Bik analysis. Applications of near-infrared spectroscopy. Revalidation or re-validation
As a manuscript
DOLBNEV DMITRY VLADIMIROVICH
IDENTIFICATION OF MEDICINES BY NEAR INFRARED SPECTROSCOPY
04/14/02 – pharmaceutical chemistry, pharmacognosy
dissertations for an academic degree
candidate of pharmaceutical sciences
Moscow – 2010
The work was carried out at the State Educational Institution of Higher Professional Education First Moscow State Medical University named after
Scientific supervisors:
Doctor of Pharmaceutical Sciences, Academician of the Russian Academy of Medical Sciences, Professor
Doctor of Pharmaceutical Sciences, Professor
Official opponents:
Lead organization:
All-Russian Scientific Center for the Safety of Biologically Active Substances (VSC BAV)
The defense will take place “___”____________________2010 at ____ o’clock at a meeting of the Dissertation Council (D 208.040.09) at the First Moscow State Medical University named after Moscow, Nikitsky Boulevard, 13.
The dissertation can be found in the library of Moscow State Medical University named after. Moscow, Nakhimovsky prospect, 49.
Scientific secretary of the dissertation
council D 208.040.09
Doctor of Pharmaceutical Sciences,
Professor
Relevance of the research topic. Over the past 15 years, near-infrared (NIR) spectroscopy has been rapidly developing and has found application in a wide variety of industries. NIR spectroscopy is known as an effective method for qualitative and quantitative analysis. This method is widely used in agriculture (to determine the quality of soils, the content of protein, fat, etc. in food products), in industry (to determine the composition of petroleum products, the quality of textile products, etc.), in medicine (to determine fat, oxygen in the blood, studies of tumor development). Currently, NIR spectroscopy is becoming one of the in-process control methods in the pharmaceutical industry in Europe and the USA.
It is used to test input raw materials, mixing uniformity, determining granulation end point, drying moisture content, tableting uniformity, and measuring coating thickness.
The NIR spectroscopy method is described in the European Pharmacopoeia and the US Pharmacopoeia, but it is still used relatively rarely in pharmacopoeial analysis: mainly when determining the water content in preparations obtained from blood.
In this regard, the development of unified methods for the analysis of pharmaceutical substances and drugs for their further use in pharmacopoeial analysis is of great importance.
This issue is of particular importance in connection with the publication of the 12th edition of the State Pharmacopoeia of the Russian Federation.
It is also necessary to note the ongoing problem of counterfeit medicines, one of the ways to solve which is the development of rapid analysis methods.
Considering the above, an urgent problem is the development of unified methods for analyzing substances and preparations and identifying counterfeit medicines using the NIR spectroscopy method.
Purpose and objectives of the study. The purpose of the study was to develop unified methods for analyzing substances and preparations and identifying counterfeit medicines using the NIR spectroscopy method.
To achieve this goal, the following tasks were solved:
– to study the possibility of obtaining NIR spectra of substances, tablets and capsules using a fiber optic sensor and an integrating sphere;
– compare the NIR spectra of substances and drugs;
– compare the NIR spectra of drugs with different contents of the active substance;
– study the possibility of using NIR spectroscopy to establish the authenticity of substances and preparations from specific manufacturers, as well as to identify counterfeit medicines;
– develop an electronic library of NIR spectra of substances and drugs.
Scientific novelty of the research results. For the first time, it has been shown that the NIR spectroscopy method can be used both to determine the authenticity of pharmaceutical substances and finished medicinal products (tablets and capsules). It has been shown that, in general, the NIR spectra of substances and drugs differ. Spectra can be obtained using a fiber optic sensor and an integrating sphere. It has been shown that if the capsule shell or tablet packaging (blister) is transparent, a spectrum can be obtained without removing the capsules or removing the tablets from the packaging. It has been shown that the NIR spectroscopy method can be used to identify counterfeit drugs, provided that the spectra of the original and test drugs are compared. Spectra of substances and drugs can be stored as an electronic library. It has been established that for a more reliable comparison of the spectrum of the test drug and the standard spectrum, the use of mathematical data processing is required.
Practical significance of the work. Developed methods for analyzing drugs using NIR spectroscopy are proposed to establish the authenticity of pharmaceutical substances, drugs in the form of tablets and capsules. The techniques allow the use of an integrating sphere and a fiber optic sensor (“gun”).
The developed methods can also be used for express identification of counterfeit medicines and for incoming and outgoing control of pharmaceutical substances and intermediates at pharmaceutical enterprises. The methods allow in some cases to carry out non-destructive quality control without opening the primary packaging.
The developed library of NIR spectra can be used to identify substances, tablets and capsules using a fiber optic sensor (“gun”) and an integrating sphere.
The results of the work have been tested and used in the quality control department.
Approbation of work. The main provisions of the dissertation work were reported and discussed at the XII Russian National Congress “Man and Medicine” (Moscow, 2005), the International Congress on Analytical Chemistry ICAS (Moscow, 2006) and the XIV Russian National Congress “Man and Medicine” (Moscow , 2007). The work was tested at a scientific and practical meeting of the Department of Pharmaceutical Chemistry with the course of toxicological chemistry of the Faculty of Pharmaceutical Sciences of Moscow State Medical University. March 22, 2010
Publications. 5 printed works have been published on the topic of the dissertation.
Linking research to the problem design of pharmaceutical sciences. The dissertation work was carried out within the framework of a complex topic of the Department of Pharmaceutical Chemistry of Moscow State Medical University named after. “Improving quality control of medicines (pharmaceutical and environmental aspects)” (state registration No. 01.200.110.54.5).
Structure and scope of the dissertation. The dissertation is presented on 110 pages of typewritten text, consists of an introduction, a literature review, 5 chapters of experimental studies, general conclusions, a list of references, and also separately includes 1 appendix. The dissertation work is illustrated with 3 tables and 54 figures. The list of references includes 153 sources, of which 42 are foreign.
Provisions for defense:
– results of studying the possibility of obtaining NIR spectra of substances, tablets and capsules using a fiber optic sensor and an integrating sphere;
– results of a comparative study of NIR spectra of substances and drugs, as well as NIR spectra of drugs with different contents of the active substance;
– results of studying the possibility of using NIR spectroscopy to establish the authenticity of substances and preparations from specific manufacturers, as well as to identify counterfeit medicines.
1. Objects of study
Substances and preparations of a number of drugs have been studied. A total of 35 substances were used in the study: aluminum hydroxide, amikacin sulfate, ascorbic acid, sodium ascorbate, warfarin sodium, vitamin B12, gemfibrozil, magnesium hydroxide, glurenorm, D-biotin, iron gluconate, zopiclone, calcium D panthenoate, clindamycin phosphate, lidocaine hydrochloride , metoprolol tartrate, nicotinamide, paracetamol, pyridoxine hydrochloride, piperacillin, ranitidine hydrochloride, riboflavin, thiamine mononitrate, tyrothricin, famotidine, folic acid, cefadroxil, cefazolin sodium salt, ceftizoxime sodium salt, ciprofloxacin hydrochloride, cyanocoblamine, various manufacturers and 59 drugs from various manufacturers containing: isoniazid, meloxicam, omeprazole, ranitidine hydrochloride, rifampicin, famotidine, ciprofloxacin, esomeprazole, ethambutol, as well as 2 falsified samples (OMEZ 20 mg, Dr. Reddy`s Lab. and Rifampicin 150 mg,).
2. Equipment and test conditions
An MPA device was used in the work - a near-infrared Fourier spectrometer (Bruker Optics GmbH, Germany). Recording parameters: spectral range from 800 nm to 2500 nm (cm-1 to 4000 cm-1), number of scans 16, spectral resolution 4 cm-1. The instrument was controlled and the obtained spectra were processed using the OPUS 6.0 software package (Bruker Optics GmbH, Germany). NIR spectra were obtained in two ways:
1) using a fiber optic sensor (“gun”),
2)
Both methods were used to obtain NIR spectra of substances, tablets and capsules.
The fiber optic sensor (“gun”) allows for reflection measurements only, while the integrating sphere allows both reflection and transmission measurements. In this work, NIR reflectance spectra were obtained.
2.1. Methods for obtaining NIR spectra:
using a fiber optic sensor (“gun”).
2.1.1. Substances . The powder substance was poured into a transparent cuvette with a layer thickness of 1 to 3 cm. Then the fiber-optic sensor was pressed perpendicular to the surface of the powder. The spectrum registration procedure was started by pressing a button on the fiber optic sensor. The measurement of the spectra was repeated 3–5 times from different areas to obtain statistically reliable analysis results.
2.1.2. Tablets removed from the blister . The fiber optic sensor was pressed perpendicular to the tablet. The spectrum registration procedure was started by pressing a button on the fiber optic sensor. The measurement of the spectra was repeated 3–5 times from different areas of the tablet to obtain statistically reliable analysis results.
2.1.3. Tablets in blister . If the blister is transparent, the measurement was carried out as follows, the fiber optic sensor was pressed perpendicular to the surface of the tablet in the blister. The spectrum registration procedure was started by pressing a button on the fiber optic sensor. The measurement of the spectra was repeated 3–5 times from different areas of the tablet in the blister to obtain statistically reliable analysis results. If the blister was opaque or aluminum, the tablet was first removed from the blister and then the NIR spectrum was obtained.
2.1.4. Capsules . If the capsule shell is transparent, then the measurement was carried out as follows: the fiber-optic sensor was pressed perpendicular to the surface of the capsule in the blister. The spectrum registration procedure was started by pressing a button on the fiber optic sensor. The measurement of the spectra was repeated 3 - 5 times from different parts of the capsule in the blister to obtain statistically reliable analysis results. If the capsule shell was not transparent, then the capsule was first opened and then the spectrum of the contents was measured in a glass cuvette.
2.2. Methods for obtaining NIR spectra:
using an integrating sphere.
Obtaining NIR spectra in reflection mode
2.2.1. Substances . The powder substance was poured into a transparent cuvette with a layer thickness of 1 to 3 cm. Then the cuvette was placed on top of the optical window of the integrating sphere. The measurement process was started on a computer using the OPUS program or directly on the device itself (the “Start” button). The measurement of the spectra was repeated 3–5 times to obtain statistically reliable analysis results.
2.2.2. Tablets removed from the blister . The tablet was placed in a special holder. A holder with a tablet was installed on top of the optical window of the integrating sphere. The measurement process was started on a computer using the OPUS program or directly on the device itself (the “Start” button). The measurement of the spectra was repeated 3–5 times from different areas of the tablet to obtain statistically reliable analysis results.
2.2.3. Capsules . If the capsule shell is transparent, then the measurement was carried out as follows: the capsule was placed in a special holder. A holder with a capsule was installed on top of the optical window of the integrating sphere. The measurement process was started on a computer using the OPUS program or directly on the device itself (the “Start” button). The measurement of the spectra was repeated 3–5 times from different parts of the capsule to obtain statistically reliable analysis results. If the capsule shell was not transparent, then the capsule was first opened, and then the spectrum of the contents in a glass cell was measured by placing the cell on top of the optical window of the integrating sphere.
3. Mathematical processing of NIR spectra.
Mathematical processing of the obtained spectra was carried out using the OPUS IDENT program, included in the OPUS 6.0 software package (Bruker Optics GmbH, Germany). The unknown spectrum was compared with the reference library spectrum by calculating the spectral distance. IDENT identifies those comparison spectra that are closest to the analyzed spectrum and determines the deviations between these spectra and the analyzed spectrum. This allows IDENT to identify unknown substances and assess the degree to which the substance meets the reference standard.
We used two methods of mathematical processing of NIR spectra: 1) Ident analysis, which correlates the spectrum and a specific substance, and 2) cluster analysis, which correlates the spectrum and a group of substances.
Once the spectra are measured, an average spectrum of each material is generated and a library of all such average spectra is created, along with statistically determined acceptance criteria (or thresholds) for all substances in the library. The test spectrum was compared with all reference spectra located in the electronic library. The result of the comparison between spectra A and B ends with the output of the spectral distance D, which is called the “match quality factor” in the IDENT program. Spectral distance indicates the degree of spectral similarity. Two spectra with a spectral distance equal to zero are completely identical. The greater the distance between two spectra, the greater the spectral distance. If the spectral distance is less than the threshold for one substance and greater than the threshold for all other substances, the unknown substance is identified.
Cluster analysis allows you to examine NIR spectra for similarity and divide similar spectra into groups. These groups are called classes or clusters. This type of analysis was carried out for a more convenient presentation of data in graphical form.
Hierarchical cluster algorithms are performed according to the following scheme:
First, calculate the spectral distances between all spectra,
· then the two spectra with the highest similarity are merged into a cluster,
· calculate the distances between this cluster and all other spectra,
· two spectra with the shortest distance merge again into a new cluster,
· calculate the distances between this new cluster and all other spectra,
· two spectra merge into a new cluster
This procedure is repeated until only one large cluster remains.
4 . Research results
The possibility of using the NIR spectroscopy method to identify substances and drugs from a number of domestic and foreign manufacturers has been studied.
As a result of the research, six different electronic libraries of NIR spectra were created:
1) NIR spectra of capsule contents obtained using a fiber optic sensor (“gun”),
2) NIR spectra of capsule contents obtained using an integrating sphere,
3) NIR spectra of tablets obtained using a fiber optic sensor (“gun”),
4) NIR spectra of tablets obtained using an integrating sphere,
5) NIR spectra of substances obtained using a fiber optic sensor (“gun”),
6) NIR spectra of substances obtained using an integrating sphere.
4.1. Dependence of the NIR spectra of substances and preparations on the method of preparation (using a “gun” and an integrating sphere).
In Fig. Figure 1 shows the NIR spectra of ranitidine hydrochloride substance from Vera Laboratories (India), obtained using a “gun” and an integrating sphere. The figure shows that the spectra differ in the intensity of the absorption bands, but the absorption bands themselves coincide in wavenumber values.
The main difference between NIR spectroscopy and mid-range IR spectroscopy is that the spectra cannot be compared visually. The fact is that, in general, there is an insufficient number of bands in the NIR spectrum, and the intensity of many bands is low (especially the second and third overtones), so mathematical processing of the spectra is required.
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Rice. 2. Result of IDENT analysis of the NIR spectrum of Ulfamid 40 mg tablets, KRKA (Slovenia), obtained using a “gun” using an electronic library of NIR spectra obtained using an integrating sphere.
Rice. 3. Result of IDENT analysis of the NIR spectrum of Ulfamid 40 mg tablets, KRKA (Slovenia), obtained using an integrating sphere using an electronic library of NIR spectra obtained using a “gun”.
4.2. Identification of the active substance by the NIR spectrum of preparations containing this substance.
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Rice. 7. Result of IDENT analysis of the NIR spectrum of Ciprofloxacin 250 mg tablets, Cypress Pharmaceutical Inc. (USA), using a library consisting of NIR spectra of various substances.
Thus, we have established that with a high content of the active substance (at least 40%) in the drug, it is possible to establish the authenticity of the drug by the NIR spectrum of the substance.
4.3. Identification of drugs with different dosages using NIR spectra.
In the third part of the study, we found that the NIR spectroscopy method can be used to determine various dosages of a particular drug, if they are available in the electronic library of NIR spectra. For this purpose, an electronic library of NIR spectra was created from drugs containing famotidine as an active ingredient, which included 27 samples from 7 different manufacturers in dosages of 10 mg, 20 mg and 40 mg (Fig. 8).
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Rice. 9. Results of IDENT analysis, quamamg tablets, 20 mg and 40 mg, Gedeon Richter Plc. (Hungary) using a library consisting of NIR spectra of various drugs in various dosages.
4.4. Identification of drugs through the blister.
To establish the possibility of identifying drugs using NIR spectroscopy through a blister, two additional libraries of NIR spectra No. 7 and No. 8 were created:
7) NIR spectra of capsules obtained using a fiber optic sensor (“gun”) directly through the blister,
8) NIR spectra of tablets obtained using a fiber optic sensor (“gun”) directly through the blister.
During the analysis, the NIR spectra of the drugs obtained through the blister were compared with the NIR spectra obtained from the surface of tablets or capsules without the blister. In Fig. Figure 10 shows such a comparison of the spectra for rifampicin capsules.
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Rice. 11. The result of IDENT analysis of the NIR spectrum of rifampicin 150 mg capsules, (Russia), obtained using a “gun” directly through the blister using an electronic library obtained through the blister.
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Rice. 13 NIR spectra of the contents of omeprazole 20 mg capsules from 14 different manufacturers in comparison with a falsified sample, obtained using an integrating sphere.
From the data obtained it is clear that without mathematical processing, only the spectrum of counterfeit can be reliably distinguished.
Using the “OPUS IDENT” software for a three-dimensional model of statistical processing of spectra (“cluster analysis”), we obtained the distribution of NIR spectra of generic omeprazole 20 mg capsules, which can be presented in the form of a dendrogram (Fig. 14).
Rice. 14. Cluster analysis of the studied samples taken in triplicate from 14 different manufacturers.
As a result of the cluster analysis, all drugs were well divided into their classes and according to their manufacturer (Fig. 14).
Mathematical processing of the results obtained using IDENT analysis showed the presence of a counterfeit drug. The OPUS program determined that this sample X is indeed falsified and its “match quality coefficient” (spectral distance) is much higher than the threshold for all drugs in this group (omeprazole, 20 mg capsules) from 14 different manufacturers, from which an electronic library was created (Fig. 15).
Rice. 15. Result of IDENT analysis for a falsified sample of OMEZ 20 mg, Dr. Reddy's Lab. (India).
As a result of the IDENT analysis, a series of all original samples of omeprazole 20 mg capsules were uniquely identified, and we compiled a summary table of results for all samples, including the falsified sample (Table 1).
Table 1. Summary table of IDENT analysis results in the omeprazole group, 20 mg capsules.
Sample name | Spectral distance | ||
Falsified sample | |||
Sample from KRKA | |||
Sample from Akrikhin company | |||
Sample from Ranbaxy Laboratories | |||
Sample from Dr. Reddy's Lab. | |||
Sample from M. J. Boipharm | |||
Sample company | |||
Sample company | |||
Sample company | |||
Sample of the company "Pharma" | |||
Sample of the Obolenskoye company" | |||
Sample company. vit. factory" |
Thus, as a result of the research carried out to identify medicinal products of omeprazole from various manufacturers using NIR spectroscopy, we were able to obtain results on identifying counterfeit products for the counterfeit drug OMEZ 20 mg, Dr. Reddy's Lab. (India), and also uniquely identify each generic according to its manufacturer. We also obtained positive IDENT analysis results for all tablets containing ranitidine hydrochloride (12 samples) and famotidine (9 samples), allowing us to uniquely identify the manufacturer of each sample.
GENERAL CONCLUSIONS
1. It was shown that NIR spectra of substances, tablets and capsules can be obtained using a fiber optic sensor and an integrating sphere. In this case, to establish authenticity, you should use an electronic library obtained in the same way as used to take the NIR spectrum of the test sample.
2. It has been shown that with a high content (at least 40%) of the active substance in the drug, it is possible to establish the authenticity of the drug based on the spectrum of the substance. However, in general, to identify drugs, one should use an electronic library compiled on the basis of the NIR spectra of the corresponding drugs.
3. It has been established that the NIR spectroscopy method can be used to differentiate drugs from a specific manufacturer that contain the same active substance in different dosages. At the same time, it is difficult in some cases to quantitatively determine the active substance in drugs from different manufacturers using the NIR spectroscopy method.
4. It has been shown that the NIR spectroscopy method can be used to identify the manufacturer of a substance or drug. In this case, a parallel analysis of the tested product of a specific series and a known product of the same series should be carried out.
5. An electronic library of NIR spectra of substances and preparations containing various active ingredients and manufactured by different manufacturers has been developed.
1. , Comparative assessment of the quality of drugs using near-infrared spectroscopy // Abstracts. report XII Russian National congr. “Man and Medicine.” – M., April 18-22. 2005.– P. 780.
2. , Detection of counterfeit medicines using NIR spectroscopy // Proc. report XIV Russian National congr. “Man and Medicine.” – M., April 16-20. 2007.– P. 17.
3. , The method of near-infrared spectroscopy as a promising direction in assessing the quality of medicines // Questions of biological, medical and pharmaceutical chemistry. – 2008. – No. 4. – P. 7-9.
4. , Application of the method of near-infrared spectroscopy for the identification of drugs // Questions of biological, medical and pharmaceutical chemistry. – 2008. – No. 6. – P. 27-30.
5. Arzamastsev A. P., Dorofeyev V. L., Dolbnev D. V., Houmoller L., Rodionova O. Ye. Analytical methods for rapid counterfeit drug detection. International Congress on Analytical Sciences (ICAS-2006), Moscow, 2006. Book of abstracts. V. 1. P. 108.
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One of the methods that has become widespread in the world for identifying counterfeits is the method of near-infrared spectroscopy with Fourier transform (NIR spectroscopy). Its main advantages are: speed of analysis, absence or minimal sample preparation (possibility of analysis without opening the package), obtaining characteristics of both physical and chemical properties of the drug (identification of components, determination of crystallinity, quantitative analysis of the active substance). Additional various research methods allow you to study samples of different physical states (transmission methods, diffuse reflection). All these advantages make it possible to reliably identify counterfeit goods, as well as identify its manufacturer. In addition, due to their design, NIR analyzers are portable and can be successfully used in mobile laboratories.
Initially, NIR spectrometers were used to control the production of drugs at all levels of its production: quality control of input raw materials, control of all production processes (drying, mixing) and quality control of output products (quality control and quantitative analysis of active components in finished products). Later, this method became widespread for identifying counterfeit goods. Since 2000, the results of identifying counterfeit products using the example of medicines from various manufacturers have been obtained and published. The same works examined various features that affect the accuracy of the analysis. Based on the experience gained, international organizations for the control of counterfeit drugs began to introduce this method to identify counterfeit products, both individually and in combination with other methods.
There are methods in which the NIR method is used for qualitative and quantitative analysis of narcotic drugs. The method allows not only to identify a suspicious sample as a drug, but also to quantify the content of the active substance.
This indicates a preference for the use of the near-infrared Fourier spectrometer method as one of the methods for the qualitative and quantitative analysis of narcotic drugs. For accurate identification of counterfeit products, quantitative determination of the active component in the drug, as well as the ability to track the manufacturer of counterfeit medicines or narcotic drugs.
At the time of the acquisition of the NIIECTS NIR analyzer at the Main Directorate of the Ministry of Internal Affairs of Ukraine in the Donetsk region, the country had a serious problem with the production and distribution of tramadol, so the first task for the NIR was to build a methodology for identifying tramadol and its manufacturer, which would allow us to determine its source. Subsequently, this method was supplemented with a technique for solving another problem - identifying counterfeit medicines.
The Antaris II near-infrared Fourier transform spectrometer from Thermo Fisher Scientific was used to develop identification methods. The appearance of the device is shown in Fig. 1.4.1.
Rice. 1.4.1. NIR spectrometer Antaris II.
The design of the spectrometer allows one device to be equipped with various devices for analyzing various types of samples.
The Antaris II spectrometer is equipped with:
· transmission module for analysis of liquid samples and plates;
· transmission detector for analysis of solid samples (tablets, capsules, powders);
· integrating sphere;
· external fiber optic probe.
The detector for solid samples is installed above the integrating sphere, which allows simultaneous analysis of the sample both by transmission, which characterizes the entire sample as a whole, and on the integrating sphere by the diffuse reflection method, which allows characterizing the surface region of the sample. The external probe is used for diffuse reflectance analysis of samples in non-standard packaging, without opening the packaging, as well as liquid samples. All of the above methods do not require sample preparation or require minimal preparation and allow you to obtain results within 3 minutes, do not require financial costs for reagents and consumables, and, most importantly, are non-destructive, which allows you to save the sample for further confirmation of the results by other methods.
Modern methods for assessing the quality of medicinal raw materials and finished products include near-infrared spectrometry. The method has a number of significant advantages, including:
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- Simultaneous analysis of various components of complex mixtures, including information about their concentrations. For example, using this method it is possible to analyze the percentage of water, organic solvents and other components in microheterogeneous systems, such as oil-in-water or water-in-oil emulsions.
- Possibility of organizing remote control of samples in real time directly in the process flow (remote control). For these purposes, stationary or portable spectrometers are used. Stationary devices are installed in production facilities of pharmaceutical enterprises, where they are integrated directly into production lines, mounting sensors above conveyor belts, in chemical reactors, and mixing chambers. This allows you to receive information online and use the received data in the automated control system. Mobile drug quality control laboratories are most often equipped with portable battery-powered NIR spectrometers.
Methods for obtaining spectra in the NIR region
In the near-infrared region, spectra are obtained using transmission or diffuse reflection.
The transmission method can be used to analyze both liquid and solid substances. In this case, liquids are placed in cuvettes or other specialized containers that are supplied with the device. Such measuring vessels can be made of ordinary or quartz glass. For transmission testing of solid samples, a probe or sphere can be used.
However, probe-based diffuse reflectance analysis has a number of significant advantages, as it provides a more detailed spectrum and more accurate results. This is achieved due to the fact that the inclined plane of the tip of the fiber optic probe minimizes the specular effect, allowing more light to be scattered. In addition, a module can be integrated into the fiber optics to read barcodes from sample packaging. It should also be noted that only with the help of a probe is it possible to identify samples remote from the device itself.
To test samples with low scattering and reflectivity, a combined transmission-reflection method is used. This requires cuvettes and sensors of a special design, thanks to which the beam flow passes through the analyzed sample twice.
In addition, “interaction” spectra can be obtained in the near-infrared region.
Problems of NIR spectrometry and ways to solve them
The main problems of this analytical method in the pharmaceutical industry for a long time have been the difficulty of analyzing the spectrum, characterized by less intense and relatively wider absorption bands compared to the fundamental bands in the mid-infrared region.
Combining mathematical methods of data processing (chemometrics) with the results of instrumental analysis made it possible to eliminate this drawback. For these purposes, modern analyzers are equipped with special software packages based on a cluster or discriminant method of processing results.
In order to be able to take into account various possible sources of changes in the spectrum in chemometric analysis, special libraries of spectra are created at pharmaceutical enterprises, taking into account the manufacturer of raw materials, the technological process of its production, the homogeneity of the material from different batches, temperature, mode of obtaining the spectrum and other factors.
According to European regulatory requirements, to compile libraries, it is necessary to study at least 3 samples of the drug substance to obtain 3 or more spectra.
Another possible problem - the possibility of a change in the spectrum due to the design features of the NIR spectrometer - is solved by qualifying the device in accordance with pharmacopoeial requirements.
Things to remember when conducting research
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Near infrared spectrometry (NIR spectrometry) is a method based on the ability of substances to absorb electromagnetic radiation in the wavelength range from 780 to 2500 nm (12500 to 4000 cm -1).
Absorption in the NIR range is usually associated with overtones of the fundamental vibrational frequencies of C-H, N-H, O-H and S-H bonds and their combinations. The most informative range is the region from 1700 to 2500 nm (6000 to 4000 cm -1).
Analysis of information extracted from NIR spectra is carried out using chemometric algorithms, which require the creation of a primary data set.
Within the scope of the applicability of the method, NIR spectrometry allows, directly or indirectly, to carry out qualitative and quantitative assessment of the chemical, physical and physicochemical characteristics of the analyzed object, including the assessment of the following characteristics:
– hydroxyl and iodine numbers, degree of hydroxylation;
– crystalline form and degree of crystallinity;
– polymorphic form or pseudopolymorphic form;
– degree of particle dispersion and others.
NIR spectrometry has the following capabilities:
– ease of sample preparation or lack of preparation;
– speed of measurements;
– non-destructive nature of the analysis;
– the possibility of simultaneous assessment of several parameters (indicators);
– the ability to carry out remote monitoring, including in process flows in real time.
Devices. Both specialized NIR spectrophotometers and other spectrophotometers capable of operating in the near-IR region of the spectrum are used.
NIR spectrophotometers consist of:
– radiation source, for example, a quartz lamp (incandescent lamp) or its analogue;
– monochromator (diffraction grating, prism, optical-acoustic filter) or interferometer (Fourier transform spectrophotometers);
– recording device – detector (based on silicon, lead sulfide, indium arsenide, indium gallium arsenide, mercury-cadmium telluride, deuterated triglycine sulfate, etc.);
– sample placement device and/or remote fiber optic sensor.
Glass or quartz cuvettes, vials, glass beakers, capsule or tablet holders and other devices are used to place samples.
Spectrophotometers can be equipped with a cell compartment, an integrating sphere (an integrating sphere is an optical component consisting of a spherical cavity coated with a highly reflective material, the sphere is designed to obtain spectra of inhomogeneous samples), external modules for measuring the transmittance of highly scattering samples, and automatic sample feeders , fiber optic probes. The choice of one or another device for analysis depends on the type of sample and the chosen measurement method. Therefore, devices that implement several measurement approaches are recommended for use.
Data processing and analysis of the results obtained are carried out using special software.
Each measurement mode (transmission, diffuse reflection and their combination) must have its own verification method, including checking the correct setting of wavelengths and checking photometric noise.
Checking that the wavelengths are set correctly. To check the correctness of the wavelength settings, record the spectrum of a standard sample that has characteristic absorption maxima and minima and compare the obtained wavelength values with the declared characteristics.
For transmission and reflection modes, to determine the correct setting of wavelengths, it is most common to use oxides of rare earth elements, water vapor in the atmosphere, methylene chloride, and others as standard samples.
In devices with Fourier transform, the wavenumber scale is linear over the entire operating range, and to check the accuracy of the installation, it is enough to use one standard sample with control of the declared characteristics in one absorption band. Devices of other types may have a nonlinear wavenumber scale and require verification of the stated metrological characteristics by at least three peaks (one or more standard samples) covering the entire operating range.
The error in setting wavelengths should be no more than ±1 nm (or an equivalent wave number) in the wavelength range up to 1900 nm and no more than ±1.5 nm for the wavelength range ≥1900 nm.
The reproducibility of the wavelength setting must comply with the requirements of the manufacturer or the requirements of regulatory documents in force in the Russian Federation.
Checking photometric linearity. To check photometric linearity, NIR spectra of standard samples with known transmission/reflection values are recorded and a graphical dependence of the obtained transmission/reflection values on the known values is plotted. The result of constructing such a relationship should be a straight line with an intersection at the center of coordinates (0.00 ± 0.05) and a tangent of the angle of inclination of the straight line (1.00 ± 0.05). To check photometric linearity in reflection mode, carbon-doped polymers or analogs are used as standard samples in an amount of at least 4 samples in the reflectance range of 10–90%. To check photometric linearity in the transmission mode, filters in the amount of 3 samples with transmission values of 10–90% and a line of 100% transmission are used as standard samples (the transmission spectrum of an empty channel is recorded).
Checking photometric noise. To estimate photometric noise when measuring transmittance, record a line of 100% in air; When measuring reflectance, record a line of 100% using suitable reference materials with a reflectivity of at least 99%. In this case, the 100% line means a measurement in which the standard sample is both the measured sample and the background. At high absorption values, photometric noise is assessed using standard samples with transmittance or reflectance values of about 10%.
Photometric noise must meet the manufacturer's specifications.
Methods of measurement. The NIR spectrum represents the dependence of the corresponding photometric quantity (optical density ( A), transmission ( T), reflection coefficient ( R) and derived quantities) from the wavelength or frequency of radiation. When measuring in the NIR region, the following methods are implemented:
– measurement of absorption (or transmission) when radiation passes through the sample;
– measurement of radiation reflected or scattered from a sample;
– a combination of the above methods.
Measurements are always taken relative to the background.
Transmittance measurement. Transmittance is a measure of the reduction in radiation intensity as it passes through a sample. This principle is implemented in most spectrophotometers in use, and the result can be expressed directly in transmittance units ( T) and/or optical density ( A).
The method is applicable for solid and liquid samples, including dispersed systems.
As a rule, special sample preparation is not required when measuring transmittance. To measure the spectrum of liquid samples, vials or cuvettes with a suitable optical path length (usually 0.5–22 mm), as well as fiber optic transmittance sensors, are used.
Diffuse reflection. In the diffuse reflectance method, the reflectance coefficient ( R), representing the ratio of the intensity of light reflected from the sample ( I), to the intensity of light reflected from the background ( I r):
or the inverse logarithmic value of this ratio ( A R):
.
A surface with a high value is used as a background. R: gold plates, perfluorinated saturated polymers, ceramic plates and other suitable materials.
The method is used for the analysis of solid samples using an integrating sphere or fiber optic sensors operating in reflection mode. In the latter case, for the reproducibility of the results obtained, it is necessary to ensure the stability of the measurement conditions, in particular the relative immobility of the sensor, the degree of pressure and other conditions.
Transmission-reflection method. This method is a combination of transmission and reflection due to the special design of cuvettes and sensors in which radiation passes through the sample twice, which allows the analysis of samples with low absorbing and scattering powers.
The double transmittance coefficient ( T*):
,
Where: I T– radiation intensity after double transmission, without sample;
I– intensity of transmitted and reflected radiation measured with the sample;
and a value similar to optical density ( A*):
.
The spectrum of air or a comparison medium is used as a background.
The method is applicable for liquid, including inhomogeneous samples.
To record the spectrum, the sample under study is placed in a cuvette with a mirror or other diffuse reflector. It is possible to use a fiber optic sensor that is immersed in the sample.