Physico-chemical methods of drug analysis. Study of the quality of medicines Methods of analysis of drugs by GF examples
The widespread introduction of the principles of evidence-based medicine into clinical practice is largely due to the economic aspect. The correct distribution of funds depends on how convincing the scientific data on the clinical and cost-effectiveness of methods of diagnosis, treatment and prevention are. In clinical practice, specific decisions should be made not so much on the basis of personal experience or expert opinion, but on the basis of rigorously proven scientific data. Attention should be paid not only to the futility, but also to the lack of evidence-based evidence of the benefits of using various methods of treatment and prevention. Currently, this provision is of particular relevance, since clinical trials are financed mainly by manufacturers of medical goods and services.
The concept of "evidence-based medicine", or "evidence-based medicine", was proposed by Canadian scientists from Mac Master University in Toronto in 1990. Evidence-based medicine is not a new science, but rather a new approach, direction or technology for collecting, analyzing, summarizing and interpreting scientific information. The need for evidence-based medicine has arisen primarily in connection with the increase in the amount of scientific information, in particular in the field of clinical pharmacology. Every year, more and more new drugs are introduced into clinical practice. They are actively studied in numerous clinical studies, the results of which are often ambiguous, and sometimes even directly opposite. To use the information received, it must not only be carefully analyzed, but also summarized.
For the rational use of new drugs, achieving their maximum therapeutic effect and preventing their adverse reactions, it is necessary to obtain a comprehensive description of the drug, data on all its therapeutic and possible negative properties already at the testing stage. One of the main ways to obtain new drugs is the screening of biologically active substances. It should be noted that this way of searching for and creating new drugs is very time consuming - on average, one drug worthy of attention falls on 5-10 thousand investigated compounds. Through screening and random observations, valuable drugs were found that entered medical practice. However, randomness cannot be the main principle in the selection of new drugs. With the development of science, it became quite obvious that the creation of drugs should be based on the identification of biologically active substances involved in vital processes, the study of pathophysiological and pathochemical processes underlying the development of various diseases, as well as an in-depth study of the mechanisms of pharmacological action. Achievements in the biomedical sciences make it possible to increasingly carry out directed synthesis of substances with improved properties and certain pharmacological activity.
Preclinical study of the biological activity of substances is usually divided into pharmacological and toxicological. Such a division is conditional, since these studies are interdependent and are based on the same principles. The results of the study of acute toxicity of medicinal compounds provide information for subsequent pharmacological studies, which, in turn, determine the intensity and duration of the study of chronic toxicity of the substance.
The purpose of pharmacological studies is to determine the therapeutic activity of the drug, as well as its effect on the main anatomical and physiological systems of the body. In the process of studying the pharmacodynamics of a substance, not only its specific activity is established, but also possible side reactions associated with the pharmacological effect. The effect of an investigational drug on sick and healthy organisms may differ, therefore, pharmacological tests should be carried out on models of the relevant diseases or pathological conditions.
In toxicological studies, the nature and severity of the possible damaging effects of drugs on experimental animals are established. There are three stages in toxicological studies:
study of acute toxicity of a substance with a single injection;
determination of chronic toxicity of the compound, which includes repeated use of the drug for 1 year, and sometimes more;
determination of the specific toxicity of the drug - oncogenicity, mutagenicity, embryotoxicity, including teratogenic effects, sensitizing properties, as well as the ability to cause drug dependence.
The study of the damaging effect of the study drug on the body of experimental animals allows us to determine which organs and tissues are most sensitive to this substance and what should be paid special attention to in clinical trials.
The purpose of clinical trials is to evaluate the therapeutic or prophylactic efficacy and tolerability of a new pharmacological agent, to establish the most rational doses and regimens for its use, as well as to compare it with existing drugs. When evaluating the results of clinical trials, the following characteristics should be taken into account: the presence of a control group, clear criteria for inclusion and exclusion of patients, inclusion of patients in studies before choosing a treatment, random (blind) choice of treatment, an adequate method of randomization, blind control, blind assessment of treatment outcomes, information about complications and side effects, information about the quality of life of patients, information about the number of patients who dropped out of the study, adequate statistical analysis indicating the names of the texts and programs used, statistical power, information about the size of the identified effect.
Clinical trial programs for different groups of drugs can vary significantly. However, some significant provisions must always be reflected. The goals and objectives of the test should be clearly stated; determine the criteria for selecting patients; indicate the method of distribution of patients into the main and control groups and the number of patients in each group; the method of establishing effective doses of the drug, the duration of the study; control method (open, blind, double, etc.), comparator drug and placebo, methods for quantitative analysis of the effect of study drugs (indicators subject to registration); methods of static data processing.
When evaluating publications on treatment methods, it should be remembered that the exclusion criteria for patients from the study are specified quite often, and the inclusion criteria are less common. If it is not clear on which patients the drug was studied, then it is difficult to assess the information content of the data obtained. Most of the research is carried out in specialized university hospitals or research centers, where patients, of course, differ from patients in district clinics. Therefore, after the initial tests, more and more research is being carried out. First - multicenter, when due to the involvement of different hospitals and the outpatient features of each of them are smoothed out. Then open. With each stage, the confidence that the research results will be applicable to any hospital increases.
The issue of establishing the dose and regimen of the study drug is very important and difficult. There are only the most general recommendations, mainly to start with a low dose, which is gradually increased until the desired or side effect is obtained. When developing rational doses and regimens for the study drug, it is desirable to establish the breadth of its therapeutic action, the range between the minimum and maximum safe therapeutic doses. The duration of use of the study drug should not exceed the duration of toxicological tests on animals.
In the process of clinical trials of new drugs, 4 interrelated phases (stages) are distinguished.
The phase of the first clinical trials is called “sighting”, or “clinico-pharmacological”. Its purpose is to establish the tolerability of the study drug and whether it has a therapeutic effect.
In phase II, clinical trials are carried out on 100-200 patients. A necessary condition is the presence of a control group that does not differ significantly in composition and size from the main group. Patients in the experimental group (main) and control should be the same in terms of gender, age, initial background treatment (it is desirable to stop it 2-4 weeks before the start of the study). The groups are randomly formed using tables of random numbers, in which each digit or each combination of digits has an equal selection probability. Randomization, or random distribution, is the main way to ensure the comparability of comparison groups.
In clinical trials, new drugs are tried to be compared with placebo, which allows you to evaluate the real effectiveness of therapy, for example, its effect on life expectancy of patients compared with no treatment. The need for a double-blind method is determined by the fact that if doctors know what treatment the patient is receiving (active drug or placebo), then they can involuntarily wishful thinking.
A necessary condition for conducting adequate clinical trials is randomization. From consideration, it is necessary to immediately exclude articles about studies in which the distribution of patients into comparison groups was not random, or the method of distribution was unsatisfactory (for example, patients were divided according to the days of the week of admission to the hospital) or there is no information about it at all. Even less informative are studies with historical control (when previously obtained data or the results of studies conducted in other medical institutions are used for comparison). In the international literature, randomization is reported in 9/10 articles on pharmacotherapy, but only 1/3 of the articles specify the method of randomization. If the quality of randomization is in doubt, then the experimental and control groups are most likely not comparable, and other sources of information should be sought.
Of great importance is the clinical significance and statistical significance of the results of treatment. The results of a clinical trial or a population study are presented in the form of information about the frequency of outcomes and the statistical significance of differences between groups of patients. Does the author present statistically significant but small differences as clinically significant? Statistically significant is what actually exists with a high probability. It is clinically significant that, by its size (for example, the magnitude of the reduction in mortality) convinces the physician of the need to change his practice in favor of a new method of treatment.
Methods, criteria for evaluating the effectiveness of the drug, the time of measurement of the relevant indicators should be agreed before the start of the test. Evaluation criteria are clinical, laboratory, morphological and instrumental. Often, the effectiveness of an investigational drug is judged by reducing the dose of other drugs. For each group of drugs there are mandatory and additional (optional) criteria.
The purpose of phase III clinical trials is to obtain additional information about the effectiveness and side effects of a pharmacological agent, clarify the features of the drug's action and determine relatively rare adverse reactions. The features of the drug in patients with circulatory disorders, kidney and liver function are being studied, interaction with other drugs is being evaluated. The results of treatment are recorded in individual registration cards. At the end of the study, the results are summarized, processed statistically and presented in the form of a report. Corresponding indicators obtained for the same period of time in the main and control groups are compared statically. For each indicator, the average difference for the studied period of time (compared to the baseline before treatment) is calculated and the reliability of the marked dynamics within each group is assessed. Then, the mean differences in the values of specific indicators of the control and experimental groups are compared to assess the difference in the effect of the study agent and placebo or comparator drug. A report on the results of clinical trials of a new drug is drawn up in accordance with the requirements of the Pharmacological Committee and submitted to the committee with specific recommendations. A recommendation for clinical use is considered justified if the new product:
More effective than known drugs of similar action;
It has better tolerance than known drugs (with the same tolerance);
Effective in cases where treatment with known drugs is unsuccessful;
More cost-effective, has a simple method of treatment or a more convenient dosage form;
In combination therapy, it increases the effectiveness of existing drugs without increasing their toxicity.
After the approval of the use of a new drug in veterinary practice and its introduction, phase IV studies begin - the effect of the drug is studied in various situations in practice.
Methods for the study of medicinal substances are divided into:
1. physical,
2. chemical,
3. physical and chemical,
4. biological.
Physical methods of analysis involves the study of the physical properties of a substance without resorting to chemical reactions. These include: determination of solubility, transparency or degree of turbidity, color; determination of density (for liquid substances), humidity, melting point, solidification point, boiling point.
Chemical research methods based on chemical reactions. These include: determination of ash content, reaction of the environment (pH), characteristic numerical indicators of oils and fats (acid number, iodine number, saponification number, etc.). For the purposes of identifying medicinal substances, only such reactions are used that are accompanied by a visual external effect, for example, a change in the color of the solution, evolution of gases, precipitation or dissolution of precipitates, etc. Chemical research methods also include weight and volume methods of quantitative analysis adopted in analytical chemistry (method of neutralization, precipitation, redox methods, etc.). In recent years, pharmaceutical analysis has included such chemical research methods as titration in non-aqueous media, complexometry. Qualitative and quantitative analysis of organic medicinal substances, as a rule, is carried out by the nature of the functional groups in their molecules.
Via physical and chemical methods study the physical phenomena that occur as a result of chemical reactions. For example, in the colorimetric method, the color intensity is measured depending on the concentration of the substance, in the conductometric analysis, the measurement of the electrical conductivity of solutions, etc.
Physical and chemical methods include: optical (refractometry, polarimetry, emission and fluorescent methods of analysis, photometry, including photocolorimetry and spectrophotometry, nephelometry, turbodimetry), electrochemical (potentiometric and polarographic methods), chromatographic methods.
biological this is an animal study (frogs, pigeons, cats). Defined in units. Subjected to: MPS containing cardiac glycosides, drugs containing hormones, enzymes, vitamins, antibiotics.
Registration of extemporaneous drugs, VAZ, VAF is carried out in accordance with the order of the Ministry of Health of the Russian Federation No. 376 and guidelines on a single design.
Labels for the design of medicines prepared individually and in the order of intra-pharmacy preparation and packaging, depending on the method of their use, are divided into:
ü labels for drugs for internal use with the inscription "Internal", "Internal for children";
ü labels for drugs for external use with the inscription "External";
ü labels for drugs for parenteral administration with the inscription "For injection";
ü labels for eye medicines with the inscription "Eye drops", "Eye ointment".
On all labels for the design of medicines, prepared individually and in the order of in-pharmacy preparation and packaging, warning labels corresponding to each dosage form must be printed in a typographical way:
ü for potions - "keep in a cool and dark place", "shake before use";
ü for ointments, eye ointments and eye drops - "keep in a cool and dark place";
ü for drops of internal use - "keep in a place protected from light";
ü for injections - "sterile".
All labels must contain the warning "Keep out of the reach of children".
The dosage form is indicated by hand.
All labels for the design of medicines prepared in the order of in-pharmacy procurement and packaging must have the following designations:
ü emblem (bowl with a snake);
ü location of the pharmacy institution (enterprise);
ü the name of the pharmacy institution (enterprise);
ü method of application (internal, external, for injection) or dosage form (ointment, eye drops, nose drops, etc.);
date of preparation...;
ü good for ...;
ü series...;
ü Keep away from children.
The text of pharmacy labels intended for the design of medicines prepared individually, as well as the method of application, must be printed in Russian or the local language.
The text of pharmacy labels intended for the design of medicines prepared in the order of in-pharmacy preparation and packaging, as well as their names and necessary warning labels, is recommended to be printed in a typographical way.
Warning labels affixed to medicines have the following text and signal colors:
ü "shake before use" - green font on a white background;
ü "keep in a place protected from light" - white font on a blue background;
ü "keep in a cool place" - white font on a blue background;
ü "childish" - white font on a green background;
ü "for newborns" - white font on a green background;
ü "handle with care" - red font on a white background;
ü "heart" - white font on an orange background;
ü "Keep away from fire" - white font on a red background.
Particularly toxic substances (<...>, cyanide and mercury oxycyanide) are issued with one black warning label with the name of the poisonous drug in Russian (or local) language in white font with the image of crossbones and a skull and the inscription "poison" and "handle with care" in accordance with the current order.
Registration of medicines prepared in pharmacies (enterprises) of various forms of ownership, in accordance with the presented Uniform Rules for the Registration of Medicines, contributes to improving the culture of drug supply to the population, strengthening control over the expiration dates of prepared medicines and their price, drawing attention to them in order to eliminate possible errors in their use.
Determination of tariffs
The payment includes:
1. The cost of drugs
2. Cost of auxiliary materials
3. Cost of dishes
4. Costs
Tariffs are approved by order of the pharmacy.
The initial data for determining production costs are the accounting and reporting data of the pharmacy for the past month.
The number of conditional production units reflects the total labor intensity of manufacturing one unit of a medicinal product and medical devices.
For one production unit, the work performed within 10 minutes is conditionally accepted.
For one unit of production of sterile and liquid dosage forms, ointments, a medicinal product is accepted, fully prepared in accordance with the current documents and intended for dispensing.
Sterile dosage forms include injectable solutions, infusion solutions, ophthalmic irrigation solutions, neonatal solutions and oils.
To ZhLF include solutions and drops for internal use and external use, oils, purified water.
Ointments include pastes, liniments, liquid plasters, suspensions, emulsions.
For one unit of powders and suppositories, a dosage form with packaging for 10 doses is conventionally accepted.
Similar information.
Pharmaceutical Analysis (FA). It is the basis of pharmaceutical chemistry and has its own characteristics that distinguish it from other types of analysis. They lie in the fact that substances of various chemical nature are analyzed: inorganic, organoelement, radioactive, organic compounds from simple aliphatic to complex natural biologically active substances. The range of concentrations of analytes is extremely wide. The objects of pharmaceutical analysis are not only individual medicinal substances, but also mixtures containing a different number of components.
The annual replenishment of the arsenal of drugs necessitates the development of new methods for their analysis. Pharmaceutical analysis methods need to be systematically improved due to the continuous increase in the requirements for both the quality of medicines and the quantitative content of biologically active substances in them. That is why pharmaceutical analysis is subject to high demands. It should be sufficiently specific and sensitive, accurate in relation to the regulatory requirements of the State Pharmacopoeia X and XI and other scientific and technical documentation (FS, GOST), carried out in short periods of time using the minimum amounts of test preparations and reagents.
Depending on the tasks set, pharmaceutical analysis includes various forms of drug quality control: pharmacopoeial analysis; step-by-step control of drug production; analysis of dosage forms of individual production; rapid analysis in a pharmacy and biopharmaceutical analysis. Its integral part is pharmacopoeial analysis, which is a set of methods for researching drugs and dosage forms set forth in the State Pharmacopoeia or other NTD (FS, FSP, GOST). Based on the results obtained during the pharmacopoeial analysis, a conclusion is made on the compliance of the medicinal product with the requirements of the State Pharmacopoeia or other scientific and technical documentation. In case of deviation from these requirements, the drug is not allowed for use.
Chemical analysis of plant raw materials. According to the technique of execution and the nature of the results obtained, chemical reactions are divided into several groups: qualitative, microchemical and histochemical, microsublimation.
To establish the authenticity of medicinal plant materials, the simplest qualitative reactions and chromatographic tests for active and related substances are used. The methodology is described in the relevant regulatory documentation for the studied type of raw material in the section "Qualitative reactions".
Qualitative reactions are performed on dry raw materials with the following types of raw materials: oak bark, viburnum, buckthorn, bergenia rhizomes, elecampane rhizomes and roots, dandelion roots, marshmallow, ginseng, barberry, linden flowers, flax seeds, ergot sclerotia (for a total of 12 types of raw materials) .
Basically, qualitative reactions are carried out with extraction (extraction) from medicinal plant materials.
Based on the properties of biologically active substances, they are extracted from raw materials with water, alcohol of various concentrations or an organic solvent, less often with the addition of alkali or acid.
An aqueous extract is prepared from raw materials containing glycosides, polysaccharides, saponins, phenol glycosides, anthraglycosides, and tannins. Acidified water is used to extract alkaloids in the form of salts from raw materials.
A large group of biologically active substances (cardiac glycosides, coumarins, lignans, flavonoids) is extracted with ethyl and methyl alcohol of various concentrations.
If the reaction is sufficiently specific and sensitive, then it is carried out with a crude extract from the raw material.
These reactions include:
general alkaloid sedimentary reactions;
reactions with a solution of aluminum chloride to flavonoids (St. John's wort, highlander bird, highlander pepper, etc.);
Synod test for flavonoids in immortelle flowers;
reaction with an alkali solution for anthracene derivatives (buckthorn bark, rhubarb roots, etc.);
reaction with a solution of iron-ammonium alum to tannins (oak bark, serpentine rhizomes, bergenia, etc.).
Accompanying substances (proteins, amines, sterols, chlorophyll) often interfere with the reaction. In this case, a purified extract is used (for example, from raw materials containing cardiac glycosides, coumarins, alkaloids, phenol glycosides, lignans).
Purify the extract by precipitating the accompanying substances with a solution of lead acetate and sodium sulfate, or using the solvent change technique or partition chromatography.
Microchemical reactions are usually carried out simultaneously with microscopic analysis, observing the results under a microscope:
on essential and fatty oils with a solution of Sudan III;
on lignified lignified elements with a solution of phloroglucinol and a 25% solution of sulfuric acid or concentrated hydrochloric acid.
Oak bark (powder) is reacted with iron ammonium alum and the reaction result is studied under a microscope.
Histochemical reactions are such reactions with the help of which it is possible to identify certain compounds directly in the cells or structures where they are localized.
According to the State Pharmacopoeia XI, histochemical reactions are carried out on mucus with a carcass solution in marshmallow roots and flax seeds.
microsublimation- direct separation from dry plant material of substances that easily sublimate when heated. The resulting sublimate is examined under a microscope, then a microchemical reaction is carried out with the appropriate reagent.
Methods for determining the authenticity of medicinal plant materials. The authenticity of raw materials is determined by macroscopic, microscopic, chemical and luminescent analyses.
macroscopic analysis. To carry it out, you need to know the morphology of plants. They study the appearance of raw materials with the naked eye or with a magnifying glass, measure the particle size using a millimeter ruler. In daylight, the color of the raw material is determined from the surface, at the fracture and in the cut. The smell is established by rubbing or breaking plants, and the taste - only in non-poisonous plants. When studying the appearance, pay attention to the morphological features of the parts of the raw material.
Microscopic analysis. Used to determine the authenticity of crushed medicinal plant materials. To do this, you need to know the anatomical structure of plants as a whole and the characteristics characteristic of a particular plant that distinguish it from other plants.
Chemical analysis. It provides for qualitative, microchemical, histochemical reactions and sublimation to determine active or related substances in raw materials. It is advisable to carry out microchemical reactions in parallel with microscopic analysis. Histochemical reactions are carried out to identify specific compounds at the sites of their localization in the plant. Sublimation is understood as the production of substances easily sublimated when heated from plant materials, followed by a qualitative reaction with sublimate.
Luminescent analysis. This is a method for studying various objects (including biological ones) based on the observation of their luminescence. Luminescence - the glow of a gas, liquid or solid, due not to the heating of the body, but to the non-thermal excitation of its atoms and molecules. Luminescent analysis is carried out to determine the substances with luminescence in medicinal raw materials.
Quality control of organotherapeutic preparations. To check the compliance of the quality of the glands with the requirements of the standard, 5% of boxes or packages are selected from each lot, but not less than five such packages. If in one of the opened boxes or packages the glands do not meet the requirements of the relevant standard in at least one of the indicators, then the entire batch is checked.
For individual types of raw materials, there are objective (laboratory) methods for assessing its quality.
Objectively, the quality of the pancreas intended for the production of insulin, according to GOST, is determined by the mass fraction of fat and the mass fraction of insulin using appropriate laboratory methods.
The mass fraction of fat is determined by a butyrometer. The mass fraction of insulin is checked at the request of the consumer by an immunoreactive method using antiserum, immunoglobulins in a homogenized gland.
The quality of the mucous membrane (epithelium) of the tongues of cattle is checked by determining the pH value of the preserving medium with the epithelium and its bacterial contamination. The essence of the method is to determine the total number of microbes in 1 ml of a preservative medium with epithelium.
The quality of the vitreous body of the eyes of cattle, pigs, sheep and goats frozen is determined by the quantitative content of hyaluronic acid (glucosamine) in the vitreous body. The principle of the method is based on the determination of glucosamine in the products of hyaluronic acid hydrolysis, which is an integral part of the hyaluronic acid molecule and is directly dependent on its content in the vitreous body.
The biological activity of the pituitary glands is determined in units of action of ACTH contained in 1 mg of acid acetonated powder (CAP) obtained from the pituitary glands.
Determination of ACTH activity is based on its ability to cause a reduction of lymphoid tissue, in particular, the goiter gland of rat pups. The unit of action of the drug is taken as the daily dose of the drug, which, when administered for five days, causes a decrease in the mass of the gland by 50 ± 5%.
The quality of the parathyroid glands is determined by the histological method. On sections of the parathyroid glands, accumulations of epithelial cells with pronounced basophilic granularity are visible. On sections of the lymphatic glands, reticular tissue is visible (in the form of a homogeneous mass), surrounded by a dense connective sheath (capsule), from which clearly visible connecting cords extend inward. The state standard stipulates that a sample of 40 glands may contain no more than one lymph node.
Methods for determining the quality of dry biological preparations. Dry biological preparations have a number of advantages over traditional liquid biological preparations due to better quality, lower weight, increased shelf life, and ease of transportation.
Physical methods. 1. Method for determining the vacuum. The essence of the method lies in the ability of a high-frequency electric current at high voltage to cause a glow in gases, the nature of which changes depending on the degree of rarefaction of the air in the ampoule (vial).
Sample selection. Sampling is carried out in accordance with the rules established in the state standards for dry biological preparations.
Apparatus and equipment. When carrying out the test, they use: an apparatus of the “D’Arsenal” or “Tesla” type, a stand for ampoules, a metal table.
Conducting a test. Test preparation:
before testing, check the appearance, the tightness of the corking of the vials, the presence of cracks, the sealing of the ampoules.
The apparatus is kept for 10 minutes after switching on. The test ampoules are placed in a tripod, then an electrode is brought to them at a distance of 1 cm. When determining the vacuum using the Tesla apparatus, one metal electrode of the apparatus is grounded through a metal table on which the ampoules are laid out, and the other is brought to the tested ampoules. Exposure no more than 1 s.
Processing of results. The appearance of glow inside the ampoules with a characteristic crackle indicates the presence of a vacuum in them.
The degree of rarefaction of air in the tested ampoules is determined by the nature of the glow of gases in the tested ampoules in accordance with the following data.
Determination of the degree of rarefaction of air in the tested ampoules
2. Method for determining humidity. The essence of the method is to determine the weight reduction of the drug sample after it has been dried for 1 hour at a temperature of 105 °C.
Sample selection. For testing, the required number of ampoules (vials) are selected from different places of packaging, taking into account the requirements for the mass of samples (in accordance with the standard).
When sampling, check the tightness of the ampoules. In vials with a lyophilized preparation, the wall and bottom are checked for integrity, as well as the completeness of the fit of the rolled cap and rubber stopper. In the presence of defects, the vial is replaced by another. Each ampoule, sealed under vacuum, is checked for leaks before removing the drug from it.
Equipment, materials and reagents. During the test, the following are used: laboratory scales, a laboratory drying cabinet, mercury thermometers, a desiccator, glass bottles, technical vaseline, anhydrous calcium chloride or dehydrated gypsum, or calcined silica gel.
Preparing for the test. The drying cabinet is checked with maximum thermometers for uniform heating.
When drying samples in weighing bottles, the lower part of the control thermometer should be at the level of weighing bottles. The readings of the control thermometer are decisive for setting the temperature in the cabinet.
The balance must be placed on a stable, vibration-free table. The results of all weighings are recorded in grams to the fourth decimal place.
The bottom of the desiccator should be filled with dehydrated calcium chloride or gypsum or silica gel. The polished edges of the vessel are lightly smeared with technical vaseline.
Three bottles of the same diameter and height should be prepared for each analysis.
Conducting a test. To determine the moisture content, three ampoules are used, if each of them contains a sample mass of at least 0.1 g. If the ampoule contains less than 0.1 g of a biological preparation, then two or more ampoules can be used.
The selected sample, crushed to a powdery state, is placed in an even layer in a pre-weighed bottle.
The bottles are installed in a drying cabinet on a shelf. The beginning of drying should be considered the time of reaching a temperature of 105 ° C according to the control thermometer. Drying time 60 min.
After drying, the weighing bottles are quickly closed with lids and transferred to a desiccator to cool to room temperature, after which the weighing bottles are weighed to the fourth decimal place and registered according to the form.
3. Method for determining the amount of oxygen. Sample selection. Sampling is carried out in accordance with the rules established in the state standards for dry biological preparations.
Equipment, materials and reagents. During the test, the following is used: a gas chromatograph of the LXM-8MD brand or other similar brands with a thermal conductivity detector and a gas chromographic column with a diameter of 3 mm and a length of 1000 mm, a muffle furnace with a heating temperature of up to 1000 ° C, a gas flow meter with a burette, a stopwatch, a medical syringe with a capacity of 1 cm 3, woven wire meshes, a measuring magnifying glass, a desiccator, a porcelain mortar, a metal ruler 30 cm long, molecular sieves - synthetic zeolite grade CaA, a medical needle, a medical rubber tube with an internal diameter of 4.2 mm, 10 m long, a bottle with a capacity 3000 cm 3 , rubber stopper, silicone oil, helium, nitrogen gas, distilled water.
Preparing for the test. Column preparation. Synthetic zeolite is crushed in a porcelain mortar, screened out on sieves, washed with distilled water, dried and calcined in a muffle furnace at a temperature of 450...500 °C for 2 hours, then cooled in a desiccator on grids to room temperature.
The chromatographic column is installed vertically and covered with synthetic zeolite. The column is not filled up by 1 cm and plugged with a mesh. The filled column is installed in the thermostat of the chromatograph and, without being connected to the detector, a flow of helium or nitrogen is passed through it for 3 hours at a temperature of 160...180 °C. Then the column is attached to the detector and helium or nitrogen continues to pass through it until the drift of the zero line stops at the maximum sensitivity of the detector.
The chromatograph is prepared for operation and switched on in accordance with the manufacturer's instructions.
Preparation of a vial with the drug for testing. To take a sample from the drug vial, equalize the gas pressure in the vial with atmospheric pressure.
Preparing a medical syringe. A metal tube is preliminarily installed on the syringe rod and the syringe is checked for leaks. With a medical syringe with a needle checked and prepared for gas sampling, a rubber tube is pierced through which helium exits the chromatograph reference column, and helium is slowly drawn in and released twice with a syringe. For the third time, by drawing helium into the syringe and placing it with the needle down, gas samples are taken from the vial with the drug.
Conducting a test. Two gas samples are taken from each vial and sequentially one after the other with an interval of 3...4 min are injected into the evaporator of the chromatograph. The sample is introduced into the evaporator by gently pressing the finger on the rod. After 110 ... 120 s after the introduction of the sample on the chromatogram, the recorder draws an oxygen peak, and then a nitrogen peak.
Processing of results. Calculate the area of the peaks of oxygen and nitrogen. To do this, the height and width of the peaks of oxygen and nitrogen are measured on the chromatograph using a metal ruler 30 cm long, a magnifying loupe, and a sharply sharpened pencil. The height of the peaks is measured from the baseline to the top of the peak, the width of the peak is measured at half its height. When measuring, take the distance from the inner thickness of the peak line to the outer one.
The peak area of oxygen (SO 2, mm 2) and nitrogen (5N 2, mm 2) is calculated by the formulas
SO 2 \u003d h 1 * b 1; SN \u003d h 2 *b 2,
where h 1 h 2 ~ height of oxygen and nitrogen peaks, mm; b 1 , b 2 - width of oxygen and nitrogen peaks, mm.
The volume fraction of oxygen (X, %) in each gas sample is calculated by the formula
X=SO 2 /(SO 2 +SN 2)
where SO 2 , SN 2 are the areas of oxygen and nitrogen peaks, mm 2 .
For the final test result, the arithmetic mean of the results of determinations in three vials of the drug is taken.
The relative reduced error of the method at a confidence level P-0.95 should not exceed 10%.
bacteriological method. Sterility control. The essence of the method lies in the microbiological assessment of the absence of growth of bacteria and fungi in the inoculation of preparations on nutrient media.
Sample selection. Samples are taken from each series of preparations in the amount of 0.15% vials, but not less than five for liquid and 10 ampoules for dry preparations.
Preparing for the test. Laboratory glassware is boiled for 15 minutes in distilled water, acidified with a solution of hydrochloric acid, and then washed with tap water and washed with a brush in a solution containing 30 g of washing powder and 50 cm 3 of aqueous ammonia per 1000 cm 3 of distilled water. After that, the dishes are thoroughly washed first with tap water, and then three times with distilled water, dried and sterilized.
Before sterilization, the dishes are placed in metal cases. Dishes are sterilized in an autoclave at 0.15 MPa for 60 minutes.
Ready-made nutrient media, tested for growth properties, are poured into 6...8 cm 3 (to determine anaerobes 10...12 cm 3) into test tubes, 50...60 cm 3 into 100 cm 3 vials.
Samples of dry biological preparations are pre-dissolved with a sterile solvent (isotonic sodium chloride solution, distilled water, etc.).
Conducting a test. 1. Testing for sterility using thioglycol medium.
From each vial of the drug, 1 cm 3 are inoculated into three test tubes containing thioglycol medium.
Two inoculated test tubes are kept in a thermostat for 14 days: one at a temperature of 21 °C, the other at a temperature of 37 °C.
The third test tube is kept for 7 days at a temperature of 37 ° C and then subcultures of 0.5 cm 3 are made from it, one test tube each for beveled casein agar, casein nutrient broth, Sabouraud medium and 1 cm 3 for casein nutrient broth under vaseline oil with pieces of meat or liver.
Transfers to casein agar, meat-peptone broth are kept for another 7 days at a temperature of 37 °C, and transfers to Sabouraud medium - at a temperature of 21 °C.
When testing samples of preparations, the sterility of media is monitored: three test tubes with each medium are kept in a thermostat for 14 days at 37 °C, with Sabouraud medium - at a temperature of 21 °C.
2. Testing for sterility without thioglycol medium.
From each sample of the preparation, inoculation is carried out on Sabouraud's liquid medium, meat-peptone agar and meat-peptone broth - three test tubes each; on the Tarozzi medium - two test tubes and two vials.
To detect aerobes, 0.5 cm 3 of seed is sown in one test tube and 1 ... 2 cm 3 in one vial, and to detect anaerobes, 1 and 5 cm 3, respectively. The crops are placed in a thermostat (at a temperature of 37 ° C; for Sabouraud - at a temperature of 21 ° C) for 7 days (15 days for anaerobes). Then re-seeding is done (except for sowing on meat-peptone agar). Subcultured on the same media. Withstand 7 days (15 days for anaerobes). Sterility control is carried out.
Evaluation of results. The results of the primary and repeated inoculations are taken into account by macroscopic, and in the case of microorganism growth - microscopic examination of all crops, they are taken into account 14 days after the initial inoculation on a thioglycol medium and 7 days after the initial inoculation without a thioglycol medium. The medium is considered sterile if no growth is observed in any of the inoculated tubes.
In cases of growth in at least one of the inoculated tubes, the sterility control is repeated on the same number of samples and microscopy of the grown microbes is carried out. The smears are Gram-stained, noting the morphology.
In the absence of growth in the repeated control, the drug is considered sterile. In the presence of growth in at least one of the test tubes and the identity of the microflora during the primary and repeated crops, the drug is considered non-sterile.
If different microflora is detected during the primary and repeated inoculations, and growth is detected only in individual test tubes, the samples are inoculated for the third time.
In the absence of growth, the drug is considered sterile. If growth is detected in at least one test tube, regardless of the nature of the microflora, the drug is considered non-sterile.
Regulatory requirements for the quality of finished dosage forms. Dosage forms are manufactured in factories, pharmaceutical factories (official drugs) and pharmacies (trunk drugs). The control of finished dosage forms at pharmaceutical enterprises is carried out in accordance with the requirements of the NTD (State Pharmacopoeia, FS, FSP, GOSTs). In accordance with the requirements of these documents, dosage forms must be subject to verification (V. D. Sokolov, 2003).
Tablets are tested for disintegration. Unless otherwise specified in a private article, tablets should disintegrate within 15 minutes, and coated tablets should not exceed 30 minutes. Enteric tablets should not disintegrate within 1 hour in hydrochloric acid solution, but should disintegrate within 1 hour in sodium bicarbonate solution. The strength of tablets for abrasion should be at least 75%. The drug contained in the tablet must be dissolved in water in 45 minutes by at least 75%. The average weight is determined by weighing 20 tablets with an accuracy of 0.001 g. Deviations from the average weight are allowed: ± 7.5% for tablets weighing 0.1 ... 0.3 g and ± 5% for tablets weighing 0.5 g and more. The tablets also control the content of talc.
Granules - determine the size using sieve analysis. The cell diameter should be 0.2...3 mm, and the number of smaller and larger granules should not exceed 5%. The disintegration test for 0.5 g granules is the same as for tablets. The disintegration time should not exceed 15 minutes. Determine moisture. To determine the content of the medicinal substance, a sample of at least 10 pounded granules is taken.
Capsules - control the average weight. The deviation of each capsule from it should not exceed ± 10%. Just as it is done with tablets, disintegration and solubility are controlled, and dosing uniformity is determined for capsules containing 0.05 g or less of the drug substance. The quantitative determination of medicinal substances is carried out according to special methods, using the contents of 20 to 60 capsules for this purpose.
Powders - establish deviations in the mass of dosed powders. They can be ± 15% with a powder weight of up to 0.1 g; ±10% - from 0.1 to 0.3 g; ±5% - from 0.3 to 1; ±3% - over 1 g.
Suppositories - visually determine uniformity in a longitudinal section. The average weight is determined by weighing with an accuracy of 0.01 g, deviations should not exceed ± 5%. Suppositories made on lipophilic bases are controlled by melting point. It must not exceed
37 °C. If this temperature cannot be established, then the time of complete deformation is determined, which should be no more than 15 minutes. Suppositories made on a hydrophilic basis are tested for solubility (indicator "dissolution"). Determine the dissolution time at a temperature of (37 ± 1) ° C, which should not exceed 1 hour. The quantitative determination of medicinal substances is carried out according to special methods.
Tinctures - determine the alcohol content or density. The content of active substances is determined using special techniques. In addition, the dry residue is determined after evaporating 5 ml of the tincture to dryness in a bottle and drying it for 2 hours at a temperature of (102.5 ± 2.5) °C. In the same volume of tincture after burning and calcining its mixture with 1 ml of concentrated sulfuric acid, the content of heavy metals is determined.
Extracts - as in tinctures, determine the density or content of alcohol, active ingredients, heavy metals. The dry weight of the residue is also established, and in thick and dry extracts - the moisture content [by drying in an oven at a temperature of (102.5 ± 2.5) ° C).
Aerosols - measure the pressure inside the cylinder using a pressure gauge at room temperature (if the propellant is a compressed gas). Check the packaging for leaks. In dosed packages, the average mass of the drug in one dose is determined, the deviation in which is allowed no more than +20%. Set the percentage of the output of the contents by removing it from the cylinder, followed by weighing. The quantitative determination of the substance is carried out in accordance with the requirements of private articles of the State Pharmacopoeia. Deviations from the stated quantities should not exceed ±15%.
Ointments - A common test is a method for determining the particle size of a drug substance in ointments. A microscope with an ocular micrometer MOV-1 is used.
Plasters. The composition, quality indicators, test methods are different and are set out in the regulatory documentation for specific products.
Eye drops are tested for sterility and the presence of mechanical impurities.
Injectable dosage forms. Injectable drug solutions administered intravenously in large quantities require special attention. They use such characteristics as appearance, including the color and transparency of solutions, the absence of mechanical impurities, apyrogenicity, sterility, the volume of the solution, the amount of active substance in it, the pH and isotonicity of blood plasma, packaging, labeling, and the volume of filling of ampoules. The norms of permissible deviations are indicated in the State Pharmacopoeia XI. In addition, the content of excipients is determined; for some of them (phenol, cresol, sulfites, chlorobutanol) allowable amounts are provided (from 0.2 to 0.5%). pH requirements vary by formulation, typically between 3.0 and 8.0. On each ampoule (bottle) indicate the name of the drug, its content (in percent) or activity (in units of action, U), volume or mass, batch number, expiration date. All tests of injectable dosage forms are regulated by the NTD.
The analysis of homeopathic medicines is very difficult due to the high dilutions of the medicinal substances. If biologically active substances are contained in tinctures, essences, ointments and other forms in dilutions up to 2 C (C - centesimal) or 0.0001, then their analysis and standardization practically do not differ from the quality control of dosage forms used in allopathic medicine. Drugs in a dilution of 2 ... 3 C (10 -4 ... 10 -6) are analyzed after special methods of concentration using evaporation, combustion of substances, followed by determination by one of the physicochemical methods, based on its resolution. At more than 3 C dilution (10 -6) it is enough to establish the authenticity of the medicinal product contained in a single single or daily dose. At very high dilutions (up to 50 C or 10 -10 ... 10 -100) it is impossible to control the quality of homeopathic remedies using existing methods. For such drugs, quality control is carried out at the stage of production, strictly controlling the technological process. The quality is controlled when adding the ingredients and recorded in the act of loading. Each ingredient is subjected to a preliminary analysis. In all these cases, chromatographic, photometric, fluorescent and other methods are used for the analysis and standardization of homeopathic medicines.
5 / 5 (votes: 1 )
Today, it is quite common to find low-quality medicines and dummy pills that cause the consumer to doubt their effectiveness. There are certain methods of drug analysis that allow to determine the composition of the drug, its characteristics with maximum accuracy, and this will reveal the degree of influence of the drug on the human body. If you have certain complaints about a drug, then its chemical analysis and objective opinion can be evidence in any legal proceeding.
What methods of drug analysis are used in laboratories?
To establish the qualitative and quantitative characteristics of a drug in specialized laboratories, the following methods are widely used:
- Physical and physico-chemical, which help determine the melting and solidification temperature, density, composition and purity of impurities, find the content of heavy metals.
- Chemical, determining the presence of volatile substances, water, nitrogen, the solubility of the medicinal substance, its acid, iodine number, etc.
- Biological, allowing you to test the substance for sterility, microbial purity, the content of toxins.
Methods for the analysis of medicines will make it possible to establish the authenticity of the composition declared by the manufacturer and determine the slightest deviations from the norms and production technology. The laboratory of ANO "Center for Chemical Expertise" has all the necessary equipment for an accurate study of any type of medicine. Highly qualified specialists use a variety of methods for analyzing medicines and will provide an objective expert opinion in the shortest possible time.
The purpose of the study of medicinal substances is to establish the suitability of the medicinal product for medical use, i.e. compliance with its regulatory document for this drug.
Pharmaceutical analysis is the science of chemical characterization and measurement of biologically active substances at all stages of production: from the control of raw materials to the assessment of the quality of the resulting medicinal substance, the study of its stability, the establishment of expiration dates and the standardization of the finished dosage form. The peculiarities of pharmaceutical analysis are its versatility and variety of substances or their mixtures, including individual chemicals, complex mixtures of biological substances (proteins, carbohydrates, oligopeptides, etc.). Methods of analysis need to be constantly improved, and if chemical methods, including qualitative reactions, prevailed in the UP Pharmacopoeia, then at the present stage, mainly physicochemical and physical methods of analysis are used.
Pharmaceutical analysis, depending on the tasks, includes various aspects of drug quality control:
1. Pharmacopoeial analysis;
2. Stage-by-stage control of the production of medicines;
3. Analysis of individual drugs.
The main and most significant is the pharmacopoeial analysis, i.e. analysis of medicines for compliance with the standard - a pharmacopoeial monograph or other ND and, thus, confirmation of its suitability. Hence the requirements for high specificity, selectivity, accuracy and reliability of the analysis.
A conclusion about the quality of a medicinal product can only be made on the basis of a sample analysis (a statistically significant sample). The sampling procedure is indicated either in a private article or in a general article of the Global Fund X1 ed. (Issue 2) p.15. To test medicines for compliance with the requirements of regulatory and technical documentation, multi-stage sampling (sampling) is carried out. With multi-stage sampling, a sample (sample) is formed in stages and the products in each stage are selected randomly in proportional quantities from the units selected in the previous stage. The number of steps is determined by the type of packaging.
Stage 1: selection of packaging units (boxes, boxes, etc.);
Stage 2: selection of packaging units in packaging (boxes, bottles, cans, etc.);
Stage 3: selection of products in primary packaging (ampoules, vials, blisters, etc.).
To calculate the selection of the number of products at each stage, use the formula:
where n- the number of packaging units of this stage.
The specific sampling procedure is described in detail in the GF X1 edition, issue 2. In this case, the analysis is considered reliable if at least four samples are reproducible.
Pharmaceutical Analysis Criteria
For various purposes of the analysis, such criteria as the selectivity of the analysis, sensitivity, accuracy, the time of the analysis, the amount of the test substance are important.
The selectivity of the analysis is essential in the analysis of complex preparations consisting of several active components. In this case, the selectivity of the analysis is very important for the quantitative determination of each of the substances.
Requirements for accuracy and sensitivity depend on the object and purpose of the study. When testing for purity or impurities, highly sensitive methods are used. For stepwise production control, the time factor spent on analysis is important.
An important parameter of the analysis method is the sensitivity limit of the method. This limit means the lowest content at which a given substance can be reliably detected. The least sensitive are chemical methods of analysis and qualitative reactions. The most sensitive enzymatic and biological methods to detect single macromolecules of substances. Of those actually used, the most sensitive are radiochemical, catalytic and fluorescent methods, which make it possible to determine up to 10 -9%; sensitivity of spectrophotometric methods 10 -3 -10 -6%; potentiometric 10 -2%.
The term "analysis accuracy" simultaneously includes two concepts: reproducibility and correctness of the results obtained.
Reproducibility - characterizes the dispersion of the results of the analysis compared to the average value.
Correctness - reflects the difference between the actual and found content of the substance. The accuracy of the analysis depends on the quality of the instruments, the experience of the analyst, etc. The accuracy of the analysis cannot be higher than the accuracy of the least accurate measurement. This means that if the titration is accurate to ±0.2 ml plus leakage error is also ±0.2 ml, i.e. in total ±0.4 ml, then when 20 ml of titrant is consumed, the error is 0.2%. With a decrease in the sample and the amount of titrant, the accuracy decreases. Thus, titrimetric analysis allows determination with a relative error of ± (0.2-0.3)%. Each method has its own accuracy. When analyzing, it is important to have an understanding of the following concepts:
Gross mistakes- are a miscalculation of the observer or a violation of the analysis methodology. Such results are discarded as unreliable.
Systematic errors - reflect the correctness of the results of the analysis. They distort the measurement results, as a rule, in one direction by some constant value. Systematic errors can be partially eliminated by introducing corrections, instrument calibration, etc.
Random errors - reflect the reproducibility of the results of the analysis. They are called by uncontrolled variables. The arithmetic mean of random errors tends to zero. Therefore, for calculations, it is necessary to use not the results of single measurements, but the average of several parallel determinations.
Absolute error- represents the difference between the result obtained and the true value. This error is expressed in the same units as the value being determined.
Relative error definition is equal to the ratio of the absolute error to the true value of the determined value. It is usually expressed as a percentage or percentage.
The values of relative errors depend on the method by which the analysis is performed and what the analyzed substance is - an individual substance and a mixture of many components.
The relative error in the study of individual substances by the spectrophotometric method is 2-3%, by IR spectrophotometry - 5-12%; liquid chromatography 3-4%; potentiometry 0.3-1%. Combined methods usually reduce the accuracy of the analysis. Biological methods are the least accurate - their relative error reaches 50%.
Methods for the identification of medicinal substances.
The most important indicator in the testing of medicinal substances is their identification or, as is customary in pharmacopoeial articles, authenticity. Numerous methods are used to determine the authenticity of medicinal substances. All the main and general are described in the GF X1 edition, issue 1. Historically, the main emphasis has been on chemical, incl. qualitative color reactions characterizing the presence of certain ions or functional groups in organic compounds, at the same time, physical methods were also widely used. In modern pharmacopoeias, the emphasis is on physico-chemical methods.
Let's focus on the main physical methods.
A fairly stable constant characterizing a substance, its purity and authenticity is the melting point. This indicator is widely used for the standardization of substances of medicinal substances. Methods for determining the melting point are described in detail in the GF X1, you yourself could try it out in laboratory classes. A pure substance has a constant melting point, however, when impurities are added to it, the melting point, as a rule, decreases very significantly. This effect is called a mixing test, and it is the mixing test that allows you to establish the authenticity of the drug in the presence of a standard sample or a known sample. There are, however, exceptions, as racemic sulphocamphoric acid melts at a higher temperature, and the various crystalline forms of indomethacin differ in melting point. Those. this method is one of the indicators that characterize both the purity of the product and its authenticity.
For some drugs, such an indicator as the solidification temperature is used. Another indicator characterizing a substance is the boiling point or temperature limits of distillation. This indicator characterizes liquid substances, for example, ethyl alcohol. The boiling point is a less characteristic indicator, it strongly depends on the pressure of the atmosphere, the possibility of the formation of mixtures or azeotropes and is used quite rarely.
Among other physical methods, it should be noted the determination density, viscosity. Standard methods of analysis are described in SP X1. The method that characterizes the authenticity of the drug is also the determination of its solubility in various solvents. According to GF X1 ed. This method is characterized as a property that can serve as an indicative characteristic of the test product. Along with the melting point, the solubility of a substance is one of the parameters by which the authenticity and purity of almost all medicinal substances are established. The pharmacopeia establishes an approximate gradation of substances by solubility from very easily soluble to practically insoluble. In this case, a substance is considered to be dissolved, in the solution of which no particles of the substance are observed in transmitted light.
Physical and chemical methods for determining authenticity.
The most informative in terms of determining the authenticity of substances are physicochemical methods based on the properties of the molecules of substances to interact with any physical factors. Physical and chemical methods include:
1.Spectral methods
UV spectroscopy
Spectroscopy in visible light
IR spectroscopy
Fluorescence spectroscopy
Atomic absorption spectroscopy
X-ray methods of analysis
Nuclear magnetic resonance
X-ray diffraction analysis
2. Sorption methods of analysis
Thin layer chromatography
Gas-liquid chromatography
High Performance Liquid Chromatography
Electrophoresis
Iontophoresis
Gel chromatography
3.Mass methods of analysis
Mass spectrometry
Chromatomass spectrometry
4. Electrochemical methods of analysis
Polarography
Electron paramagnetic resonance
5. Use of standard samples
Let us briefly consider the methods of analysis applicable in pharmacy. All these methods of analysis will be read to you in detail at the end of December by Professor V.I. Myagkikh. Some spectral methods are used to determine the authenticity of medicinal substances. The most reliable is the use of the low-frequency region of IR spectroscopy, where the absorption bands most reliably reflect this substance. I also call this area the fingerprint area. As a rule, comparison of IR spectra taken under standard conditions of a standard sample and a test sample is used to confirm authenticity. The coincidence of all absorption bands confirms the authenticity of the drug. The use of UV and visible spectroscopy is less reliable, because the nature of the spectrum is not individual and reflects only a certain chromophore in the structure of an organic compound. Atomic absorption spectroscopy and X-ray spectroscopy are used to analyze inorganic compounds, to identify chemical elements. Nuclear magnetic resonance makes it possible to establish the structure of organic compounds and is a reliable method for authenticating, however, due to the complexity of the instruments and the high cost, it is used very rarely and, as a rule, only for research purposes. Fluorescence spectroscopy is applicable only to a certain class of substances that fluoresce when exposed to UV radiation. In this case, the fluorescence spectrum and the fluorescence excitation spectrum are quite individual, but strongly depend on the medium in which the given substance is dissolved. This method is more commonly used for quantitation, especially of small quantities, as it is one of the most sensitive.
X-ray diffraction analysis is the most reliable method for confirming the structure of a substance, it allows you to establish the exact chemical structure of a substance, however, it is simply not suitable for stream analysis of authenticity and is used exclusively for scientific purposes.
Sorption methods of analysis found a very wide application in pharmaceutical analysis. They are used to determine authenticity, the presence of impurities, and quantification. You will be given a lecture in detail about these methods and the equipment used by Professor V.I. Myagkikh, a regional representative of Shimadzu, one of the main manufacturers of chromatographic equipment. These methods are based on the principle of sorption-desorption of substances on certain carriers in a carrier stream. Depending on the carrier and sorbent, they are divided into thin-layer chromatography, liquid column (analytical and preparative, including HPLC), gas-liquid chromatography, gel filtration, iontophoresis. The last two methods are used to analyze complex protein objects. A significant drawback of the methods is their relativity, i.e. Chromatography can characterize a substance and its quantity only when compared with a standard substance. However, it should be noted as a significant advantage - the high reliability of the method and accuracy, because. in chromatography, any mixture must be separated into individual substances and the result of the analysis is precisely the individual substance.
Mass spectrometric and electrochemical methods are rarely used to confirm authenticity.
A special place is occupied by methods for determining authenticity in comparison with a standard sample. This method is used quite widely in foreign pharmacopoeias to determine the authenticity of complex macromolecules, complex antibiotics, some vitamins, and other substances containing especially chiral carbon atoms, since it is difficult or even impossible to determine the authenticity of an optically active substance by other methods. A standard sample should be developed and issued on the basis of a developed and approved pharmacopoeial monograph. In Russia, only a few standard samples exist and are used, and the so-called RSOs are most often used for analysis - working standard samples prepared immediately before the experiment from known substances or corresponding substances.
Chemical methods of authentication.
The identification of medicinal substances by chemical methods is used mainly for inorganic medicinal substances, since other methods are most often not available or they require complex and expensive equipment. As already mentioned, inorganic elements are easily identified by atomic absorption or X-ray spectroscopy. Our Pharmacopoeia Monographs usually use chemical authentication methods. These methods are usually divided into the following:
Precipitation reactions of anions and cations. Typical examples are the precipitation reactions of sodium and potassium ions with (zincuranyl acetate and tartaric acid), respectively:
Such reactions are used in great variety and they will be discussed in detail in a special section of pharmaceutical chemistry regarding inorganic substances.
Redox reactions.
Redox reactions are used to reduce metals from oxides. For example, silver from its formalin oxide (silver mirror reaction):
The oxidation reaction of diphenylamine is the basis for testing the authenticity of nitrates and nitrites:
Reactions of neutralization and decomposition of anions.
Carbonates and hydrocarbonates under the action of mineral acids form carbonic acid, which decomposes to carbon dioxide:
Similarly, nitrites, thiosulfates, and ammonium salts decompose.
Changes in the color of a colorless flame. Sodium salts color the flame yellow, copper green, potassium purple, calcium brick red. It is this principle that is used in atomic absorption spectroscopy.
Decomposition of substances during pyrolysis. The method is used for preparations of iodine, arsenic, mercury. Of the currently used, the reaction of basic bismuth nitrate is most characteristic, which decomposes when heated to form nitrogen oxides:
Identification of organoelement medicinal substances.
Qualitative elemental analysis is used to identify compounds containing arsenic, sulfur, bismuth, mercury, phosphorus, and halogens in an organic molecule. Since the atoms of these elements are not ionized, preliminary mineralization is used to identify them, either by pyrolysis, or again by pyrolysis with sulfuric acid. Sulfur is determined by hydrogen sulfide reaction with potassium nitroprusside or lead salts. Iodine is also determined by pyrolysis by the release of elemental iodine. Of all these reactions, the identification of arsenic is of interest, not so much as a drug - they are practically not used, but as a method for monitoring impurities, but more on that later.
Testing the authenticity of organic medicinal substances. The chemical reactions used to test the authenticity of organic medicinal substances can be divided into three main groups:
1. General chemical reactions of organic compounds;
2. Reactions of formation of salts and complex compounds;
3. Reactions used to identify organic bases and their salts.
All these reactions are ultimately based on the principles of functional analysis, i.e. the reactive center of the molecule, which, when reacting, gives the appropriate response. Most often, this is a change in some properties of a substance: color, solubility, state of aggregation, etc.
Let us consider some examples of the use of chemical reactions for the identification of medicinal substances.
1. Reactions of nitration and nitrosation. They are used quite rarely, for example, to identify phenobarbital, phenacetin, dicain, although these drugs are almost never used in medical practice.
2. Diazotization and azo coupling reactions. These reactions are used to open primary amines. Diazotized amine combines with beta-naphthol to give a characteristic red or orange color.
3. Halogenation reactions. Used to open aliphatic double bonds - when bromine water is added, bromine is added to the double bond and the solution becomes colorless. A characteristic reaction of aniline and phenol is that when they are treated with bromine water, a tribromo derivative is formed, which precipitates.
4. Condensation reactions of carbonyl compounds. The reaction consists in the condensation of aldehydes and ketones with primary amines, hydroxylamine, hydrazines and semicarbazide:
The resulting azomethines (or Schiff bases) have a characteristic yellow color. The reaction is used to identify, for example, sulfonamides. The aldehyde used is 4-dimethylaminobenzaldehyde.
5. Oxidative condensation reactions. The process of oxidative cleavage and the formation of azomethine dye underlies ninhydrin reaction. This reaction is widely used for the discovery and photocolorimetric determination of α- and β-amino acids, in the presence of which an intense dark blue color appears. It is due to the formation of a substituted salt of diketohydrindylidene diketohydramine, a condensation product of excess ninhydrin and reduced ninhydrin with ammonia released during the oxidation of the test amino acid:
To open phenols, the reaction of the formation of triarylmethane dyes is used. So phenols interacting with formaldehyde form dyes. Similar reactions include the interaction of resorcinol with phthalic anhydride leading to the formation of a fluorescent dye - fluorescein.
Many other reactions are also used.
Of particular interest are reactions with the formation of salts and complexes. Inorganic salts of iron (III), copper (II), silver, cobalt, mercury (II) and others for testing the authenticity of organic compounds: carboxylic acids, including amino acids, derivatives of barbituric acid, phenols, sulfonamides, some alkaloids. The formation of salts and complex compounds occurs according to the general scheme:
R-COOH + MX = R-COOM + HX
The complex formation of amines proceeds similarly:
R-NH 2 + X = R-NH 2 X
One of the most common reagents in pharmaceutical analysis is a solution of iron (III) chloride. Interaction with phenols, it forms a colored solution of phenoxides, they are colored blue or purple. This reaction is used to discover phenol or resorcinol. However, meta-substituted phenols do not form colored compounds (thymol).
Copper salts form complex compounds with sulfonamides, cobalt salts with barbiturates. Many of these reactions are also used for quantitative determination.
Identification of organic bases and their salts. This group of methods is most often used in ready-made forms, especially in the study of solutions. So salts of organic amines, when alkalis are added, form a precipitate of a base (for example, a solution of papaverine hydrochloride) and vice versa, salts of organic acids, when a mineral acid is added, give a precipitate of an organic compound (for example, sodium salicylate). To identify organic bases and their salts, the so-called precipitation reagents are widely used. More than 200 precipitating reagents are known, which form water-insoluble simple or complex salts with organic compounds. The most commonly used solutions are given in the second volume of the SP 11th edition. An example is:
Scheibler's reagent - phosphotungstic acid;
Picric acid
Styphnic acid
Picramic acid
All these reagents are used for the precipitation of organic bases (for example, nitroxoline).
It should be noted that all these chemical reactions are used for the identification of medicinal substances not by themselves, but in combination with other methods, most often physicochemical, such as chromatography, spectroscopy. In general, it is necessary to pay attention to the fact that the problem of the authenticity of medicinal substances is a key one, because this fact determines the harmlessness, safety and effectiveness of the drug, so this indicator needs to be given great attention and it is not enough to confirm the authenticity of the substance by one method.
General requirements for purity tests.
Another equally important indicator of the quality of a medicinal product is purity. All medicinal products, regardless of the method of their preparation, are tested for purity. This determines the content of impurities in the preparation. It is conditionally possible to divide impurities into two groups: the first, impurities that have a pharmacological effect on the body; the second, impurities, indicating the degree of purification of the substance. The latter do not affect the quality of the drug, but in large quantities reduce its dose and, accordingly, reduce the activity of the drug. Therefore, all pharmacopoeias set certain limits for these impurities in drugs. Thus, the main criterion for the good quality of the drug is the absence of impurities, which is impossible by nature. The concept of the absence of impurities is associated with the detection limit of one method or another.
The physical and chemical properties of substances and their solutions give an approximate idea of the presence of impurities in drugs and regulate their suitability for use. Therefore, in order to assess good quality, along with the establishment of authenticity and determination of the quantitative content, a number of physical and chemical tests are carried out to confirm the degree of its purity:
Transparency and degree of turbidity carried out by comparison with a turbidity standard, and transparency is determined by comparison with a solvent.
Chromaticity. A change in the degree of color may be due to:
a) the presence of an extraneous colored impurity;
b) a chemical change in the substance itself (oxidation, interaction with Me +3 and +2, or other chemical processes that occur with the formation of colored products. For example:
Resorcinol turns yellow during storage due to oxidation under the action of atmospheric oxygen to form quinones. In the presence of, for example, iron salts, salicylic acid acquires a purple color due to the formation of iron salicylates.
Color assessment is carried out by comparing the main experience with color standards, and colorlessness is determined by comparison with a solvent.
Very often, a test is used to detect impurities of organic substances, based on their interaction with concentrated sulfuric acid, which can act as an oxidizing or dehydrating agent. As a result of such reactions, colored products are formed. The intensity of the resulting color should not exceed the corresponding color standard.
Determination of the degree of whiteness of powdered drugs– physical method, first included in GF X1. The degree of whiteness (hue) of solid medicinal substances can be assessed by various instrumental methods based on the spectral characteristics of the light reflected from the sample. To do this, reflectances are used when the sample is illuminated with white light obtained from a special source, with a spectral distribution or passed through light filters (with a transmission max of 614 nm (red) or 439 nm (blue)). You can also measure the reflectance of light passed through a green filter.
A more accurate assessment of the whiteness of medicinal substances can be carried out using reflection spectrophotometers. The value of the degree of whiteness and the degree of brightness are characteristics of the quality of whites and whites with shades of medicinal substances. Their permissible limits are regulated in private articles.
Determination of acidity, alkalinity, pH.
The change in these indicators is due to:
a) a change in the chemical structure of the medicinal substance itself:
b) the interaction of the drug with the container, for example, exceeding the permissible limits of alkalinity in a novocaine solution due to glass leaching;
c) absorption of gaseous products (CO 2 , NH 3) from the atmosphere.
Determination of the quality of medicines according to these indicators is carried out in several ways:
a) by changing the color of the indicator, for example, an admixture of mineral acids in boric acid is determined by methyl red, which does not change its color from the action of weak boric acid, but turns pink if it contains impurities of mineral acids.
b) titrimetric method - for example, to establish the permissible limit for the content of hydriodic acid formed during storage of a 10% alcohol solution of I 2, titration is carried out with alkali (no more than 0.3 ml of 0.1 mol / l NaOH by volume of the titrant). (Formaldehyde solution - titrated with alkali in the presence of phenolphthalein).
In some cases, the Global Fund sets the volume of titrant to determine the acidity or alkalinity.
Sometimes two titrated solutions are added in succession: first an acid and then an alkali.
c) by determining the pH value - for a number of drugs (and necessarily for all injection solutions) according to the NTD, it is envisaged to determine the pH value.
Techniques for preparing a substance in the study of acidity, alkalinity, pH
- Preparation of a solution of a certain concentration specified in the NTD (for substances soluble in water)
- For those insoluble in water, a suspension of a certain concentration is prepared and the acid-base properties of the filtrate are determined.
- For liquid preparations immiscible with water, agitation with water is carried out, then the aqueous layer is separated and its acid-base properties are determined.
- For insoluble solids and liquids, the determination can be carried out directly in suspension (ZnO)
The pH value approximately (up to 0.3 units) can be determined using indicator paper or a universal indicator.
The colorimetric method is based on the property of indicators to change their color at certain ranges of pH values. To perform the tests, buffer solutions with a constant concentration of hydrogen ions are used, differing from each other by a pH value of 0.2. To a series of such solutions and to the test solution add the same amount (2-3 drops) of the indicator. According to the coincidence of color with one of the buffer solutions, the pH value of the medium of the test solution is judged.
Determination of volatile substances and water.
Volatile substances can enter drugs either due to poor purification from solvents or intermediates, or as a result of the accumulation of degradation products. Water in the medicinal substance can be contained in the form of capillary, absorbed bound, chemically bound (hydrated and crystalline) or free.
Drying, distillation and titration with Fischer's solution are used to determine volatile substances and water.
drying method. The method is used to determine the loss in weight on drying. Losses can be due to the content of hygroscopic moisture and volatile substances in the substance. Dried in a bottle to constant weight at a certain temperature. More often, the substance is kept at a temperature of 100-105 ºС, but the conditions for drying and bringing to a constant mass may be different.
The determination of volatile substances can be carried out for some products by the method of ignition. The substance is heated in a crucible until the volatile substances are completely removed. then gradually increase the temperature until complete calcination at red heat. For example, the GPC regulates the determination of sodium carbonate impurities in the sodium bicarbonate medicinal substance by the calcination method. Sodium bicarbonate decomposes into sodium carbonate, carbon dioxide and water:
Theoretically, the weight loss is 36.9%. According to GPC, the loss in mass should be at least 36.6%. The difference between the theoretical and specified in the GPC mass loss determines the allowable limit of sodium carbonate impurities in the substance.
distillation method in GF 11 is called "Definition of water", it allows you to determine hygroscopic water. This method is based on the physical property of the vapors of two immiscible liquids. A mixture of water and an organic solvent distills at a lower temperature than either of these liquids. GPC1 recommends using toluene or xylene as the organic solvent. The water content in the test substance is determined by its volume in the receiver after the end of the distillation process.
Titration with Fisher's reagent. The method allows to determine the total content of both free and crystalline water in organic, inorganic substances, solvents. The advantage of this method is the speed of execution and selectivity with respect to water. Fisher's solution is a solution of sulfur dioxide, iodine and pyridine in methanol. Among the disadvantages of the method, in addition to the need for strict adherence to tightness, is the impossibility of determining water in the presence of substances that react with the components of the reagent.
Ash definition.
The ash content is due to mineral impurities that appear in organic substances in the process of obtaining auxiliary materials and equipment from the initial products (primarily metal cations), i.e. characterizes the presence of inorganic impurities in organic substances.
but) total ash- is determined by the results of combustion (ashing, mineralization) at high temperature, characterizes the sum of all inorganic substances-impurities.
Ash composition:
Carbonates: CaCO 3, Na 2 CO 3, K 2 CO 3, PbCO 3
Oxides: CaO, PbO
Sulphates: CaSO4
Chlorides: CaCl 2
Nitrates: NaNO 3
When obtaining medicines from plant materials, mineral impurities can be caused by dust pollution of plants, absorption of trace elements and inorganic compounds from soil, water, etc.
b) Ash insoluble in hydrochloric acid, obtained after treatment of total ash with dilute HCl. The chemical composition of the ash is heavy metal chlorides (AgCl, HgCl 2, Hg 2 Cl 2), i.e. highly toxic impurities.
in) sulfate ash- Sulphated ash is determined in assessing the good quality of many organic substances. Characterizes impurities Mn + n in a stable sulfate form. The resulting sulfate ash (Fe 3 (SO 4) 2, PbSO 4, CaSO 4) is used for the subsequent determination of heavy metal impurities.
Impurities of inorganic ions - C1 -, SO 4 -2, NH 4 +, Ca +2, Fe +3 (+2) , Pv +2, As +3 (+5)
Impurities:
a) impurities of a toxic nature (an admixture of CN - in iodine),
b) having an antagonistic effect (Na and K, Mg and Ca)
The absence of impurities that are not allowed in the medicinal substance is determined by a negative reaction with the appropriate reagents. Comparison in this case is carried out with a part of the solution, to which all reagents are added, except for the main one that opens this impurity (control experiment). A positive reaction indicates the presence of an impurity and the poor quality of the drug.
Permissible impurities - impurities that do not affect the pharmacological effect and the content of which is allowed in small quantities established by the NTD.
To establish the permissible limit for the content of ion impurities in medicines, reference solutions are used that contain the corresponding ion in a certain concentration.
Some medicinal substances are tested for the presence of impurities by titration, for example, the determination of the impurity of norsulfazole in the drug fthalazol. The admixture of norsulfazole in phthalazole is determined quantitatively by nitritometrically. Titration of 1 g of phthalazole should consume no more than 0.2 ml of 0.1 mol/l NaNO 2 .
General requirements for reactions that are used in tests for acceptable and unacceptable impurities:
1. sensitivity,
2. specificity,
3. reproducibility of the reaction used.
The results of reactions proceeding with the formation of colored products are observed in reflected light on a dull white background, and white precipitates in the form of turbidity and opalescence are observed in transmitted light on a black background.
Instrumental methods for determining impurities.
With the development of analysis methods, the requirements for the purity of medicinal substances and dosage forms are constantly increasing. In modern pharmacopeias, along with the considered methods, various instrumental methods are used, based on the physicochemical, chemical and physical properties of substances. The use of UV and visible spectroscopy rarely gives positive results and this is due to the fact that the structure of impurities, especially organic drugs, as a rule. It is close to the structure of the drug itself, so the absorption spectra differ little, and the impurity concentration is usually ten times lower than that of the main substance, which makes differential analysis methods unsuitable and allows one to estimate the impurity only approximately, i.e. as it is commonly called semi-quantitatively. The results are somewhat better if one of the substances, especially the impurity, forms a complex compound, while the other does not, then the maxima of the spectra differ significantly and it is already possible to determine the impurities quantitatively.
In recent years, IR-Fourier instruments have appeared at enterprises that allow determining both the content of the main substance and impurities, especially water, without destroying the sample, but their use is constrained by the high cost of instruments and the lack of standardized analysis methods.
Excellent impurity results are possible when the impurity fluoresces under UV light. The accuracy of such assays is very high, as is their sensitivity.
Wide application for testing for purity and quantitative determination of impurities both in medicinal substances (substances) and in dosage forms, which, perhaps, is no less important, because. many impurities are formed during the storage of drugs, obtained by chromatographic methods: HPLC, TLC, GLC.
These methods make it possible to determine impurities quantitatively, and each of the impurities individually, in contrast to other methods. The methods of HPLC and GLC chromatography will be discussed in detail in a lecture by prof. Myagkikh V.I. We will focus only on thin layer chromatography. The method of thin layer chromatography was discovered by the Russian scientist Tsvet and at the beginning existed as chromatography on paper. Thin layer chromatography (TLC) is based on the difference in the speeds of movement of the components of the analyzed mixture in a flat thin layer of the sorbent when the solvent (eluent) moves through it. Sorbents are silica gel, alumina, cellulose. Polyamide, eluents - organic solvents of different polarity or their mixtures with each other and sometimes with solutions of acids or alkalis and salts. The separation mechanism is due to the distribution coefficients between the sorbent and the liquid phase of the substance under study, which in turn is associated with many, including the chemical and physicochemical properties of the substances.
In TLC, the surface of an aluminum or glass plate is covered with a sorbent suspension, dried in air, and activated to remove traces of solvent (moisture). In practice, commercially manufactured plates with a fixed layer of sorbent are usually used. Drops of the analyzed solution with a volume of 1-10 μl are applied to the sorbent layer. The edge of the plate is immersed in the solvent. The experiment is carried out in a special chamber - a glass vessel, closed with a lid. The solvent moves through the layer under the action of capillary forces. Simultaneous separation of several different mixtures is possible. To increase the separation efficiency, multiple elution is used either in the perpendicular direction with the same or a different eluent.
After the completion of the process, the plate is dried in air and the position of the chromatographic zones of the components is set in various ways, for example, by irradiation with UV radiation, by spraying with coloring reagents, and kept in iodine vapor. On the resulting distribution pattern (chromatogram), the chromatographic zones of the mixture components are arranged in the form of spots in accordance with their sorbability in the given system.
The position of the chromatographic zones on the chromatogram is characterized by the value of R f . which is equal to the ratio of the path l i traversed by the i-th component from the starting point to the path Vп R f = l i / l.
The value of R f depends on the coefficient of distribution (adsorption) K і and the ratio of the volumes of the mobile (V p) and stationary (V n) phases.
Separation in TLC is affected by a number of factors: the composition and properties of the eluent, the nature, fineness and porosity of the sorbent, temperature, humidity, the size and thickness of the sorbent layer, and the dimensions of the chamber. Standardization of experimental conditions allows setting R f with a relative standard deviation of 0.03.
Identification of the components of the mixture is carried out by the values of R f . The quantitative determination of substances in the zones can be carried out directly on the sorbent layer by the area of the chromatographic zone, the fluorescence intensity of the component or its combination with a suitable reagent, by radiochemical methods. Automatic scanning devices are also used to measure the absorption, transmission, reflection of light, or radioactivity of chromatographic zones. The separated zones can be removed from the plate together with the sorbent layer, the component can be desorbed into the solvent, and the solution can be analyzed spectrophotometrically. Using TLC, substances can be determined in quantities from 10 -9 to 10 -6; the error of determination is not less than 5-10%.