetate solution as the mobile phase in an isocratic elution mode. A postcolumn compensation technology was used to improve the ionization efficiency of impurities in the spray chamber. Results: Three polar impurities (disodium cytidine tetraphosphate, disodium cytidine diphosphate, disodium cytidine monophosphate) were detected in CTP-2Na for injection. The former one is probably the overreaction product during the production of CTP-2Na, the latter two were reported as degradation products. The fragmentation patterns of cytidine phosphate compounds in negative ion mode are summarized. Conclusion: This study provides a good reference for the separation and identification of polar impurities in nucleotide drugs.

Supercritical Fluid Chromatography-Tandem Mass Spectrometry for Rapid Quantification of Pentacyclic Triterpenoids in Plant Extracts.

Pentacyclic triterpenoids (PCTs) are a widely distributed class of plant secondary metabolites. These compounds have high bioactive properties, primarily antitumor and antioxidant activity. In this study, a method was developed for the quantitative analysis of pentacyclic triterpenoids in plants using supercritical fluid chromatography–tandem mass spectrometry (SFC-MS/MS). Separation of ten major PCTs (friedelin, lupeol, β-amyrin, α-amyrin, betulin, erythrodiol, uvaol, betulinic, oleanolic and ursolic acids) was studied on six silica-based reversed stationary phases. The best results (7 min analysis time in isocratic elution mode) were achieved on an HSS C18 SB stationary phase using carbon dioxide—isopropanol (8%) mobile phase providing decisive contribution of polar interactions to the retention of analytes. It was shown that the use of atmospheric pressure chemical ionization (APCI) is preferred over atmospheric pressure photoionization (APPI). The combination of SFC with APCI-MS/MS mass spectrometry made it possible to achieve the limits of quantification in plant extracts in the range of 2.3–20 μg·L−1. The developed method was validated and tested in the analyses of birch outer layer (Betula pendula) bark, and licorice (Glycyrrhiza glabra) root, as well as lingonberry (Vaccinium vitis-idaea), cranberry (Vaccinium oxycoccos), apple (Malus domestica “Golden Delicious” and Malus domestica “Red Delicious”) peels.

Response surface optimization of a vortex-assisted dispersive liquid–liquid microextraction method for highly sensitive determination of repaglinide in environmental water by HPLC/UV

A vortex-assisted dispersive liquid–liquid microextraction (DLLME) method, mated to chemometrics and combined with HPLC/UV detection was optimized and validated for enrichment and determination of repaglinide in environmental samples using nateglinide as an internal standard (IS). A phosphate buffer (10 mM, pH 2.5): acetonitrile (45:55, v/v) was used as a mobile phase with a flow rate of 1 mL/min in an isocratic elution mode. Chemometrics-assisted optimization was performed using a quadratic integrated d-optimal design. The developed model assessed the statistical significance of the independent variables and their interactions to attain the optimum conditions revealing that extractant type, extractant volume and pH are the most influential factors. Optimization of the extraction procedures was performed with the aid of Design Expert 8® software, which suggested 58 different experiments. The optimal conditions were 30 µL of 1-octanol as extractant, 100 µL of acetonitrile as a disperser at pH 8. Under the optimized conditions, the method showed linearity over the range of 1–100 ng/mL with a limit of detection of 0.4 ng/mL. The accuracy, the intra- and inter-day precision were assessed, the %recoveries were found to be between 98.48 and 100.81% with %RSD lower than 1.3. Using chemometrics in method optimization helped achieve the maximum possible enrichment with the least effort, time, and reagents while considering all possible interactions between variables.

A Vancomycin HPLC Assay for Use in Gut Microbiome Research.

ABSTRACTThe human microbiome project has revolutionized our understanding of the interaction between commensal microbes and human health. By far, the biggest perturbation of the microbiome involves use of broad-spectrum antibiotics excreted in the gut. Thus, pharmacodynamics of microbiome changes in relation to drug exposure pharmacokinetics is an emerging field. However, reproducibility studies are necessary to develop the field. A simple and fast high-performance liquid chromatography-photodiode array detector (HPLC) method was validated for quantitative fecal vancomycin analysis. Reproducibility of results were tested based on sample storage time, homogeneity of antibiotic within stool, and concentration consistency after lyophilization. The HPLC method enabled the complete elution of vancomycin within ~4.2 min on the reversed-phase C18 column under the isocratic elution mode, with excellent recovery (85% to 110%) over a 4-log, quantitative range (0.4–100 μg/mL). Relative standard derivations (RSD) of intra-day and inter-day results ranged from 0.4% to 5.4%. Using sample stool aliquots of various weights consistently demonstrated similar vancomycin concentrations (mean RSD: 6%; range: 2–16%). After correcting for water concentrations, vancomycin concentrations obtained after lyophilization were similar to the concentrations obtained from the original samples (RSD less than 10%). These methodologies establish sample condition standards for a quantitative HPLC to enable vancomycin pharmacokinetic studies with the human microbiome.IMPORTANCE Research on antibiotic effect on the gut microbiome is an emerging field with standardization of research methods needed. In this study, a simple and fast high-performance liquid chromatography method was validated for quantitative fecal vancomycin analysis. Reproducibility of results were tested to standardize storage time, homogeneity of antibiotic within stool, and concentration consistency after lyophilization. These methodologies establish sample condition standards for a quantitative HPLC to enable vancomycin pharmacokinetic studies with the human microbiome.

Development and Validation of an RP-HPLC-FLD Method for the Determination of Biogenic Amines after Pre-column Derivatization with a New Pyrazoline Based Reagent, 3- Naphthyl-1-(4-trifluoromethyl)-5-(4-carboxy phenyl)-2-pyrazoline

Application of 3-Naphthyl-1-(4-trifluoromethyl)-5-(4-carboxy phenyl)-2-pyrazoline (NFCP) as a novel fluorigenic label for the derivatization of biogenic amines (BAs) and separation by liquid chromatography (LC) was investigated. Optimum conditions for the pre-column derivatization and chromatographic separation were investigated. The resulting derivatives are fluorescent at λex/λem: 380/460 nm. For the sake of evaluating the efficacy of the proposed analysis of BAs of different nature, the formation of nine NFCP-BAs derivatives was tested individually. Simultaneous derivatization and separation of four BAs (histamine, tyramine, tryptamine, and phenylethylamine) were achieved under the isocratic elution mode. Furthermore, the derivatives were identified by the characteristic product ion obtained during LC-MS/MS analysis. The viability of the method was established by measuring levels of histamine in fresh and spoiled fish samples. Histamine concentrations were found to be 0.4 µgg-1 and 4.1 µgg-1 in fresh and spoiled tuna fish samples respectively. As a simple, reliable, and sensitive pre-column derivatization method, this work provides for future research to develop a novel HPLC method for the analysis of BAs from food samples such as fish and various other matrices by utilizing the new pyrazoline compound.

Development of the Validated Stability-Indicating Method for the Determination of Vortioxetine in Bulk and Pharmaceutical Formulation by HPLC-DAD, Stress Degradation Kinetics Studies and Detection of Degradation Products by LC-ESI-QTOF-MS.

Vortioxetine (VOR) is a new antidepressant drug used to treat major depressive disorder. In this work, a novel, simple, rapid, accurate, precise, selective, stability-indicating, and fully validated high-performance liquid chromatography method with diode array detection (HPLC-DAD) was developed to determine VOR in bulk and pharmaceutical formulations. A Polar-RP column was used, with a mobile phase consisting of acetonitrile (ACN), methanol (MeOH), acetate buffer pH 3.5, and addition of diethylamine (DEA) in the isocratic elution mode. Assessing the stability of the VOR is fundamental to guarantee the efficacy, safety, and quality of drug products. In this study, the VOR active pharmaceutical ingredient (API) and tablets were subjected to a detailed study of forced degradation, using several degrading agents (acid, alkaline, water, heat, light, and oxidation agents). The developed HPLC-DAD method allows the collection of all the essential data to determine degradation kinetics. It was found that the decomposition of vortioxetine is fragile towards oxidative conditions and photolysis, yielding the first-order and second-order kinetic reaction in the above stress conditions, respectively. The degradation products (DPs) were identified by the high-resolution liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-QTOF-MS) method. The HPLC-DAD method was successfully applied for the quantification of VOR in tablets. Additionally, in silico toxicity prediction of the DPs was performed.

DEVELOPMENT OF AN HPLC METHOD FOR SIBUTRAMINE IDENTIFICATION. SEARCH FOR UNDECLARED SIBUTRAMINE IN BIOLOGICALLY ACTIVE ADDITIVES APPETITE SUPPRESSANTS

In this study an available reversed-phase HPLC method for the identification of sibutramine with UV detection and isocratic elution mode was developed, the evaluation of the method applicability and determination of validation indicators were carried out. It has been shown that the developed method meets the current international requirements. To search for sibutramine, the developed method has been used in some biologically active additives ("Lipocarnit", "Pineapple extract", "Turboslim night" and "Turboslim day").

Implementation of Quality by Design Approach for Optimization of RP-HPLC Method for Quantification of Abiraterone Acetate in Solid Dispersion in Forced Degradation Studies

Background: Abiraterone acetate is a derivative of steroidal progesterone, used as a first-line therapy for metastatic castration of prostate cancer. Objective: The present study encompasses the design of an experiment approach for developing a simple, reliable, and rapid RP-HPLC method for the estimation of abiraterone acetate. Method: The chromatographic separation was efficiently conducted on a Hypersil Gold C18 (50 x 4.6 mm, 5 µm) HPLC column, using the mobile phase composition of acetonitrile: dibasic potassium phosphate (0.01 mM) in the ratio of 80:20 (%v/v) at pH 6.5 with an isocratic elution mode. Furthermore, the different force degradation study including hydrolysis, oxidation, thermal, and photolytic was performed for abiraterone acetate. Result: The dynamic linearity was established in the concentration range of 0.5-10 µg/mL with r2 of 0.9998. Furthermore, the limit of detection and the limit of quantitation were 0.0978 µg/mL and 0.3260 µg/mL. The degradation of abiraterone acetate was shown in both acidic (54.16 ± 0.247 after 24 hrs) and basic conditions (35.06 ± 0.458 after 24 hrs). Furthermore, the developed method was successfully employed to quantify abiraterone acetate in bulk powder and the solid dispersion did not show any change in the retention time. Conclusion: The developed method was validated according to the ICH Q2 (R1) specification, which was found to be sensitive, accurate, precise, robust, linear, and selective compared to the reported chromatographic method.

Development and Validation of a RP-UHPLC Method for estimation of Gliclazide loaded Microsphere

Objective: The objective of the current work is to develop and validate a simple yet effective, efficient and reproducible RP-UHPLC method for the determination of Gilcazide loaded in a microsphere formulation. Methods: The chromatographic analysis was carried out on Thermo Scientific (Dionex Ultra 3000plus) UHPLC using a stainless steel YMC C8 column (25cm × 4mm) packed with endcapped octylsilane bonded to porous silica (4µm) in isocratic elution mode using phosphate buffer pH 3.4 and HPLC grade acetonitrile in the ratio of 20: 80 (v/v) as eluent. The flow rate was adjusted as 1ml/min and eluent was detected at 230 nm. The retention time was observed at 1.9 min. Results: The linear dynamic range for the developed UHPLC method was found in the concentration range of 2-10µg/ml and percentage recovery was in the range of 94%-98%. Drug Entrapment Efficiency Data of Gliclazide loaded microsphere was found to be 93.72% Conclusion: In this RP–UHPLC method, the linearity was within the range of 2–10μg/mL and the validation parameters were within the limits was performed as per ICH guidelines. The % Drug entrapment Efficiency of the Gliclazide loaded microsphere was found to be 93.72 % which was satisfactory.

Simple Eco-Friendly RP-LC Method for the Synchronous Separation of Six Widely Used Drugs in the Treatment of Cerebrovascular and Vestibular Disorders: Application for Analysis in Their Single and Combined Drug Products.

Reverse phase liquid chromatography (RP-LC) is considered the most extensively used chromatographic technique in drug separation and quantification, yet its routine use shows increasing environmental challenges about energy consumption, solvent used, and waste engendered. From this perception, efforts for greening the developed RP-LC methods are focused on using green solvents and reducing the amount of solvent used and waste produced. The target of this work is to develop an eco-friendly RP-LC method for the synchronous separation of six widely used drugs in the treatment of cerebrovascular and vestibular disorders, namely neurotropic piracetam, antihistamines dimenhydrinate, cinnarizine, and fluranizine, and vasodilators vincamine and vinpocetine. Separation and quantification are accomplished at ambient temperature by isocratic elution mode using acetonitrile-0.2% triethylamine (85:15 by volume) as a mobile phase at a flow rate of 1.5 mL/min on a Hypersil C18 column. UV detection and quantification are carried out at 225 nm. The method is fully validated per ICH guidelines, where all analytical parameters are within the acceptable range (r > 0.9999), accuracy (≥99.55%), and precision (0.02-0.97%). The method is applied for the routine analysis of the cited drugs in their single and combined drug products, and it is considered excellent in terms of greenness with respect to Eco-Scale assessment. As the routine use of the RP-LC technique shows increasing environmental challenges, efforts for greening are attempted focusing on the use of green solvents and reducing the amount of solvent used and waste produced. A simple eco-friendly RP-LC method for the synchronous separation of six widely used drugs in the treatment of cerebrovascular and vestibular disorders is developed. The method is fully validated per ICH guidelines, where all analytical parameters are within the acceptable range. The method is applied for the routine analysis of the cited drugs in their single and combined drug products.

A Stability-Indicating UPLC Method for Simultaneous Quantification of Epalrestat and Pregabalin in Bulk and Marketed Formulations

A specific, precise, rapid and reliable stability indicating UPLC method has been developed and validated for simultaneous separation and estimation of Epalrestat and Pregabalin in bulk drugs and marketed formulations. Chromatographic separation was achieved on a agilent Zorbax SB C18 (2.1 x 100mm, 1.8µm) column using isocratic mode of elution with 0.1% Formic acid and Acetonitrile in the ratio 60:40 v/v at a flow rate of 1.0 mL/min and analytes were monitored at 226nm. The retention times of Epalrestat and Pregabalin were about 0.97 and 1.27 minutes respectively. The developed method was validated for specificity, linearity, precision, accuracy, ruggedness and stress degradation studies were established. The results of specificity and stability studies indicate that there was no interference of diluent, excipients, and degradation products at respective retention times of analytes. The detector response was linear in the range of 10-150% level with respect to test concentration of Epalrestat and Pregabalin. Correlation coefficient (R2) was found not less than 0.999 for both analytes. The percentage assay of Epalrestat and Pregabalin were about 100.99% and 100.77% respectively. Hence, the developed method was rapid, specific, accurate and economical, and it can be used for routine analysis of these drugs in bulk and marketed formulations.

High-performance liquid chromatography quantitative analysis of ephedrine alkaloids in Ephedrae Herba on a perfluorooctyl stationary phase.

Ephedrae Herba is one of the most commonly used herbal medicines, and it has been shown that most of the clinical efficacy for cold and asthma is exerted by its alkaloidal components. A simple and sensitive high-performance liquid chromatography method was developed using a perfluorooctyl column for the simultaneous determination of five alkaloids (norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, and methylephedrine) in Ephedrae Herba. The mobile phase comprising acetonitrile and 15mM ammonium trifluoroacetate was used to elute the targets in isocratic elution mode. The method was validated for linearity (R2 >0.999), repeatability, intraday and interday precision, recoveries with trueness (93.87-110.99%), limits of detection (5.35-5.76µg/mL), and limits of quantification (20µg/mL). The quantitative results revealed that the developed method was precise and accurate. Then it was successfully applied to determine the difference in the contents ofthree batches of Ephedrae Herba from three pharmaceutical companies.

Development and validation of an LC-MS/MS method for pharmacokinetic study of lobetyolin in rats

A simple and selective liquid chromatography tandem with mass spectrometry (LC-MS/ MS) method for quantification of lobetyolin in rat plasma was developed and validated. Chromatographic separation was achieved on a Thermo ODS C18 reversed-phase column using 0.1% aqueous formic acid-methanol (50:50, v/v) in an isocratic elution mode at a flow rate of 0.4 mL.min-1. LC/MS performance was done in a positive ion ESI mode and the MS/MS transitions were monitored at m/z 419.3 [M+Na]+ → m/z 203.1 for lobetyolin and m/z 394.9 [M+Na]+ → m/z 231.9 for IS, respectively. The assay exhibited a linear dynamic range over 1.0-500 ng.mL-1 for lobetyolin in plasma. Both the precision (%RSD) and accuracy (RE%) were within acceptable criteria (<15%). Recoveries ranged from 87.0% to 95.6%, and the matrix effects were from 91.0% to 101.3%. After oral administration, the peak plasma concentration of lobetyolin was obtained as 60.1 ng.mL-1 at 1.0 h. The proposed LC-MS/MS method could be applied to a pharmacokinetic study employing 66 samples from 6 Wistar rats.

Fast Determination of a Novel Iron Chelate Prototype Used as a Fertilizer by Liquid Chromatography Coupled to a Diode Array Detector.

The environmental risk of the application of synthetic chelates has favored the implementation of new biodegradable ligands to correct Fe-deficient plants. This study developed and validated an analytical method for determination of a new prototype iron chelate—Fe(III)-benzeneacetate, 2-hydroxy-α-[(2-hydroxyethyl)amino]—(BHH/Fe3+) based on liquid chromatography with diode array detection, as a potential sustainable alternative. Chromatographic analysis was performed on a LiChrospher RP-18 in reverse-phase mode, with a mobile phase consisting of a mixture of acetonitrile (solvent A) and sodium borate buffer 0.20 mM at pH = 8 (solvent B) at a flow rate of 1.0 mL/min in isocratic elution mode. This method was fully validated and found to be linear from the limit of quantification (LOQ) to 50 mg/L and precise (standard deviation below 5%). The proposed method was demonstrated to be selective, precise, and robust. The developed methodology indicated that it is suitable for the quantification of iron chelate BHH/Fe3+.

A strategy to process hundred-gram level complex sample using liquid-liquid-refining extraction and consecutive counter-current chromatography: Toona sinensis case study

Large-scale preparation of target compounds from complex samples is facing great challenges. In the present study, an efficient strategy for large-scale preparation of target compound was proposed and successfully applied in the separation of active components from Toona sinensis. The pretreatment technology of liquid-liquid refining extraction (LLRE) combined with consecutive high-speed counter-current chromatography (HSCCC) was used to process hundred grams of extractions. Firstly, two phase solvent systems composed of n-hexane–ethyl acetate–methanol–water (5:5:5:5, v/v) and (2:5:2:5, v/v) were used to remove low polar and high polar impurities from 100 g crude extracts of T. sinensis, respectively, and 9.25 g of crude sample was obtained. And then, n-hexane–ethyl acetate–methanol–water (2.5:5:2.5:5, v/v) was used as the solvent system for HSCCC separation. The isocratic elution mode with max loading and consecutive injections mode were investigated to obtain more target compound. As a result, ethyl gallate with purity of 97% was successfully separated by 5 times consecutive counter-current chromatography. The separation was repeated once. Finally, ethyl gallate (3.73 g) was isolated from 9.25 g of crude sample (100 g crude extracts). The results demonstrated that the yield increased from 0.26 g/h/L of untreated crude extract to 0.93 g/h/L of LLRE pre-treated sample for single injection, and further increased to 1.62 g/h/L for 5 consecutive injections mode with the present method.

Development and validation of a sensitive and high throughput UPLC-MS/MS method for determination of paraquat and diquat in human plasma and urine: application to poisoning cases at emergency departments of hospitals.

Paraquat and diquat are well-known toxic herbicides, at least responsible for hundreds of fatal poisoning events worldwide. However, the determination of diquat and paraquat in plasma and urine is very challenging because of their high polarity and double charge characteristics. In this study, we aim to develop a rapid and reliable method for the determination of paraquat and diquat in human plasma and urine by ultraperformance liquid chromatography-tandem mass spectrometry. The chromatographic separation of paraquat and diquat was tested with different chromatographic columns and different mobile phase conditions. The mass parameters were optimized by product ions, source gas flow, cone flow, desolvation temperature, and capillary voltage. The isocratic elution mode gave rapid appearance of peak of paraquat and diquat. The sharp peak shapes for paraquat and diquat were achieved with CORTECS® UPLC® HILIC (100× 2.1mm, 1.6μm) column by adding formic acid and ammonium acetate in mobile phase in isocratic elution mode. The lower limit of quantification of 1.0ng/mL for paraquat and diquat were achieved using only 50 μL of human plasma or urine. The running time for analysis of both paraquat and diquat was as short as 3.5min per sample. A rapid and reliable method for the determination of paraquat and diquat was developed and applied to 387 clinical poisoning cases and 22 poisoning cases were found to be paraquat or diquat poisoning.

A novel stability-indicating HPLC-DAD method for determination of favipiravir, a potential antiviral drug for COVID-19 treatment; application to degradation kinetic studies and in-vitro dissolution profiling

Modern pharmaceutical analysis is paying a lot of attention to the stability of novel drug formulations as well as establishment of suitable stability-indicating approaches. In the current work, a comprehensive stability-indicating HPLC-DAD method has been developed and validated for determination of favipiravir (FAV) which is a novel and emerging antiviral option in COVID-19 treatment. The stability of FAV was examined under different stress conditions. FAV was found to be susceptible to acid, base hydrolysis and oxidative degradation. Structure elucidation of the forced degradation products was carried out using mass spectrometry (MS) operated in electrospray ionization mode. Effective separation of FAV and its induced degradation products was achieved using isocratic elution mode on Zorbax C18 column maintained at 30 °C. The mobile phase used was comprised of 25.0 mM phosphate buffer (pH 3.5 ± 0.05) containing 0.1% (w/v) heptane sulphonic acid sodium salt-methanol–acetonitrile (62:28:10, by volume), delivered at flow rate of 1.0 mL/min. The diode array detector signal for FAV was monitored at 321.0 nm over a concentration range of 6.25–250.00 µg/mL. The potential mechanisms for generation of degradation products were postulated through comparison of MS1 fragmentation pattern of FAV and its degradation products. Moreover, the proposed method was also extended to study the degradation kinetics. Additionally, dissolution profiling of FAV in different media was monitored. Clearly, the suggested approach is accurate, reliable, time-saving, and cost-effective. As a result, it may be utilized for regular quality control and stability assessment of FAV in its tablet dosage form.

Determination of Folic Acid in Multivitamin Preparations by Reversed Phase HPLC

A great variety of components in multivitamin preparations containing folic acid, and a variety of test methods and conditions of folic acid determination proposed by manufacturers, require alignment of test procedures for products with similar composition.The aim of the study was to compare the results of experimental verification of folic acid determination procedures which use reversed phase high-performance liquid chromatography (RP HPLC) with isocratic elution mode. Materials and methods: The Agilent 1260 Infinity II LC system with a diode array detector (280 nm), isocratic elution mode, C8- and C18-bonded silica gel chromatographic columns, model mixtures containing folic acid, cyanocobalamin, ferrous sulfate, and potassium iodide, were used in the study.Results: The lowest relative standard deviation of the folic acid peak area (RSD=0.09%), and the lowest asymmetry factor (As=1.04) for folic acid were observed for the model mixture “ferrous sulfate+folic acid+cyanocobalamin” and the following test conditions. Column: 250×4.0 mm, silica gel for chromatography, octylsilyl (C8), endcapped; mobile phase: methanol‒phosphate buffer (12:88), pH 6.6; column temperature: 25ºС. The study demonstrated the feasibility of using these conditions for determination of pteroic acid impurity with simultaneous precipitation of interfering ferrous ions, using ethylenediaminetetraacetic acid solution, pH 9.5, as a solvent.Conclusions: RP HPLC can be recommended as an optimal aligned test procedure for determination of folic acid in combination products. It is recommended to use a solution containing folic and pteroic acids for system suitability testing.

Analysis of Vitamin A in Multivitamin Products

Vitamin A is present in multivitamin products mainly in the form of retinol esters: retinyl acetate, retinyl palmitate, and beta carotene—retinol precursor (dimer) found in plants, which is capable of converting into retinol in liver cells. Retinol is determined in medicinal products primarily by high performance liquid chromatography (HPLC), with preliminary purification and vitamin isolation by liquid-liquid extraction. However, scientific literature also describes other methods of sample preparation and analysis of such compounds. An important issue is differentiation of vitamin A from other fat-soluble vitamins often included as components in multivitamin products. The aim of the study was to analyse and summarise data on current methods used for determination of vitamin A and its derivatives in medicinal products. The authors analysed the range of vitamin A products authorised in the Russian Federation, and the test methods described in their product specification files. The study demonstrated that the test method most often used for determination of retinol esters was HPLC with isocratic elution mode using octadecylsilyl packing in the reverse-phase mode, and, less frequently, aminopropylsilyl packing in the normal phase mode. Determination of beta carotene in medicinal products is most often performed using spectrophotometry.

A Simple and Efficient Two-Dimensional High-Speed Counter-Current Chromatography Linear Gradient and Isocratic Elution Modes for the Preparative Separation of Coumarins from Roots of Toddalia asiatica (Linn.) Lam.

Toddalia asiatica (L.) Lam. (Rutaceae) has shown a broad spectrum of biological properties, such as anti-inflammatory, antioxidant, antimicrobial, anti-HIV, and anticancer properties. The present study is concerned with the separation of the main components with broad partition coefficients (KD values) from T. asiatica, using linear gradient high-speed counter-current chromatography (LGCCC) combined with an off-line two-dimensional (2D) mode. Similar to the binary gradient HPLC, the LGCCC mode is operated by the adjustment of the proportion between the mobile phase of 5:5:1:9 (v/v) (pump A) and 5:5:4.5:5.5 (v/v) (pump B) in an n-hexane/ethyl acetate/methanol/water solvent system. The off-line 2D-CCC mode was used in this study for the secondary separation of two similar KD value compounds with n-hexane/ethyl acetate/methanol/water (5:5:4:6, v/v). Notably, six coumarins, namely, tomentin (1), toddalolactone (2), 5,7,8-trimethoxycoumarin (3), mexoticin (4), isopimpinellin (5), and toddanone (6), were efficiently separated. The structures of the pure compounds were elucidated by spectral techniques and compared with the literature.