UV visible spectroscopy. Spectroscopy in the UV and visible ranges
GENERAL PHARMACOPEIAN AUTHORIZATION
Instead of OFS GF X, OFS GF XI, OFS 42-0042-07 GF XII, part 1
The decrease in the intensity of monochromatic radiation passing through a homogeneous absorbing medium is quantitatively described by the Bouguer-Lambert-Beer law:
log 10 (1/T) = BUT= ε ∙ c∙ b,(1)
- T- transmission, the ratio of the intensity of the light flux passing through the substance to the intensity of the light flux incident on the substance; T= I/I 0 ;
- I is the intensity of the transmitted monochromatic radiation;
- I 0 is the intensity of the incident monochromatic radiation;
- ε is the molar absorption index;
- With is the molar concentration of a substance in solution;
- b
Value log 10 (1/T) is called optical density, denoted by the letter BUT and is a measurable quantity. In the absence of other physicochemical factors, the measured absorbance ( BUT) is proportional to the concentration of the substance in the solution ( With) and layer thickness ( b).
Value is the specific absorption rate, i.e. optical density of a solution of a substance with a concentration of 10 g/l (1 g/100 ml) in a cuvette with a layer thickness of 1 cm. The values and ε are related by the relation:
Mm. - molecular mass the substance under study.
Optical density measurement
Unless otherwise specified in the monograph, the optical density measurement is carried out at the specified wavelength using cuvettes with a layer thickness of 1 cm and at a temperature of (20 ± 1) °C compared with the same solvent or the same mixture of solvents in which the substance is dissolved . When measuring the optical density of a solution at a given wavelength, the optical density of the solvent cuvette, measured against air at the same wavelength, should not exceed 0.9 and preferably not less than 0.2.
The absorption spectrum is presented in such a way that the optical density or some function of it is given along the ordinate axis, and the wavelength or some function of the wavelength is given along the abscissa axis.
If only one wavelength is indicated in the monograph for the maximum absorption, this means that the obtained value of the maximum should not differ from the indicated one by more than ± 2 nm.
Devices
Spectrophotometers designed for measurements in the ultraviolet and visible regions of the spectrum consist of an optical system that emits monochromatic radiation in the range from 190 to 800 nm and ensures its passage through the sample, and a device for measuring optical density.
The main parts of these devices are: a radiation source, a dispersive device (prism or grating), a slit for isolating a wavelength band, sample cuvettes, an emitted energy detector, built-in amplifiers and measuring instruments.
Verification of the wavelength scale in the ultraviolet and visible region. The calibration accuracy of the instrument on the wavelength scale in the spectral series is checked according to those given in Table. 1 to the spectral lines of a hydrogen (Hβ) or deuterium (Dβ) discharge lamp, to the lines of mercury vapor (Hg) of a quartz-mercury arc lamp, as well as to the absorption maxima of a solution of holmium perchlorate (Ho) (the ready reagent for calibrating the spectrophotometer is a 4% solution of holmium oxide in 14.1% perchloric acid solution). The tolerance is ± 1 nm for ultraviolet and ± 3 nm for visible.
Table 1– Absorption maxima to check the wavelength scale
241.15 nm (HO) | 404.66 nm (Hg) |
253.7 nm (Hg) | 435.83 nm (Hg) |
287.15 nm (HO) | 486.0 nm (Dβ) |
302.25 nm (Hg) | 486.1 nm (Hβ) |
313.16 nm (Hg) | 536.3 nm (HO) |
334.15 nm (Hg) | 546.07 nm (Hg) |
361.5 nm (HO) | 576.96 nm (Hg) |
365.48 nm (Hg) | 579.07 nm (Hg) |
The wavelength scale can also be calibrated using suitable glass filters that have fixed absorption bands in the visible and ultraviolet regions, as well as standard glasses containing didymium (a mixture of praseodymium and neodymium) and glasses containing holmium.
Checking the optical density scale. To check the optical density scale, use standard inorganic glass filters or a solution of potassium dichromate at the wavelengths indicated in Table. 2, where for each wavelength the exact value of the specific absorption index and the allowable limits are given.
A solution of potassium dichromate to check the optical density scale at 235, 257, 313 and 350 nm is prepared as follows: from 57.0 to 63.0 mg (accurately weighed) of potassium dichromate, previously dried to constant weight at a temperature of 130 ° C, is dissolved in 0.005 M sulfuric acid solution and bring the volume of the solution with the same solvent to 1000 ml. To check the optical density at 430 nm, dissolve 57.0-63.0 mg (accurately weighed) of potassium dichromate in a 0.005 M solution of sulfuric acid and bring the volume of the solution to the mark with the same solvent.
Table 2 - Specific absorption rate of standards at different wavelengths
Wavelength, in nanometers | Specific indicatortakeovers | Permissible limits for |
235 | 124,5 | 122.9 to 126.2 |
257 | 144,5 | 142.8 to 146.2 |
313 | 48,6 | 47.0 to 50.3 |
350 | 107,3 | 105.6 to 109.0 |
430 | 15,9 | 15.7 to 16.1 |
Scattered light limit. Scattered light can be detected at a given wavelength using appropriate filters or solutions: for example, the absorbance of a 12 g/l solution of potassium chloride in a cuvette with a layer thickness of 1 cm increases sharply between 220 and 200 nm and should be greater than 2 at 198 nm at using water as a reference solution.
Resolution(for qualitative analysis). If there is an indication in the monograph, determine the resolution of the spectrophotometer as follows. Record the spectrum of a 0.02% (v/v) solution of toluene in hexane. Minimum permissible value the ratio of optical density at the absorption maximum at 269 nm to the optical density at the absorption minimum at 266 nm is indicated in the pharmacopoeial article.
Spectral slit width(for quantitative analysis). In the case of using a spectrophotometer with a variable width of the spectral slit at a selected wavelength, errors associated with the width of this slit are possible. To eliminate them, the slit width should be small compared to the half-width of the absorption band (width at half the optical density) and, at the same time, should be as large as possible to obtain a high intensity of the incident monochromatic radiation (I0). Thus, the width of the slit should be such that its further decrease does not change the value of the measured optical density.
Cuvettes. Permissible deviations in the layer thickness of the used cuvettes should be no more than ± 0.005 cm. The cuvettes intended for the test solution and the reference solution should have the same transmission (or optical density) when filled with the same solvent. Otherwise, this difference should be taken into account.
solvent requirements. For determinations made in the ultraviolet and visible regions, the sample of the analyte is dissolved in an appropriate solvent, which must be optically transparent in the wavelength range used. Many solvents are suitable for these wavelength ranges, including water, alcohols, chloroform, lower hydrocarbons, ethers, and dilute solutions of strong acids and bases.
Identification
Absorption spectrophotometry in the ultraviolet and visible regions of the spectrum is used to determine the authenticity medicines by:
— comparison of the absorption spectra of the test solution and the standard sample solution; in the indicated region of the spectrum, the positions of the maxima, minima, shoulders, and inflection points should coincide;
- indications of the positions of maxima, minima, shoulders and inflection points of the absorption spectrum of the test solution; the difference between the observed and indicated wavelengths at the absorption maxima and minima should not normally exceed ± 2 nm.
There are other options for use, specified in the pharmacopoeial articles.
quantitation
Determination of the concentration of substances by the spectrophotometric method is based on the use of the Bouguer-Lambert-Beer law:
FROM is the concentration of the substance in g/100 ml;
BUT is the optical density of the test solution;
is the specific absorption rate of a substance;
b is the optical path length or layer thickness, in centimeters.
In some cases, even when monochromatic radiation is used, deviations from the Bouguer-Lambert-Beer law can be observed, due to the processes of dissociation, association, and complex formation. Therefore, one should first check the linearity of the dependence of the optical density of the solution on the concentration in the analytical region. In the presence of deviations from the linear dependence, one should use not formula (3), but the experimentally found dependence.
Usually, the determination of the concentration by the spectrophotometric method is carried out using a standard sample. The concentration calculation is based on the equation:
FROM and FROM 0 are the concentrations of the test solution and the standard sample solution, respectively;
BUT and BUT 0 are the optical densities of the test solution and the standard sample solution, respectively.
The concentrations of the test solution and the standard solution should be close.
First, the optical density of the standard sample solution prepared as indicated in the Pharmacopoeia Monograph is measured, then the optical density of the test solution is measured. The second measurement is carried out immediately after the first, using the same cuvette, under the same experimental conditions.
The standard sample method is more accurate and reliable. The possibility of applying the value of the specific absorption index in each specific case should be justified. Generally, the method using the SAR value is applicable with tolerances of the analyte content of at least ±10% of the nominal content.
Multi-component spectrophotometric analysis
Multicomponent spectrophotometric analysis (analysis of mixtures) is used for the simultaneous quantitative determination of several components of drugs, each of which obeys the Bouguer-Lambert-Beer law.
Quantification in multicomponent spectrophotometric analysis is usually based on the use of the equation:
BUT i is the optical density of the test solution at i-th wavelength;
E ij - absorption rates (depending on the way the concentration is expressed) j-th component of the sample at i-th analytical wavelength;
c j - concentration j th component of the sample.
Appropriate methods of analysis and calculation formulas are indicated in pharmacopoeial monographs.
Derivative spectrophotometry
In derivative spectrophotometry, the original absorption spectra (zero order) are converted into the spectra of derivatives of the first, second and higher orders.
The spectrum of the first derivative is a plot of the gradient of the absorption curve (rate of change in absorbance versus wavelength, dA/dλ) on the wavelength.
The spectrum of the second derivative is a plot of the curvature of the absorption spectrum ( d 2 A/dλ 2) on the wavelength. The second derivative at any wavelength is related to the concentration as follows:
BUT is the optical density at wavelength λ;
is the specific absorption index at wavelength λ;
With is the concentration of the substance in the solution, in grams/100 ml;
l is the thickness of the layer, in centimeters.
Derived spectrophotometry can be used both for the purposes of identifying substances and for their quantitative determination in multicomponent mixtures, as well as in cases where there is background absorption caused by the presence of substances whose content is not regulated.
Devices
Use spectrophotometers that meet the above requirements and are equipped with an analog RC differentiating module or digital differentiator, or other means of obtaining spectrum derivatives, in accordance with the instructions for the instrument. Some methods for obtaining spectra of the second derivative lead to a shift in wavelengths relative to the original spectrum, which should be taken into account where necessary.
Resolution
If indicated in monographs, record the spectrum of the second derivative for a solution of 0.2 g/l toluene in methanol, using methanol as a reference solution. The spectrum should have a small negative extremum located between two large negative extrema at 261 nm and 268 nm, in accordance with Fig. 1. If there are no other indications in pharmacopoeial monographs, the A/B ratio must be at least 0.2.
Methodology
The analysis procedure is similar to that used in conventional spectrophotometry, but derivatives are used instead of optical densities. Prepare a solution of the test sample, set up the device in accordance with the manufacturer's instructions and calculate the amount of the analyte, as indicated in the monograph.
Picture 1– Spectrum of the second derivative of a solution of toluene (0.2 g/l) in methanol
Methods for the analysis of antibiotics
Activity set
Unit of action (U)
cardiac glycosides
vitamins
Under expiration date
Oxidation
30. Refractometry
Refractometry
Aldehyde group
1. + phenylhydrazine hydrochloride in the form of a hydrochloric acid solution - the formation of a yellow flocculent residue of phenylhydrazone.
2. formation of a Schiff base upon interaction with aromatic amines.
per tertiary nitrogen atom
1. with precipitation(general alkaloid) reagents: Wagner, Mayer, Dragendorff, picric acid solution, and also with potassium dichromate solution.
per phosphorus atom
1. Phosphate ions form a yellow precipitate of phosphorus molybdate with a solution of ammonium molybdate.
Quantification
Phenolic hydroxyl
1. + iron (III) chloride. Solutions (water, alcohol or acetone) acquire a green color.
2. Azo combination.
3. Aurine dye sample
Nitro group
1. After the hydrogenation of the nitro group in the nitroxoline molecule to an aromatic amino group, the reaction of diazotization and azo coupling with an alkaline solution of β-naphthol is performed. A red-orange color appears.
2. + diphenylamine in the presence of concentrated sulfuric acid (blue color).
3. + sodium hydroxide - acisols are formed (red-orange color).
Tertiary nitrogen atom
1. when heated in a solution of citric acid and acetic anhydride, a purple-red color appears.
2. with precipitation(general alkaloid) reagents: Wagner, Mayer, Dragendorff, picric acid solution, and also with potassium dichromate solution (yellow precipitate).
Nitroxalin forms colored intracomplex compounds with metal cations: magnesium, cadmium, copper (II), zinc, aluminum.
Quantification
Nitroxoline is determined by non-aqueous titration using acetic anhydride as a solvent and 0.1 M perchloric acid as a titrant. The determination of nitroxoline is performed in the presence of formic acid and malachite green indicator, and the determination of chlorquinaldol is carried out with a crystal violet indicator.
Ester group
1. hydroxam test
2. + sodium hydroxide
Phenolic hydroxyl
1. + iron (III) chloride and a,a-dipyridyl in a mixture of ethanol and benzene. A red color appears.
2. oxidation reactions accompanied by the formation of colored substances.
1 - when heated to 80 C with concentrated nitric acid, a red-orange color is formed.
2 - when potassium hexacyanoferrate (III) is added in an alkaline medium, a colored product is formed.
3 - salts of cerium (IV), iron (III), tocopherol is oxidized to o-, n-tocopherylquinone, the formation of which causes a yellow color.
This chemical reaction is used to quantification tocopherol acetate. The definition is based on acid hydrolysis (refluxing in the presence of sulfuric acid). Then the released tocopherol is titrated with cerium (IV) sulfate (diphenylamine indicator) until a blue-violet color appears.
Pteridine derivatives
Pteridine is a heterocyclic system consisting of two fused heterocycles of pyrimidine and pyrazine:
This group includes: Folic acid.
Folic acid is stored in a well-closed container, in a dry, dark place, as it is hygroscopic and decomposes under the influence of light. The decomposition process is especially fast in an acidic environment in solutions under the influence of ultraviolet radiation.
Requirements for storage conditions of various groups of drugs depend on their physical and chemical properties and the impact of various environmental factors. They are regulated by the "Instructions for the organization of storage in pharmacies of various groups of medicines and medical devices", approved by order of the Ministry of Health of the Russian Federation No. 377 of November 13, 1996.
Deposition method
A weighed portion of the analyte is dissolved in water or another solvent, and the element to be determined is precipitated with a reagent in the form of a poorly soluble compound. The precipitate obtained is filtered off, washed, dried, calcined and weighed. Knowing the mass of the sediment, calculate the content of the element to be determined in mass fractions or percentage of the sample taken.
besieged form is a compound in the form of which the component to be determined precipitates from solution.
Gravimetric (weight) form name the compound being weighed.
Selection method
It is based on the isolation of the analyte from the analyte and its precise weighing.
Stripping method
In this method, the analyte is isolated as a volatile compound by the action of an acid or high temperature.
Direct distillation (the component to be determined is isolated from the sample in the form of a gaseous product, captured and then its mass is determined).
· Indirect distillation (the mass of the gaseous product is determined by the difference between the masses of the analyzed component before and after heat treatment).
In the practice of pharmaceutical analysis, this method is widely used in determining the moisture content of drugs, plant materials.
Methods for the analysis of antibiotics
Activity set diffusion or turbidimetric methods. SP XI recommends the agar diffusion method for quantitative determination, which consists in comparing the effect of certain concentrations of the test and the standard sample of the antibiotic on the test microorganism.
Since the composition of the agar medium and the conditions for performing a biological test are the same, the size of the diffusion zone (in which the development of the test microorganism is inhibited by the antibiotic) depends only on the chemical nature of the antibiotic and its concentration.
Unit of action (U) is a measure of the biological activity of antibiotics. For ED take the minimum amount of antibiotic that suppresses the development of the test microorganism in a certain amount growth medium.
Accelerated microbiological methods include methods based on the suppression of changes in the pH of the nutrient medium during the growth of test microorganisms (the urease method).
cardiac glycosides - nitrogen-free compounds of plant origin, characterized by cardiotonic action. These drugs play an extremely important role in the treatment of patients with acute and chronic heart failure of any origin. When determining the activity of medicinal raw materials and many preparations of cardiac glycosides, biological standardization is used. Most often, the activity of cardiac glycosides is expressed in frog action units (ICE) and feline action units (CED). One ICE corresponds to the minimum dose of a standard drug in which it causes cardiac arrest in the majority of experimental frogs, cats, pigeons. So, the crushed powder of digitalis leaves according to activity corresponds to the following proportion: one gram of leaf powder is equal to 50-66 ICE or 10-13 KED. During storage, the activity of the leaves decreases.
vitamins are a group of substances of different chemical structure, necessary in small quantities for the normal functioning of the body. A number of vitamins are part of enzyme systems and are a kind of biological catalysts for chemical or photochemical processes occurring in a living cell (thiamine, riboflavin, pyridoxine, pantothenic acid, etc.).
For the qualitative and quantitative assessment of vitamins in natural sources, both biological and physicochemical methods are used. The principle of assessing biological activity is that animals (rats, pigeons, guinea pigs) are transferred to a diet containing proteins, fats, carbohydrates, mineral salts and all vitamins, except for the one under study. Then it is established how much of the test vitamin can cure or protect the animal from beriberi. In parallel, conduct a similar test with a standard drug.
The biological method for assessing the activity of vitamins is very laborious, its accuracy is relatively low. Therefore, physical, chemical and physico-chemical methods are commonly used to test for the authenticity and quantification of vitamins.
28. Stability and shelf life of drugs (influence of moisture, CO 2 , light, air oxygen, impurities).
Under expiration date medicines understand the period of time during which they must fully maintain their therapeutic activity, safety and, in terms of qualitative and quantitative characteristics, comply with the requirements of the Global Fund or FS (FSP), in accordance with which they were released and stored under the conditions provided for by these articles.
After the expiration date, the medicinal product cannot be used without quality control and a corresponding change in the established expiration date. There is a certain relationship between the concept of "shelf life", which has a temporary meaning, and the concept of "stability", which determines the quality of drugs (its stability).
Temperature - with an increase, the reaction rate increases; with a decrease (the activity of MgSO 4, CaCl 2, adrenaline solution decreases).
Light - the rate of decomposition increases; crystalline solids are more stable than solutions; color change with prolonged exposure; some substances retain their activity (containing iron, while their stability increases).
Moisture - reduces pharmacological activity; + and - affects LP; hygroscopicity.
Oxidation- a process that is one of the reasons for the decomposition of drugs. Some of them (derivatives of phenols) are oxidized, being in a crystalline state. The oxidation process is noticeably activated upon dissolution. Drugs exhibiting active reducing properties (aldehydes, hydrazides, phenothiazine derivatives, etc.) are especially easily oxidized.
The system of measures aimed at protecting the drug from oxidation is primarily reduced to reducing the influence of atmospheric oxygen or to the maximum removal of impurities that catalyze the oxidation process. Using oxidizing agents, it is possible to model the oxidation process. If we then compare the resulting oxidation products of the standard sample and the decomposition products of the drug, we can draw a conclusion about the mechanism of the oxidation process. This makes it possible to solve the problem of ways of stabilization, since the factors influencing the rate of the oxidation reaction will become known.
Methods to improve stability:
1) physical (solids - in tightly closed containers; suspensions - in a dry state; injections - in sealed ampoules);
2) chemical (oxidation, metals).
29. Pharmacokinetics and bioavailability.
Pharmacokinetics - a section of pharmacology about the absorption, distribution, deposition, metabolism and release of drugs.
Carrying out pharmacokinetic studies is possible only on the basis of the use of modern methods of biopharmaceutical analysis, which make it possible to trace the process of absorption and distribution of drugs in organs and tissues. They include elucidation of the influence of various biopharmaceutical factors on the therapeutic efficacy of drugs; study of their bioavailability and development of methods for its determination; creation of methods for the determination of drugs and their metabolites in biological fluids.
The pharmacokinetics of drugs are influenced by various factors: age, genetic, sex, body weight, nutrition, pregnancy, as well as various pathological processes, such as diseases of the liver, kidneys, cardiovascular system, gastrointestinal tract, endocrine, infectious and other diseases.
Bioavailability is the amount of unchanged substance that has reached the blood plasma, relative to the initial dose of the drug.
One of the main stages of any study of the bioavailability of drugs is the use of biopharmaceutical analysis to determine the concentration of a drug (metabolite) in biological fluids.
30. Refractometry
Refractometry is based on the dependence of the value of the refractive index of light on the concentration of the solution of the test substance. The refractive index also depends on the temperature, the wavelength of the light, the concentration of the substance, and the nature of the solvent. Refractometry is used to identify medicinal substances by molar refraction. For quantitative determination choose the interval of linear relationship between the concentration of the solution and the refractive index. In this interval, the concentration (x) is calculated by the formula: x = (n - n O) / F, where n is the refractive index of the substance solution; n O is the refractive index of the solvent; F is a factor equal to the increase in the refractive index with an increase in the concentration of a substance by 1% (set experimentally).
Refractometric determinations are performed on refractometers, at a stable temperature (20 ± 0.3 ° C) and the wavelength of the D line of the sodium spectrum (589.3 nm) in the refractive index range from 1.3 to 1.7. The device is adjusted according to reference liquids or purified water, for which n D 20 = 1.3330.
Spectrophotometry in the UV, visible, IR regions of the spectrum in assessing the quality of drugs.
Use spectrophotometric methods of analysis by absorption of substances monochromatic electromagnetic radiation.
Photometric methods of analysis are based on the use of the Bouguer-Lambert-Beer law:
In case of discrepancy with the law, first, using a standard solution, the dependence of optical density on concentration is established, and then a calibration graph is built, with the help of which calculations are performed.
Light Ranges:
Spectrophotometry in the UV and visible regions- 1 of the widely used physico-chemical methods in pharmaceutical analysis.
The analyzed drugs should have chromophoric groups in the structure of the molecule (conjugated bonds, aromatic nucleus, etc.), which determine various electronic transitions in molecules and the absorption of electromagnetic radiation.
The curve of dependence of the light absorption intensity on the wavelength (nm) is called the absorption spectrum of a substance and is its specific characteristic. Measurement of the absorption spectra of solutions of analyzed substances in the UV (190-380 nm) and visible (380-780 nm) regions is carried out using spectrophotometers of various brands (SF-26, SF-46, etc.). As solvents, water free from impurities, solutions of acids and alkalis, ethanol, chloroform and other organic solvents are used.
The specific absorption index is the value of the optical density of a solution containing 1.0 g of a substance in 100 ml of a solution, measured in a cuvette with a working length of 1 cm. ±2%.
The constant is measured in different units; in moles - molar absorption coefficient, in % - specific absorption index
Identification of drugs can be carried out according to, E, the nature of the spectral curves in various solvents, the position of the maximum and minimum of light absorption or their ratio (at different wavelengths). For quantitative spectrophotometric analysis, the choice of the analytical absorption band is important. The latter should be free from overlapping absorption bands of other components of the mixture and have a sufficiently high specific absorption rate of the analyte.
Spectrophotometry in the IR region. The nature of the absorption bands in the IR region is associated with vibrational transitions and changes in the vibrational states of the nuclei included in the molecule of the absorbing substance. Therefore, absorption in the IR region is possessed by molecules whose dipole moments change upon excitation of oscillatory motions of the nuclei. The scope of IR spectroscopy is similar, but wider than that of the UV method. The IR spectrum unambiguously characterizes the entire structure of the molecule, including its minor changes. Important advantages of IR spectroscopy are high specificity, objectivity of the obtained results, and the possibility of analyzing substances in the crystalline state. To measure IR spectra on single-beam or double-beam IR spectrophotometers, suspensions of substances in liquid paraffin are used or the substance to be analyzed is placed between plates of potassium bromide.
Each IR spectrum is a series of absorption bands, the maxima of which are determined by the wave number, measured in cm -1 and a certain intensity. For the analysis of LB usually use the spectral region from 4000 to 400 cm -1 .
GF XI recommends two methods of authentication by IR spectra. One of them is based on a comparison of the IR spectra of the test drug and its standard sample recorded under identical conditions. The second way is to compare the IR spectrum of the tested medicinal product with its standard spectrum attached to the PS and registered in accordance with the requirements specified in it.
GOU VPO Irkutsk State medical University
ROSZDRAVA RF
Tyzhigirova V.V., Filippova S.Yu.
APPLICATIONS OF IR- AND UV- SPECTROSCOPIC
METHODS IN PHARMACEUTICAL ANALYSIS
Tutorial in pharmaceutical chemistry for students
Faculty of Pharmacy
Senior Lecturer, Department of Pharmaceutical and Toxicological Chemistry, ISMU, Ph.D. Tyzhigirova V.V., Assistant of the Department of Pharmaceutical and Toxicological Chemistry, ISMU, Ph.D. Filippova S.Yu.
Reviewers:
Head Department of Pharmacognosy with a Botany Course of the State Medical University, Doctor of Pharmaceutical Sciences, Professor Fedoseeva G.M., Professor of the Department of Chemical Technology of the State Technical University, Doctor of Chemical Sciences Shaglaeva N.S.
Published by the decision of the Central Committee of the MSMU (protocol No. dated) Introduction This manual has been prepared for students of the Faculty of Pharmacy with the aim of mastering the analysis of drugs by IR and UV spectroscopic methods.
Modern regulations for the analysis of drugs suggest the widespread use of these methods. IR spectroscopy is the main method in testing medicinal substances for authenticity. UV spectrophotometry is used to assess the quality of both medicinal substances and preparations made from them in terms of authenticity, good quality and quantitative content. In addition, the method is widely used in assessing the quality of solid dosage forms in terms of "Dissolution" and "Uniformity of dosage".
The manual briefly outlines the basics of the methods, their capabilities and limitations. Material is given on the application of methods in the analysis of drugs for various purposes. The presented material is accompanied by specific examples on the use of methods in pharmaceutical analysis. At the end of the manual for self-control of the development of the material are given test questions, test tasks, situational tasks with explanations. A list of tasks for independent work students and a standard solution for one of them.
The manual was compiled in accordance with the standard program in pharmaceutical chemistry (2001) and is intended for self-preparation of students for a cycle of classes on the analysis of drugs by spectrophotometric methods.
1. Characteristics of spectroscopic methods of analysis analysis methods include physical methods based on the interaction of electromagnetic radiation with matter.
Electromagnetic radiation has a dual nature: wave and corpuscular, so it can be characterized by wave and energy parameters. Wave parameters include:
wavelength - the distance traveled by a wave during one complete oscillation. The wavelength is usually expressed in nanometers nm 110 m or micrometers µm 110 m;
9 frequency - the number of times per second when the electromagnetic field reaches its maximum value. Hertz is used to measure frequency;
wave number - the number of wavelengths that fit into a unit length: 1. The wave number is measured in reciprocal centimeters cm 1.
The corpuscular nature of light is characterized by the energy of electromagnetic radiation quanta. In the SI system, energy is measured in joules.
is described by the Planck equation:
- change in the energy of the elementary system as a result of the absorption of a photon with energy h ;
c is the speed of light (3 1010 cm s-1).
When light quanta are absorbed, the internal energy of the particle increases, which is the sum of the energy of electron motion EE, the vibrational energy of the atoms of the molecule EV and the rotational energy. The magnitude of these energies decreases in the order: EE EV ER, and their numerical values are related as: 103:102:1.
As can be seen from the presented relationship, depending on the energy of electromagnetic radiation in a molecule, various energy transitions are possible. If, in accordance with equation (1), we take into account that the wavelength and radiation energy are inversely proportional, then certain sections can be distinguished in the electromagnetic spectrum (table 1).
energy transition processes corresponding to them Interaction of electromagnetic radiation with matter in the optical (ultraviolet, visible, infrared) region underlies the spectrophotometric method, which is widely used in pharmaceutical analysis.
The absorption of electromagnetic radiation in the UV, visible and IR regions of the spectrum is quantitatively described by the Bouguer-Lambert-Beer law, which expresses the dependence of the intensity of a monochromatic light flux passing through an absorbing substance layer (I) on the intensity of the light flux incident on it (I concentration of the absorbing substance (s ), the thickness of the absorbing layer (L) and from the molar absorption index (), characterizing the absorbing substance:
To measure the degree of absorption of electromagnetic radiation, devices have been designed that make it possible to determine not the intensity of the electromagnetic flux, but its weakening due to the absorption of the analyzed substance. And to characterize the degree of absorption of electromagnetic radiation, such photometric quantities as transmission and optical density are introduced.
Transmission (T) is the ratio of the intensity of the light flux passing through the absorbing layer to the intensity of the incident light flux:
Based on formulas (2) and (3), we can write:
Transmission varies from 0 to 1 and is usually expressed as a percentage (%) from 0 to 100.
The inconvenience of calculations led to the introduction of another photometric quantity - optical density (D) as the decimal logarithm of the reciprocal of transmission:
practically measured in the range from 0 to 2. Formula (5) clearly shows that the absorption of electromagnetic radiation by a substance does not depend on the intensity of the light flux, but depends on the nature of the substance and is directly proportional to the concentration of the substance and the thickness of the absorbing layer.
It can be seen from formula (5) that, based on the measured optical density, the absorption index can be calculated using the formula:
The concentration (C) can be expressed in moles per 1 liter or in grams per 100 ml of solution, and depending on this, the molar or specific absorption rate is calculated using formula (6):
– molar absorption index is the optical density of a one-molar solution of a substance with an absorbing layer thickness of 10 mm.
optical density of a 1% solution with the thickness of the absorbing layer, see.
The absorption coefficient in the UV region can reach large values (up to 105 l cm-1 mol-1). In the IR region, the value has insignificant values and is usually not determined.
3. Characteristics of spectrophotometers Regardless of the region of the spectrum, instruments for measuring transmission or absorption consist of 5 main components:
1 - source of energy radiation; 2 - a dispersing device that allows you to select a limited region of wavelengths; 3 – cuvettes for sample and solvent; 4 – detector that converts radiation energy into a measured signal; 5 – signal indicator with a scale.
The source of radiation in the UV region is a hydrogen or deuterium lamp. In a hydrogen lamp, hydrogen glows during discharge, and almost continuous radiation occurs in the 200-nm region.
IR radiation is obtained from an inert solid body, heated electric shock to very high temperatures. So, for example, a silicon carbide rod, called a globar, when heated to 1500 0 C between two electrodes, radiates energy in the region of 1 - 40 microns.
A monochromator is a dispersive device that decomposes radiation into its constituent waves of different wavelengths. The most versatile UV monochromators are prisms made of quartz or glass. For IR spectroscopy, prisms of alkali or alkaline earth metal halides are used. A system of lenses, mirrors, and slits is connected to the dispersing element, which directs radiation with the required wavelength from the monochromator to the instrument's detector.
Detectors - in the UV region, photocells are usually used to convert light energy into electrical energy.
IR radiation is detected by the rise in temperature of blackened material placed in the path of the flow.
The measuring scale of the spectrophotometer is graduated in percent transmission T (I 1 0 0) and in optical density D (lg I), and the scale of wavelengths or wave numbers is in nanometers or reciprocal centimeters, respectively.
Spectrophotometers are a combination of the main components discussed above and vary in complexity and performance. Spectrophotometers are single- and double-beam.
The most commonly used are two-beam devices, in which the luminous flux is divided into two - the main and the comparison flux. With this method of measurement, most of the random noise from the source and detector is compensated, which provides a smaller error in the determination.
The fundamental difference between UV and IR spectrometers lies in the different location of the cuvettes: between the dispersing device and the photodetector in UV spectrophotometers or between the radiation source and the dispersing device in IR spectrometers. This is explained by the fact that in the UV region the absorption can reach large values, which makes it possible to accurately measure the absorption of a monochromatic light flux. In the IR region, absorption takes on insignificant values, which makes it difficult to measure it directly. Therefore, to register IR spectra, the so-called inverted design of devices is used, in which the entire spectrum of radiation that has passed through the substance is recorded. Then the IR spectrum will have high transmission values in the entire region except for the area in which absorption occurred. Therefore, the scale of the recording device in IR spectrometers is calibrated for transmission. UV spectrophotometers are calibrated for both transmission and absorbance.
4. Characteristics of absorption spectra The most important characteristic of electromagnetic radiation is its spectrum. The absorption spectra in the UV and IR regions are of different nature and are characterized as electronic and vibrational spectra, respectively.
If an organic molecule interacts with radiation in the UV region of the spectrum, then at a certain frequency, an energy quantum will be absorbed, accompanied by a transition of valence electrons from the ground to the excited level.
That's why physical nature absorption bands in the UV region are associated with electronic transitions: when a molecule absorbs electromagnetic radiation in the UV region, a transition occurs between the electronic levels of the molecule.
Different electronic transitions require different energy, so the absorption bands are located at different wavelengths.
Types of electronic transitions from the ground state from bonding and orbitals and from non-bonding n orbitals to an excited state to antibonding and orbitals are presented in Table 2.
Table 2. Types of electronic junctions Presence in the structure single bonds(–C–C–) and isolated chromophore groups (-CH=N; -N=N-; -N=O, etc.) determines absorption in the far UV region (100–200 nm.). However, absorption in the far UV region (up to 200 nm) has no analytical significance, since modern spectrophotometers operate in the spectral region starting from 180–200 nm. For the purposes of spectrophotometric analysis, electronic transitions of conjugated bonds are used. The conjugation of sublevels, the transitions of electrons in which require a significantly longer wavelength region of the spectrum and has a high intensity.
The position and intensity of the absorption bands are greatly affected by electron-donating (-NH2, -OH, -SH) and electron-withdrawing (-N=O, -NO2, etc.) substituents, which play the role of auxochromes. They enter into p, and, pairing with -electronic system chromophore and cause a shift in its electron density, thereby reducing the energy of the corresponding transitions. The absorption bands are shifted to the long wavelength region of the spectrum (the so-called bathochromic effect). In addition, electron delocalization increases the intensity of absorption bands (the so-called hyperchromic substituent effect).
Thus, in the UV-region absorb molecules that have in their structure chromophore groups conjugated to each other. The longer the conjugation system, the longer the substance absorbs in the longer wavelength region of the spectrum.
The absorption spectrum in the UV region is expressed as a graphical dependence of the optical density (D) or molar absorption coefficient () on the wavelength () of the incident light.
Instead of D or often use their logarithms. The wavelength can be expressed in various units - nm or microns. The construction of the spectrum in different coordinates will affect its character, therefore, it requires regulation in regulatory documents.
The UV spectrum is characterized as electronic, but when electrons are excited, the energy of the vibrational motion of atoms and the energy of the rotational motion of the molecule will change, so a number of lines appear in the spectrum, which, merging, form broad absorption bands (Fig. 1).
Absorption bands in the UV spectrum, as a rule, are characterized by the location of max and intensity, expressed through the specific absorption index (E1cm).
The absorption bands in the UV region tend to broaden, so the UV spectra are not very selective. However, they provide reliable information about the presence of a system of conjugated bonds in the structure of the analyte.
a chromophore system that includes a double bond –C=C– conjugated with a carbonyl group –C=O, and the enol hydroxyl located at the end of the conjugation chain plays the role of an auxochrome.
the characteristic absorption maximum max = 243 nm and the value of the specific absorption index E1cm = 543, which are used to determine its authenticity.
Rice. Fig. 1. UV spectrum of 0.001% ascorbic acid solution The bands associated with the excitation of vibrational energy levels are located in the spectral region from about 300 to 4000-5000 cm-1, which corresponds to the energy of infrared radiation quanta (3-60 kJ/mol).
The energy of IR radiation is insufficient for the implementation of electronic transitions; Under the influence of IR radiation, only vibrational and rotational transitions are possible.
As a result, the physical nature of absorption bands in the IR region is associated with vibrations of atoms in a molecule: when a molecule absorbs electromagnetic radiation in the IR region, a transition occurs between the vibrational energy levels of one electronic state. In this case, the rotational energy levels also change, so the IR spectra are vibrational-rotational.
oscillatory movements. Normal vibrations are usually subdivided into valence vibrations, characterized by the movement of atoms along the bond axes, and deformation vibrations, in which the bond angles change, while the bond lengths practically do not change.
During normal vibration, all the nuclei of the molecule vibrate with the same frequency and phase, although the amplitudes of their vibrations can vary significantly. Therefore, in a normal vibrational state in a molecule, the centers of gravity of positive and negative charges coincide and, therefore, the molecule will be generally non-polar, although each chemical bond may be polarized.
When absorbing IR radiation, the amplitude of vibrations of atoms into vibrational quantum levels. In this case, the oscillatory process is accompanied general change dipole of the molecule.
Thus, in the IR region, molecules absorb, in which, when the vibrational motions of atoms are excited, the electric moment of the dipole changes.
The oscillation frequency depends on the mass of the atoms in the molecule and the forces acting between them. And the number of vibrational states of a molecule is largely determined by the number of atoms and, consequently, the number of bonds formed by them.
The absorption spectrum in the IR region is expressed as a graphical dependence of transmission (T) on frequency (), expressed in reciprocal centimeters.
The IR spectrum is characterized by a series of closely spaced absorption bands, which are described by their position in the spectrum and relative intensities: strong, medium, weak (Fig. 2).
In the spectra, characteristic bands and the area of "fingerprints" are distinguished. In the region of 1300 - 400 cm-1, there are absorption bands corresponding to vibrations of single bonds С–С, C–N, C–O. As a result of the fact that the C, N, and O atoms are close in mass and are connected by bonds of approximately the same energy, it is impossible to assign the bands to individual groups and bonds. However, the entire set of bands in this region of the spectrum is a characteristic of the nuclear skeleton of the molecule as a whole. This area is called the "fingerprint" area.
If in an atomic grouping the bonds and masses of atoms differ greatly from the parameters of the rest of the molecule, then vibrations are observed in a narrow frequency range and appear in the spectra of all compounds containing this grouping. Such oscillations are called characteristic (group) and they appear in the region of 4000 - 1300 cm-1. Thus, vibrations of groups containing a light hydrogen atom (C–H, O–H, N–H, etc.) and vibrations of groups with multiple bonds (C = C, C = C, C = N, C = O , N = N, etc.). As can be seen, the characteristic vibrations correspond to the atoms that make up the functional groups. The position of the characteristic bands in the spectrum is practically independent of the carbon skeleton to which the group is associated, and provides valuable information regarding the general structure of the molecule.
For structural analysis substances according to their vibrational spectra, there are special correlation tables.
Table 3. Characteristic absorption maxima of some bonds of atoms having a characteristic frequency IR spectra even relatively simple connections consist of a huge number of sharp highs and lows. However, it is precisely this set of peaks that partly determines the specificity of the spectrum. So, in the IR spectrum of ascorbic acid (Fig. 2) there is an intense corresponding double C=C bond; absorption band in the region of the unsaturated ring - lactone. In addition, a series of characteristic absorption bands in the region of 3500–3200 cm 1 is observed, due to stretching vibrations of alcohol and en-diol hydroxyl OH groups. In the area of "fingerprints", absorption bands are pronounced, which characterize single C–C and C–O bonds.
Interpretation of IR spectra is rather complicated, therefore, an IR spectrum of a standard sample of ascorbic acid is obtained in parallel. The spectrum of the analyte should have absorption bands matching in position and relative intensities with the standard spectrum.
5. Sample preparation for photometric determinations by preparing a solution of the appropriate concentration. Since the spectrophotometric method is highly sensitive, solutions with a very low concentration of 10-6 - 10-8 g/ml are photometered.
To reduce the error at the stage of taking a micro-sample, it is increased to macro, and then the dilution technique is used.
reasonable choice of solvent for spectrophotometric determinations. First of all, it must be transparent in the measured region of the spectrum, for which its transmission limit is taken into account (Table 4).
solvents used in photometry cause the ionization of a substance, which leads to a redistribution of the electron density in the conjugation chain and, consequently, to a change in the pattern of the spectrum. Acid-type ionization produces an additional lone pair of electrons in the molecule, which leads to intensity. Ionization by the main type (protonization) can often lead to the opposite effect, since the lone electron pair binds to the proton, which leads to a decrease in the influence of the substituent.
A good example of the influence of the nature of the solvent on the pattern of the spectrum is the position of the absorption band in the spectrum of folic acid (max = 320 nm in an acid solution, max = 365 nm in an alkali solution). Folic acid has in its structure functional groups of both acidic and basic nature, which make it possible to use solutions of acids and alkalis as solvents for spectrophotometric determinations:
The largest bathochromic shift of the absorption band in the spectrum of folic acid is observed in a sodium hydroxide solution, since the dissolution of a substance in an alkali solution is accompanied by acid-type ionization. Moreover, the main contribution to conjugation is made by the anionic oxygen atom at C 4 of the heterocyclic system, pterin.
The preparation of the analyzed sample in IR spectroscopy is associated with additional difficulties due to the fact that most solvents are not transparent in the IR region, and therefore the choice of solvent requires special care. In this case, one should take into account not only its transparency in the IR region of the spectrum, but also the possibility of influencing the absorbing system. Thus, for example, water is generally excluded, and not only because of the strong absorption, but also because of the impact on the materials from which the cuvettes and the optical part of the devices are made. Of all the solvents, carbon tetrachloride and carbon disulfide are the most suitable, the use of which also has limitations: the first is used in the region up to 7.6 microns, the second in the range of 7.6 - 15 microns. To reduce the absorption of radiation by the solvent, it is necessary to use narrow cuvettes with a thickness of 0.1–mm. At the same time, it is necessary to increase the concentration of solutions to -4.5%, so that the transmission value during measurements in the IR region takes optimal values.
The most commonly analyzed sample for IR spectrometry is prepared by obtaining tablets, when the analyzed sample is crushed, mixed with spectroscopically pure potassium bromide and pressed; or by obtaining a paste, when the test sample is triturated with vaseline or other mineral oil transparent in the IR region, and then the resulting paste is squeezed between two plates of sodium chloride.
6. Comparative characteristics absorption methods The most important characteristics of any method, including photometric. are its sensitivity and accuracy.
Quantitatively, the sensitivity of spectrophotometric determinations can be characterized by the sensitivity coefficient S, which determines how much the optical density of a solution changes with a very small change in the concentration of the analyte.
Mathematically, it is expressed by the first derivative of optical density with respect to concentration:
Thus, the sensitivity is proportional to the molar absorption index and the larger it is, the smaller the amount of the substance, other things being equal, can be determined.
The value of the molar absorption coefficient in the UV and visible regions of the spectrum is ten times greater than in the IR range. The thickness of the absorbing layer used in measurements is 1 cm for the UV region of the spectrum, and 0.5–5.0 cm for the visible region; for the IR region, see. Therefore, the sensitivity of photometric determination in the UV and visible range is much higher than in the IR range, and for the UV region it is 10-4–10-6 of the molar mass of the analyte.
The error in the spectrophotometric determination of the concentration (C) can be characterized by expressing it as a function of the optical density and thickness of the absorbing layer:
Thus, the error in determining the concentration C will be the smaller, the larger and l, which is typical for the UV and visible regions of the spectrum.
Based on the foregoing, it follows that for the purposes of quantitative analysis, spectrophotometry in the UV and visible regions of the spectrum has advantages over IR spectroscopy. At the same time, as was shown in the characterization of the spectra, IR spectroscopy is a more selective and informative method and, therefore, is widely used for the purposes of qualitative analysis.
7. Application of Spectrophotometry in Pharmaceutical Analysis IR spectroscopy is the most widely used method in pharmaceutical analysis to determine identity. This is due to the high specificity of the vibrational spectrum.
Identification of a medicinal substance can be carried out by comparing the IR spectrum of the substance under study with the similar spectrum of its standard sample or with the drawing of the standard spectrum given in the monograph.
In practice, when interpreting the spectra, the position of the absorption bands and their intensity (strong, medium, weak) are determined.
It is recommended to begin the comparison of IR spectra with an analysis of the characteristic bands, which are usually well manifested in the spectra, and only if they coincide, the low-frequency region is compared. The coincidence of the spectral curve of the test substance with the pattern of the standard spectrum indicates the identity of the two substances. The absence in the spectrum of the substance under study of the bands observed in the spectrum of the standard sample clearly indicates that these substances are different. The presence in the spectrum of the test substance of a larger number of bands, compared with the spectrum of the standard, can be explained both by contamination of the test substance, and by the difference between both substances.
Thus, the IR spectrum of the test sample must have complete coincidence of the absorption bands with the absorption bands of the standard spectrum in position and relative intensity.
Pharmaceutical analysis can consider the IR spectra of structurally similar steroid compounds: cortisone acetate, hydrocortisone acetate and prednisolone (Fig. 6 - 8).
The most characteristic for all three substances is the 1600 - cm 1 region, which accounts for the stretching vibrations of the C \u003d C group at C 4 of medium intensity (1606 - 1626 cm 1), the stretching vibrations of the C \u003d O groups at C 3 and C 11 (1656 - 1684 cm 1), groups C \u003d O at C 20 (1706 - 1733 cm 1). All spectra show maxima in the range from 3200 to 3500 cm 1, which correspond to vibrations of the free hydroxyl group.
Cortisone acetate and hydrocortisone acetate are esters, which appears on their IR spectra as characteristic bands in the region of 1219 - 1279 cm 1. These absorption bands are absent in the spectrum of prednisolone. But for the IR spectrum of prednisolone, as 3-keto-1, pregnadiene, there is a band of a strong degree of intensity of stretching vibrations of the C \u003d C bond at C 1 (1595 cm 1).
identification of steroids of similar structure by the position of the main bands in the spectrum and their relative intensities.
In pharmaceutical analysis for the purposes of quantitative determination of the difficulties that do not allow for comparable accuracy. These include the need to measure in a very narrow cuvette, the length of which is difficult to reproduce; high probability of overlapping absorption bands; a small width of the absorption band at the maximum, which leads to deviations from the basic law of light absorption.
tour of steroid compounds. So, in fig. 6 - 8 shows the IR spectra of cortisone acetate, hydrocortisone acetate and prednisolone.
7.2. Application of UV-spectrophotometry in analysis UV-spectroscopy in pharmaceutical analysis is used for various purposes.
structure, it is advisable to use UV spectrophotometry in order to use such spectral characteristics as the position and intensity of absorption bands.
Determining the authenticity of the UV spectrophotometric method can be carried out in various ways.
One of them is based on constructing a spectral curve and determining on it the characteristic, so-called analytical wavelengths at which the maximum (max), minimum (min) is observed, not strictly defined values of max and min are regulated, but their allowable intervals. This circumstance is explained by the permissible error in the calibration of the wavelength scale on various instruments.
Since the UV spectrum has one, two, less often three wide bands should be used. However, the FS strictly regulates the determination conditions (solvent, concentration of the working solution), and the spectral curve must be plotted in the coordinates or D specified in the FS.
absorption at a given analytical wavelength, expressed in terms of the specific absorption index E 1%. The essence of the determination is reduced to measuring the optical density of the analyzed sample at max and compared with the value of the specific absorption index, which, in turn, is determined by the standard sample for the analyzed medicinal substance and is given in the FS as an acceptable interval.
spectrophotometry in order to determine the authenticity of substances that have a system of conjugated bonds in the structure is mandatory, but due to low selectivity it is considered as an additional method in the test block. So, substances with the same type of system of conjugated bonds are characterized by absorption in the same region of the spectrum.
A good example of this is the spectral characteristics of steroid compounds: prednisolone, cortisone acetate and hydrocortisone acetate (Fig. 3-5).
As can be seen from fig. 3-5, in the structure of these substances there is the same type of chromophore system, which arises as a result of the conjugation of the carbonyl group at C 3 and the double bond at C 4. Therefore, these substances absorb in the same region of the spectrum at wavelengths of 238 - 242 nm. In general, the absorption due to the chromophoric system of 4–en–3–one bonds is analytical for steroid compounds and can be considered as a group-wide test for this class of substances.
Ergocalciferol and retinol acetate, belonging to the same group of alicyclic vitamins, differ in the number of conjugated double bonds and therefore absorb in different regions of the spectrum.
Ergocalciferol has in its structure a system of three conjugated double bonds. The conjugation of double -C=C-bonds causes the absorption of ergocalciferol at 265 nm with a specific index of 480 - 485.
Retinol acetate is also based on an alicyclic structure:
However, unlike ergocalciferol, retinol has a pentaene conjugation chain. An increase in the number of conjugated bonds leads to a decrease in the energy of electronic transitions and, as a consequence, to a shift of the absorption band to the long wavelength region with an increase in its intensity. Retinol acetate has a pronounced absorption maximum in the longer wavelength region of the spectrum, compared to ergocalciferol, at 326 nm, and the specific absorption index takes a value of 1550.
Photometric characteristics of other medicinal substances from the class of vitamins, alkaloids, steroid hormones and antibiotics used for analytical purposes are given in tables 5-9.
specific impurities in medicinal substances.
absorption (), the smaller the amount of substance can be determined.
The use of the method for determining impurities is justified only in terms of the absorption coefficient. Such an admixture is called light-absorbing.
The determination of impurities by the spectrophotometric method is reduced to two cases. If an impurity absorbs in a region of the spectrum that is different from the absorption region of the medicinal substance, then the presence of an impurity is judged by the appearance of an additional absorption band in the spectrum. An example of hydrotartrate:
The conjugation of the aromatic ring with two -OH groups located in the ortho position with respect to each other causes the absorption of adrenaline in the UV region at a wavelength of 279 nm. Adrenolone, being a product of the oxidation of adrenaline, has a quinoid structure, which causes absorption in the longer wavelength region of the spectrum at 310 nm.
The impurity can absorb in the region of the spectrum characteristic of the medicinal substance. In this case, the presence of an impurity is judged by the increase in optical density at the analytical wavelength.
The use of this technique is possible subject to the law of additivity, according to which the optical density of the sum of substances is equal to the sum of the optical densities of individual substances, subject to independent absorption of these substances: D A D B D A B :.
For example, this technique is used in the determination of absorbing impurities in cyanocobalamin. The optical density of the drug solution is determined at 278 nm, 361 nm and 548 nm.
Then the ratios of optical densities are calculated, which should be included in the intervals given in the FS:
Table 5. Photometric characteristics of some drugs Table.6. Photometric characteristics of steroid hormones acetate Table 7. Photometric characteristics of some phenylamines Table 8. Photometric characteristics of some drugs Drotaverine hydrochloride 0.1 mol/l Table 9. Photometric characteristics of some drugs Benzylpenicillin Water Nitrophenylalkylamine derivatives are used quite widely. The application of the method is based on the existence of a directly proportional dependence of the absorption value on the concentration of a substance in the analyzed solution:
spectrophotometric method:
graphic according to the calibration schedule;
comparative relative to the standard sample;
calculated according to the specific absorption index (E1% 1cm).
The first method is the most rational when conducting serial analyses. Its essence is as follows: a series of dilutions of a standard sample is prepared in the concentration range at which compliance with the Bouguer–Lambert–Beer law is observed. The optical density of solutions of a standard sample is measured and a calibration curve is built. Then, a solution of the analyzed sample is prepared at a concentration approximately corresponding to the middle of the calibration curve, and its optical density (DX) is measured on the same device, the CX value, g/ml, is determined (Fig. 6).
Rice. 6. Calibration graph of the analyzed sample and its dilution method:
The second quantification method is as follows:
in parallel, solutions of the analyzed and standard samples of approximately the same concentration (CX and CC.O) are prepared and their optical density is measured (DX and DС.О.) under equal conditions (max, l.) In accordance with the basic law of light absorption, we can write:
Given that and l are the same, combining both equations, we get:
DCO CCO DCO
Further, in the calculation formula, the value of the macro-weights of the standard and analyzed samples and the method of their dilution are taken into account:This method is more accurate, therefore it is widely used when performing single analyzes.
If standard samples are not available in the laboratory, calculations in quantitative analysis can be made using known value E1%1cm according to the formula:
However, this method of analysis is the least preferable, since in this case the role of errors due to the individual characteristics of the instruments increases.
To ensure the required accuracy of the analysis, it is necessary to scientifically substantiate the conditions for quantitative determination.
Choice of analytical wavelength. For this purpose, a spectral curve of dependence of optical density on wavelength is built according to a solution of a standard sample. On the spectral curve determine the analytical wavelengths corresponding to the wavelengths of maximum absorption. Of all the absorption bands in the spectrum, for the purposes of quantitative analysis, one is chosen that is characterized by light absorption and provides the highest sensitivity of the determination. On the other hand, gentle maxima are more preferable, because in this case the error in setting the wavelength is less affected.
obedience to the Bouguer–Lambert–Beer law. To do this, a series of dilutions of the standard sample is prepared and the values of their optical densities are measured at the selected analytical wavelength. Based on the data obtained, a calibration graph is built - a graphical dependence of optical density on concentration. The absorption of electromagnetic radiation by a substance obeys the basic law of light absorption for the concentration range in which the graph is a straight line emerging from the origin (Fig. 7).
Rice. Fig. 7. Calibration curve Cn - Cm - the concentration range in which the Bouguer–Lambert–Beer law is observed.
This can be clearly demonstrated using Figure 8.
Rice. 8. Calibration chart:
1 - if the Bouguer-Lambert-Beer law is observed, 2 - if the Bouguer-Lambert-Beer law is not observed. Lambert-Beer exceeds the C1 error when the law is satisfied.
Choice of working range of optical density (D). It has been established that the relative error of optical density measurement takes the minimum values at D = 0.434. Therefore, they try to work in the range of optical densities from 0.3 to 0.8, in which the device is calibrated with the greatest accuracy. Since the optical density is directly proportional to the concentration of the substance in the analyzed sample and the thickness of the absorbing layer, it is these parameters that should be varied to select the optimal values of the optical density. At the same time, the concentration is chosen in such a way that its value falls within the interval at which compliance with the Bouguer–Lambert–Beer law is observed.
Choice of standard sample (RS). Spectrophotometry is a relative method and therefore requires the use of reference materials, which can be either national reference materials (GRS) or working reference materials (RSS). When performing the analysis of substances, GSO is used, and when analyzing drugs, the use of RSO is allowed.
Preparation of the analyzed sample. Absorption measurement in the UV region is carried out in solutions. In view of high sensitivity spectrophotometric method, the working concentration of the CX solution has low values. Therefore, in the method of spectrophotometric determination, the scientifically substantiated value of the macro-load and the measuring utensils used for its dilution should be regulated.
Choice of reference solution. Photometric determinations in any region of the spectrum involve the use of reference solutions - these are solvents or solutions containing all components of the analyzed sample, except for the substance being determined. Photometric instruments are designed in such a way that the use of cuvettes with a reference solution allows you to bring the optical density scale to zero and thereby level out the absorption due to the walls of the cuvette, solvent and other reagents used to prepare the analyzed sample.
Due to its high sensitivity, UV spectrophotometry is widely used in testing solid dosed drugs for dosing uniformity. This test is mandatory when the content of the active substance is 0.05 g or less. Highly sensitive methods are required to estimate this amount. One of them is UV spectrophotometry.
The high sensitivity of the method also makes it possible to estimate the amount of the active substance released from the dosage form into the dissolving medium. Therefore, UV spectrophotometry is often used in determining the "Dissolution" test adopted by the Global Fund for solid drugs.
Thus, one of the advantages of UV spectrophotometry is its versatility, which makes it possible to use the method for solving various analytical problems.
1. Phenomenon underlying spectroscopic methods of analysis.
2. Classification of spectroscopic methods of analysis. The principle of classification.
3. Nature of absorption in the UV and IR regions of the spectrum.
4. Basic law of light absorption.
5. Basic photometric quantities.
6. Characteristics of the main units of spectrophotometers.
Fundamental difference between UV spectrophotometers and IR spectrometers.
7. Characteristics of the absorption spectra in the UV and IR regions of the spectrum.
8. Comparative characteristics of the applicability of UV and IR spectroscopy for solving pharmaceutical problems.
9. Features of sample preparation for spectrophotometric determinations in the UV and IR regions of the spectrum.
10. Application of UV spectrophotometry to determine the authenticity of medicinal substances.
11. Possibilities of using UV spectrophotometry to determine impurities. Definition methods.
12. Application of UV spectrophotometry in quantitative analysis.
Choice of quantitation conditions. Methods for calculating the results of analysis.
13. Application of IR spectroscopy in pharmaceutical analysis.
1. The spectrophotometric method is based on a) selective absorption of electromagnetic radiation by the analyzed substance b) emission of electromagnetic radiation by excited atoms or molecules c) reflection of electromagnetic radiation by the analyzed substance 2. The absorption of electromagnetic radiation by a substance depends on a) the intensity of the light flux b) the nature of the substance c) the thickness of the absorbing layer d) the content of the substance in the analyzed solution 3. Set the correspondence of electromagnetic radiation 4. The absorption spectrum 1) in the UV region is a) a graphical dependence of the optical density (D) or molar absorption coefficient () on the wavelength () of the incident light b) graphical dependence of transmission (T) on frequency (), expressed in reciprocal centimeters the presence in the structure of the system of conjugated bonds 6. Absorption bands in the spectrum 1) in the UV region are characterized by a) the location of the analytical wavelengths max, min b) the position in the analytical region of the spectrum of the entire set of absorption bands c) the absorption intensity expressed in terms of the specific absorption index (E1cm) d) the relative intensity characterized by as a small, medium and high degree 7. Establish a correspondence 1) area 1300 - 400 cm 1 a) characteristic of the nuclear skeleton 2) area 4000 - 1300 cm 1 of the molecule as a whole 8. More selective and informative for the purposes of determining the authenticity of medicines is a) spectrophotometry in the UV region b) spectrophotometry in the IR region 9. Identification of a medicinal substance by IR spectra can be carried out a) by the coincidence of the absorption bands and relative intensity with the spectrum of the standard sample b) by the coincidence of absorption bands and relative intensity with the spectrum pattern, given in the FS c) according to the position and intensity of analytical wavelengths, reg lamented in FS 10. When testing for the authenticity of medicinal substances, the UV-spectrophotometric method is considered as a) the main b) additional Determination of the authenticity of medicinal substances UV-11.
spectrophotometric method can be carried out a) according to the spectral curve b) according to the calibration graph c) according to the value of the specific absorption index at an analytical wavelength of 12. The sensitivity of the determination is higher, and the error in measuring the absorption value is less a) in the UV region b) in the IR region 13. In the quantitative analysis of medicinal substances, a) spectrophotometry in the UV region b) spectrophotometry in the IR region is used 14.
spectrophotometric determination involves a) taking a macro-weight of the medicinal substance, followed by its dissolution and dilution with an appropriate solvent using volumetric flasks b) rubbing the medicinal substance with vaseline oil or other liquid and placing the resulting suspension between two plates of potassium bromide c) rubbing the medicinal substance with potassium bromide and subsequent pressing 15. The choice of the concentration of the analyte solution in UV-spectrophotometric determinations is carried out a) according to the spectral curve b) according to the calibration graph c) based on the concentration of the standard solution 16. In the method of quantitative determination of medicinal substances by the UV-spectrophotometric method, a) b) volumetric utensils for sample dilution c) concentration of the solution of the analyte d) concentration of the standard solution or method of its preparation e) analytical wavelength f) solution c Equation 17. In the longer wavelength part of the spectrum, there are absorption bands
S NH N S NH N
18. It is possible to distinguish medicinal substances using the method a) spectrophotometry in the UV region b) spectrophotometry in the IR region 19. For two derivatives of 5 - nitrofuran, the absorption bands in the UV region of the spectrum substances 20. The use of UV-spectrophotometric method in the analysis of glucose is justified for the purpose of a) determining the authenticity of glucose b) determining the impurity of hydroxymethylfurfural c) quantitative determination of glucose b) characteristic absorption bands of the IR spectrum 2. b, c, d 9. a, b 10. b 11. a, c 12. a 13. a 14. a 15. b, c 16. a, b, d, e, f 17. b 18. b 19. a 20. b 21. b 1. UV spectrum of a 0.002% solution of dibazole in 95% alcohol in the range from 225 nm to 300 nm has maxima at wavelengths of 244 ± 2 nm ;275 ± 1 nm; 281 ± 1 nm and minima at wavelengths of 230 ± 2 nm;
253 ± 2 nm; 279 ± 1 nm.
How to prepare an alcoholic solution of dibazol and obtain its spectrum?
2. The specific absorption index of furacilin in an alcoholic solution at = 365 nm is 850 - 875. To determine the specific index, the analyst prepared a 0.0005% solution of furacilin.
hydrochloric at \u003d 243 nm has a specific absorption index of E11cm \u003d 542.5. To determine the indicator, the analyst prepared a 0.001% solution of ascorbic acid according to the following procedure: about 0.05 g (accurately weighed) of ascorbic acid was placed in a volumetric flask with a capacity of 100 ml and dissolved in a 0.001 M solution of hydrochloric acid, brought the volume of the solution to the mark. 2 ml of the resulting solution was diluted with a solvent in a volumetric flask with a capacity of 100 ml, resulting in a 0.001% solution. Check the correctness of the calculation of the concentration of the solution and evaluate the method of preparing the solution from the standpoint of metrology.
4. The analyst prepared a 0.001% solution of papaverine hydrochloride using 0.1 M hydrochloric acid as a solvent. I measured the optical density of the prepared solution on the device at = 310 nm in a cuvette with a layer thickness of 1 cm relative to the solvent. The optical density of the solution was D = 0.23. Then, using the formula, I calculated the specific absorption rate:
In accordance with the ND, the specific indicator should be 211 - 220.
Based on the data obtained, the analyst concluded that the medicinal substance did not comply with the requirements of the RD in terms of E11cm. Evaluate the actions of the analyst.
analytical chemical reactions. With concentrated sulfuric acid, a bright yellow oxonium salt is obtained. The reaction with a solution of silver nitrate in nitrate is confirmed by the melting point. When preparing a new draft FSP, it was decided to use the spectral characteristics of diphenhydramine instead of analytical reactions. The following change was made to the “Testing for Authenticity” section: the UV spectrum of a 0.05% solution of diphenhydramine in 95% alcohol in the region from 230 nm to 280 nm has maxima at wavelengths of 253 ± 2 nm; 258 ± 2 nm; 264 ± 2 nm and minima at wavelengths Is the decision made correct?
6. When developing a new draft RD for ascorbic acid, the spectral characteristics of the substance obtained by UV and IR spectroscopy.
spectroscopy and analytical chemical reactions. The IR spectrum of novocaine, obtained in tablets with potassium bromide in the range from 4000 to 600 cm 1, should have a complete coincidence of the absorption bands with the absorption bands of the attached spectrum.
Analytical chemical reactions confirm the presence of a primary aromatic amino group and a chlorine ion in the structure of novocaine.
novocaine for authenticity.
8. The impurity of adrenolone in the medicinal substance of adrenaline hydrotartrate is determined by the spectrophotometric method. In hydrochloric at = 310 nm in a cell with a layer thickness of 10 mm should not exceed 0.2.
The analyst prepared a 0.2% solution of the drug substance and measured its optical density, observing the conditions specified in the RD. The optical density of the analyte was 0.26. When repeating the analysis, similar results were obtained. On the basis of the obtained data, the analyst concluded that the medicinal substance did not meet the requirements of the RD in terms of the content of the adrenolone impurity.
9. In the draft FSP for tablets of acetylsalicylic acid 0.5 g, in the section "Testing for authenticity", along with analytical reactions, spectral characteristics were included by the spectrophotometric method. The same method is recommended for the determination of the "Dissolution" test and quantitative analysis.
10. Quantitative determination of the substance of riboflavin, according to the FS, is carried out by the spectrophotometric method according to the method:
about 0.07 g of riboflavin (accurately weighed) is placed in a 500 ml volumetric flask, 5 ml of water are added and stirred until the sample is completely moistened. Add dropwise (no more than 5 ml) 1 M sodium hydroxide solution and stir until complete dissolution of the sample. Immediately add 100 ml of water and 2.5 ml of glacial acetic acid, mix and bring the volume of the solution with water to the mark. 20 ml of this solution is transferred to a volumetric flask with a capacity of 200 ml, 3.5 ml of 0.1 M sodium acetate solution is added and the volume of the solution is adjusted to the mark with water. Measure the optical density of the resulting solution at = 444 nm in a cuvette with a layer thickness of 10 mm.
D is the optical density of the test solution;
a - riboflavin weighed in g;
328 - specific absorption index at 444 nm.
riboflavin by specific absorption rate. Check the correctness of the hitch calculation.
Quantitative determination of dibazol solution 1% for 11.
injections are carried out in accordance with the RD by the spectrophotometric method according to the following method:
2 ml of the drug is placed in a volumetric flask with a capacity of 100 ml, the volume of the solution is brought to the mark with 95% alcohol and mixed.
with a capacity of 50 ml, add 30 ml of 95% alcohol, 1 ml of 0.1 M sodium hydroxide solution, bring the volume of the solution with alcohol to the resulting solution on a spectrophotometer at = 244 nm in a cuvette with a layer thickness of 10 mm. Alcohol 95% is used as a reference solution. In parallel, the optical density of the standard sample solution (RSO) of dibazole is measured.
1 ml of RSO solution contains about 0.00002 g of dibazol.
Check the calculations of the weighed portion of the dibazol preparation.
12. In accordance with the FSP, the quantitative determination of picamilon 20 mg tablets is carried out by the UV spectrophotometric method according to the following method: about 0.08 g (accurately weighed) of the powder of crushed tablets is quantitatively transferred with water into a 500 ml volumetric flask, the volume of the solution is adjusted to the mark with water, mixed and filtered through a paper filter (red ribbon).
Measure the optical density of the resulting solution on a spectrophotometer at the absorption maximum at a wavelength of ± 2 nm in a cuvette with a layer thickness of 10 mm. In parallel, the optical density of a solution of a standard sample of picamilon is measured. Water is used as a reference solution.
Is the quantitation method selected correctly?
1. To prepare a 0.002% alcohol solution of dibazol, dissolve 0.2 g of dibazol in a volumetric flask with a capacity of ml in 95% alcohol, bring the volume of the solution to the mark. You will get a 0.2% solution, which must be diluted 100 times. To do this, 1 ml of the prepared solution is placed in a volumetric flask with a capacity of ml and brought to the mark with alcohol.
Then, the optical density of a 0.002% dibazole solution in a cuvette with a layer thickness of 10 mm is measured on a spectrophotometer relative to the solvent in the region from 225 nm to 300 nm after 5 nm, and near the maxima and minima after 1 nm. Based on the obtained values, a spectral curve of the dependence of the optical density (D) on the wavelength () is built.
The wavelengths corresponding to the maximum and minimum absorptions are marked on the spectral curve. They must correspond to the wavelengths given in the RD.
The task is greatly facilitated when working on modern spectrophotometers with an automatic device for recording spectra.
2. Specific absorption rate is the absorption of 1% solution with a layer thickness of 1 cm. This indicator is calculated by the formula:
E1 cm of furacilin is 850 - 875. This means that its 1% solution has an optical density D = 850 - 875. It is almost impossible to measure such a solution density on a spectrophotometer, since its scale is graduated from 0 to 2. Moreover, the smallest calibration error is in the area of 0.3 - 0.8. And the optimal optical density for measurement is D = 0.43. Therefore, the test solution is prepared in such a concentration that its optical density is close to 0.43.
Thus, the analyst's calculations are correct.
3. The concentration of ascorbic acid solution to determine the specific absorption rate E11cm is calculated by the formula:
The analyst prepared a 0.001% solution instead of 0.0008%. This is quite acceptable, since the prepared solution will have an optical density:
This density is included in the range of optical densities recommended for measurement of 0.3 - 0.8. Consequently, the analyst of correct metrology prepared the solution not accurately enough, taking a sample of the substance equal to 0.05 g. To ensure the accuracy of weighing, it is better to take the sample of the substance as much as possible, in extreme cases, equal to 0.1 g.
A 0.1% solution should be prepared from it first, using a 100 ml volumetric flask, and then diluted 100 times to obtain a solution with the required concentration of 0.001%. To do this, you can take 2 ml of a 0.1% solution and a volumetric flask with a capacity of 200 ml.
4. The analyst made an unreasonable conclusion. He prepared a solution of low concentration (0.001%). When measuring the optical density (D) of such a solution, a significant error was made, since D = 0.23 does not correspond to the optimal value of D = 0.43.
The inaccuracy of optical density measurements affected the calculations of the specific absorption index.
prepare a new solution with a concentration of 0.002%, measure its absorption, and only then draw a conclusion.
5. The decision to establish the authenticity of Diphenhydramine only on the basis of its UV spectrum is unreasonable.
The UV spectrum of diphenhydramine characterizes only aromatic rings in the structure of the substance:
Similar chromophore groups are found in a number of medicinal substances (ephedrine g/chl, atropine sulfate, etc.).
Therefore, the UV spectra of a substance do not provide reliable information about its authenticity.
The identity test must be supplemented with analytical chemical reactions confirming other structural fragments of diphenhydramine, in particular, an ether bond and a chlorine ion.
spectrophotometric methods is rational in assessing the authenticity of Diphenhydramine.
6. Methods of UV and IR spectroscopy provide reliable testing of ascorbic acid for authenticity. Therefore, analytical chemical reactions can be excluded. Accepted ND reactions, as a rule, confirm the presence of an ascorbic en-diol group in the structure of the acid, which determines the reducing properties of the substance.
However, in the structure of the substance there are also primary and secondary alcohol groups, an internal ester group, which are not evaluated by the chemical method.
Only the IR spectrum of ascorbic acid provides complete information about the structure of a substance by the presence of characteristic absorption bands of enol and alcohol OH groups, a double bond in the ring and a lactone group, as well as by a set of absorption bands in the "fingerprint" region. Reliability is ensured by comparing the spectrum of ascorbic acid with the spectrum of its standard sample or spectrum pattern.
The UV spectrum of ascorbic acid reflects the presence of only conjugated double bonds in the structure. Therefore, UV spectroscopy is an additional method, and IR spectroscopy is the main method in authenticity testing.
Thus, the analyst's proposal to use a set of UV and IR spectroscopy methods in testing ascorbic acid for authenticity is justified.
7. The complex of testing novocaine for authenticity using IR spectroscopy and the chemical method is scientifically substantiated and rational. IR spectroscopy is a specific method of functional analysis that makes it possible to detect all functional groups in the structure of novocaine: the primary aromatic amino group, the ester group, the substituted ammonium cation, by the presence of characteristic absorption bands in the IR spectrum in the region of 3500 - 1300 cm 1. The region of skeletal vibrations ( below 1300 cm 1) is characterized by many absorption bands and is purely individual for novocaine.
An analytical reaction proves the presence of a chlorine ion, the azo dye is a group dye for aromatic amines and allows the medicinal substance to be attributed to the group of local anesthetics.
8. When determining the impurity of adrenolone by the spectrophotometric method, it must be borne in mind that the method for determining the impurity by is explained by the fact that the absolute value of the optical density is poorly reproduced on different devices. Therefore, it is advisable to determine the ratio of optical densities at different wavelengths () and normalize the relative value, which is more or less constant and is better reproduced on different devices.
For a reasonable conclusion about the adrenolone impurity content, the analyst must be sure that the spectrophotometer readings are correct. Therefore, instruments in the laboratory must be verified by the metrological service. If the devices are verified, then it is possible to measure the optical density of the test solution on different spectrophotometers and compare the obtained values. With the reproducibility of the value D = 0.26 on different devices, it can be confidently stated that adrenaline hydrotartrate does not meet the requirements of the RD in terms of the adrenolone impurity content.
9. The choice of UV spectrophotometric method for testing acetylsalicylic acid tablets for authenticity and determining the "Dissolution" test is scientifically sound. The UV spectrum of acetylsalicylic acid complements the analytical reactions for authenticity, since the absorption band in the spectrum indicates the aromatic nature of the substance.
It is quite logical to use the method in determining the “Dissolution” test, which shows the amount of a substance that has passed into the dissolving medium from the dosage form in 45 minutes at 37O C. The “Rotating basket” device is used for testing. One tablet is placed in a basket and lowered into the dissolution medium - an acetate buffer solution with a pH of 4.5 and a volume of 700 ml. After 45 minutes, a sample is taken and the content of acetylsalicylic acid is determined.
Since the amount of active substance in the dissolution medium will be approximately:
a highly sensitive method would be required to determine it in a sample.
This method is UV spectrophotometry.
For quantitative purposes, it is better to use the titrimetric method, which is absolute and does not require comparison with a standard sample. The dosage of acetylsalicylic acid tablets, equal to 0.5 g, allows the use of this method.
highly sensitive methods. Therefore, the determination of substances of medicinal substances, as a rule, is carried out by means of titration.
However, the substance of riboflavin does not have analytical reactions that meet the requirements of titrimetry. For this reason, for the quantitative determination of riboflavin, not chemical, but a spectrophotometric method is chosen, since riboflavin absorbs intensively in the UV and visible regions of the spectrum.
quantitative goals is reasonable. However, the method of determination given in the FS by the specific absorption index needs to be improved, since it is accompanied by a significant error.
More correct and accurate is the method of comparison with a standard sample of the GSO category.
The calculation of the riboflavin weight is carried out according to the specific absorption index E11cm = 328.
percent, taking into account the optimal value of the optical density D = 0.43:
increase, then use the dilution technique. According to the FS method, the sample is increased by 5000 times and obtained. Thus, the sample of riboflavin is calculated correctly. However, from the point of view of metrology, it is better to increase the micro-weight by a factor of 8000 and obtain a sample equal to 0.1 g.
11. Choice of spectrophotometric method for quantitative science-based. The content of the active substance in the preparation is low, therefore, its determination requires a highly sensitive method, which is UV spectrophotometry. In addition, according to its chemical structure, dibazol belongs to the heteroaromatic series and actively absorbs UV radiation, which makes it possible to use the method for quantitative purposes.
The spectrophotometric method is relative and requires comparison with a standard sample. The analyzed solution and the standard sample solution are prepared at approximately the same concentration.
The concentration of the standard sample solution is indicated in the ND. This is the basis for calculating the sample weight. In our case, the concentration of the working standard sample solution is 0.00002 g/ml.
The test solution must be prepared with the same dibazol content.
CX \u003d CC.O \u003d 0.00002 g / ml Then they recalculate for a dibazol solution:
Since the sample is small, it is increased by a factor of 1000 and the dilution technique is used:
Thus, the sample of dibazol preparation was calculated correctly.
12. Picamilon is a medicinal substance of the heterocyclic series, aminobutyric acids:
It is used as a nootropic in the form of tablets with a dosage of 20 mg. In the structure of the substance there is a pyridine chromophore conjugated with the amide group and causing the absorption of picamilon in the UV region. Therefore, the choice of the spectrophotometric method for quantitative purposes is fully justified. In addition, the content of the active substance in tablets is negligible (20 mg), so a highly sensitive method is required, such as UV spectrophotometry.
There is no doubt about the choice of water as a solvent, since the active substance, being the sodium salt of a carboxylic acid, dissolves in water.
Pre-treatment is provided in order to separate excipients that are insoluble in water and therefore interfere with the determination.
standard sample ensures the correctness and accuracy of the analysis.
The disadvantage of the proposed method is that the analyzed portion of the tablet mass is small (0.08 g). From the standpoint of metrology, it is better to work with a sample of 0.1 g or more. The larger the sample, the smaller the weighing error. An increase in the weight in this case is quite possible, since there is no need to save the analyzed material, since the weight is taken from 20 tablets, ground into powder.
Give justification for the use of UV - spectrophotometric quantitative analysis with a full calculational reasoning. When completing the task, use the algorithm and an example of solving the problem.
1. Solution of anaprilin 0.25% in CCO ampoules 0.00002 g ml 2. Nicotinic acid solution 1% in CCO ampoules 0.00001 g ml 4. Ointment hydrocortisone eye 0.5% - 3.0 CCO 0.00001 g ml 7. Rectal suppositories with diclofenac sodium 50 and 100 mg 9. Cortisone acetate tablets 0.025 g 10. Prednisolone tablets 0.001 g 11. Ethinylestradiol tablets 0.00001 g Average weight 0.056 g 12. Pregnin tablets 0.01 g Average weight 0.108 g 13. Pyridoxine tablets 0.002 g Average weight 0.205 g 14. Thiamine chloride tablets 0.002 g Average weight 0.212 g to compiling a method for quantitative analysis of drugs by UV spectrophotometric method 1. Justify the choice of method.
3. Solve the issue of pre-treatment.
4. Draw up methods for preparing a solution of the test drug and a solution of a standard sample.
5. Draw up a technique for spectrophotometric analysis.
6. Make a calculation formula for the content of the active substance.
The object of analysis is an injection solution with a low content of the drug. The latter circumstance requires the use of the most sensitive method in the quantitative analysis. These methods include UV spectrophotometry. In addition, this method does not require laborious and lengthy analytical operations.
spectrophotometric method is possible if there is a system of conjugated bonds in its structure.
electromagnetic radiation in the UV region due to the presence in the spectrophotometric method.
Sample calculation: the starting point for calculating the sample dosage form in its spectrophotometric analysis is which will be determined.
In the condition of the problem, the concentration of the solution of the working standard sample (RSO) CCO 0.00005 g ml is given.
The analyzed solution is 0.1%, therefore, it is possible to make a proportion:
0.1 g of adrenaline - 100 ml of solution The calculated sample can be increased 100 times. This will allow you to use a 5 ml pipette for its measurement, and a 100 ml volumetric flask for subsequent dilution.
carried out in a 0.1 M solution of hydrochloric acid. The choice of solvent is determined by ensuring the stability of the medicinal substance in its solution.
additional analytical operations to extract the substance from its dosage form are not required.
Methodology:
5 ml of a solution of adrenaline hydrochloride is placed in a volumetric flask with a capacity of 100 ml and the volume of the solution is brought to the mark with a 0.1 M solution of hydrochloric acid. Measure the optical density of the resulting solution on a spectrophotometer at an analytical wavelength in a cuvette with a layer thickness of 10 mm. In parallel, the optical density of the working standard solution (WRS) of epinephrine hydrochloride is measured.
As a reference solution, a 0.1 M solution of hydrochloric acid is used.
Calculation of results.
DX; DCO are the optical densities of the test solution and the RCO solution of adrenaline hydrochloride, respectively.
medicinal substances. M.: "Medicine", 1978. - 248 p.
"Medicine", 1975. - 151 p.
Belikov V.G. pharmaceutical chemistry. At 2 p.m. / V. G.
Belikov. - Pyatigorsk, 2003. - 720 p.
State Pharmacopoeia of the USSR. / Ministry of Health of the USSR. – 11th edition. - M.: Medicine, 1987. - Issue. 1. - 336 p.
State Pharmacopoeia of the USSR. / Ministry of Health of the USSR. – 11th edition. - M.: Medicine, 1989. - Issue. 2. - 400 s.
State Pharmacopoeia Russian Federation/ 12 - edition. - "Publishing house" NTsESMP ", 2008. - 704 p.
Kazitsina L.A., Kupletskaya N.B. Application of UV -, IR -, NMR - and mass - spectroscopy in organic chemistry. M., ed. Moscow un - ta, 1979. - 240 p.
Methods for drug analysis / N.P. Maksyutina and others - Kyiv:
Health, 1984. – 224 p.
Fundamentals of analytical chemistry. In 2 books. Book. 2. Methods of Yu.A. Zolotov. - 2nd ed. - M .: Higher. school; 2002. - 494 p.
Otto M. Modern methods analytical chemistry. / M.
Otto. - M.: Technosfera, 2006. - 416 p.
Pharmaceutical Chemistry: Textbook / Ed. A.P.
Arzamastsev. - M.: GEOTAR - MED, 2004. - 640 p.
FSP 42 - 0035225102 Ascorbic acid.
Introduction
1. Characteristics of spectroscopic methods of analysis
2. Basic law of light absorption Photometric quantities ............... 3. Characteristics of spectrophotometers
4. Characterization of the absorption spectra
5. Sample preparation for photometric determinations
6. Comparative characteristics of absorption methods
7. Application of spectrophotometry in pharmaceutical analysis ............... 7.1. Application of IR - spectroscopy in the analysis of drugs 7.2. Application of UV - spectrophotometry in the analysis of drugs
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Photometric (absorption) methods of analysis are based on the ability of the analyte to selectively absorb light.
The analysis of substances based on the measurement of light absorption includes spectrophotometry and photocolorimetry.
Spectrophotometry is based on the absorption of monochromatic light, i.e., light of a certain wavelength (1-2 nm) in the visible, ultraviolet and infrared regions of the spectrum.
This kind of light absorption measurement is carried out using spectrophotometers of various brands, which always use a monochromatic light energy flux obtained by means of an optical system called a monochromator.
Absorption in the ultraviolet (UV) and visible regions of the spectrum is associated mainly with the excitation of electrons.
The absorption of light in the infrared region of the spectrum (IR) is due to molecular vibrations.
Depending on the wavelength range at which the light absorption of solutions of chemical substances is measured, methods based on measuring light absorption are divided into spectrophotometry in the UV region of the spectrum with a wavelength range of 200-400 nm, spectrophotometry in the visible spectral region (400-760 nm) and spectrophotometry in the infrared region of the spectrum (760-20,000 nm). But usually the unit of measurement for the wavelengths of IR spectra is a micron (1 micron = = 10 -4 cm) or a wave number (cm -1), i.e., the number of waves in 1 cm.
In pharmaceutical analysis, spectroscopy in the UV and visible regions of the spectrum is more often used.
The UV spectroscopy method is included in the SP IX, SP X and MF II, as well as in the latest editions of the pharmacopoeias of almost all countries to determine the authenticity, purity and quantification of a substance in preparations.
The absorption spectrum or absorption spectrum is a graphical representation of the amount of light absorbed by a substance at certain wavelengths.
To construct a characteristic absorption curve - the magnitude of the wavelengths (R,) in UV spectroscopy or wave numbers (cm -1) in IR spectroscopy - is applied to the abscissa axis, and the repayment value (L) 1 or the percentage of transmission (G) (at IR spectroscopy) - on the y-axis (Fig. 5, 6).
When constructing curves for the extinction spectra in the UV and visible parts of the spectra, you can use the values of the specific extinction rates (Ј 1% i CM) or the molar absorption index (e) 2, where e is the optical density of a 1 M solution of a substance with a layer thickness in 1 cm; J 1% i CM - the value of the repayment of a solution containing 1 g of a substance in 100 ml of a solution with a layer thickness of 1 cm.
These quantities are determined experimentally; for many substances they are given in the literature.
A characteristic of the absorption spectrum is the position of the maxima (minimums) of the absorption of light by a substance, as well as the absorption intensity, which is characterized by the optical density (D) or specific absorption index (Ј 1% 1cm) at certain wavelengths.
UV spectrophotometric measurement is usually carried out in solutions. As solvents, distilled
bath water, acids, alkalis, alcohols (ethyl, methyl) and some other organic solvents.
The solvent should not absorb light in the same region of the spectrum as the substance under study. The nature of the spectrum can change in various solvents, as well as with a change in the pH of the medium.
The factors that determine the absorption of light by the substances under study are the presence in their molecules of the so-called
Each functional group in a molecule of a substance is characterized by the absorption of light in a certain region of the spectrum, which is used for the purposes of identifying and quantifying the substance in the preparation.
In addition to chromophores, the molecule can include functional groups that do not absorb in the near ultraviolet themselves, but can affect the behavior of the chromophore associated with them. Such groups, called auxochromes, usually cause absorption at longer wavelengths and with a higher extinction factor than is typical for a given chromophore. Examples of auxochromes: -SH, -NH 2, -OH.
IR spectra for most organic compounds, unlike UV spectra, are characterized by the presence of a larger number of absorption peaks (see Fig. 6). Therefore, the PC spectroscopy method makes it possible to obtain the most complete information about the structure and composition of the analyte, which makes it possible to identify compounds that are very similar in structure.
In SP X and MF II, the IR spectroscopy method was adopted for the identification of many organic medicinal substances with polyfunctional groups in their molecules by comparison with the spectra of standard samples taken under the same conditions. In original literature recent years are given! IR spectra of antibiotics, hormones, coumarins and many other medicinal substances of organic nature. In connection with the increasing requirements for the quality of drugs, IR spectroscopy, as one of the reliable methods of identification, is becoming increasingly important.
STATE EDUCATIONAL INSTITUTION OF HIGHER PROFESSIONAL EDUCATION
"KUBAN STATE MEDICAL UNIVERSITY"
MINISTRIES OF HEALTH OF THE RUSSIAN FEDERATION
CHAIR OF PHARMACY
COURSE WORK IN PHARMACEUTICAL CHEMISTRY
Topic: " Preparations of estrogen hormones and their synthetic analogues.
Pharmaceutical analysis.
Performed:
student
pharmaceutical
faculty
V course, 2 groups
Buslaeva N.P.
Checked:
teacher, Ph.D.
ON THE.
Krasnodar city,
2014
Introduction ................................................ ................................................. .............3
Chapter I. Theoretical part............................................... ..................................5
- Classification of representatives of estrogen hormone preparations
And their synthetic analogues .............................................................. ......................5
- Physical properties used for
establishing the good quality of drugs .............................................. 8
- Chemical methods of identification .................................................................. .......ten
- Purity Test Methods .................................................................. ................fourteen
- Chemical Quantification Methods...............................................15
- Physical and physico-chemical methods of quantitative
definitions ................................................. ..............................................19
- Storage conditions of medicines, use and
release form .................................................................. .........................................twenty
Chapter II. Experimental part................................................ ...................21
- Application of spectrophotometry in the UV region of the spectrum
in the analysis of diethylstilbestrol and sinestrol in tablets of 0.001 ..... 21
Conclusion................................................. ................................................. .......29
Bibliography................................................ ...............................................31
Introduction
The breakthrough of the medical scientific community in solving the problem of increasing the average life expectancy of a woman and improving the quality of life was the use of estrogen hormone replacement therapy in medical practice.
Estrogen hormones in the female body are produced in the ovarian follicles.
Estrogen-containing drugs have been used since the 40s of the last century to correct estrogen-deficient conditions caused by age-related or surgical “turning off” of ovarian function.
Estrogens belong to the group of steroid hormones and are derivatives of the hydrocarbon estran:
There are three natural estrogenic hormones: estrone, estradiol and estriol:
For a long time, the natural hormone estrone has been used in medicine in the form of oily solutions. Estradiol has twice the activity, but due to rapid inactivation, it has not been used. Subsequently, it was proved that estradiol esters are more stable substances than estrone. In addition, they have a prolonged action.
Of the semi-synthetic analogues of estradiol, ethinylestradiol, mestranol and estradiol dipropionate are used as medicines. Ethinyl estradiol and mestranol are characterized by the presence of an ethynyl radical in position 17 in the molecule, which led to an increase in estrogenic activity several times compared to estrone and its preservation during oral administration.
Substances with estrogenic activity were found not only among steroids, but also in a number of aromatic compounds, in particular, phenanthrene derivatives, diphenyl derivatives, and others. It is assumed that the estrogenic effect depends on the presence of aromatic nuclei in the molecule.
Synthetic analogues of non-steroidal estrogens used in medical practice include sinestrol and diethylstilbestrol.
Thus, the effectiveness of the use of estrogen drugs in the correction of the hormonal status of patients, proven by many years of world clinical practice, determines the relevance of studying the properties and characteristics of the pharmaceutical analysis of drugs in this group.
Chapter I . Theoretical part
1. Classification of representatives of estrogen hormone preparations and their synthetic analogues
Natural estrogens (follicular hormones) are derivatives of the hydrocarbon estran.
The most important representatives are estrone (Fig. 1) and estradiol (Fig. 2).
Rice. 1. Structural formula of estrone
(3-hydroxyestratri-1, 3, 5(10)-en-17-one)
Rice. 2. Structural formula of estradiol
(3, 17β-dihydroxyestratri-1, 3, 5(10)-ene)
Unlike androgens, in the molecules of estrone and estradiol, the A ring is aromatic, and there is no angular methyl group at carbon 10.
Of the semi-synthetic analogues of estradiol, ethinyl estradiol (Fig. 3), estradiol dipropionate (Fig. 4) and mestranol (Fig. 5) are used.
Rice. 3. Structural formula of ethinyl estradiol
(17α-ethynylestratriene-1,3,5-diol-3,17β)
Rice. 4. Structural formula of estradiol dipropionate
(estratriene-1,3,5(10)-diol-3,17β dipropionate)
Rice. 5. Structural formula of mestranol
(17α-ethynylestratrien-1,3,5-diol-3,17β-3-methyl ester)
At present, a number of nonsteroidal estrogens, such as sinestrol (Fig. 6) and diethylstilbestrol (Fig. 7), have been synthesized.
Rice. 6. Structural formula of sinestrol
(meso-3,4-bis-(p-oxyphenyl)-hexane)
Rice. 7. Structural formula of diethylstilbestrol
(trans-3,4-bis-(p-oxyphenyl)-hexene-3)
2. Physical properties used to establish the good quality of drugs
According to physical properties, estradiol derivatives are white or slightly creamy crystalline substances. Practically insoluble in water, slightly or moderately (ethinylestradiol) soluble in chloroform, moderately or slightly soluble (ethinylestradiol) in ethanol. Estradiol dipropionate is moderately and slowly soluble in vegetable oils. Estradiol derivatives have four asymmetric carbon atoms in the molecule, that is, they differ from each other and from other steroid hormones in specific rotation.
Synthetic estrogens sinestrol and diethylstilbestrol are white, odorless crystalline powders in terms of their physical properties. Practically insoluble in water, freely soluble in ethanol and ether, slightly soluble in chloroform. Sinestrol is slightly soluble in peach and olive oils.
To physical and physico-chemical methods of identification based on the use physical properties This group of drugs includes:
- Melting point determination:
- t sq. (ethinylestradiol) = 181-186 ° C;
- t sq. (mestranol) = 149-154 ° C;
- t sq. (estradiol dipropionate) = 104-108 ° C;
- t sq. (sinestrol) = 184-187 ° C;
- t sq. (diethylstilbestrol) = 168-174 °C.
- Determination of the specific angle of rotation:
- 0.4% solution in pyridine for ethinylestradiol = -27 to -31°;
- 2% solution in chloroform for mestranol = + 2 to + 8°;
- 1% solution in dioxane for estradiol dipropionate = + 37 to 41 °.
- UV and IR spectroscopy:
- The UV absorption spectrum of a solution of ethinylestradiol in a mixture of ethanol and sodium hydroxide in the region of 220-330 nm has absorption maxima at 241 and 299 nm and absorption minima at 226 and 271 nm, and a solution in ethanol has an absorption maximum at a wavelength of 280 nm.
- Estradiol dipropionate - 0.01% solution in ethanol, which in the region of 220-235 nm should have two absorption maxima at 269 and 276 nm.
- The authenticity of ethinyl estradiol, mestranol and estradiol dipropionate is confirmed by IR spectra taken in vaseline oil in the region of 4000 - 200 cm-1 .
- In an ethanol solution in the region of 230-250 nm, a 0.005% solution of sinestrol has absorption maxima at 280 nm, a minimum at 247 nm and a shoulder at from 283 nm to 287 nm,
- 0.01% diethylstibestrol solution - absorption maximum at 242 nm and shoulder at 276 to 280 nm.
3. Chemical identification methods
General group reactions to the steroid core:
- Under the action of concentrated sulfuric acid, the solution in the presence of ethinylestradiol acquires an orange-red color with yellowish-green fluorescence, after adding the resulting solution to 10 ml of water, the color changes to purple and a purple precipitate precipitates.
- Mestranol with concentrated sulfuric acid forms a blood-red color with a yellowish-green fluorescence.
Private identification reactions:
- Acid hydrolysis under the action of concentrated sulfuric acid of estradiol dipropionate with the formation of estradiol and propionic acid:
Estradiol dipropionate estradiol
Subsequent heating in the presence of ethanol leads to the formation of propionic acid ethyl ester, which has a characteristic odor:
C 2 H 5 -COOH + C 2 H 5 OH \u003d C 2 H 5 -COO-C 2 H 5 + H 2 O
- The presence of phenolic hydroxyl in the ethinylestradiol molecule is confirmed by the reaction of the formation of ethinylestradiol benzoate, which has t sq. \u003d 199-202 ° С.
Also, according to the reaction of the formation of an azo dye with diazotized sulfanilic acid:
A dark red solution is formed.
- The presence of unsubstituted phenolic hydroxyls in the molecules of sinestrol and diethylstilbestrol can be detected using ferric chloride ( III ). Alcohol solutions of diethylstilbestrol are stained in green color, gradually turning into yellow.
- The reaction is the formation of bromine derivatives of sinestrol: under the action of bromine water, a yellow precipitate of tetrabromosinestrol is released on its solution in glacial acetic acid:
Diethylstilbestrol, when performing the same reaction in the presence of liquid phenol, acquires an emerald green color that appears when heated.
- Sinestrol nitration reaction: when nitric acid is added and heated in a water bath, a yellow color gradually appears:
- When concentrated sulfuric acid acts on a chloroform solution of sinestrol in the presence of formalin, the chloroform layer turns cherry red. A solution of diethylstilbestrol in concentrated sulfuric acid has a bright orange color, which gradually disappears after dilution with water.
- Heating diethylstilbestrol with acetic acid and vanillin followed by addition of hydrochloric acid, boiling and addition of chloramine (after cooling) produces a blue color
- Solution of diethylstilbestrol in glacial acetic acid after addition phosphoric acid and heated in a water bath acquires an intense yellow color, which almost disappears when diluted with glacial acetic acid.,
4. Test methods for purity
Impurities of extraneous steroids in preparations of estrogen hormones are determined by TLC on Silufol UV-254 plates. The SOVS of estrone, estradiol, etc. are used as witnesses. The total content of steroid impurities is allowed - no more than 2%, incl. ethinyl estradiol contains no more than 1% estrone.
The presence of impurities in synthetic analogues of non-steroidal estrogens is determined by TLC on plates with a layer of silica gel or on Silufol UV-254 by the ascending method, using the solvent system benzene-hexane-acetone (sinestrol) or chloroform-methanol (diethylstilbestrol). The developer is phosphomolybdic acid.
In diethylstilbestrol, the presence of an impurity of 4,4 - dihydroxystilbene and related esters is determined by the optical density (not more than 0.5) of a 1% solution in ethanol at 325 nm.
5. Chemical methods of quantitative determination
- For the quantitative determination of estradiol dipropionate, an alkaline hydrolysis reaction is used with an accurately measured amount of a 0.1 M alcoholic solution of potassium hydroxide, the excess of which is titrated with 0.1 M hydrochloric acid. The indicator is phenolphthalein.
KOH + HCl \u003d KCl + H 2 O
- The quantitative determination of sinestrol in the substance is performed by the method of indirect neutralization. Acetic anhydride in pyridine is added to the substance of sinestrol, when heated, a diacetylated derivative of sinestrol (ester) is obtained. The excess of acetic anhydride, which has turned into acetic acid, is titrated with 0.5 M sodium hydroxide solution. Phenolphthalein indicator. In parallel, a control experiment is performed with the same amount of acetic anhydride.
A similar process occurs in the determination of diethylstilbestrol.
- Sinestrol can also be quantified by the reverse bromide-bromatometric method. Bromine, released due to the interaction of a 0.1 M solution of potassium bromate and potassium bromide, precipitates sinestrol in the form of a tetrabromo derivative. The titrant excess is determined by the iodometric method:
- Ethinylestradiol is quantified by an indirect neutralization method. Purified tetrahydrofuran is used as the solvent. released after the addition of silver nitrate nitric acid titrated with 0.1 M sodium hydroxide solutions. The equivalence point is determined potentiometrically with a glass electrode. Ethinylestradiol forms a double salt with silver nitrate, which consists of the silver salt of ethinylestradiol and six molecules of silver nitrate. [ 3]
An example of the quantitative determination of sinestrol by the titrimetric method:
Give a conclusion on the quality of sinestrol (M.m. = 270.37 g / mol) in terms of quantitative content, taking into account the requirements of the Global Fund X (there must be at least 98.5% sinestrol in the substance), if 5 ml of a solution of acetic anhydride in anhydrous pyridine is taken for acetylation for acetylation, and 17.60 ml of acetic anhydride is used to titrate the excess of acetic anhydride and the released acetic acid, 17.60 ml of 0, 5 mol/l sodium hydroxide solution with K = 1.0013. 24.88 ml of titrant solution was used for the control experiment.
Method for indirect non-aqueous alkalimetric determination of sinestrol.
Chemistry of reactions:
After the acetylation reaction, unreacted acetic anhydride undergoes hydrolysis to form acetic acid:
2CH 3 COOH + 2NaOH = 2CH 3 COONa + 2H 2 O
K steh. = 2:2 = 1:1 = 1. The titrant solution is prepared from real particles.
But 1 mol of sinestrol interacts with 2 mol of acetic anhydride.
Therefore, F equiv. = 1:2 = ½.
M.e. (sinestrol) = ½ × M.m. (sinestrol) = ½ × 270.37 g/mol = 135.185 g/mol eq.
T = M.e. ×C / 1000 \u003d 135.185 g / mol equiv × 0.5 mol / l / 1000 \u003d 0.06759 g / ml.
C \u003d (V control. × K 1 - V × K 2) × T × 100% / a \u003d (24.88 ml × 1 - 17.60 ml × 1.0013) × 0.06759 g / ml × 100% / 0.4988 g \u003d 98.38%.
Conclusion: the sinestrol substance in terms of the quantitative content of sinestrol does not meet the requirements of the RD, since the content is below the standard - it must be at least 98.5%.
6. Physical and physico-chemical methods of quantitative analysis
- The photocolorimetric determination of ethinyl estradiol is based on the use of a diazo reagent (a mixture of sulfanilic acid, sodium nitrite and hydrochloric acid). In an alkaline medium, a biazo derivative of ethinyl estradiol is formed, which is colored red. As a reference solution, a solution of the same derivative with a known concentration and a known optical density is used.
- Sinestrol and diethylstilbestrol can also be quantified photoelectrocolorimetrically by the red-colored product of the biazo coupling with diazotized sulfanilic acid.
7. Storage conditions of medicinal products, use and forms of release
Estradiol derivatives are stored according to list B. Ethinylestradiol is stored in well-closed orange glass jars, and mestranol and estradiol dipropionate are stored in a dry, dark place.
Used as an estrogen. Given the prolonged action of estradiol dipropionate, it is administered intramuscularly in 1 ml of a 0.1% solution in oil 2-3 times a week. Ethinylestradiol is administered orally in the form of tablets of 0.00001 and 0.00005 g.
Mestranol is one of the components of Infecundin tablets, an active oral contraceptive containing 0.0001 g of mestranol and 0.0025 g of norethinodrel.
Ethinylestradiol is part of such contraceptives as "Marvelon", "Non-ovlon", "Ovidon", used in the form of tablets.
Synthetic estrogen preparations are stored according to list B, in a well-closed container, protected from light.
By pharmacological action, they are close to natural estrogenic hormones. When administered orally, they are not destroyed in the gastrointestinal tract, are rapidly absorbed. Assign inside in the form of tablets of 1 mg and intramuscularly in the form of oily solutions of 0.1% and 2-3% concentration. Solutions of high concentration (2-3%) are prescribed in the treatment of malignant neoplasms.
Chapter II . experimental part
1. Application of spectrophotometry in the UV region of the spectrum
in the analysis of diethylstilbestrol and sinestrol in tablets of 0.001
Absorption UV spectrophotometry is based on measuring the amount of absorbed electromagnetic radiation in a certain narrow wavelength region.
Usually, approximately monochromatic radiation in the region from 190 to 380 nm is used for UV measurements.
Basic concepts
Absorption (It ) is the decimal logarithm of the reciprocal of the transmittance ( J ). The GF uses the terms "optical density"(D), as well as "extinction" (E).
Transmittance (J ) is the quotient of dividing the intensity of the light passing through the substance by the intensity of the light falling on the substance.
absorbency (a t ) - frequent from the division of absorption ( D ) on the concentration of the substance (C), expressed in grams per liter, and the length of the absorption layer in centimeters(L):
In pharmacopoeias, the term "specific absorption rate" is more often used.when the concentration (C) is expressed in grams per 100 ml; thus = 10 × a t .
Molar extinction rate (ε) is the absorption quotient ( I t ) on the concentration of the substance (C), expressed in moles per liter, and the length of the absorption layer in centimeters.
Absorption spectrum is a graphical expression of the ratio of absorption (or any function) to wavelength (or any function of wavelength).
Devices. The Pharmacopoeia does not indicate the specific types of instruments recommended for performing measurements. In our country, both domestic and imported devices are used. To ensure the uniformity of measurements, it is recommended that the specified operating conditions be strictly adhered to during operation of the device. It is especially important to provide metrological services for instruments with regard to their calibration both on the wavelength scale and on the photometric scale. This service is usually carried out by the relevant state metrological organizations.
Factors affecting the reproducibility and correctness of the results.
To obtain reliable data, it is necessary to strictly follow the instructions for the care of the device and its operation, pay attention to such factors as the accuracy of the cuvette thickness and their spectral transmittance.
The cuvettes used for test and control solutions must be identical and have the same spectral transmittance if they contain only one solvent. Otherwise, an appropriate amendment must be made.
Particular attention should be paid to the cleanliness of the cuvettes. Do not touch the outer surfaces of the cuvette with your fingers, they should not get liquid (solvent or test solution). Consideration should also be given to possible limitations associated with the use of solvents.
The sensitivity of the method is determined mainly by the ability of the substance to absorb and is expressed, as mentioned above, by the molar absorption coefficient. The limiting concentrations of substances analyzed by spectrophotometry are usually lower than in titrimetric or gravimetric methods. This explains the use of spectrophotometry in the determination of small amounts of substances, especially in various dosage forms.
The main condition for quantitative analysis is the observance of the law of Bouguer - Lambert - Beer within the limits of the corresponding concentrations. To check compliance with the law, plot the dependence (absorption - wavelength) or calculate the factor for each standard solution and determine the concentration range within which the A / C value remains constant.
Two fundamentally different methods of spectrophotometric quantitative determinations exist and are used. According to one of them, the content of the substance in percent(From research ) is calculated on the basis of a pre-calculated absorption value, more often from the value of E 1% 1cm.
Where
V - breeding, ml. See above for other designations.
The main drawback of this definition is a well-known fact: different spectrophotometers (even different instruments of the same model and production) give significant deviations in absorbance for the same standard solution.
More reliable and reproducible results are provided by comparing the absorbance of the test substance with the absorbance of a standard sample determined under the same conditions. This takes into account numerous factors that affect spectrophotometric measurements, such as wavelength setting, slit width, cuvette and solvent absorption, etc.
Spectrophotometric quantitative determination of the content of a drug substance in the analysis of individual substances should be associated with the use of a specially prepared standard sample of this substance.
Standard Samples- are substances thatcompare the tested medicinal products in their analysis using physico-chemical methods. These samples are subdivided into State standard samples (GSO) and working standard samples (RSO). GSO is an especially pure sample of the substance of the medicinal substance.
The release of the GSO is carried out in accordance with the pharmacopoeial article. A pharmacopoeial monograph on the GSO is developed and revised by enterprises (organizations) that produce or develop medicinal products, and is agreed with the State research Institute for Standardization of Medicines and approved in the prescribed manner.Samples of serial medicinal substances that meet the requirements of a pharmacopoeial article are used as RSO. When calculating the quantitative content of the analyte in the dosage form, the actual content of this substance in the RSO is taken into account.
Determination of the substance content at
using a standard sample
Calculation of the quantitative content of an individual substance in percent(X ) when using a standard sample is carried out according to the formula:
If the concentration of the PCO standard solution is expressed as a percentage (C std. = %), then the formula for calculating the content in g has the form:
If we know the optical density of the standard drug solution and calculate the specific absorption rate of the drug solution, we can also calculate the content of the drug in the tablet (in grams), considering the average weight of the tablet:
The concentration of the solution using a standard sample of the medicinal substance (in%) is expressed by the formula:
Where
V 1 is the volume of the first dilution, ml;
V 2 is the volume of the second dilution, ml.
See above for other designations.
For substance, g:
For solid dosage forms (tablets, dragees), g:
Where
100 - conversion factor.
Example #1
Do the sinestrol tablets satisfy the requirements of the FS in terms of quantitative content, if for a solution obtained by dissolving 0.3005 g of powder of crushed tablets in ethyl alcohol in a volumetric flask with a capacity of 100 ml, the optical density is 0.550, for a solution of sinestrol GSO with a content of 0.00003 g / ml , the optical density is 0.560 (ɣ=280 nm, in a 1 cm layer). The content of sinestrol should be 0.0009 - 0.0011 g based on the average tablet weight (P = 0.101 g).
× a = 0.550 × 0.00003 g/ml × 100 ml × 0.101 g / 0.560 × 0.3005 g = 0.00099 g ≈ 0.001 g
Example #2
Assess the quality of 0.001 g sinestrol tablets if the following results are obtained during spectrophotometric determination (ɣ=280 nm): standard solution absorbance = 0.385, standard solution concentration 0.00003 g/ml, test solution absorbance = 0.392. The mass of powder of crushed tablets 0.3204 g was dissolved in 100 ml of absolute ethyl alcohol. Calculate the content in g based on the average tablet weight (20 tablets weigh 2.040 g). According to the FS, it should contain 0.0009 - 0.0011 g in terms of one tablet.
First, calculate the average weight of one tablet:
2.040 g / 20 = 0.102 g.
From research = D research. × With std. × V × P / D std. × a = 0.392 × 0.00003 g/ml × 100 ml × 0.102 g / 0.385 × 0.3204 g = 0.00097 g ≈ 0.001 g
Conclusion: according to the quantitative content of sinestrol in tablets of 0.001 g, they meet the requirements of the Federal Assembly.
Conclusion
modern science and society dictate fundamentally new requirements for the entire healthcare system, in particular, for the sector of pharmaceutical preparations.
On the one hand, the value of health is growing in the system of society's priorities, and new medical, technological and social challenges are emerging associated with changes in the demographic structure of the population. On the other hand, thanks to the development of medical technologies, the ability to really influence the health of the population is significantly increased, as evidenced by the significant success in the fight against the most life-threatening diseases achieved in Western countries over the past 2-3 decades.
It should also be taken into account that the global global trend is the continued growth in the consumption of medicines, which is associated, on the one hand, with an increase in the standard of living of the population, and on the other hand, with its aging.
Promising direction in relation to estrogenic drugs is the improvement of existing and the development of new methods for obtaining semi-synthetic and synthetic analogues of estrogenic hormones.
The great advantage of synthetic estrogens is the availability of their synthesis due to the simplicity of the chemical structure. The formation of ethers and esters does not reduce the activity of estrogen, but increases the duration of action.
It is assumed that the estrogenic effect depends on the presence of aromatic nuclei in the molecule. Important role belongs to hydroxyl and ketone groups capable of forming hydrogen bonds and interacting with proteins in the body.
Estrogen hormone preparations are used to treat a large number serious pathologies, including malignant neoplasms.
Hormonal contraception, which has gained wide popularity in the last decade, is also based on the widespread use of estrogenic hormone preparations in its composition.
The study of the features of the pharmaceutical analysis of this group of drugs confirms the advantages of using physicochemical methods, namely UV spectrophotometry, which allows the identification of substances, the presence of impurities and the quantitative determination of steroidal estrogens and their synthetic analogues of a non-steroidal structure.
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