Modern methods of drug analysis. Research work "analysis of drugs". A medicinal substance is pure if further purification does not change its pharmacological activity, chemical stability, physical characteristics
Currently, for the quantitative determination of medicinal substances in regulatory documentation (GF CHYY), classical (titrimetric) methods of analysis are quite widely used, but in this case the determination is not carried out based on the pharmacologically active part of the molecule.
Nitrometry is a titrimetric analysis method in which a sodium nitrite solution is used as a titration reagent.
It is used for the quantitative determination of compounds containing a primary or secondary aromatic amino group, for the determination of hydrazines, as well as aromatic nitro compounds after preliminary reduction of the nitro group to an amino group. An exact weighed sample of the drug, specified in the private pharmacopoeial monograph, is dissolved in a mixture of 10 ml of water and 10 ml of hydrochloric acid, diluted 8.3%. Add water to a total volume of 80 ml, 1 g of potassium bromide and, with constant stirring, titrate with 0.1 M sodium nitrite solution. At the beginning of the titration, add sodium nitrite solution at a rate of 2 ml/min, and at the end (0.5 ml before the equivalent amount) - 0.05 ml/min.
Titration is carried out at a solution temperature of 15-20°C, but in some cases cooling to 0-5°C is required.
The equivalence point is determined by electrometric methods (potentiometric titration, amperometric titration) or using internal indicators.
In potentiometric titration, a platinum electrode is used as an indicator electrode, while silver chloride or saturated calomel electrodes are used as reference electrodes.
A potential difference of 0.3-0.4 V is applied to the electrodes, unless otherwise indicated in a private pharmacopoeial monograph.
As internal indicators, use tropeolin 00 (4 drops of solution), tropeolin 00 mixed with methylene blue (4 drops of tropeolin 00 solution and 2 drops of methylene blue solution), neutral red (2 drops at the beginning and 2 drops at the end of titration).
Titration with tropeolin 00 is carried out until the color changes from red to yellow, with a mixture of tropeolin 00 with methylene blue - from red-violet to blue, with neutral red - from red-violet to blue. The dwell time at the end of the titration with neutral red is increased to 2 minutes. At the same time, a control experiment is carried out.
Nitrometry is used to determine: chloramphenicol, novocaine hydrochloride, paracetamol, sulfadimethoxine. The determination is based on the aromatic amino group.
The method of non-aqueous titration determines arbidol, articaine hydrochloride, atenolol, acyclovir, diazolin, diphenhydramine, droperidol, drotaverine hydrochloride, isoniazid, ketamine hydrochloride, clotrimazole, clonidine hydrochloride, codeine, codeine phosphate, caffeine, caffeine anhydrous, metronidazole , diclofenac sodium, nicotinamide, nitrazepam , papaverine hydrochloride, pyridoxine hydrochloride, piroxicam, fenpiverinium bromide, chloropyramine hydrochloride, verapamil hydrochloride, haloperidol, gliclazide, diazepam, itraconazole, clemastine fumarate, meloxicam, meldonium, metformin hydrochloride, sodium cromoglycate, ti amine chloride, tinidazole, thioridazine, thioridazine hydrochloride, phenazepam . This method is used to quantify more than half of the medicinal substances included in the Global Fund ChYY. The disadvantage of this method is that the decomposition products of drugs, which have basic properties, can also be titrated with perchloric acid along with undecomposed drugs.
Quantitative determination of analgin according to GF ChYY is carried out using the iodometric method. About 0.15 g (exactly weighed) of the substance is placed in a dry flask, 20 ml of 96% alcohol, 5 ml of 0.01 M hydrochloric acid solution are added and immediately titrated with 0.1 M iodine solution with stirring until a yellow color appears, which does not disappear in for 30 s. . The method is based on the oxidation of sulfur plus 4 to sulfur plus 6. The disadvantage of the method is that the determination is not carried out based on the pharmacologically active part of the molecule (1-phenyl-2,3-dimethyl-4-methylamino pyrazolone-5).
The alkalimetry method is used to determine acetylsalicylic acid, glutamic acid, doxazosin mesylate, methyluracil, naproxen, nicotinic acid, pitofenone hydrochloride, theophylline, furosemide - the equivalence point is established using an indicator. Bromhexine hydrochloride, lidocaine hydrochloride, lisinopril, ranitidine hydrochloride - with potentiometric termination. Standardization of these substances is carried out mainly according to HCl, which is not a pharmacologically active substance.
HPLC method GF ChYY recommends using for the determination of guaifenesin, carbamazepine, ketorolac, riboxin, simvastatin, ondansetron hydrochloride. The determination is carried out based on the pharmacologically active part of the drug molecule.
The spectrophotometric method is used to determine hydrocortisone acetate, spironolactone, and furazolidone. The method is not selective enough, since the decomposition products and the substance under study may have the same light absorption maxima.
At the present stage of development of pharmaceutical chemistry, physicochemical methods of analysis have a number of advantages over classical ones, since they are based on the use of both physical and chemical properties of medicinal substances and in most cases are characterized by rapidity, selectivity, high sensitivity, the possibility of unification and automation.
The GLC method is universal, highly sensitive, and reliable. This method for the qualitative and quantitative determination of dimexide ointment 50% was used by M.V. Gavrilin, E.V. Kompantseva and others.
A.G. Vitenberg, in the course of studying chlorinated tap water, found that the content of impurities of volatile halogenated hydrocarbons does not remain constant and increases as the water remains in the water supply system. This indicates the incompleteness of the chemical transformations of humic substances after chlorination of water. Existing certified methods based on headspace gas chromatographic analysis do not take this feature into account and provide for the determination of only free halogenated hydrocarbons. A comparative assessment of official methods was carried out, and sources of errors exceeding acceptable values were identified. Ways have been proposed to optimize all stages of analysis to create methods that provide a minimum of error and reliable information on the content of volatile halogenated hydrocarbons in tap and waste waters.
Gas chromatography was used to determine amphetamines, barbiturates, benzodiazepines, and opiates in urine using high-temperature solid-phase microextraction of drugs.
Ion chromatography was used by Siang De-Wen to determine anion exchangers in drinking water. The method turned out to be simple, fast and accurate (all anions are detected simultaneously with a standard deviation of ? 3%, regeneration 99.7% and 102%). The analysis lasted 15 minutes.
A number of authors have calculated: the differences in the gas chromatographic retention indices of the chlorination products of aliphatic ketones and the starting carbonyl compounds are constant. Their numerical values depend on the number and position of chlorine atoms in the molecule. We have developed a variant of additive schemes for assessing retention indices for the identification of chlorinated carbonyl compounds. I.G. Zenkevich established the order of chromatographic elution of diastomer b-b"-dichloro-K-alkanes (K?2).
I.V. Gruzdyev and co-authors studied 2- and 4-chloroaniline, 2,4- and 2,6-dichloroaniline, 2,4,5- and 2,4,6-trichloroaniline and unsubstituted aniline, developed methods for determining their trace amounts in drinking water, including the preparation of bromo derivatives, liquid extraction with toluene, as well as for the determination of diphenhydramine hydrochloride and its base in the presence of decomposition products.
V.G. Amelin et al used gas chromatography with a time-of-flight mass spectrometry detector to identify and determine pesticides and polycyclic aromatic hydrocarbons (46 ingredients) in water and food.
Potapova T.V., Shcheglova N.V. When studying the equilibrium reactions of the formation of cyclohexadiaminetetraacetate, ethylenediaminetetraacetate, diethylenetriaminepentaacetate complexes of some metals, the method of ion exchange chromatography was used.
Using analytical systems (liquid chromatography, mass spectrometry), Sony Weihua and a number of authors found that in processes involving OH radicals of active electrolytes, pharmaceutical drugs were almost completely destroyed.
Vitaliev A.A. and others have studied the conditions for isolating ketorolac and diclofenac from biological fluids. They proposed a method of extraction with organic solvents at different pH values. The TLC method was used to identify the analytes.
The use of planar chromatography using the example of amino acids and amlodipine was demonstrated by Pakhomov V.P., Checha O.A. for the study and separation of optically active medicinal substances into individual stereoisomers with subsequent identification.
The method of capillary gas chromatography in combination with mass spectrophotometry showed that the extraction of steroids from the blood was the most complete (~100%).
Using recirculation HPLC, the scientists isolated eight noncytotoxic bacterial drug resistance modifications.
N.N. Dementieva, T.A. Zavrazhskaya used gas chromatographic methods for the analysis of various drugs in injection solutions and eye drops.
Liquid chromatography was used to determine hyperacin and pseudohyperacin in pharmaceutical preparations with fluorescence detection. The same method identified valproic acid in human serum, the detection limit was 700 mmol/l. The HPLC method was used to determine disodium cromoglycate in pharmaceuticals. Using this method, it was possible to detect 98.2-100.8% of the analyte added to the sample.
M.E. Evgeniev and his colleagues established the influence of the nature and polarity of the eluent, the content of the aqueous phase in a water-non-aqueous mixture and its pH on the mobility of 5,7-dinitrobenzofurazine derivatives of a number of aromatic amines under UV-HPLC conditions. The ZORBAX SB-C18 column has been developed to separate a mixture of six aromatic amines.
When developing methods for assessing the quality of novocaine, cyclometazidine, sydnocarb A.S. Kvach and co-authors used HPLC and microcolumn adsorption chromatography methods in combination with a photometric method of analysis, allowing for the quantitative determination of novocaine in the substance and liquid dosage forms by the pharmacologically active part of the molecule.
I.A. Kolychev, Z.A. Temerdashev, N.A. Frolov developed an HPLC method for the determination of twelve phenolic compounds in plant materials using reverse-phase HPLC with UV detection and eluent elution mode. We studied the influence of various separation factors of gallic, trans-ferulic, protocatechinic, trans-caffeic acids, quercetin, rutin, dihydroquercetin and epicatechin.
ON THE. Epstein used the HPLC method for the simultaneous determination of drug substances in suspensions. A number of authors have used this method to determine the simultaneous content of paroxetine, risperidone and 9-hydroxyrepiredone in human plasma (with coulometric detection. Using HPLC with a UV detector in column reloading mode, a method for the determination of clotrimazole and mometasone furate in a wide range of concentrations is described.
A.M. Martynov, E.V. Chuparina developed a non-destructive technique for X-ray fluorescent analysis of ions in plants using a spectrometer. It was found that reducing plant weight from 6 to 1 gram increases the sensitivity of element determination. Using this technique, the elemental composition of violets used in medicine was determined.
A.S. Saushkina, V.A. Belikov performed spectrophotometry to identify chloramphenicol in dosage forms. Using the UV spectrophotomery method, a method for the quantitative determination of paracetamol and mefenamic acid in tablets has been proposed. The optimal conditions for the spectrophotometric analysis of metazide, ftivazid, isoniazid, chloramphenicol and synthomycin were established based on the study of UV spectra. In the spectrophotometric determination of ketorolac, the relative error is ±1.67%.
IN AND. Vershinin and co-authors identified deviations from additivity of light-absorbing mixtures and predicted them using statistical models obtained in the course of a full factorial experiment. The models relate variations and composition of mixtures, allowing optimization of spectrophotometric analysis techniques.
Zh.A. Kormosh and his co-authors determined piroxicam based on the extraction of its ionic associate with polymethine dye using the SFM method. Maximum extraction with toluene is achieved at pH=8.0-12.0 of the aqueous phase. To control the quality of medicinal products containing piroxicam, an extraction-spectrophotometric determination method has been developed.
A promising method for studying a medicinal substance is extraction photometry. This method is characterized by high sensitivity due to the formation of reaction products with reagents, leading to the appearance of additional chromophores, increased conjugation, and also due to the concentration of reaction products in the organic phase. Sufficient accuracy, comparative ease of implementation and the ability to determine the active substance from the pharmacologically active part of the molecule are another advantage of extraction photometry.
E.Yu. Zharskaya, D.F. Nokhrin, T.P. Churin used extraction photometry to determine verapamil hydrochloride, mezapam by the pharmacologically active part of the molecule based on the reaction with the copper salicylate complex (CI).
N.T. Bubon et al. used bromocresol purple as a reagent for medicinal substances. Based on this reaction, extraction-photometric methods for the determination of fluoroacyzine and acephen in tablets were developed.
G.I. Lukyanchikova and colleagues used extraction photometry in the analysis of aceclidine, oxylidine for the pharmacologically active part of the molecule based on the reaction with bromothymol blue. A number of authors have used the extraction-photometric method for the quantitative determination of metamizil in a 0.25% injection solution.
Studying the influence of pH and temperature on the stability of aqueous solutions of antispasmodic, G.I. Oleshko developed an extraction-photometric method for its analysis of the pharmacologically active part of the molecule based on the complexation reaction with bromothallic acid.
A.A. Litvin and his co-authors developed an extraction-photometric method for analyzing novocaine in injection solutions and ointments and studied the possibility of using it in the study of drugs containing novocaine during storage.
T.A. Smolyanyuk proposed a method for the extraction-photometric determination of diphenhydramine hydrochloride using tropeolin 000-1, which allows it to be analyzed in the presence of impurities.
In practical pharmacy, photometry and turbidimetry are widely used. L.V. Kadzhonyan, I.A. Kondratenko was quantitatively determined by the photometric method by the pharmacologically active part of the molecule of diphenhydramine hydrochloride and trimecaine. V.A. Popkov and others used differential scanning colorimetry in pharmaceutical analysis for nicotinic acid, isoniazid, and ftivazid. A.I. Sichko used phototurbidimetry to quantify teturam. The disadvantage of photometric methods is that they do not always allow the determination of the active substance in the presence of destruction products.
The fluorimetric method has also been used for the quantitative determination of medicinal substances. V.M. Ivanov, O.A. Grigoriev, A.A. Khabarov used fluorescent analysis in quality control of medicinal products containing furocoumarins of the psoralen group and folic acid. Column chromatography is also widely used. D.E. Bodrina, S.K. Eremin, B.N. Izotov used a microcolumn on a Melichrome liquid chromatograph to determine benzodiazepines in biological objects.
Recently, the chromato-spectrophotometric method for the quantitative determination of a substance based on the pharmacologically active part of the molecule has become widespread. It combines the high sensitivity of ultraviolet spectroscopy and the separation power of thin layer chromatography. S.A. Valevko, M.V. Mishustina developed a method for the chromatographic-spectrophotometric determination of papaverine hydrochloride, and D.S. Lazaryan and E.V. Kompantseva used it to determine chlorpropamide in the presence of their breakdown products.
The spectrophotometric method does not always allow objective control of the quantitative content of the active component. This is due to the fact that breakdown products sometimes have an absorption maximum in the same spectral region as drugs.
Mass spectrometry, atomic absorption spectrophotometry, NMR, IR, and PMR spectroscopy offer great opportunities in the analysis of a drug substance and its conformations. A gas chromatography-mass spectrometric method was used to identify diphenhydramine hydrochloride. It was established that the drug substance contains four impurities: benzophenone, 9-methylene fluorene, 9-fluorenyl dimethyl aminoethyl ether and diphenyl methyl ether. The diphenhydramine content was 96.80%.
A method for the determination of atropine in belladonna extracts using atmospheric pressure chemical ionization mass spectrometry is described. Terbutamine was used as an internal standard. L.V. Adeishvili and co-authors studied the spectra of diphenhydramine hydrochloride and mebedrol, and proposed their use for drug identification.
V.S. Kartashov used the NMR method to identify drugs derived from quinoline and isoquinoline. The characteristic signals in the NMR spectra of drugs allow their reliable identification using a personal computer.
High magnetic field strength NMR spectroscopy was used to quantify propranolol.
T.S. Chmilenko, E.A. Galimbievskaya, F.A. Chmilenko showed that the interaction of phenol red with polyhexamethylene guanidinium chloride produces an ionic associate and several forms of aggregates, the composition of which was determined by spectrophotometric, turbidimetric, refractometric and conductometric methods. A redistribution of absorption bands occurs, extreme points are observed that correspond to areas of maximum accumulation of formed aggregates. A method has been developed for determining PHMG in the Biopag-D disinfectant using extreme points.
Non-aqueous solvents have become widely used in modern pharmaceutical analysis. If previously the main solvent in the analysis was water, now various non-aqueous solvents (glacial or anhydrous acetic acid, acetic anhydride, dimethylformamide, dioxane, etc.) are simultaneously used, which make it possible to change the strength of basicity and acidity of the analyzed substances. The micromethod has been developed, in particular the droplet method of analysis, convenient for use in in-pharmacy quality control of medicines.
In recent years, research methods have been widely developed in which a combination of various methods is used in the analysis of medicinal substances. For example, gas chromatography-mass spectrometry is a combination of chromatography and mass spectrometry. Physics, quantum chemistry, and mathematics are increasingly penetrating modern pharmaceutical analysis.
The analysis of any medicinal substance or raw material must begin with an external examination, paying attention to the color, smell, shape of crystals, containers, packaging, and color of glass. After an external examination of the object of analysis, an average sample is taken for analysis in accordance with the requirements of the State Fund X (p. 853).
Methods for studying medicinal substances are divided into physical, chemical, physicochemical, and biological.
Physical methods of analysis involve studying 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, boiling. The corresponding methods are described in the Global Fund X. (p. 756-776).
Chemical research methods are based on chemical reactions. These include: determination of ash content, reaction of the medium (pH), characteristic numerical indicators of oils and fats (acid number, iodine number, saponification number, etc.).
For the purpose of identifying medicinal substances, only those reactions are used that are accompanied by a visible external effect, for example, a change in the color of the solution, the release of gases, the precipitation or dissolution of precipitation, etc.
Chemical research methods also include gravimetric and volumetric methods of quantitative analysis adopted in analytical chemistry (neutralization method, precipitation method, redox methods, etc.). In recent years, pharmaceutical analysis has included such chemical research methods as titration in non-aqueous media and complexometry.
Qualitative and quantitative analysis of organic medicinal substances is usually carried out according to the nature of the functional groups in their molecules.
Physicochemical methods are used to study 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 conductometric analysis, the electrical conductivity of solutions is measured, etc.
Physicochemical methods include: optical (refractometry, polarimetry, emission and fluorescence analysis methods, photometry, including photocolorimetry and spectrophotometry, nephelometry, turbodimetry), electrochemical (potentiometric and polarographic methods), chromatographic methods.
Introduction
1.2 Errors possible during pharmaceutical analysis
1.3 General principles for testing the authenticity of medicinal substances
1.4 Sources and causes of poor quality of medicinal substances
1.5 General requirements for purity tests
1.6 Methods of pharmaceutical analysis and their classification
Chapter 2. Physical methods of analysis
2.1 Testing physical properties or measuring physical constants of medicinal substances
2.2 Setting the pH of the medium
2.3 Determination of transparency and turbidity of solutions
2.4 Estimation of chemical constants
Chapter 3. Chemical methods of analysis
3.1 Features of chemical methods of analysis
3.2 Gravimetric (weight) method
3.3 Titrimetric (volumetric) methods
3.4 Gasometric analysis
3.5 Quantitative elemental analysis
Chapter 4. Physico-chemical methods of analysis
4.1 Features of physicochemical methods of analysis
4.2 Optical methods
4.3 Absorption methods
4.4 Methods based on radiation emission
4.5 Methods based on the use of a magnetic field
4.6 Electrochemical methods
4.7 Separation methods
4.8 Thermal methods of analysis
Chapter 5. Biological methods of analysis1
5.1 Biological quality control of medicinal products
5.2 Microbiological control of medicinal products
List of used literature
Introduction
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 assessing the quality of the resulting drug substance, studying its stability, establishing expiration dates and standardizing the finished dosage form. Pharmaceutical analysis has its own specific features that distinguish it from other types of analysis. These features lie in the fact that substances of various chemical natures are subjected to analysis: inorganic, organoelement, radioactive, organic compounds from simple aliphatic to complex natural biologically active substances. The range of concentrations of the analyzed substances is extremely wide. The objects of pharmaceutical analysis are not only individual medicinal substances, but also mixtures containing different numbers of components. The number of medicines is increasing every year. This necessitates the development of new methods of analysis.
Methods for pharmaceutical analysis require systematic improvement due to the continuous increase in requirements for the quality of drugs, and the requirements for both the degree of purity of drugs and their quantitative content are growing. Therefore, it is necessary to widely use not only chemical, but also more sensitive physicochemical methods to assess the quality of drugs.
There are high demands on pharmaceutical analysis. It must be quite specific and sensitive, accurate in relation to the standards stipulated by the State Pharmacopoeia XI, VFS, FS and other scientific and technical documentation, carried out in short periods of time using minimal quantities of test drugs and reagents.
Pharmaceutical analysis, depending on the objectives, includes various forms of drug quality control: pharmacopoeial analysis, step-by-step control of drug production, analysis of individually manufactured dosage forms, express analysis in a pharmacy and biopharmaceutical analysis.
An integral part of pharmaceutical analysis is pharmacopoeial analysis. It is a set of methods for studying drugs and dosage forms set out in the State Pharmacopoeia or other regulatory and technical documentation (VFS, FS). Based on the results obtained during the pharmacopoeial analysis, a conclusion is made about the compliance of the medicinal product with the requirements of the Global Fund or other regulatory and technical documentation. If you deviate from these requirements, the medicine is not allowed for use.
A conclusion about the quality of a medicinal product can only be made based on the analysis of a sample (sample). The procedure for its selection is indicated either in a private article or in the general article of the Global Fund XI (issue 2). Sampling is carried out only from undamaged packaging units, sealed and packaged in accordance with the requirements of the normative and technical documentation. In this case, the requirements for precautionary measures for working with poisonous and narcotic drugs, as well as for the toxicity, flammability, explosion hazard, hygroscopicity and other properties of drugs must be strictly observed. To test for compliance with the requirements of the normative and technical documentation, multi-stage sampling is carried out. The number of stages is determined by the type of packaging. At the last stage (after control by appearance), a sample is taken in the amount necessary for four complete physical and chemical analyzes (if the sample is taken for regulatory organizations, then for six such analyses).
From the Angro packaging, spot samples are taken, taken in equal quantities from the top, middle and bottom layers of each packaging unit. After establishing homogeneity, all these samples are mixed. Bulk and viscous drugs are taken with a sampler made of inert material. Liquid drugs are thoroughly mixed before sampling. If this is difficult to do, then point samples are taken from different layers. The selection of samples of finished medicinal products is carried out in accordance with the requirements of private articles or control instructions approved by the Ministry of Health of the Russian Federation.
Performing a pharmacopoeial analysis makes it possible to establish the authenticity of the drug, its purity, and determine the quantitative content of the pharmacologically active substance or ingredients included in the dosage form. Although each of these stages has its own specific purpose, they cannot be viewed in isolation. They are interconnected and mutually complement each other. For example, melting point, solubility, pH of an aqueous solution, etc. are criteria for both the authenticity and purity of the medicinal substance.
Chapter 1. Basic principles of pharmaceutical analysis
1.1 Pharmaceutical analysis criteria
At various stages of pharmaceutical analysis, depending on the tasks set, criteria such as selectivity, sensitivity, accuracy, time spent on performing the analysis, and the amount of the analyzed drug (dosage form) are used.
The selectivity of the method is very important when analyzing mixtures of substances, since it makes it possible to obtain the true values of each of the components. Only selective analytical techniques make it possible to determine the content of the main component in the presence of decomposition products and other impurities.
Requirements for the accuracy and sensitivity of pharmaceutical analysis depend on the object and purpose of the study. When testing the degree of purity of a drug, methods are used that are highly sensitive, allowing one to establish the minimum content of impurities.
When performing step-by-step production control, as well as when conducting express analysis in a pharmacy, the time factor spent on performing the analysis plays an important role. To do this, choose methods that allow analysis to be carried out in the shortest possible time intervals and at the same time with sufficient accuracy.
When quantitatively determining a drug substance, a method is used that is distinguished by selectivity and high accuracy. The sensitivity of the method is neglected, given the possibility of performing the analysis with a large sample of the drug.
A measure of the sensitivity of a reaction is the detection limit. It means the lowest content at which, using this method, the presence of the analyte component can be detected with a given confidence probability. The term "detection limit" was introduced instead of such a concept as "opening minimum", it is also used instead of the term "sensitivity". The sensitivity of qualitative reactions is influenced by factors such as volumes of solutions of reacting components, concentrations of reagents, pH of the medium, temperature, duration experience. This should be taken into account when developing methods for qualitative pharmaceutical analysis. To establish the sensitivity of reactions, the absorption indicator (specific or molar) is increasingly used, determined by the spectrophotometric method. In chemical analysis, sensitivity is determined by the value of the detection limit of a given reaction. Physicochemical methods are distinguished by high sensitivity. analysis. The most highly sensitive are radiochemical and mass spectral methods, allowing the determination of 10 -8 -10 -9% of the analyte, polarographic and fluorimetric methods 10 -6 -10 -9%; the sensitivity of spectrophotometric methods is 10 -3 -10 -6%, potentiometric 10. -2%.
The term “analytical accuracy” simultaneously includes two concepts: reproducibility and correctness of the results obtained. Reproducibility characterizes the dispersion of test results compared to the average value. Correctness reflects the difference between the actual and found content of a substance. The accuracy of the analysis for each method is different and depends on many factors: calibration of measuring instruments, accuracy of weighing or measuring, experience of the analyst, etc. The accuracy of the analysis result cannot be higher than the accuracy of the least accurate measurement.
4.2 Optical methods
This group includes methods based on determining the refractive index of a light beam in a solution of a test substance (refractometry), measuring the interference of light (interferometry), and the ability of a substance solution to rotate the plane of a polarized beam (polarimetry).
Optical methods are increasingly used in the practice of intrapharmacy control due to the rapidity and minimal consumption of the analyzed drugs.
Refractometry is used to test the authenticity of medicinal substances that are liquids (nicotinic acid diethylamide, methyl salicylate, tocopherol acetate), and in intrapharmacy control - to analyze dosage forms, including double and triple mixtures. Volumetric refractometric analysis and refractometric analysis by the method of complete and incomplete extraction are also used.
Various variants of methods for analyzing drugs, titrated solutions, and distilled water using the interferometric method have been developed.
Polarimetry is used to test the authenticity of medicinal substances whose molecules contain an asymmetric carbon atom. Among them, most of the drugs are from the groups of alkaloids, hormones, vitamins, antibiotics, and terpenes.
Analytical chemistry and pharmaceutical analysis use X-ray powder refractometry, spectropolarimetric analysis, laser interferometry, rotational dispersion and circular dichroism.
In addition to the indicated optical methods for the identification of individual medicinal substances in pharmaceutical and toxicological analysis, chemical microscopy does not lose its importance. The use of electron microscopy is promising, especially in phytochemical analysis. Unlike optical microscopy, the object is exposed to a beam of high-energy electrons. The image formed by the scattered electrons is observed on a fluorescent screen.
One of the promising rapid physical methods is radiographic analysis. It allows you to identify drugs in crystalline form and distinguish their polymorphic state. Various types of microscopy and methods such as Auger spectrometry, photoacoustic spectroscopy, computed tomography, radioactivity measurements, etc. can also be used to analyze crystalline medicinal substances.
An effective non-destructive method is reflectance infrared spectroscopy, which is used to determine impurities of various decomposition products and water, as well as in the analysis of multicomponent mixtures.
4.3 Absorption methods
Absorption methods are based on the properties of substances to absorb light in various regions of the spectrum.
Atomic absorption spectrophotometry is based on the use of ultraviolet or visible radiation of a resonant frequency. Absorption of radiation is caused by the transition of electrons from the outer orbitals of atoms to higher energy orbitals. Objects that absorb radiation are gaseous atoms, as well as some organic substances. The essence of determinations by atomic absorption spectrometry is that resonant radiation from a hollow cathode lamp passes through the flame in which the analyzed sample solution is sprayed. This radiation hits the entrance slit of the monochromator, and only the resonance line of the element under test is distinguished from the spectrum. The photoelectric method measures the decrease in the intensity of the resonance line, which occurs as a result of its absorption by atoms of the element being determined. The concentration is calculated using an equation that reflects its dependence on the attenuation of the radiation intensity of the light source, the length of the absorbing layer and the light absorption coefficient at the center of the absorption line. The method is highly selective and sensitive.
The absorption of resonance lines is measured on atomic absorption spectrophotometers such as “Spektr-1”, “Saturn”, etc. The accuracy of determinations does not exceed 4%, the detection limit reaches 0.001 μg/ml. This indicates the high sensitivity of the method. It is increasingly used to assess the purity of drugs, in particular the determination of minimal heavy metal impurities. The use of atomic absorption spectrophotometry for the analysis of multivitamin preparations, amino acids, barbiturates, some antibiotics, alkaloids, halogen-containing drugs, and mercury-containing compounds is promising.
It is also possible to use X-ray absorption spectroscopy in pharmacy, based on the absorption of X-ray radiation by atoms.
Ultraviolet spectrophotometry is the simplest and most widely used absorption analysis method in pharmacy. It is used at all stages of pharmaceutical analysis of medicinal products (testing of authenticity, purity, quantitative determination). A large number of methods have been developed for qualitative and quantitative analysis of dosage forms using ultraviolet spectrophotometry. For identification, atlases of the spectra of medicinal substances can be used, systematizing information about the nature of the spectral curves and the values of specific absorption indices.
There are various options for using the UV spectrophotometry method for identification. When testing for authenticity, medicinal substances are identified by position maximum light absorption. More often, pharmacopoeial monographs provide the positions of the maximum (or minimum) and the corresponding values of optical densities. Sometimes a method is used that is based on calculating the ratio of optical densities at two wavelengths (they usually correspond to two maxima or a maximum and a minimum of light absorption). A number of medicinal substances are also identified by the specific absorption rate of the solution.
It is very promising for the identification of medicinal substances to use such optical characteristics as the position of the absorption band on the wavelength scale, the frequency at the absorption maximum, the value of the peak and integral intensity, the half-width and asymmetry of the bands, and the oscillator strength. These parameters make the identification of substances more reliable than establishing the wavelength of maximum light absorption and the specific absorption index. These constants, which make it possible to characterize the presence of a relationship between the UV spectrum and the structure of the molecule, were established and used to assess the quality of medicinal substances containing an oxygen heteroatom in the molecule (V.P. Buryak).
An objective choice of optimal conditions for quantitative spectrophotometric analysis can only be carried out by a preliminary study of ionization constants, the influence of the nature of solvents, pH of the medium and other factors on the nature of the absorption spectrum.
The NTD provides various methods of using UV spectrophotometry for the quantitative determination of medicinal substances, such as vitamins (retinol acetate, rutin, cyanocobalamin), steroid hormones (cortisone acetate, prednisone, pregnin, testosterone propionate), antibiotics (sodium salts of oxacillin and methicillin, phenoxymethylpencillin, chloramphenicol stearate, griseofulvin). The solvents commonly used for spectrophotometric measurements are water or ethanol. Calculation of concentration is carried out in various ways: according to a standard, specific absorption rate or calibration curve.
It is advisable to combine quantitative spectrophotometric analysis with identification by UV spectrum. In this case, a solution prepared from one sample can be used for both of these tests. Most often, in spectrophotometric determinations, a method is used that is based on comparing the optical densities of the analyzed and standard solutions. Certain analytical conditions require medicinal substances capable of forming acid-base forms depending on the pH of the environment. In such cases, it is necessary to first select conditions under which the substance in solution will be completely in one of these forms.
To reduce the relative error of photometric analysis, in particular to reduce systematic error, the use of standard samples of medicinal substances is very promising. Considering the difficulty of obtaining and high cost, they can be replaced by standards prepared from available inorganic compounds (potassium dichromate, potassium chromate).
In SP XI, the scope of application of UV spectrophotometry has been expanded. The method is recommended for the analysis of multicomponent systems, as well as for the analysis of medicinal substances that themselves do not absorb light in the ultraviolet and visible regions of the spectrum, but can be converted into light-absorbing compounds using various chemical reactions.
Differential methods make it possible to expand the scope of photometry in pharmaceutical analysis. They make it possible to increase its objectivity and accuracy, as well as analyze high concentrations of substances. In addition, these methods can analyze multicomponent mixtures without prior separation.
The method of differential spectrophotometry and photocolorimetry is included in the State Fund XI, issue. 1 (p. 40). Its essence lies in measuring the light absorption of the analyzed solution relative to a reference solution containing a certain amount of the test substance. This leads to a change in the working area of the instrument scale and a reduction in the relative error of the analysis to 0.5-1%, i.e. the same as for titrimetric methods. Good results were obtained when neutral filters with known optical density were used instead of reference solutions; included in the set of spectrophotometers and photocolorimeters (V.G. Belikov).
The differential method has found application not only in spectrophotometry and photocolorimetry, but also in phototurbidimetry, photonephelometry, and interferometry. Differential methods can be extended to other physicochemical methods. Methods of chemical differential analysis based on the use of such chemical influences on the state of the drug substance in solution, such as changing the pH of the environment, changing the solvent, changing the temperature, the influence of electric, magnetic, ultrasonic fields, etc., also have great prospects for the analysis of drugs.
One of the variants of differential spectrophotometry, the ?E-method, opens up wide possibilities in quantitative spectrophotometric analysis. It is based on the transformation of the analyte into a tautomeric (or other) form that differs in the nature of light absorption.
New opportunities in the field of identification and quantification of organic substances are opened by the use of derivative UV spectrophotometry. The method is based on the isolation of individual bands from UV spectra, which are the sum of overlapping absorption bands or bands that do not have a clearly defined absorption maximum.
Derivative spectrophotometry makes it possible to identify medicinal substances or their mixtures that are similar in chemical structure. To increase the selectivity of qualitative spectrophotometric analysis, a method for constructing second derivatives of UV spectra is used. The second derivative can be calculated using numerical differentiation.
A unified method for obtaining derivatives from absorption spectra has been developed, which takes into account the peculiarities of the nature of the spectrum. It was shown that the second derivative has a resolution approximately 1.3 times greater compared to direct spectrophotometry. This made it possible to use this method for the identification of caffeine, theobromine, theophylline, papaverine hydrochloride and dibazole in dosage forms. Second and fourth derivatives are more effective in quantitative analysis compared to titrimetric methods. The duration of determination is reduced by 3-4 times. Determination of these drugs in mixtures turned out to be possible regardless of the nature of absorption of accompanying substances or with a significant reduction in the influence of their light absorption. This eliminates labor-intensive operations for separating mixtures.
The use of a combined polynomial in spectrophotometric analysis made it possible to exclude the influence of nonlinear background and to develop methods for the quantitative determination of a number of drugs in dosage forms that do not require complex calculations of analysis results. The combined polynomial has been successfully used in the study of processes occurring during the storage of medicinal substances and in chemical toxicological studies, as it allows reducing the influence of light-absorbing impurities (E.N. Vergeichik).
Raman spectroscopy (RSS) differs from other spectroscopic methods in sensitivity, a large selection of solvents and temperature ranges. The presence of a domestic Raman spectrometer of the DSF-24 brand makes it possible to use this method not only for establishing the chemical structure, but also in pharmaceutical analysis.
The method of spectrophotometric titration has not yet received proper development in the practice of pharmaceutical analysis. This method makes it possible to perform indicator-free titration of multicomponent mixtures with similar values rK based on a sequential change in optical density during the titration process depending on the volume of added titrant.
The photocolorimetric method is widely used in pharmaceutical analysis. Quantitative determination by this method, in contrast to UV photometry, is carried out in the visible region of the spectrum. The substance being determined is converted into a colored compound using some reagent, and then the color intensity of the solution is measured using a photocolorimeter. The accuracy of the determination depends on the choice of optimal conditions for the chemical reaction.
Very widely used in photometric analysis are methods for the analysis of drugs derived from primary aromatic amines, based on the use of diazotization and azo coupling reactions. Widely used as azo component N-(1-naphthyl)-ethylenediamine. The reaction of formation of azo dyes underlies the photometric determination of many drugs derived from phenols.
The photocolorimetric method is included in the technical documentation for the quantitative determination of a number of nitro derivatives (nitroglycerin, furadonin, furazolidone), as well as vitamin preparations (riboflavin, folic acid) and cardiac glycosides (celanide). Numerous methods have been developed for the photocolorimetric determination of drugs in dosage forms. Various modifications of photocolorimetry and methods for calculating concentration in photocolorimetric analysis are known.
Polycarbonyl compounds such as bindon (anhydro-bis-indanedione-1,3), alloxan (tetraoxohexa-hydropyrimidine), sodium salt of 2-carbethoxyindanedione-1,3 and some of its derivatives have proven promising for use as color reagents in photometric analysis. Optimal conditions have been established and unified methods have been developed for the identification and spectrophotometric determination in the visible region of medicinal substances containing a primary aromatic or aliphatic amino group, a sulfonyl urea residue, or being nitrogen-containing organic bases and their salts (V.V. Petrenko).
Widely used in photocolorimetry are coloring reactions based on the formation of polymethine dyes, which are obtained by breaking the pyridine or furan rings or by certain condensation reactions with primary aromatic amines (A.S. Beisenbekov).
For identification and spectrophotometric determination in the visible region of the spectrum of medicinal substances, derivatives of aromatic amines, thiols, thioamides and other mercapto compounds are used as color reagents N-chlorine-, N-benzenesulfonyl- and N-benzenesulfonyl-2-chloro-1,4-benzoquinone imine.
One of the options for unifying methods of photometric analysis is based on indirect determination from the residue of sodium nitrite introduced into the reaction mixture in the form of a standard solution taken in excess. The excess nitrite is then determined photometrically by diazotization reaction using ethacridine lactate. This technique is used for indirect photometric determination of nitrogen-containing medicinal substances by nitrite ion formed as a result of their transformations (hydrolysis, thermal decomposition). The unified methodology allows for quality control of more than 30 such medicinal substances in numerous dosage forms (P.N. Ivakhnenko).
Phototurbidimetry and photonephelometry are methods that have great potential, but are still of limited use in pharmaceutical analysis. Based on the measurement of light absorbed (turbidimetry) or scattered (nephelometry) by suspended particles of the analyte. Every year the methods are improved. For example, chronophototurbidimetry is recommended in the analysis of medicinal substances. The essence of the method is to establish changes in light extinction over time. The use of thermonephelometry, based on establishing the dependence of the concentration of a substance on the temperature at which cloudiness of the drug solution occurs, is also described.
Systematic studies in the field of phototurbidimetry, chronophototurbidimetry and phototurbidimetric titration have shown the possibility of using phosphotungstic acid for the quantitative determination of nitrogen-containing drugs. In phototurbidimetric analysis, both direct and differential methods were used, as well as automatic phototurbidimetric titration and chronophototurbidimetric determination of two-component dosage forms (A.I. Sichko).
Infrared (IR) spectroscopy is characterized by broad information content, which makes it possible to objectively assess the authenticity and quantitative determination of medicinal substances. The IR spectrum unambiguously characterizes the entire structure of the molecule. Differences in chemical structure change the nature of the IR spectrum. Important advantages of IR spectrophotometry are specificity, speed of analysis, high sensitivity, objectivity of the results obtained, and the ability to analyze a substance in a crystalline state.
IR spectra are measured using usually suspensions of medicinal substances in liquid paraffin, the intrinsic absorption of which does not interfere with the identification of the analyzed compound. To establish authenticity, as a rule, the so-called “fingerprint” region (650–1500 cm -1), located in the frequency range from 650 to 1800 cm -1, as well as stretching vibrations of chemical bonds are used
С=0, С=С, С=N
The State Fund XI recommends two methods for establishing the authenticity of medicinal substances using IR spectra. One of them is based on a comparison of the IR spectra of the test substance and its standard sample. The spectra must be taken under identical conditions, i.e. samples must be in the same state of aggregation, in the same concentration, the registration rate must be the same, etc. The second method is to compare the IR spectrum of the test substance with its standard spectrum. In this case, it is necessary to strictly comply with the conditions provided for the removal of the standard spectrum, given in the relevant technical documentation (GF, VFS, FS). Complete coincidence of absorption bands indicates the identity of the substances. However, polymorphic modifications can give different IR spectra. In this case, to confirm the identity, it is necessary to recrystallize the test substances from the same solvent and take the spectra again.
The intensity of absorption can also serve as confirmation of the authenticity of the drug substance. For this purpose, constants such as the absorption index or the value of the integral absorption intensity, equal to the area that the curve in the absorption spectrum encircles, are used.
The possibility of using IR spectroscopy to identify a large group of medicinal substances containing carbonyl groups in the molecule has been established. Identity is determined by characteristic absorption bands in the following areas: 1720-1760, 1424-1418, 950-00 cm -1 for carboxylic acids; 1596-1582, 1430-1400, 1630-1612, 1528-1518 cm -1 for amino acids; 1690--1670, 1615--1580 cm -1 for amides; 1770--1670 cm -1 for barbituric acid derivatives; 1384--1370, 1742--1740, 1050 cm -1 for terpenoids; 1680--1540, 1380--1278 cm -1 for tetracycline antibiotics; 3580-3100, 3050-2870, 1742-1630, 903-390 cm -1 for steroids (A.F. Mynka).
The IR spectroscopy method is included in the pharmacopoeias of many foreign countries and in MF III, where it is used to identify more than 40 medicinal substances. Using IR spectrophotometry, it is possible to carry out not only a quantitative assessment of medicinal substances, but also the study of such chemical transformations as dissociation, solvolysis, metabolism, polymorphism, etc.
4.4 Methods based on radiation emission
This group of methods includes flame photometry, fluorescent and radiochemical methods.
SP XI includes emission and flame spectrometry for the purpose of qualitative and quantitative determination of chemical elements and their impurities in medicinal substances. The radiation intensity of the spectral lines of the tested elements is measured using domestic flame photometers PFL-1, PFM, PAZH-1. Photocells connected to digital and printing devices serve as recording systems. The accuracy of determinations using emission, as well as atomic absorption, flame spectrometry methods is within 1-4%, the detection limit can reach 0.001 μg/ml.
Quantitative determination of elements by flame emission spectrometry (flame photometry) is based on establishing the relationship between the intensity of the spectral line and the concentration of the element in solution. The essence of the test is to spray the analyzed solution into an aerosol in the burner flame. Under the influence of the flame temperature, the evaporation of the solvent and solid particles from the aerosol droplets, the dissociation of molecules, the excitation of atoms and the appearance of their characteristic radiation occur. Using a light filter or monochromator, the radiation of the element being analyzed is separated from others and, when it hits a photocell, it causes a photocurrent, which is measured using a galvanometer or potentiometer.
Flame photometry was used for the quantitative analysis of sodium-, potassium- and calcium-containing drugs in dosage forms. Based on a study of the effect on the emission of determined cations, organic anions, auxiliary and accompanying components, methods for the quantitative determination of sodium bicarbonate, sodium salicylate, PAS-sodium, bilignost, hexenal, sodium nucleinate, calcium chloride and gluconate, bepaska, etc. were developed. Methods for simultaneous determination of two salts with different cations in dosage forms, for example, potassium iodide - sodium bicarbonate, calcium chloride - potassium bromide, potassium iodide - sodium salicylate, etc.
Luminescent methods are based on the measurement of secondary radiation resulting from the action of light on the analyte. These include fluorescent methods, chemiluminescence, X-ray fluorescence, etc.
Fluorescent methods are based on the ability of substances to fluoresce in UV light. This ability is due to the structure of either the organic compounds themselves or the products of their dissociation, solvolysis and other transformations caused by the action of various reagents.
Organic compounds with a symmetrical molecular structure, which contain conjugated bonds, nitro-, nitroso-, azo-, amido-, carboxyl or carbonyl groups, usually have fluorescent properties. The intensity of fluorescence depends on the chemical structure and concentration of the substance, as well as other factors.
Fluorimetry can be used for both qualitative and quantitative analysis. Quantitative analysis is performed using spectrofluorimeters. The principle of their operation is that light from a mercury-quartz lamp, through a primary light filter and a condenser, falls onto a cuvette with a solution of the test substance. The concentration is calculated using the scale of standard samples of a fluorescent substance of known concentration.
Unified methods have been developed for the quantitative spectrofluorimetric determination of p-aminobenzenesulfamide derivatives (streptocide, sulfacyl sodium, sulgin, urosulfan, etc.) and p-aminobenzoic acid (anesthesin, novocaine, novocainamide). Aqueous alkaline solutions of sulfonamides have the greatest fluorescence at pH 6-8 and 10-12. In addition, sulfonamides containing an unsubstituted primary aromatic amino group in the molecule, after heating with o-phthalaldehyde in the presence of sulfuric acid, acquire intense fluorescence in the region of 320-540 nm. In the same region, derivatives of barbituric acid (barbital, barbital sodium, phenobarbital, etaminal sodium) fluoresce in an alkaline environment (pH 12-13) with a fluorescence maximum at 400 nm. Highly sensitive and specific methods for the spectrofluorimetric determination of antibiotics have been proposed: tetracycline, oxytetracycline hydrochloride, streptomycin sulfate, passomycin, florimycin sulfate, griseofulvin and cardiac glycoside celanide (F.V. Babilev). Studies have been carried out on the fluorescence spectra of a number of drugs containing natural compounds: derivatives of coumarin, anthraquinone, flavonoids (V.P. Georgievsky).
Complex-forming groups have been identified in 120 medicinal substances, derivatives of hydroxybenzoic, hydroxynaphthoic, anthranilic acids, 8-hydroxyquinoline, oxypyridine, 3- and 5-hydroxyflavone, pteridine, etc. These groups are capable of forming fluorescent complexes with cations of magnesium, aluminum, boron, zinc, scandium when fluorescence is excited from 330 nm and above and emitted at wavelengths exceeding 400 nm. The research carried out made it possible to develop fluorimetric techniques for 85 drugs (A.A. Khabarov).
Along with derivative spectrophotometry in pharmaceutical analysis, the possibility of using derivative spectrofluorimetry has been substantiated. Spectra are recorded on an MPF-4 fluorescent spectrophotometer with a thermostatic cell, and derivatives are found by similar differentiation using a computer. The method was used to develop simple, accurate and highly sensitive methods for the quantitative determination of pyridoxine and ephedrine hydrochlorides in dosage forms in the presence of decomposition products.
Prospects for use X-ray fluorescence for determining small amounts of impurities in drugs is due to high sensitivity and the ability to perform analysis without preliminary destruction of the substance. Method X-ray fluorescence spectrometry turned out to be promising for the quantitative analysis of substances containing heteroatoms such as iron, cobalt, bromine, silver, etc. in the molecule. The principle of the method is to compare the secondary X-ray radiation of the element in the analyzed and standard sample. X-ray fluorescence spectrometry is one of the methods that does not require preliminary destructive changes. The analysis is performed on a domestic spectrometer RS-5700. Analysis duration 15 min.
Chemiluminescence is a method that involves using the energy generated during chemical reactions.
This energy serves as a source of excitement. It is emitted during oxidation by some barbiturates (especially phenobarbital), aromatic acid hydrazides and other compounds. This creates great opportunities for using the method to determine very low concentrations of substances in biological material.
Radiochemical methods are increasingly used in pharmaceutical analysis. Radiometric analysis, based on the measurement of?- or?-radiation using spectrometers, is used (along with other parameters to assess the quality of pharmacopoeial radioactive drugs. Highly sensitive methods of analysis using radioactive isotopes (labeled atoms) are widely used in various fields of technology and especially in analytical chemistry To detect traces of impurities in substances, activation analysis is used; to determine difficult-to-separate components in mixtures, the isotope dilution method is also used. An original option for combining radioisotope and chromatographic methods is the study of diffusion-sedimentary chromatograms. layer of gelatin gel using radioactive tracers.
4.5 Methods based on the use of a magnetic field
The methods of NMR and PMR spectroscopy, as well as mass spectrometry, are distinguished by high specificity and sensitivity and are used for the analysis of multicomponent mixtures, including dosage forms, without their preliminary separation.
The NMR spectroscopy method is used to test the authenticity of medicinal substances, which can be confirmed either by a full set of spectral parameters characterizing the structure of a given compound, or by the most characteristic signals of the spectrum. Authenticity can also be established using a standard sample by adding a certain amount of it to the analyzed solution. Complete coincidence of the spectra of the analyzed substance and its mixture with the standard sample indicates their identity.
NMR spectra are recorded on spectrometers with operating frequencies of 60 MHz or more, using such basic characteristics of the spectra as chemical shift, resonance signal multiplicity, spin-spin interaction constant, and resonance signal area. The most extensive information about the molecular structure of the analyte is provided by 13 C and 1 H NMR spectra.
Reliable identification of preparations of gestagenic and estrogenic hormones, as well as their synthetic analogues: progesterone, pregnin, ethinyl estradiol, methyl estradiol, estradiol dipropionate, etc. - can be carried out by 1 H NMR spectroscopy in deuterated chloroform on an UN-90 spectrometer with an operating frequency of 90 MHz (internal standard - tetramethylsilane).
Systematic studies have made it possible to establish the possibility of using 13 C NMR spectroscopy for the identification of medicinal substances of 10-acyl derivatives of phenothiazine (chloracyzine, fluoroacyzine, ethmosine, ethacyzine), 1,4-benzodiazepine (chloro-, bromo- and nitro derivatives), etc. Using 1 H NMR spectroscopy and 13 C, identification and quantitative assessment of the main components and impurities in preparations and standard samples of natural and semi-synthetic antibiotics aminoglycosides, penicillins, cephalosporins, macrolides, etc. was carried out. This method was used to identify a number of vitamins under unified conditions: lipoic and ascorbic acids, lipamide, choline and methylmethionine sulfonium chlorides, retinol palmitate, calcium pantothenate, ergocalciferol. The 1H NMR spectroscopy method made it possible to reliably identify such natural compounds with a complex chemical structure as cardiac glycosides (digoxin, digitoxin, celanide, deslanoside, neriolin, cymarin, etc.). A computer was used to speed up the processing of spectral information. A number of identification techniques are included in the FS and VFS (V.S. Kartashov).
Quantitative determination of a drug substance can also be performed using NMR spectra. The relative error of quantitative determinations by the NMR method depends on the accuracy of measurements of the areas of resonant signals and is ±2-5%. When determining the relative content of a substance or its impurity, the areas of the resonance signals of the test substance and the standard sample are measured. The amount of the test substance is then calculated. To determine the absolute content of a drug or impurity, the analyzed samples are prepared quantitatively and an accurately weighed mass of the internal standard is added to the sample. After this, the spectrum is recorded, the signal areas of the analyte (impurity) and the internal standard are measured, and then the absolute content is calculated.
The development of pulsed Fourier spectroscopy technology and the use of computers have made it possible to sharply increase the sensitivity of the 13 C NMR method and extend it to the quantitative analysis of multicomponent mixtures of bioorganic compounds, including medicinal substances, without their preliminary separation.
The spectroscopic parameters of PMR spectra provide a whole range of diverse and highly selective information that can be used in pharmaceutical analysis. The conditions for recording spectra should be strictly observed, since the values of chemical shifts and other parameters are influenced by the type of solvent, temperature, pH of the solution, and concentration of the substance.
If a complete interpretation of PMR spectra is difficult, then only the characteristic signals are isolated, by which the test substance is identified. PMR spectroscopy is used to test the authenticity of many medicinal substances, including barbiturates, hormonal agents, antibiotics, etc.
Since the method provides information about the presence or absence of impurities in the main substance, PMR spectroscopy is of great practical importance for testing medicinal substances for purity. Differences in the values of certain constants allow one to draw a conclusion about the presence of impurities of decomposition products of the drug substance. The sensitivity of the method to impurities varies widely and depends on the spectrum of the main substance, the presence of certain groups containing protons in the molecules, and solubility in the corresponding solvents. The minimum impurity content that can be determined is usually 1-2%. Particularly valuable is the ability to detect isomer impurities, the presence of which cannot be confirmed by other methods. For example, an admixture of salicylic acid was found in acetylsalicylic acid, morphine in codeine, etc.
Quantitative analysis based on the use of PMR spectroscopy has advantages over other methods in that when analyzing multicomponent mixtures there is no need to isolate individual components to calibrate the device. Therefore, the method is widely applicable for the quantitative analysis of both individual medicinal substances and solutions, tablets, capsules, suspensions and other dosage forms containing one or more ingredients. The standard deviation does not exceed ±2.76%. Methods for analyzing tablets of furosemide, meprobamate, quinidine, prednisolone, etc. are described.
The range of applications of mass spectrometry in the analysis of medicinal substances for identification and quantitative analysis is expanding. The method is based on the ionization of molecules of organic compounds. It is highly informative and extremely sensitive. Mass spectrometry is used to determine antibiotics, vitamins, purine bases, steroids, amino acids and other drugs, as well as their metabolic products.
The use of lasers in analytical instruments significantly expands the practical application of UV and IR spectrophotometry, as well as fluorescence and mass spectroscopy, Raman spectroscopy, nephelometry and other methods. Laser excitation sources make it possible to increase the sensitivity of many analysis methods and reduce the duration of their implementation. Lasers are used in remote analysis as detectors in chromatography, bioanalytical chemistry, etc.
4.6 Electrochemical methods
This group of qualitative and quantitative analysis methods is based on electrochemical phenomena occurring in the medium under study and associated with changes in the chemical structure, physical properties or concentration of substances.
Potentiometry is a method based on measuring the equilibrium potentials that arise at the boundary between the test solution and the electrode immersed in it. SP XI includes a method of potentiometric titration, which consists in establishing the equivalent volume of titrant by measuring the EMF of the indicator electrode and the reference electrode immersed in the analyzed solution. The direct potentiometry method is used to determine pH (pH-metry) and determine the concentration of individual ions. Potentiometric titration differs from indicator titration in the ability to analyze highly colored, colloidal and turbid solutions, as well as solutions containing oxidizing agents. In addition, several components in a mixture can be titrated sequentially in aqueous and non-aqueous media. The potentiometric method is used for titration based on reactions of neutralization, precipitation, complexation, oxidation - reduction. The reference electrode in all of these methods is calomel, silver chloride or glass (the latter is not used in the analysis by neutralization). The indicator electrode for acid-base titration is a glass electrode, for complexometric titration it is mercury or ion-selective, for the precipitation method it is silver, and for redox titration it is platinum.
The EMF that occurs during titration due to the potential difference between the indicator electrode and the reference electrode is measured using high-resistance pH meters. The titrant is added from a burette in equal volumes, constantly stirring the titrated liquid. Near the equivalence point, the titrant is added in increments of 0.1-0.05 ml. The value of the EMF at this point changes the most strongly, since the absolute value of the ratio of the change in EMF to the increment in the volume of the added titrant will be maximum. Titration results are presented either graphically, by establishing an equivalence point on the titration curve, or by calculation. Then the equivalent volume of the titrant is calculated using the formulas (see SP XI, issue 1, p. 121).
Amperometric titration with two indicator electrodes, or titration until the current stops, is based on the use of a pair of identical inert electrodes (platinum, gold) that are under low voltage. The method is most often used for nitrite and iodometric titration. The equivalence point is found by a sharp increase in the current passing through the cell (within 30 s) after adding the last portion of the reagent. This point can be established graphically by the dependence of the current on the volume of the added reagent, just as with potentiometric titration (SP XI, issue 1, p. 123). Methods for biamperometric titration of medicinal substances using nitritometry, precipitation and oxidation-reduction methods have also been developed.
Particularly promising is ionometry, which uses the relationship between the EMF of a galvanic network with an ion-selective electrode and the concentration of the analyzed ion in the electrode cell of the circuit. Determination of inorganic and organic (nitrogen-containing) medicinal substances using ion-selective electrodes differs from other methods in their high sensitivity, rapidity, good reproducibility of results, simple equipment, available reagents, suitability for automated monitoring and study of the mechanism of action of drugs. As an example, we can cite methods for the ionometric determination of potassium, sodium, halides and calcium-containing medicinal substances in tablets and in saline blood replacement fluids. Using domestic pH meters (pH-121, pH-673), an I-115 ionometer and potassium selective electrodes, potassium salts of various acids (orotic, aspartic, etc.) are determined.
Polarography is an analysis method based on measuring the current generated at a microelectrode during the electroreduction or electrooxidation of the analyte in solution. Electrolysis is carried out in a polarographic cell, which consists of an electrolyzer (vessel) and two electrodes. One of them is a mercury dripping microelectrode, and the other is a macroelectrode, which is either a layer of mercury on the electrolyzer or an external saturated calomel electrode. Polarographic analysis can be performed in an aqueous environment, in mixed solvents (water - ethanol, water - acetone), in non-aqueous media (ethanol, acetone, dimethylformamide, etc.). Under identical measurement conditions, a half-wave potential is used to identify a substance. Quantification is based on measuring the limiting diffuse current of the test drug substance (wave height). To determine the content, the method of calibration curves, the method of standard solutions and the method of additives are used (SP XI, issue 1, p. 154). Polarography is widely used in the analysis of inorganic substances, as well as alkaloids, vitamins, hormones, antibiotics, and cardiac glycosides. Due to their high sensitivity, modern methods are very promising: differential pulse polarography, oscillographic polarography, etc.
The possibilities of electrochemical methods in pharmaceutical analysis are far from exhausted. New variants of potentiometry are being developed: inversion currentless chronopotentiometry, direct potentiometry using a gas ammonium-selective electrode, etc. Research is expanding in the field of application in pharmaceutical analysis of methods such as conductometry, based on the study of the electrical conductivity of solutions of analytes; coulometry, which consists in measuring the amount of electricity spent on the electrochemical reduction or oxidation of the ions being determined.
Coulometry has a number of advantages over other physicochemical and chemical methods. Because this method is based on measuring the amount of electricity, it allows one to directly determine the mass of a substance rather than any property proportional to concentration. This is why coulometry eliminates the need to use not only standard but also titrated solutions. As for coulometric titration, it expands the scope of titrimetry through the use of various unstable electrogenerated titrants. The same electrochemical cell can be used to perform titrations using different types of chemical reactions. Thus, the neutralization method can determine acids and bases even in millimolar solutions with an error of no more than 0.5%.
The coulometric method is used to determine small quantities of anabolic steroids, local anesthetics and other medicinal substances. Tablet fillers do not interfere with the determination. The methods are distinguished by their simplicity, expressiveness, speed and sensitivity.
The method of dielectric measurements in the range of electromagnetic waves is widely used for express analysis in chemical technology, food industry and other fields. One of the promising areas is dielcometric monitoring of enzymes and other biological products. It allows for a quick, accurate, reagent-free assessment of parameters such as humidity, degree of homogeneity and purity of the drug. Dielcometric control is multi-parameter, the tested solutions can be opaque, and measurements can be performed in a non-contact manner with the results recorded on a computer.
4.7 Separation methods
Of the physicochemical separation methods in pharmaceutical analysis, chromatography, electrophoresis and extraction are mainly used.
Chromatographic methods for separating substances are based on their distribution between two phases: mobile and stationary. The mobile phase can be a liquid or gas, the stationary phase can be a solid or liquid adsorbed on a solid carrier. The relative speed of movement of particles along the separation path depends on their interaction with the stationary phase. This results in each substance traveling a certain length on the carrier. The ratio of the speed of movement of the substance to the speed of movement of the solvent is denoted by this value. This value is a constant of the substance for given separation conditions and is used for identification.
Chromatography makes it possible to most effectively carry out the selective distribution of the components of the analyzed sample. This is of significant importance for pharmaceutical analysis, in which the objects of study are usually mixtures of several substances.
According to the mechanism of the separation process, chromatographic methods are classified into ion exchange, adsorption, sedimentation, partition, and redox chromatography. According to the form of the process, column, capillary and plane chromatography can be distinguished. The latter can be done on paper and in a thin (fixed or unfixed) layer of sorbent. Chromatographic methods are also classified according to the state of aggregation of the analyzed substance. These include various methods of gas and liquid chromatography.
Adsorption chromatography is based on the selective adsorption of individual components from a solution of a mixture of substances. Adsorbents such as aluminum oxide, activated carbon, etc. serve as the stationary phase.
Ion exchange chromatography uses ion exchange processes occurring between the adsorbent and electrolyte ions in the analyzed solution. The stationary phase is cation exchange or anion exchange resins; the ions they contain are capable of being exchanged for similarly charged counterions.
Sediment chromatography is based on the difference in solubility of substances formed during the interaction of the components of the mixture being separated with the precipitant.
Partition chromatography consists in the distribution of mixture components between two immiscible liquid phases (mobile and stationary). The stationary phase is a carrier impregnated with a solvent, and the mobile phase is an organic solvent that is practically immiscible with the first solvent. When performing the process in a column, the mixture is divided into zones containing one component each. Partition chromatography can also be performed in a thin layer of sorbent (thin layer chromatography) and on chromatography paper (paper chromatography).
Before other separation methods in pharmaceutical analysis, ion exchange chromatography began to be used for the quantitative determination of drugs: salts of sulfuric, citric and other acids. In this case, ion exchange chromatography is combined with acid-base titration. Improvements in the method have made it possible to separate some hydrophilic organic compounds using reverse-phase ion-pair chromatography. It is possible to combine complexometry with the use of cation exchangers in Zn 2+ form for the analysis of amino derivatives in mixtures and alkaloids in extracts and tinctures. Thus, the combination of ion exchange chromatography with other methods expands its scope of application.
In 1975, a new version of chromatography was proposed, used for the determination of ions and called ion chromatography. To perform the analysis, columns measuring 25 X 0.4 cm are used. Two-column and single-column ion chromatography have been developed. The first is based on ion-exchange separation of ions on one column, followed by a decrease in the background signal of the eluent on the second column and conductometric detection, and the second (without suppression of the background signal of the eluent) is combined with photometric, atomic absorption and other methods of detecting the ions being determined.
Despite the limited number of works on the use of ion chromatography in pharmaceutical analysis, the promise of this method is obvious for the simultaneous determination of the anionic composition of multicomponent dosage forms and saline solutions for injection (containing sulfate, chloride, carbonate, and phosphate ions), for the quantitative determination of heteroelements in organic medicinal substances (containing halogens, sulfur, phosphorus, arsenic), to determine the level of contamination of water used in the pharmaceutical industry with various anions, to determine certain organic ions in dosage forms.
The advantages of ion chromatography are the high selectivity of determination of ions, the possibility of simultaneous determination of organic and inorganic ions, a low limit detected (up to 10 -3 and even 10 -6 μg/ml), small sample volume and ease of preparation, speed of analysis (in 20 min, separation of up to 10 ions is possible), simplicity of equipment, the possibility of combination with other analytical methods and expansion of the scope of chromatography in relation to objects that are similar in chemical structure and difficult to separate by TLC, GLC, HPLC.
The most widely used methods in pharmaceutical analysis are paper chromatography and thin layer chromatography.
In paper chromatography, the stationary phase is the surface of special chromatography paper. The distribution of substances occurs between the water located on the surface of the paper and the mobile phase. The latter is a system that includes several solvents.
In pharmaceutical analysis, when performing tests using paper chromatography, they are guided by the instructions of the State Fund XI, no. 1 (p. 98) and private pharmacopoeial monographs for the corresponding medicinal substances (dosage forms). When testing authenticity, the test substance and the corresponding standard sample are chromatographed on the same sheet of chromatographic paper. If both substances are identical, then the corresponding spots on the chromatograms have the same appearance and equal Rf values. If a mixture of the test substance and the standard sample is chromatographed, then if they are identical, only one spot should appear on the chromatogram. To exclude the influence of chromatography conditions on the obtained R f values, you can use a more objective value of R S , which is the ratio of the R f values of the test and standard samples.
When testing for purity, the presence of impurities is judged by the size and color intensity of the spots on the chromatogram. The impurity and the main substance must have different R f values. For a semi-quantitative determination of the impurity content, a chromatogram of the test substance taken in a certain amount and several chromatograms of a standard sample taken in precisely measured quantities are simultaneously obtained on one sheet of paper under the same conditions. Then the chromatograms of the test and standard samples are compared with each other. A conclusion about the amount of impurity is made from the size of the spots and their intensity.
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Pharmaceutical analysis (PA). It is the basis of pharmaceutical chemistry and has its own characteristics that distinguish it from other types of analysis. They consist in the fact that substances of various chemical natures are analyzed: inorganic, organoelement, radioactive, organic compounds from simple aliphatic to complex natural biologically active substances. The range of concentrations of the analyzed substances is extremely wide. The objects of pharmaceutical analysis are not only individual medicinal substances, but also mixtures containing different numbers of components.
The annual replenishment of the arsenal of drugs necessitates the development of new methods for their analysis. Methods for pharmaceutical analysis require systematic improvement due to the continuous increase in requirements both for the quality of medicines and for the quantitative content of biologically active substances in them. This is why high demands are placed on pharmaceutical analysis. It must be quite 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 minimal quantities of test drugs and reagents.
Depending on the tasks, pharmaceutical analysis includes various forms of quality control of medicines: pharmacopoeial analysis; step-by-step control of drug production; analysis of individually manufactured dosage forms; rapid analysis in a pharmacy and biopharmaceutical analysis. Its integral part is pharmacopoeial analysis, which is a set of methods for studying drugs and dosage forms set out in the State Pharmacopoeia or other scientific and technical documentation (FS, FSP, GOST). Based on the results obtained during the pharmacopoeial analysis, a conclusion is made about the compliance of the medicinal product with the requirements of the State Pharmacopoeia or other technical documentation. If you deviate from these requirements, the medicine is not allowed for use.
Chemical analysis of plant 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 set out in the relevant regulatory documentation for the type of raw material under study 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, marshmallow, ginseng, barberry roots, linden flowers, flax seeds, ergot sclerotia (for a total of 12 types of raw materials) .
Basically, qualitative reactions are carried out with extraction (extract) from medicinal plant materials.
Based on the properties of biologically active substances, they are extracted from raw materials with water, alcohol of varying concentrations or an organic solvent, less often with the addition of alkali or acid.
The aqueous extract is prepared from raw materials containing glycosides, polysaccharides, saponins, phenologlycosides, anthraglycosides, and tannins. Alkaloids are extracted from raw materials in the form of salts using acidified water.
A large group of biologically active substances (cardiac glycosides, coumarins, lignans, flavonoids) are extracted with ethyl and methyl alcohol of varying concentrations.
If the reaction is sufficiently specific and sensitive, then it is carried out with a crude extract from the raw material.
Such reactions include:
general alkaloid sedimentary reactions;
reactions with a solution of aluminum chloride on flavonoids (St. John's wort, knotweed, peppermint, etc.);
Synod test for flavonoids in immortelle flowers;
reaction with an alkali solution on anthracene derivatives (buckthorn bark, rhubarb roots, etc.);
reaction with a solution of ferroammonium alum on tannins (oak bark, serpentine rhizomes, bergenia, etc.).
Often the reaction is interfered with by accompanying substances (proteins, amines, sterols, chlorophyll). In this case, a purified extract is used (for example, from raw materials containing cardiac glycosides, coumarins, alkaloids, phenol glycosides, lignans).
The extraction is purified by precipitation of accompanying substances with a solution of lead acetate and sodium sulfate or using the method of changing solvents or the method of partition chromatography.
Microchemical reactions are usually carried out simultaneously with microscopic analysis, observing the results under a microscope:
for essential and fatty oils with Sudan III solution;
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 ferroammonium alum and the result of the reaction is studied under a microscope.
Histochemical reactions are reactions that can be used to detect certain compounds directly in the cells or structures where they are localized.
According to State Pharmacopoeia XI, histochemical reactions are carried out on mucus with a solution of ink in marshmallow roots and flax seeds.
Microsublimation- direct isolation from dry plant material of substances that easily sublime 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 the raw material with the naked eye or using a magnifying glass, and measure the particle sizes 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 is established only in non-poisonous plants. When studying the appearance, pay attention to the morphological characteristics of 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 in general and the characteristics characteristic of a particular plant that distinguish it from other plants.
Chemical analysis. Provides for carrying out 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 their locations in the plant. Sublimation is understood as the production from plant raw materials of substances that easily sublime when heated, followed by a qualitative reaction with the sublimate.
Luminescent analysis. This is a method for studying various objects (including biological ones), based on the observation of their luminescence. Luminescence is the glow of a gas, liquid or solid, caused not by the heating of the body, but by the non-thermal excitation of its atoms and molecules. Luminescent analysis is carried out to determine substances with luminescence in medicinal raw materials.
Quality control of organotherapeutic drugs. To check whether the quality of the iron meets the requirements of the standard, 5% of the boxes or packages are selected from each batch, 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 for 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 their 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 consumer’s request using an immunoreactive method using antiserum and immunoglobulins in a homogenized gland.
The quality of the mucous membrane (epithelium) of cattle tongues is checked by determining the pH value of the preservative 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 preservative medium with epithelium.
The quality of the vitreous body of frozen eyes of cattle, pigs, sheep and goats 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 hydrolysis products of hyaluronic acid, 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 acidic acetonated powder (AAP) obtained from the pituitary glands.
Determination of ACTH activity is based on its ability to cause a reduction of lymphoid tissue, in particular the thymus gland of rats. One unit of action of the drug is taken to be the daily dose of the drug that, when administered over five days, causes a decrease in the weight of the gland by 50±5%.
The quality of the parathyroid glands is determined histologically. On sections of the parathyroid glands, accumulations of epithelial cells with pronounced basophilic granularity are visible. On sections of the lymph glands, reticular tissue is visible (in the form of a homogeneous mass), surrounded by a dense connective membrane (capsule), from which clearly visible connective 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, lighter weight, increased shelf life, and ease of transportation.
Physical methods. 1.Method for determining vacuum. The essence of the method lies in the ability of high-frequency electric current at high voltage to cause a glow in gases, the nature of which varies depending on the degree of rarefaction of the air in the ampoule (bottle).
Sample selection. Sampling is carried out in accordance with the rules established in state standards for dry biological preparations.
Hardware and equipment. When carrying out the test, the following equipment is used: a “D’Arsenal” or “Tesla” type apparatus, a stand for ampoules, and a metal table.
Carrying out the test. Preparation for the test:
Before testing, check the appearance, tightness of the sealing of the vials, the presence of cracks, and sealing of the ampoules.
The device is kept for 10 minutes after switching on. The test ampoules are installed in a tripod, then an electrode is brought to them at a distance of 1 cm. When determining vacuum using a Tesla apparatus, one metal electrode of the device is grounded through a metal table on which the ampoules are laid out, and the other is brought to the ampoules being tested. Exposure is no more than 1 s.
Processing the results. The appearance of a glow inside the ampoules with a characteristic crackling sound indicates the presence of a vacuum in them.
The degree of air rarefaction 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 air rarefaction in the tested ampoules
2. Method for determining humidity. The essence of the method is to determine the decrease in the mass of a drug sample after drying it for 1 hour at a temperature of 105 °C.
Sample selection. For testing, the required number of ampoules (vials) is selected from different places in the packaging, taking into account the requirements for sample weight (in accordance with the standard).
When taking samples, check the tightness of the ampoules. For bottles with a lyophilized drug, the wall and bottom are checked for integrity, as well as the complete fit of the rolled-up cap and rubber stopper. If there are defects, the bottle is replaced with another. Each ampoule, sealed under vacuum, is checked for leaks before removing the drug from it.
Equipment, materials and reagents. When carrying out the test, use: laboratory scales, laboratory drying cabinet, mercury thermometers, desiccator, glass bottles, technical petroleum jelly, 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 bottles, the lower part of the control thermometer should be at the level of the bottles. The readings of the control thermometer are decisive for setting the temperature in the cabinet.
The scale must be installed on a stable table without vibration. The results of all weighings are recorded in grams accurate 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 lubricated with technical petroleum jelly.
For each analysis, three bottles of identical diameters and heights must be prepared.
Carrying out the test. To determine humidity, 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 placed in a drying cabinet on a shelf. The beginning of drying should be considered the time when the temperature reaches 105 °C according to the control thermometer. Drying time 60 min.
After drying is completed, the bottles are quickly closed with lids and transferred to a desiccator to cool to room temperature, after which the bottles are weighed to the fourth digit and recorded according to their shape.
3. Method for determining the amount of oxygen. Sample selection. Sampling is carried out in accordance with the rules established in state standards for dry biological preparations.
Equipment, materials and reagents. When carrying out the test, use: gas chromatograph brand LXM-8MD or other similar brands with a thermal conductivity detector and a gas chromatography 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 mesh, measuring magnifying glass, desiccator, porcelain mortar, metal ruler 30 cm long, molecular sieves - synthetic zeolite grade CaA, medical needle, medical rubber tube with an internal diameter of 4.2 mm, length 10 m, 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, sifted 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 meshes to room temperature.
The chromatographic column is installed vertically and filled with synthetic zeolite. The column is not topped up by 1 cm and is sealed with a mesh. The filled column is installed in the chromatograph thermostat and, without connecting to the detector, a flow of helium or nitrogen is passed through it for 3 hours at a temperature of 160...180 °C. The column is then connected to the detector and helium or nitrogen continues to flow through it until the zero line drift stops at maximum detector sensitivity.
The chromatograph is prepared for operation and turned on in accordance with the factory instructions.
Preparing a bottle with the drug for testing. To take a sample from a bottle with the drug, the gas pressure in the bottle is equalized with atmospheric pressure.
Preparing a medical syringe. First install a metal tube on the syringe rod and check the syringe for leaks. A medical syringe with a needle, tested and prepared for gas sampling, pierces the rubber tube through which helium comes out of the chromatograph reference column, and the helium is drawn in and released twice slowly with the syringe. The third time, by drawing helium into the syringe and placing it with the needle down, gas samples are taken from the bottle with the drug.
Carrying out the test. Two gas samples are taken from each bottle and sequentially introduced one after another with an interval of 3...4 minutes into the chromatograph evaporator. The sample is introduced into the evaporator by gently pressing the rod with your finger. 110... 120 s after introducing the sample, the recorder draws an oxygen peak on the chromatogram, and then a nitrogen peak.
Processing the results. The area of the oxygen and nitrogen peaks is calculated. To do this, measure the height and width of the oxygen and nitrogen peaks on a chromatograph using a 30 cm long metal ruler, a magnifying glass and a 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 using the formulas
SO 2 = h 1 *b 1; SN = 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 using the formula
X=SO 2 /(SO 2 +SN 2)
where SO 2, SN 2 are the peak areas of oxygen and nitrogen, mm 2.
The arithmetic mean of the results of determinations in three bottles of the drug is taken as the final test result.
The relative reduced error of the method with a confidence probability of P-0.95 should not exceed 10%.
Bacteriological method. Sterility control. The essence of the method is the microbiological assessment of the absence of growth of bacteria and fungi in seeding preparations on nutrient media.
Sample selection. From each series of drugs, samples are taken in the amount of 0.15% of bottles, but not less than five for liquid and 10 ampoules for dry drugs.
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 this, the dishes are thoroughly washed first with tap water, and then three times with distilled water, dried and sterilized.
Before sterilization, dishes are placed in metal cases. Sterilize the dishes in an autoclave at 0.15 MPa for 60 minutes.
Ready-made nutrient media, tested for growth properties, are poured into test tubes of 6...8 cm 3 (for determining anaerobes, 10...12 cm 3), and 50...60 cm 3 into bottles with a capacity of 100 cm 3.
Samples of dry biological preparations are pre-dissolved with a sterile solvent (isotonic sodium chloride solution, distilled water, etc.).
Carrying out the test. 1. Carrying out a sterility test using thioglycollate medium.
From each bottle of the drug, 1 cm 3 is inoculated into three test tubes containing thioglycollate 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 tube is kept for 7 days at a temperature of 37 °C and then subcultured with 0.5 cm 3 of one tube on slanted casein agar, casein nutrient broth, Sabouraud's medium and 1 cm 3 per casein nutrient broth under Vaseline oil with pieces of meat or liver.
Subcultures on casein agar and meat-extract broth are maintained for another 7 days at a temperature of 37 °C, and subcultures on Sabouraud's medium are maintained at a temperature of 21 °C.
When testing drug samples, the sterility of the media is monitored: three tubes with each medium are kept in a thermostat for 14 days at 37 °C, with Sabouraud’s medium - at a temperature of 21 °C.
2. Conducting a sterility test without thioglycollate medium.
Each sample of the drug is inoculated onto Sabouraud's liquid medium, meat-extract agar and meat-extract broth - three tubes each; on Wednesday Tarozzi - two test tubes and two bottles.
To identify aerobes, 0.5 cm 3 of seed material is sown in one test tube and 1...2 cm 3 in one bottle, and to identify 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 reseeding is done (except for sowing on meat peptone agar). Subculture on the same media. Leave for 7 days (15 days for anaerobes). Carry out sterility control.
Evaluation of results. The results of primary and repeated inoculations are taken into account by macroscopic, and in the case of microbial growth, microscopic examination of all inoculations, taken into account 14 days after the initial inoculation on a thioglycollate medium and 7 days after the initial inoculation without a thioglycollate medium. The medium is considered sterile if growth is not observed in any of the inoculated tubes.
In cases of growth in at least one of the inoculated tubes, sterility control is repeated on the same number of samples and microscopy of the grown microbes is carried out. Smears are Gram stained to note morphology.
If there is no growth in repeated control, the drug is considered sterile. If there is growth in at least one of the tubes and the microflora is identical during the initial and repeated cultures, the drug is considered non-sterile.
If during the initial and repeated cultures different microflora are identified, and growth is detected only in separate test tubes, the samples are inoculated a third time.
If there is no 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 produced in factories, pharmaceutical factories (official medicines) and pharmacies (mainstream medicines). Control of finished dosage forms at pharmaceutical enterprises is carried out in accordance with the requirements of the NTD (State Pharmacopoeia, FS, FSP, GOST). In accordance with the requirements of these documents, dosage forms must be tested (V.D. Sokolov, 2003).
Tablets are tested for disintegration. If there are no other instructions in a private article, then tablets should disintegrate within 15 minutes, and coated tablets should disintegrate no more than 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 abrasion strength of the tablets must be at least 75%. The medicine contained in the tablet must dissolve in water by at least 75% within 45 minutes. 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 talc content.
Granules - determined by 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%. Testing the disintegration of granules from a 0.5 g sample is the same as for tablets. The disintegration time should not exceed 15 minutes. Determine moisture. To determine the content of the medicinal substance, take a sample of at least 10 ground granules.
Capsules - control average weight. The deviation of each capsule from it should not exceed ±10%. In the same way as with tablets, disintegration and solubility are monitored, and dosage uniformity is determined for capsules containing 0.05 g or less of the drug substance. Quantitative determination of medicinal substances is carried out using special methods, using for these purposes the contents of 20 to 60 capsules.
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 temperature. It should 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 (dissolution indicator). The dissolution time is determined at a temperature of (37±1) °C, which should not exceed 1 hour. Quantitative determination of medicinal substances is carried out using 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 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 determined, and in thick and dry extracts the moisture content is determined [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 compressed gas). Check the packaging for leaks. In dosed packages, the average weight of the drug in one dose is determined, the deviation in which is allowed no more than +20%. The percentage of contents released is determined by removing it from the container and then weighing it. Quantitative determination of a 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 of determining the particle size of drug substances in ointments. A microscope with an MOV-1 eyepiece micrometer is used.
Plasters. The composition, quality indicators, and 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 inclusions.
Injectable dosage forms. Injection medicinal solutions administered intravenously in large quantities require special attention. They use such characteristics as appearance, including the color and transparency of solutions, absence of mechanical impurities, pyrogen-freeness, sterility, volume of solution, amount of active substance in it, pH and isotonicity of blood plasma, packaging, labeling, filling volume of ampoules. The norms of permissible deviations are indicated in 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 depend on the drug, usually its value can range from 3.0 to 8.0. Each ampoule (bottle) indicates the name of the drug, its content (in percentage) or activity (in action units, ED), volume or weight, batch number, expiration date. All tests of injectable dosage forms are regulated by normative and technical documentation.
The analysis of homeopathic medicines is very difficult due to high dilutions of medicinal substances. If biologically active substances are contained in tinctures, essences, ointments and other forms in dilutions up to 2 C (C is a hundredth) or 0.0001, then their analysis and standardization are practically no different from quality control of dosage forms used in allopathic medicine. Medicines at a dilution of 2...3 C (10 -4 ...10 -6) are analyzed after special concentration techniques using evaporation, combustion of substances, followed by determination by one of the physicochemical methods, based on its resolution. With a dilution of more than 3 C (10 -6), it is sufficient to establish the authenticity of the drug contained in one 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 production stage, strictly controlling the technological process. Quality is controlled when ingredients are loaded and recorded in the loading report. Each ingredient is subjected to preliminary analysis. In all of these cases, chromatographic, photometric, fluorescent and other methods are used to analyze and standardize homeopathic medicines.
