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Specialists in pharmacognosy are facing an important task of improving quality control methods for combination herbal medicinal products in order to enable reliable assessment of the content of active substances that are responsible for the drug’s pharmacological effect.The aim of the study was to investigate the possibility of using the European Pharmacopoeia method to determine the total content of anthracene derivatives in the antihaemorrhoidal medicinal herb mixture Proctophytol® and to propose limit values for the total content of anthracene derivatives.Materials and methods: individual mixture components containing anthracene derivatives, such as senna leaves and frangula bark, as well as model mixtures containing these individual components in a 1:1 ratio, and a model mixture imitating Proctophytol® were used in the study. The determination of the anthracene derivatives content was carried out using spectrophotometry.Results: it was demonstrated that the European Pharmacopoeia method could be used to assess the quality of Proctophytol® in terms of anthracene derivatives total content. The spectrophotometric method described in the European Pharmacopoeia has advantages over the methods described in manufacturer specifications for Russian products, because it allows for thorough extraction of the active substances and is standardized for anthracene derivatives.Conclusions: the European Pharmacopoeia method was adjusted to determine anthracene derivatives in the medicinal herb mixture Proctophytol®. It was proposed to use 70% ethanol instead of 70% methanol as extraction solvent. The authors identified optimum sample weights and test solution dilutions, and calculated and verified the limit for anthracene derivatives content—“Not less than 1.9%”. The adjusted method can be recommended for inclusion in the monographs of the State Pharmacopoeia of the Russian Federation for medicinal herb mixtures similar to Proctophytol®.

INTRODUCTION. The safe use of lily-of-the-valley medicinal products requires adequately controlling the content of cardiac glycosides. The State Pharmacopoeia of the Russian Federation stipulates that the content of cardiac glycosides in lily-of-the-valley medicinal products should be quantified using bioassays and spectrophotometry. However, the determination of cardiac glycosides needs more accurate and selective physicochemical methods, such as high-performance liquid chromatography (HPLC).AIM. This study aimed to develop and validate an HPLC analytical procedure for the quantitative determination of cardiac glycosides (convallatoxin) in lily-of-the-valley herbal medicinal products.MATERIALS AND METHODS. The study examined the Lily-of-the-Valley Tincture, Zelenin Drops, Valocormid, Carniland®, and Lily-of-the-Valley Extract Reference Standard samples. The content of cardiac glycosides was determined by comparison with a reference standard for convallatoxin by HPLC and spectrophotometry. The samples were prepared as outlined in Monograph 3.4.0003.18 Lily-of-the-Valley Herb Tincture of the State Pharmacopoeia of the Russian Federation. Mixed cardiac glycosides were separated on a Luna 5 µm C18(2) column by gradient elution with 0.1% orthophosphoric acid in water and acetonitrile. The analysis was performed using an autoinjector with sample cooling to 5 °C.RESULTS. The developed analytical procedure met the acceptance criteria for specificity, intermediate precision, linearity (correlation coefficient of 0.99985), and repeatability (relative standard deviation of convallatoxin measurements of 1.61%). The analytical procedure is suitable for the quantitative determination of convallatoxin in lily-of-the-valley herbal medicinal products because it produces reliable and repeatable results.CONCLUSIONS. The authors developed a highly sensitive and selective HPLC analytical procedure for the quantitative determination of convallatoxin in lily-of-the-valley herbal medicinal products. The content of convallatoxin ranged from 0.012 to 0.018 mg/mL in the liquid active substance Lily-of-the-Valley Tincture, from 0.004 to 0.013 mg/mL in the medicinal product Zelenin Drops, and from 0.005 to 0.007 mg/mL in the medicinal products Carniland® and Valocormid. For the Lily-of-the-Valley Extract Reference Standard, the content of convallatoxin amounted to 0.029 mg/mL.

Scientific relevance. The State Pharmacopoeia of the Russian Federation, edition XIV, requires the identification of aniseed and fennel fruits by the same thin-layer chromatography (TLC) procedure using Sudan III and menthol. These markers are neither specific nor related to the therapeutic effects of these herbal drug products. The visual interpretation of chromatograms is complicated because of the low intensity of adsorption zones. Moreover, the corresponding compendial monographs do not characterise the adsorption zones specific to each of the plants. The most abundant component in aniseed and fennel essential oils, trans-anethole, would make a better reference standard from a methodological point of view.Aim. This study aimed to optimise the TLC procedure for essential oil determination in herbal drugs and herbal medicinal products of aniseed and fennel fruits and subsequently recommend it for inclusion in the relevant compendial monographs.Materials and methods. The study examined samples from several batches of herbal drugs, including aniseed and common fennel fruits, sourced from Russian manufacturers. The reference standards comprised commercial reagents, including trans-anethole, anise oil, and linalool, as well as fresh essential oils that had been steam distilled from the test samples in a Clevenger apparatus. The study was conducted by TLC. Sample preparation involved using Merck aluminium TLC plates, an IKA KS-501 digital orbital shaker, and a CAMAG Linomat 5 semi-automatic sample application system. The authors heated the plates in either a Binder ED53 drying oven or a CAMAG TLC plate heater. For visualisation, the authors selected a CAMAG TLC VISUALIZER 2 UV imaging and documentation system.Results. The informational and experimental research showed the feasibility of using trans-anethole as an identification reference standard for common anise and fennel fruits. The authors selected the optimal solvent for extracting active substances from the test products (hexane) and a detection reagent for improving visual evaluation of the chromatograms (1% sulfovanillin). The authors established characteristic adsorption zones for differentiating between aniseed and fennel fruits.Conclusions. The optimised procedure identifies the main components in the essential oils of aniseed and fennel fruits with a specific reference standard. This procedure may be recommended for inclusion in draft monographs for the corresponding herbal medicinal products.

Ascorbic acid (vitamin C) is involved in many vital biochemical processes in the human body. Rose hips are a natural source of ascorbic acid. Rose hips are used in decoctions and many vitamin herbal teas. Russian compendial methods for the quantitative determination of ascorbic acid have certain limitations precluding its objective measurement in herbal drugs, herbal drug preparations, and herbal medicinal products.The aim of the study was to develop an analytical procedure for the quantitative determination of ascorbic acid in herbal drugs, herbal medicinal preparations, and herbal medicinal products using high performance liquid chromatography (HPLC).Materials and methods. The study involved two herbal medicinal products, Rose Hips (rose hips) and Vitamin Herbal Tea 2 (rose hips and rowan fruits), and reference standards for ascorbic acid, rutoside, quercetin, gallic acid, caffeic acid, chlorogenic acid, luteolin-7-glucoside, citric acid, DL-malic acid, thiamine, and pyridoxine hydrochloride. The authors used a 1260 Infinity II chromatograph with a diode array detector by Agilent and an Inertsil ODS-3 chromatographic column (250 mm × 4.6 mm, 5 µm) by GL Sciences. The HPLC system was operated in gradient elution mode, and the detector was set at 244 nm.Results. Ascorbic acid content in herbal drugs, herbal medicinal preparations, and herbal medicinal products can be determined by HPLC. Adequate sample preparation and chromatography conditions allow for inhibiting ascorbic acid oxidation in aqueous solutions for a period sufficient to complete testing (not less than 8 h).Conclusions. The authors developed a highly sensitive and selective HPLC procedure for the quantitative determination of ascorbic acid intended for the standardisation of herbal drugs, herbal medicinal preparations, and herbal medicinal products. The procedure is worthy of inclusion in the Rose Hips and Vitamin Herbal Tea 2 monographs of the State Pharmacopoeia of the Russian Federation. Switching to HPLC should not require lowering the limit for ascorbic acid (not less than 0.2%) established in the Rose Hips and Vitamin Herbal Tea 2 monographs for the titration method. The ascorbic acid content was below this limit in the vast majority of study samples. However, this discrepancy may be explained by the presence of dog rose (Rosa canina) species low in vitamin C, which should normally be used only as choleretics.

When used to quantify alkaloids in thermopsis dry extract, acidimetric titration has several limitations, including insufficient specificity of the method and arbitrary selection of a titration endpoint. Different parts of the Thermopsis lanceolata plant produce alkaloids in different proportions: the herb is rich in thermopsine, whereas the seeds are rich in cytisine. Since thermopsine and cytisine have different pharmacological effects, it is important to identify and quantify individual alkaloids in thermopsis dry extract.The aim of the study was to develop and validate an analytical procedure for identifying and quantifying total alkaloids in thermopsis dry extract by high-performance thin-layer chromatography (HPTLC) with densitometric detection.Materials and methods. The study included samples from two pilot-scale batches of thermopsis dry extract and reference standards for cytisine and thermopsine. The authors used Merck HPTLС Silica Gel 60 F254 20×10 cm plates for chromato-graphic separation and analysed the results with a CAMAG® TLC Scanner 4 densitometer and the winCATS software.Results. The authors identified thermopsis dry extract using HPTLC separation followed by spectrodensitometry. The alkaloid-specific solution applied to chromatography plates helped to identify the three most abundant and four minor alkaloids of thermopsis. The most abundant alkaloids were thermopsine, cytisine, and an unidentified alkaloid with a retention factor of approximately 0.2. These three alkaloids accounted for almost 80% of the total alkaloid content of the dry extract. The authors quantified cytisine, thermopsine, and total alkaloids expressed as thermopsine.Conclusions. The authors developed and validated an analytical procedure for identifying and quantifying total alkaloids in thermopsis dry extract. This procedure offers the possibility of reducing the analysis time from 4–5 hours to 2–2.5 hours.

Currently, there is an increase in preand post-approval testing of medicinal products containing diosmin and hence a need to unify approaches to standardisation of this group of pharmaceuticals. Moreover, the State Pharmacopoeia of the Russian Federation lacks a monograph for these products.The aim of the study was to determine an approach to standardisation of medicinal products containing diosmin.Materials and methods: the study analysed scientific publications, as well as monographs of leading foreign pharmacopoeias. Experimental work was carried out using samples of diosmin-containing pharmaceuticals in the form of 500 and 1000 mg film-coated tablets produced by Russian and foreign manufacturers. The study involved high performance liquid chromatography with UV detection using an Agilent 1260 Infinity II liquid chromatography system with a diode array detector. The following reference standards were used: a diosmin RS, USP grade; a hesperidin CRS, Ph. Eur. Grade; and a diosmin CRS for testing chromatography system suitability for identification of impurities A, B, C, D, E, and F, Ph. Eur. grade.Results: the authors reviewed quality requirements for pharmaceutical products containing diosmin and analysed experimental data obtained during preand post-approval testing of Russian and foreign medicines. The comparison of regulatory documents for registered diosmin-containing medicinal products showed a difference in approaches to assessing the contents of related substances and active pharmaceutical ingredients. Having analysed the literature, experimental data and regulatory requirements for standardisation of diosmin-containing pharmaceuticals, the authors recommended an approach to standardisation. According to the approach, concomitant flavonoids (hesperidin, isorchoifolin, linarin, and diosmetin) contributing to the pharmacological activity of a medicinal product are specified as part of Assay, and process-related by-products (impurities A and D) are specified and evaluated as part of Related substances tests.Conclusion: the authors propose to evaluate the contents of concomitant flavonoids (hesperidin, isorchoifolin, linarin, diosmetin) under Assay and to specify impurities A and D, as well as single unidentified impurities and total amount of impurities under Related substances.

Arbutin is the main active ingredient in many herbal medicinal products that have diuretic, antimicrobial, bactericidal, and antioxidant effects. Many of these products are mixtures of different herbal substances. Therefore, the approaches to quality control of HMPs can vary significantly. The aim of the study was to compare arbutin assay procedures used in quality control of arbutin-containing products. Materials and methods. Samples of the following HMPs were used in the study: monocomponent Bearberry Leaf and multicomponent Brusniver® (herbal mixture, powder). The test methods used were titrimetry, ultraviolet-visible spectrophotometry, and high-performance liquid chromatography (HPLC). Results. The authors compared five arbutin assay procedures described in the monographs and product files for arbutin-containing HMPs. Conclusions. It has been established that the analysed procedures cannot be used interchangeably as equivalent test methods; the limits for arbutin have to be established for each specific procedure. Iodometric titration is the most labour- and time-consuming method, and the determined titration endpoint is a subjective assessment. Spectrophotometric methods do not require the use of an arbutin reference standard, but they can give overestimated results as compared to the HPLC and titrimetry methods. HPLC methods are more selective, but they require the use of reference standards. The recommended test methods for HMP quality control are HPLC and visible spectrophotometry; the titrimetric method is recommended for replacement.

Abstract. Papaverine hydrochloride products are used as anticonvulsants in routine medical practice. Most of the approved product specification files include thin-layer chromatography for assessment of product-related impurities and UV spectrophotometry for determination of active pharmaceutical ingredients. An HPLC assay is not used for determination of papaverine hydrochloride in drug dosage forms.The aim of the study was to develop an HPLC test method for determination of product-related impurities and for quantification of papaverine hydrochloride in solutions for injection, tablets, and rectal suppositories.Materials and methods: samples of the following Russian-made papaverine products were used in the study: Papaverine, solution for injection, 20 mg/mL; Papaverine, rectal suppositories, 20 mg; Papaverine, tablets, 40 mg. The Agilent 1260 Infinity II DAD System was used for the HPLC assay, and the Agilent 8453Е UV-Vis System was used for recording UV spectra. The determination of product-related impurities and the assay of active ingredients were performed simultaneously by HPLC using a reversed-phase column Kromasil 100-5-C18, 250×4.6 mm, 5 μm, the gradient elution mode, and detection at 238 nm. Papaverine Hydrochloride USP RS, 99% purity, and Noscapine EP CRS were used as reference standards.Results: the study demonstrated that determination of product-related impurities and assay of active ingredients in papaverine products can be performed simultaneously using HPLC.Conclusions: the authors proposed an HPLC test method for determination of active ingredients in papaverine products, which is aligned with the “consistent standardisation” principle and can be recommended for inclusion into draft monographs for papaverine products.

Abstract. Identification of hellebore (Veratrum Lobelianum Bernh.) herbal substance, as well as hellebore-based herbal preparation and herbal medicinal product by the same group of biologically active substances using the same test method is in line with the so-called “consistent standardisation” principle.The aim of the study was to develop a harmonised approach to identification of steroidal alkaloids in hellebore products (hellebore water, hellebore tincture) and hellebore herbal substance (hellebore rhizomes with roots).Materials and methods: samples of hellebore water, hellebore tincture, and hellebore rhizomes with roots were analysed by high-performance thin-layer chromatography (HPTLC) using an HPTLC plate.Results: the authors developed a harmonised identification procedure for products made from hellebore rhizomes with roots (herbal substance, herbal preparation, and herbal medicinal product) based on HPTLC detection of steroidal alkaloids. The results of the study will be used to prepare amendments to the Identification part of monograph FS.2.5.0104.18 “Hellebore rhizomes with roots”. The developed test procedure is proposed for inclusion into draft monographs “Hellebore rhizomes with roots, tincture” and “Hellebore rhizomes with roots, tincture, solution for external use”.Conclusions: the developed test procedure can be used as an identification test for a range of products from the hellebore herbal substance to hellebore-based herbal medicinal products, which is based on the detection of the same group of biologically active substances.

The quality control of the «Valerian rhizome and roots» herbal substance is carried out using high performance liquid chromatography (HPLC) according to the State Pharmacopoeia of the Russian Federation, XIV edition. The quantitative analysis of the active ingredients in valerian tincture is performed using a non-specific and non-selective spectrophotometric method. Therefore, it is important to introduce in Russia a more modern test procedure for quantitative determination of active ingredients in valerian tincture.The aim of the study was to develop a selective and sensitive HPLC procedure for quantitative determination of the total content of sesquiterpenic acids, expressed as valerenic acid, for the purpose of valerian tincture standardisation.Materials and methods: valerian tincture samples produced by seven Russian manufacturers were used as test samples, and valerenic acid was used as the reference standard. The quantitative analysis of the active ingredients was performed by two methods: spectrophotometry at 512 nm following the reaction of valerenic acid ethylester with hydroxyalamine and ferric chloride, and by HPLC using a Nucleosil C18 column, 125×4.6 mm, 5 µm particle size, in gradient elution mode, with detection at 220 nm.Results: the spectrophotometric technique was shown to be insufficiently specific. The authors of the study validated the chromatographic test procedure, established system suitability criteria, and compared the results obtained by the two test procedures. They also determined a tentative standard of the total content of sesquiterpenic acids, expressed as valerenic acid, obtained by HPLC.Conclusions: the HPLC assay developed for quantitative determination of active ingredients in valerian tincture is more specific as compared to the spectrophotometric technique, as the sum of the peaks of valerenic and acetoxyvalerenic acids and the results for the reference standard are taken into account during calculations. The new test procedure is in line with the cross-cutting standardisation principle and can be recommended for inclusion into the draft monograph «Valerian tincture».

The process of harmonisation of Russian and foreign approaches and requirements in the field of medicines quality assurance calls for revision of quality control procedures included in various regulations and guidelines. The monograph FS.2.5.0062.18 “Hawthorn flowers” of the State Pharmacopoeia of the Russian Federation XIV edition includes a test procedure for determination of flavonoids by a chromatospectrophotometric method. This procedure does not take into account current scientific capabilities and has a number of shortcomings, therefore it was necessary to revise the existing test procedure and develop a new approach to the standardisation of the hawthorn flower herbal substance.The objective of the study was to develop an assay method for standardisation and evaluation of hawthorn flower using high performance liquid chromatography (HPLC).Materials and methods: the study was performed using samples of hawthorn flowers by Russian manufacturers. Quercetin (USP RS) and Hyperoside (HWI, primary standard) were used as the reference standards. The HPLC analysis was performed using an Infinity II 1260 DAD LC system (Agilent), and the UV spectra were recorded on a Cary 100 Varian spectrophotometer. A TLC Visualizer (CAMAG) was used to obtain digital images of thin layer chromatography plates.Results: the authors developed an HPLC test procedure for quantitative determination of total flavonoids, expressed as hyperoside, in hawthorn flowers. The developed procedure gives reliable and reproducible results and is characterised by high sensitivity and selectivity. The results of quantitative determination of the total flavonoid content in hawthorn flowers were used to propose the standard for the total content of flavonoids, expressed as hyperoside, of “not less than 0.5%”.Conclusions: the developed assay method for determination of active pharmaceutical ingredients in hawthorn flower products by HPLC can be recommended for inclusion into the Assay part of the “Hawthorn Flowers” monograph of the State Pharmacopoeia of the Russian Federation.

The use of assay methods with different test conditions for determination of anthracene derivatives in herbal substances and herbal medicinal products requires adjustment of the established limits for total anthracene derivatives content based on the test method used.The aim of the study was to compare different pharmacopoeial methods of quantitative determination of anthracene derivatives, and established limits for the active substance content, and to analyse the possibility of adjusting the limits based on the test method used.Materials and methods: the total content of anthracene derivatives was measured in the samples of frangula bark (fragmented bark and powder) and senna leaves (fragmented leaves and powder) using the test methods described in the State Pharmacopoeia of the Russian Federation (XI and XIII editions), and the European Pharmacopoeia.Results: the analysis of Russian and foreign pharmacopoeial requirements for determination of anthracene derivatives in laxative herbal medicinal products demonstrated differences in extraction solvents, extraction conditions, and calculation methods used. The experiments showed that the optimal extraction solvent was 70 % alcohol solutions, and that an indirect cobaltic chloride reference standard should be replaced by specific absorbance of glucofrangulin A and sennoside B. It was shown that the limits for total anthracene derivatives content should be chosen based on the extraction conditions used.Conclusions: comparative theoretical research and experimental studies helped to determine optimal assay conditions and respective limits for total anthracene derivatives content to be used in quality control of herbal substances and herbal medicinal products.

The paper describes the basic quality parameters of the starting materials of herbal origin and herbal drugs, taking into account the requirements of State Pharmacopeia XI and draft general chapters of State Pharmacopeia XIII. It enlists the main terms and definitions, and the requirements to presentation of the quality parameters. It provides the examples of typical criticism of the normative documents on herbal drugs in terms of various quality parameters, including “Appearance”, “Identification tests”, “Chromatography”, “Degree of fineness”, “Assay”. The criticism has been leveled at the quality of both draft normative documents and the samples submitted for testing. The survey conducted allows for explaining the expert requirements to the quality of samples and the presentation of several test methods.

The review contains summary information about possible impurities, determination of which is described by draft monograph «fixed vegetable oils» and state standards of Russian Federation. The review briefly describes methods for determination of soaps, unsaponifiable components, residual solvents, volatile compounds, foreign fatty oils, aldehydes, moisture, proteins, paraffin, wax, tar and mineral oils, heavy metals, phosphorus-containing substances, cyanides and hydrogen cyanides.