Beak spectrometry. GPM.2.1.0001.15 Spectrometry in the near infrared region
As a manuscript
DOLBNEV DMITRY VLADIMIROVICH
IDENTIFICATION OF DRUGS BY METHOD OF NEAR INFRARED SPECTROSCOPY
14.04.02 - pharmaceutical chemistry, pharmacognosy
dissertations for a degree
candidate of pharmaceutical sciences
Moscow - 2010
The work was carried out at the 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 (VNTs BAV)
The defense will take place on "___" ____________________ 2010 at ____ hours 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 the Moscow State Medical University. Moscow, Nakhimovsky prospect, 49.
Scientific secretary of the dissertation
Council D 208.040.09
Doctor of Pharmacy,
Professor
Relevance of the research topic. In the last 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, tumor development studies). Currently, NIR spectroscopy is becoming one of the methods of in-process control in the pharmaceutical industry in Europe and the USA.
It is used to check incoming raw materials, mixing uniformity, granulation end point, drying moisture content, tabletting uniformity, coating thickness measurement.
The method of NIR spectroscopy is described in the European Pharmacopoeia and the US Pharmacopoeia, however, it is still relatively rarely used 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 release of the 12th edition of the State Pharmacopoeia of the Russian Federation.
It is also necessary to note the continuing problem of counterfeit medicines, one of the ways to solve which is the development of express methods of analysis.
Considering the above, an urgent problem is the development of unified methods for the analysis of substances and preparations and the detection of counterfeit medicines using the NIR spectroscopy method.
Purpose and objectives of the study. The aim of the study was to develop unified methods for the analysis of substances and preparations and the detection of 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;
– to compare the NIR spectra of substances and preparations;
– to compare the NIR spectra of preparations with different contents of the active substance;
– explore the possibility of using NIR spectroscopy to identify substances and preparations of specific manufacturers, as well as to identify counterfeit medicines;
– to develop an electronic library of NIR spectra of substances and preparations.
Scientific novelty of the research results. It has been shown for the first time that the NIR spectroscopy method can be used both for establishing the authenticity of pharmaceutical substances and for finished drugs (tablets and capsules). It is shown that, in general, the NIR spectra of substances and preparations differ. Spectra can be obtained using a fiber optic sensor and an integrating sphere. It has been shown that if the shell of the capsule or the package of tablets (blister) is transparent, it is possible to obtain a spectrum without removing the capsules or removing the tablets from the package. It has been shown that the NIR spectroscopy method can be used to detect counterfeit drugs, provided that the spectra of the original and the tested drugs are compared. Spectra of substances and preparations can be stored in the form of 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.
The practical significance of the work. The developed methods for the analysis of medicinal products using the NIR spectroscopy method are proposed for establishing the authenticity of pharmaceutical substances, preparations 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 input and output control of pharmaceutical substances and intermediates at pharmaceutical enterprises. The techniques 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 in the identification of 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). Approbation of the work was carried out at the scientific and practical meeting of the Department of Pharmaceutical Chemistry with the course of toxicological chemistry of the Pharmaceutical Faculty of Moscow State Medical University. March 22, 2010
Publications. On the topic of the dissertation, 5 publications were published.
Relation of research to the problematic plan of the pharmaceutical sciences. The dissertation work was carried out within the framework of the complex topic of the Department of Pharmaceutical Chemistry of the Moscow State Medical University. "Improving the quality control of medicines (pharmaceutical and environmental aspects)" (state. reg. No. 01.200.110.54.5).
The structure and scope of the dissertation. The dissertation is presented on 110 pages of typewritten text, consists of an introduction, 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, 42 of which 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 preparations, as well as NIR spectra of preparations with different content of the active substance;
– the results of studying the possibility of using NIR spectroscopy to establish the authenticity of substances and preparations of specific manufacturers, as well as to identify counterfeit medicines.
1. Objects of study
Substances and preparations of a number of medicines have been studied. A total of 35 substances were used in the study: aluminum hydroxide, amikacin sulfate, ascorbic acid, sodium ascorbate, sodium warfarin, 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, ceftizoxime sodium, ciprofloxacin hydrochloride, cyanocoblamin, various manufacturers and 59 drugs from various manufacturers containing: isoniazid, meloxicam, omeprazole, ranitidine hydrochloride, rifampicin, famotidine, ciprofloxacin, esomeprazole, ethambutol, as well as 2 adulterated samples (OMES 20 mg, Dr. Reddy`s Lab. and Rifampicin 150 mg,).
2. Equipment and test conditions
In the work, we used an MPA device - Fourier spectrometer of the near IR range (Bruker Optics GmbH, Germany). Recording parameters: spectral range from 800 nm to 2500 nm (samp-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 measuring only reflection, the integrating sphere – both reflection and transmission. In the work, NIR reflectance spectra were obtained.
2.1. Methods for obtaining NIR spectra:
using a fiber optic sensor ("gun").
2.1.1. Substances . The substance-powder 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 powder surface. The spectrum registration procedure was started by pressing the button on the fiber-optic sensor. Spectra measurements were repeated 3–5 times from different areas to obtain statistically significant analysis results.
2.1.2. Tablets taken from the blister . The fiber optic sensor was pressed perpendicular to the tablet. The spectrum registration procedure was started by pressing the button on the fiber-optic sensor. The measurement of the spectra was repeated 3-5 times from different parts of the tablet to obtain statistically significant results of the analysis.
2.1.3. Tablets in a 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 the button on the fiber-optic sensor. Measurement of the spectra was repeated 3-5 times from different parts of the tablet in the blister to obtain statistically significant results of the analysis. If the blister is opaque or aluminium, the tablet is first removed from the blister and then the NIR spectrum is 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 the button on the fiber-optic sensor. The measurements of the spectra were repeated 3-5 times from different parts of the capsule in the blister to obtain statistically significant results of the analysis. If the capsule shell is not transparent, then the capsule was first opened, and then the content spectrum 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 substance-powder 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 significant results of the analysis.
2.2.2. Tablets taken from the blister . The tablet was placed in a special holder. The holder with the 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 parts of the tablet to obtain statistically significant results of the analysis.
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. The holder with the 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). Measurement of the spectra was repeated 3-5 times from different parts of the capsule to obtain statistically significant results of the analysis. If the capsule shell is not transparent, then the capsule was first opened, and then the spectrum of the contents in a glass cuvette was measured by placing the cuvette on top of the optical window of the integrating sphere.
3. Mathematical processing of NIR spectra.
The obtained spectra were mathematically processed using the OPUS IDENT program included in the OPUS 6.0 software package (Bruker Optics GmbH, Germany). The unknown spectrum was compared with the library comparison 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, where the statistically determined acceptable criteria (or thresholds) for all substances in the library are entered. The tested spectrum was compared with all comparison spectra available in the electronic library. The result of the comparison between spectra A and B ends up with the output of the spectral distance D, which in the IDENT program is called the "coincidence quality factor". The spectral distance indicates the degree of spectral similarity. Two spectra with 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 has been identified.
Cluster analysis allows one to study 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 executed 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,
the two spectra with the smallest 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 for the identification of substances and drugs of a number of domestic and foreign manufacturers was studied.
As a result of the study, six different electronic libraries of NIR spectra were created:
1) NIR spectra of the contents of the capsules, obtained using a fiber optic sensor ("gun"),
2) NIR spectra of the contents of the capsules, 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).
On fig. Figure 1 shows the NIR spectra of the substance ranitidine hydrochloride 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 terms of the values of the wave numbers.
The main difference between NIR spectroscopy and mid-range IR spectroscopy is that the spectra cannot be visually compared with each other. The fact is that, in general, an insufficient number of bands is observed in the NIR spectrum, and the intensity of many bands is low (especially the second and third overtones), so it is necessary to carry out mathematical processing of the spectra.
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Rice. Fig. 2. The result of IDENT analysis of the NIR spectrum of tablets Ulfamid 40 mg, KRKA (Slovenia), obtained using a "gun" using an electronic library of NIR spectra obtained using an integrating sphere.
Rice. Fig. 3. The result of IDENT analysis of the NIR spectrum of tablets Ulfamid 40 mg, 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. IDENT analysis result of NIR spectrum of Ciprofloxacin 250 mg tablets, Cypress Pharmaceutical Inc. (USA), using a library consisting of NIR spectra of various substances.
Thus, we found 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 by 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 in the electronic library of NIR spectra. For this purpose, an electronic library of NIR spectra was created from preparations containing famotidine as an active ingredient, which included 27 samples from 7 different manufacturers at dosages of 10 mg, 20 mg, and 40 mg (Fig. 8).
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Rice. 9. Results of IDENT analysis of Quamamg 20 mg and 40 mg tablets, Gedeon Richter Plc. (Hungary) using a library consisting of NIR spectra of various drugs at various dosages.
4.4. Identification of drugs through a blister.
To establish the possibility of identifying drugs by NIR spectroscopy through a blister, two libraries of NIR spectra No. 7 and No. 8 were additionally 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 drugs obtained through the blister were compared with the NIR spectra obtained from the surface of tablets or capsules without a blister. On fig. 10 shows such a comparison of the spectra for rifampicin capsules.
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Rice. Fig. 11. The result of the 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 compared to a adulterated sample, obtained using an integrating sphere.
From the data obtained, it can be seen that without mathematical processing, only the spectrum of the counterfeit can be reliably distinguished.
Using the software "OPUS IDENT" for a three-dimensional model of statistical processing of spectra ("cluster analysis"), we obtained the distribution of NIR spectra of generics of omeprazole 20 mg capsules, which can be represented as a dendrogram (Fig. 14).
Rice. 14. Cluster analysis of test samples taken in triplicate from 14 different manufacturers.
As a result of the cluster analysis, all drugs were well divided according to their classes and according to their manufacturer (Fig. 14).
Mathematical processing of the obtained results by IDENT analysis showed the presence of a counterfeit drug. The OPUS program determined that this sample X is indeed falsified and its “coincidence quality factor” (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. fifteen).
Rice. 15. The 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 falsified samples (Table 1).
Tab. 1. Summary table of the results of IDENT analysis in the omeprazole group, 20 mg capsules.
Sample name | Spectral distance | ||
falsified sample | |||
Sample from KRKA | |||
Akrikhin's sample | |||
Sample from Ranbaxy Laboratories | |||
Sample from Dr. Reddy's Lab. | |||
Sample from M. J. Boipharm | |||
Firm sample | |||
Firm sample | |||
Firm sample | |||
Sample company -Pharma» | |||
Sample of the Obolenskoye company» | |||
Company sample. vit. plant" |
Thus, as a result of the studies on the identification of omeprazole drugs from various manufacturers using NIR spectroscopy, we were able to obtain results on the identification of counterfeit products for the counterfeit drug OMEP 20 mg, Dr. Reddy's Lab. (India), as well as to uniquely identify each generic according to its manufacturer. We also obtained positive IDENT 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 is shown that the NIR spectra of substances, tablets and capsules can be obtained using a fiber-optic sensor and an integrating sphere. In this case, for authentication, you should use an electronic library obtained in the same way that is used to take the NIR spectrum of the test sample.
2. It has been shown that at a high content (at least 40%) of the active substance in the preparation, it is possible to establish the authenticity of the preparation by the spectrum of the substance. However, in the general case, to identify drugs, an electronic library compiled on the basis of the NIR spectra of the corresponding drugs should be used.
3. It has been established that the NIR spectroscopy method can be used to differentiate preparations of a specific manufacturer containing one active substance in different dosages. At the same time, it is difficult in some cases to carry out a quantitative determination of the active substance in preparations from different manufacturers using the NIR spectroscopy method.
4. It has been shown that NIR spectroscopy can be used to identify the manufacturer of a substance or preparation. In this case, a parallel analysis of the tested agent of a particular series and the known agent of the same series should be carried out.
5. An electronic library of NIR spectra of substances and preparations containing various active substances and manufactured by different manufacturers has been developed.
1. , Comparative assessment of the quality of preparations by the method of near infrared spectroscopy // Proceedings. report XII Russian nat. congr. "Man and medicine". - M., April 18-22. 2005.– P. 780.
2. , Detection of counterfeit medicines by NIR spectroscopy // Tez. report XIV Russian nat. 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.
MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION
GENERAL PHARMACOPEIAN AUTHORIZATION
Cspectrometry in near OFS.1.2.1.1.0001.15
infrared Introduced for the first time
Spectrometry in the near infrared (NIR) region is a method based on the ability of substances to absorb electromagnetic radiation in the wavelength range from 780 to 2500 nm (from 12500 to 4000 cm-1).
Absorption in the NIR range is associated, as a rule, with the overtones of the fundamental vibrational frequencies of the 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 (from 6000 to 4000 cm -1).
Spectrometry in the NIR region is characterized by ease of sample preparation or lack of sample preparation, rapid measurements, non-destructive nature of the analysis (without opening the drug package), simultaneous evaluation of several parameters (indicators), remote monitoring, including in real-time process flows.
NIR spectrometry allows you to directly or indirectly conduct a qualitative and quantitative assessment of the chemical, physical and physico-chemical characteristics of the analyzed object, including:
– hydroxyl and iodine number, degree of hydroxylation;
– crystal form and degree of crystallinity;
– polymorphic form or pseudopolymorphic form;
– dispersion of particles and others.
The analysis of information extracted from the NIR spectra is carried out using chemometric algorithms.
Equipment
NIR spectrometers consist of:
- radiation source, for example, a quartz lamp (incandescent lamp) or its equivalent;
- monochromator (diffraction grating, prism, optical-acoustic filter) or interferometer (for Fourier spectrometers);
- a recording device - a 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 probe.
The spectrometers can be equipped with a cuvette 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 reflectance spectra of inhomogeneous samples), external modules for measuring the transmission of highly scattering samples, automatic feeding devices samples, fiber optic probes, etc. The choice of one or another device for analysis depends on the type of sample and the chosen method of measurement.
Samples are placed in glass or quartz cuvettes, vials, glass beakers, capsule or tablet holders, and other devices.
Data processing and analysis of the obtained results are carried out using special software.
Each measurement mode (transmission, diffuse reflection, and combinations thereof) shall have its own verification procedure, including verification of wavescale accuracy and reproducibility, linearity, response stability, and photometric noise.
Checking the accuracy of the wave scale. To check the accuracy of the wave scale, the spectrum of the standard is recorded, which has characteristic maxima and absorption minima, and the obtained wavelength values are compared with the declared characteristics. Oxides of rare earth elements, water vapor in the atmosphere, methylene chloride, and others are used as standards.
In devices with a Fourier transform, the scale of wave numbers is linear over the entire operating range, and to check the accuracy of the wave scale, it is sufficient to use one standard with the control of the declared characteristics by one absorption band. Instruments of other types may have a non-linear nature of the wave number scale and require verification of the declared metrological characteristics for at least three peaks (one or more standards) covering the entire operating range.
The error in setting the wavelengths should be no more than ± 1 nm (or its equivalent value of the wave number) in the wavelength range up to 1900 nm and no more than ± 1.5 nm for the wavelength range ≥ 1900 nm.
Wavelength reproducibility must comply with the requirements of the manufacturer or the requirements of regulatory documents in force on the territory of the Russian Federation.
Verification of photometric linearity and stability of responses. To check photometric linearity, the NIR spectra of standards with known transmission or reflectance values are recorded and a graphical dependence of the obtained transmission or reflection values on the known values is plotted. The result of constructing such a dependence should be a straight line with a cutoff (0.00 ± 0.05) and a tangent of the slope of the straight line (1.00 ± 0.05).
To check photometric linearity in reflection mode, carbon-doped polymers or equivalents are used as standards. If the instrument is used to measure samples with an absorbance of 1.0 or less, then it is sufficient to use 4 standards in the range of reflectance values from 10 to 90%, for example, 10, 20, 40 and 80% with corresponding absorbance values of 1.0; 0.7; 0.4 and 0.1. When measuring a sample with an absorbance greater than 1.0, a 2% and/or 5% reflectance standard is added to the specified set of standards.
To check the photometric linearity in transmission mode, 3 filters with transmission values in the range from 10 to 90% and a line of 100% transmission are used as standards, i.e. recording the transmission spectrum of an empty channel.
To check the stability of the response, periodically measure the standard with unchanged physical and chemical properties. The background measurement must be carried out using the same internal or external standard. The deviation of the photometric response should not exceed ± 2%.
Checking photometric noise. To estimate photometric noise when measuring transmission, a 100% air line is recorded; when measuring reflectance, record a line of 100% using suitable standards with at least 99% reflectance. In this case, the 100% line means a measurement in which the standard is the measured sample and the background at the same time. At high absorption values, the photometric noise is evaluated using standards with transmission or reflectance values of about 10%.
The photometric noise must meet the requirements specified in the manufacturer's specification.
Measurement methods
The NIR spectrum is the dependence of the corresponding photometric quantity [optical density ( BUT), transmittance ( T), reflection coefficient ( R) and derivatives] on the wavelength or frequency of the radiation. When measuring in the NIR region, the following methods are implemented:
– measurement of transmission (or absorption) during the passage of radiation through the sample;
– measurement of radiation reflected or scattered from the sample;
- a combination of the above methods.
Measurements are always carried out relative to the background.
Transmission measurement. Transmission is a measure of the reduction in intensity of radiation as it passes through a sample. This principle is implemented in most used spectrometers, and the result can be presented directly in transmission units ( T) and/or optical density ( A).
I 0 is the intensity of the incident light;
I is the intensity of light transmitted through the sample;
The method is applicable to solid and liquid samples, including dispersed systems.
As a rule, special preparation of samples for transmission measurements is not required. To measure the spectrum of liquid samples, use vials or cuvettes with a suitable optical path length (usually 0.5 - 22 mm), as well as fiber optic probes with a special nozzle.
diffuse reflection. In the diffuse reflection method, the reflectance is measured ( 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 reciprocal logarithmic value of this ratio ( A R):
A surface with a high magnitude is used as the background. R: gold plates, perfluorinated saturated polymers, ceramic plates and other suitable materials.
The method is used to analyze solid samples using an integrating sphere or fiber optic probes 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 probe, the degree of contact of the sensor with the sample, and other conditions.
Transmission - reflection. This method is a combination of transmission and reflection due to the special design of the cuvettes and sensors, in which the radiation passes through the sample twice, which allows the analysis of samples with low absorbing and scattering power.
As a photometric quantity, the double transmission coefficient is used ( T*):
I T is the radiation intensity after double transmission, without a sample;
I is the intensity of transmitted and reflected radiation measured with the sample;
and a value similar to the optical density ( BUT*):
The spectrum of air or reference medium is used as a background.
The method is applicable to liquid, including inhomogeneous samples.
To record the spectrum, the test sample is placed in a cuvette with a mirror or other diffuse reflector. It is possible to use a fiber optic probe with a special nozzle, which is immersed in the sample.
Factors affecting measurement results
Sample temperature. The temperature of a sample can affect both its transmission and its reflection. Temperature control is important in the analysis of thermally labile objects, in the case of which a difference of several degrees can lead to significant spectral changes, including solid samples containing water, dispersed systems, amorphous objects, etc.
Moisture and residual solvents. The presence of water and residual solvents can affect the nature of the spectrum and the results of the analysis. The need and conditions for drying should be specified in pharmacopoeial monographs.
Sample thickness determines the degree of transmission. With an increase in the layer thickness, an increase in absorption is observed. Therefore, in comparative measurements of transmission, the thickness of the sample must be the same or taken into account. When measuring reflection, the layer thickness is not of fundamental importance, but it must be taken into account that the layer thickness must be comparable to the depth of penetration of the beam into the sample. In case of insufficient thickness, an additional reflective material is placed behind the sample, for example, a stamp with a gold coating.
Optical properties of the sample. When analyzing solid samples, it is necessary to ensure that the sample is as homogeneous as possible, since differences in particle density or particle size affect the nature of the spectrum. The spectra of physically, chemically or optically heterogeneous samples should be recorded either with an increased beam size or using devices that rotate the samples during measurements. In this case, it is desirable to measure each sample several times with subsequent averaging of the spectra.
Polymorphism. The difference in the crystal structure (polymorphism) affects the spectrum, which makes it possible to distinguish crystalline or amorphous forms from each other based on their NIR spectra. When carrying out the analysis, it is necessary to take into account the crystal structure (modification) of the reference spectrum used in the analysis method.
Sample age. Sample properties can change over time, and these changes can cause spectral differences for the same samples. These changes should be taken into account when building calibration models, both for identification purposes and for quantitative analysis purposes.
QUALITATIVE ANALYSIS
Qualitative analysis (qualification and identification) in NIR spectrometry is based on the similarity of the spectra of the same substance.
To conduct a qualitative analysis, a library of standard spectra is initially created, an optimal mathematical model is selected for processing the spectra and implementing algorithms for their comparison. Next, the library is validated in conjunction with the selected mathematical model (see the Validation of Qualitative Methods section). Qualitative analysis is carried out by comparing the spectrum of the test sample with the spectra in the library (see section "Data Analysis").
Creation of spectrum library
The library represents sets of spectra containing characteristic information about each object of analysis. For each set of spectra, the optimal identification parameters are determined using appropriate methods and algorithms. The specified settings are valid for the entire library. For nearby objects that are indistinguishable at given settings, sublibraries are created in which other methods of spectrum preprocessing and analysis algorithms can be used. The number of spectra in the library is not limited.
The library includes spectra of substances that meet the requirements, the quality of which is confirmed by pharmacopoeial or other certified methods.
To take into account possible variations in the properties of each type of analyzed objects, the spectra of several series (batches) are recorded. The registration of the spectra is carried out under similar measurement conditions and perform the same pre-processing. The selected pre-processing of the spectra included in the library remains unchanged during subsequent measurements.
Spectrum preprocessing methods
It is recommended to pre-process the spectra in order to increase the information content of the results obtained and reduce the effect of spectral variations. Raw data processing may include calculation of the first or second derivative, normalization, multiplicative scatter correction, and other methods, or combinations thereof. When choosing methods for spectrum preprocessing, it should be taken into account that they can lead to loss of information or the appearance of errors-artifacts.
Data analysis
Comparison of the spectra of the tested samples during qualitative analysis is carried out with individual or averaged spectra in the library, including using various mathematical methods.
The library can be used to build classification algorithms. It is possible to use different algorithms, for example, the principal component method (PCA), combined with cluster analysis, the SIMCA method (soft independent modeling of class analogy - independent modeling of class analogies), as well as other algorithms, both included in the mathematical software of NIR spectrometers, and and developed by a third party. The reliability of the method used must be verified. For example, correlation coefficient, sum of squared deviations, distances within the model and other indicators should be consistent with the decision level presented in the validation procedure.
The method of analysis must be validated.
Qualitative Method Validation
Method validation is intended to demonstrate its suitability for the purposes of the analysis.
The validation of the method is carried out on a test set of objects that did not participate in the construction of the method, and involves checking the specificity, sensitivity and stability (robustness).
Sensitivity shows what part of the objects of the test set, similar to the objects of the library, are correctly recognized as "own".
Specificity indicates how much of the non-library objects in the validation set are correctly recognized as "foreign".
Particular attention is paid to the results of classification of objects whose spectra are visually similar to the spectra of library objects, but differ from them in composition or chemical structure. Such samples should be correctly identified as "foreign".
Stability indicates that minor changes in conditions (eg temperature, air humidity, vibrations, sample temperature, material compaction, probe immersion depth, layer thickness, etc.) do not affect the results and reliability of identification or qualification.
QUANTITATIVE ANALYSIS
Calibration Model Development
When developing the model, the dependence of the change in the intensity of absorption or reflection in the spectrum of samples on changes in the properties and/or composition of substances is established. In this case, the spectra of samples with known values of their composition and/or their properties, confirmed by certified methods, are recorded. Since chemometric algorithms do not allow extrapolations, it is necessary that the range of calibration concentrations be not less than the expected range of analyzed concentrations or other quantitative characteristics. Calibration samples should be as evenly distributed as possible within the working concentration range.
The registration of the spectra is carried out while observing the experimental parameters, factors affecting the results of measurements and primary processing, which are pre-optimized for all analyzed objects and remain constant during subsequent measurements.
The calibration model is optimized using an appropriate spectrum preprocessing technique, spectral domain selection, and a mathematical algorithm.
Spectra Preprocessing
Carried out in the same way as described in the "Qualitative Analysis" section.
Data analysis. Any reasonable mathematical algorithm can be used to construct a calibration model. Since there is a strong overlap of absorption bands in the NIR range, quantitative analysis is carried out mainly by chemometric algorithms, for example, such as the method of projections onto latent structures (PLS, English PLS), the method of regression to principal components (RGK, English PCR) and others .
Calibration Model Validation
Validation of a calibration model involves demonstrating its suitability for a given purpose. In this case, the following validation characteristics should be determined: specificity (selectivity), linearity, working range of concentrations (analytical area), correctness, precision and stability (robustness).
When constructing calibration models using chemometric methods of analysis, the calibration quality is estimated by the rms calibration residual ( RMSEC) and the rms residual of the forecast ( RMSEP).
Alternative statistical methods (paired t-test, bias estimation, etc.).
Emissions
When analyzing the NIR method, one should take into account, correct and reasonably exclude outlier results.
All outliers are subject to analysis and, if they are informative or validated using a validated methodology, they can be included in the model.
Revalidation or revalidation
A qualitative or quantitative method that has been validated and found suitable for use needs periodic revalidation or revalidation. If deviations are found, the method must be corrected.
The NIR method is revalidated if:
- a new object has been added to the library (for qualitative analysis);
- there are prerequisites for changing the characteristics of objects whose spectra are already included in the library (changing the production technology (synthesis), composition, quality of the packaging feedstock, etc.);
- other changes and/or inconsistencies were found in the properties of the analyzed objects or the methodology.
Transferring Models
When transferring models of qualitative and quantitative analysis from one instrument to another, the spectral characteristics of the spectrometers used (resolution, range of wave numbers, etc.) should be taken into account. Model transfer procedures are understood as various chemometric algorithms (mathematical and statistical). After transferring to another device, to confirm the operability of the model, it must be revalidated.
Data storage
Data storage is carried out in electronic form in accordance with the requirements of the software. In this case, it is necessary to save the original spectra that have not been subjected to mathematical processing, with the aim of their possible further use in optimizing libraries or methods.
6. Spectroscopy in the near infrared region (NIR)
Spectrometry in the near infrared region (NIR spectrometry, NIR) is a method based on the ability of substances to absorb electromagnetic radiation in the wavelength range from 780 to 2500 nm (from 12500 to 4000 cm -1).
Absorption in the NIR range is associated, as a rule, with the overtones of the fundamental vibrational frequencies of the 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 (from 6000 to 4000 cm -1).
The analysis of information extracted from the NIR spectra is carried out using chemometric algorithms that require the creation of a primary data array. As part of the applicability of the method, NIR spectrometry allows you to directly or indirectly conduct a qualitative and quantitative assessment of the chemical, physical and physico-chemical characteristics of the analyzed object, including the evaluation of the following characteristics:
Hydroxyl and iodine number, degree of hydroxylation;
Crystal form and degree of crystallinity;
Polymorphic form or pseudopolymorphic form;
The degree of dispersion of particles and others.
NIR spectrometry has the following capabilities:
Ease of sample preparation or no preparation;
Speed of measurements;
Non-destructive nature of the analysis;
Possibility of simultaneous evaluation of several parameters (indicators);
Possibility of carrying out remote control, including in technological streams 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 equivalent;
Monochromator (diffraction grating, prism, optical-acoustic filter) or interferometer (spectrophotometers with Fourier transform);
Recording device - detector (based on silicon, lead sulfide, indium arsenide, indium-gallium arsenide, mercury telluride, cadmium, deuterated triglycine sulfate, etc.);
Sample placement devices and/or remote fiber optic sensor.
Samples are placed in glass or quartz cuvettes, vials, glass beakers, capsule or tablet holders, and other devices. Spectrophotometers can be equipped with a cuvette 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 transmission of highly scattering samples, automatic sample feeders , fiber optic probes. The choice of one or another device for analysis depends on the type of sample and the chosen method of measurement. Therefore, devices that implement several approaches to measurement are recommended for use. Data processing and analysis of the obtained results are carried out using special software. Each measurement mode (transmission, diffuse reflection, and their combination) must have its own verification procedure, including verification of the correct wavelength setting and photometric noise verification.
Checking the correct setting of the wavelengths. To check the correctness of the wavelength setting, the spectrum of a standard sample is recorded, which has characteristic absorption maxima and minima, and the obtained wavelength values are compared 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 a Fourier transform, the scale of wave numbers is linear over the entire operating range, and to check the accuracy of the installation, it is sufficient to use one standard sample with the control of the declared characteristics by one absorption band. Instruments of other types may have a non-linear nature of the wave number scale and require verification of the declared metrological characteristics for at least three peaks (one or more standard samples) covering the entire operating range. The error in setting the wavelengths should be no more than ±1 nm (or its equivalent value of the wave number) in the wavelength range up to 1900 nm and no more than ±1.5 nm for the wavelength range?1900 nm.
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One of the methods that have become widespread in the world for identifying counterfeit goods is the method of near infrared spectroscopy with Fourier transform (NIR spectroscopy). Its main advantages are: the speed of analysis, the absence or minimum sample preparation (the possibility of analysis without opening the package), obtaining characteristics of both the 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 (methods for transmission, diffuse reflection). All these advantages make it possible to reliably identify a counterfeit, as well as to identify its manufacturer. In addition, NIR analyzers due to their design 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). In the future, this method has been widely used to identify counterfeit goods. Since 2000, the results of the identification of counterfeit products have been obtained and published on the example of medicines from various manufacturers. In the same works, various features affecting the accuracy of the analysis were considered. Based on the experience gained, international organizations for the control of counterfeit drugs began to implement 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 the 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 using 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, quantitative determination of the active ingredient 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 NIR analyzer by the NIIECC at the Main Directorate of the Ministry of Internal Affairs of Ukraine in the Donetsk region, there was a serious problem with the production and distribution of tramadol in the country, so the first task for the NIR was to develop a methodology for identifying tramadol and its manufacturer, which would make it possible to determine its source. Subsequently, this method was supplemented by a technique for solving another problem - the identification of counterfeit medicines.
To develop identification methods, an Antaris II near infrared Fourier transform spectrometer manufactured by Thermo Fisher Scientific was used. 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 instrument to be equipped with various devices for the analysis of various types of samples.
The Antaris II spectrometer is equipped with:
· a transmission module for the analysis of liquid samples and plates;
· a transmission detector for the 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 for transmission, which characterizes the entire sample as a whole, and on the integrating sphere by the diffuse reflection method, which allows characterizing the surface area of the sample. The external probe is used for diffuse reflectance analysis of samples in non-standard packaging, without opening the package, as well as liquid samples. All of the above methods do not require sample preparation or require minimal preparation and allow you to get a result 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.
WHAT IS NEAR IR?
The near infrared (NIR) range of the electromagnetic spectrum extends from 800 nm to 2500 nm (12500 to 4000 cm-1 ) and lies between the mid-IR region with longer wavelengths and the visible region with shorter wavelengths. The middle and near ranges can be used for vibrational spectroscopy. While the mid-IR spectra record mainly atomic vibrations in the individual chemical bonds of most molecules, the corresponding NIR spectra show the so-called overtones and combination bands.
On the wavenumber scale (cm-1 ) these overtones appear as something less than the component fundamental frequencies. For example, the main vibration of the C-H bond (n) of the trichloromethane (CHCl3) molecule occurs at 3040 cm-1 , the first three overtones (2n, 3n and 4n) are observed at 5907cm-1 , 8666cm -1 and 11338cm -1 respectively.
At the same time, the absorption capacity decreases with increasing overtone number, for example, a series of these values for CHCl3 is 25000, 1620, 48,
1.7 cm-1 /mol, respectively.
Due to the sharp decrease in the intensity of higher overtones, NIR spectra are usually suppressed by overlapping overtones and combination bands of structurally lighter groups (eg C-H, N-H and O-H). Within these NIR spectra, significant information about the molecular structure of the sample under study is contained, and this information can be extracted by modern data processing methods.
Benefits of NIR spectroscopy
Speed (usually 5 - 10s)
No sample pretreatment required
Ease of measurement
High accuracy and reproducibility of analysis
No pollution
Process control
Possibility of measurements through glass and plastic packaging
Measurement automation
Transferring a method from one instrument to another
Analysis of physical and chemical properties
Compared to liquid chemical analysis methods, NIR spectroscopy analysis is faster, simpler and more accurate. Measurements can be carried out very quickly, usually the analysis time is only 5-10 seconds. It does not require preliminary preparation of the sample and special training of personnel. These spectra may contain information about the physical properties of the material, such as particle size, thermal and mechanical pretreatment, viscosity, density, and the like.
COMPARISON OF IR SPECTROSCOPY
near and medium ranges
The reduction in sample preparation time is one of the main advantages of near IR compared to mid IR. This is primarily due to the relatively low absorption coefficient for most materials in the NIR range. Measurements in the middle range of powdered samples are traditionally performed either by the diffuse reflection method or by pressing the samples into tablets and measuring the spectra in the transmission mode. In both cases, the samples must first be ground to a fine powder and then mixed with a non-absorbent material such as KBr. The crushed and mixed with KBr powders are placed in a mold and compressed into tablets at high pressure using a hydraulic or manual press. In the case of measurements in diffuse reflectance mode, the ground and mixed with KBr sample is placed directly into the sample cup, the surface of the sample is leveled and then inserted into the diffuse reflection attachment for measurement. These sample preparation methods have been widely and successfully used, but have disadvantages such as longer sample preparation times, higher potential for sample contamination, possibly reduced sample-to-sample and user-to-user reproducibility due to sample preparation differences, and additional cost of KBr diluent.
In addition, the advantage of NIR spectroscopy is that rather inexpensive optical fiber is used to measure solid and liquid samples. Comparable accessories for the mid-IR region are either limited by their physical reach, or by their fragility and difficulty in working with them. All this makes NIR spectroscopy much more attractive for use in a manufacturing process.
COMPARISON spectroscopy
and dispersing devices
Fourier spectrometers in the near infrared range differ significantly from dispersive spectrometers in the near infrared range by the method of obtaining the spectrum. Dispersive devices use a narrow slit and a dispersive element, such as a grating, to convert light into a spectrum. This spectrum is projected onto a sensor, or a plurality of sensors, where the light intensity at each wavelength is determined. The spectral resolution of dispersive devices is determined by a fixed slit width, usually 6-10nm (from 15cm-1 to 25cm -1 , at 2000nm). Resolution cannot be selected by software, and increasing resolution requires a narrower slit and attenuates the resulting signal. Thus, for all dispersive devices, there is a problem of choosing between resolution and signal-to-noise ratio.
A Fourier transform spectrometer, in contrast, uses an interferometer to view combinations of wavelengths of light emerging from a wide band of a near infrared source and directs these combinations into a single detector.
In each scan of the interferometer, data is collected in the form of an interferogram in which the signal intensity is correlated with the displacement of the moving part of the interferometer. This offset of the interferometer is directly related to the wavelength, and a mathematical transformation (Fourier transform) is applied to plot the signal intensity as a function of wavelength, from which the spectral absorption measure or spectrum transmittance is calculated.
Simultaneously, the HeNe laser beam passes through the interferometer and is directed towards its own detector. The displacement of the interferometer results in maxima and minima of the signal on this laser detector, which occur at precisely defined intervals, multiples of the laser wavelength. The zero-crossing points of this signal are used as collection points for digitization of the NIR detector signal. Thus, due to the control of digitization, the Fourier spectrometer's wavelength accuracy is significantly higher than that of any other dispersive instrument. This length accuracy has a direct impact on the stability conditions of the calibration models developed on Fourier systems, as well as on the ability to transfer the calibration model to other Fourier instruments, which will be described later.
The spectral resolution for Fourier spectrometers is determined by the degree of mobility of the interferometer, which is controlled by software, which allows you to greatly increase the resolution compared to a dispersive spectrometer, and, with the help of software, choose the resolution in the course of research. In addition, the wide NIR beam in the Fourier instrument is directed through large circular apertures instead of the narrow rectangular slit used in the dispersive document, which illuminates a larger area of the sample and increases the light intensity in the detector. This performance advantage results in a higher signal-to-noise ratio for Fourier spectrometers compared to dispersive instruments. A better signal-to-noise ratio leads to a significant reduction in the detection time and, as a result, to higher quality spectra on a Fourier instrument at any spectral resolution.
FTIR NIR SPECTROSCOPY for Qualitative and Quantitative Analysis
Today, many manufacturers strive not only to deliver the highest quality end product, but also to improve production efficiency through laboratory analysis and use in production. By gaining tighter control over technology, it is possible to optimize the use of substances by adding or removing them to produce specified products, minimizing distribution or processing costs.
NIR is a spectroscopic technique ideal for processing measurements due to its ability to quickly perform remote measurements through high performance silica optical fiber. Signal attenuation within such fibers is very low (eg 0.1 dB/km), and NIR fiber optic cables and sensors are robust, relatively inexpensive, and widely available. Processing sensors can be hundreds of meters away from the spectrometer, and multiple sensors can be connected to a single spectrometer.
NIR MEASUREMENT METHODS
NIR sampling methods for solids are based on either diffuse reflectance or simple transmission measurement. Diffuse reflectance measurements are generally made with a fiber optic sensor or an integrating sphere.
On fig. 2 shows an MPA Fourier NIR spectrometer (manufactured by Bruker Optik GmbH, Germany), which has 2 fiber optic sensor ports and a separate sample compartment, allowing the direct transmission method to be used.
This photograph shows a common reflectance sensor used to analyze powder samples in test tubes.
The samples are analyzed when the sensor contacts the material sample. The end of the analysis is signaled by luminous LEDs.
The integrating sphere (Fig. 3) allows you to collect spectral data from inhomogeneous substances, such as mixed powders, grains, polymer granules, etc. The resulting spectra represent a spatial average of the entire material within the circular measurement window (diameter 25 mm).
For better averaging, a rotating beaker and autosamplers can be used.
BIK REVOLUTION
IN PHARMACEUTICAL
INDUSTRY.
QUALITY CONTROL ISSUES
The pharmaceutical industry is known to be one of the most heavily regulated industries in the world, and Bruker manufactures quality assurance devices for pharmaceutical consumers, with which consumers can verify that drugs meet requirements. The OPUS software package controls all functions of the spectrometer. This software package includes a comprehensive check of a set of programs and hardware. OPUS will fully check the correct functioning by pressing the key. This includes testing the internal verification device built into the spectrometer.
The software can be run in a password-protected "GLP" mode, with full administrator control over the user, their access to menus, settings, and customized macro programs. The data block provides full and automatic control of all actions performed with spectra. An pictogram-based programming language is built into the software to automate complex procedures. As a result, there is an increase in repeatability and a reduction in potential errors.
Bruker is an ISO9000 company, and all software and hardware undergoes strict quality control, several stages of final testing and verification before delivery to the customer. The installation of the instrument at the customer's site is carried out by our experienced technical engineers, who provide the customer with a working instrument upon delivery and then continuously throughout the life of the instrument.
RAW IDENTIFICATION
One of the first steps in the production of any pharmaceutical product is to identify and verify that the various input raw materials meet the required requirements. NIR spectroscopy through fiber optic sensors is rapidly becoming the standard method for performing this conformance check, providing unparalleled identification speed for both solids and liquids.
To perform this type of analysis, a calibration model must be created that affects the substances of interest to us. First, it is necessary to obtain several spectra for each raw material, taking into account all possible variations that may occur. This usually includes types of raw materials obtained from various sellers, from various places, etc. Once the spectra have been measured, an average spectrum of each material is generated, and a library of all such average spectra is created, along with the statistically determined acceptable criteria (or thresholds) for all substances in the library.
The library then verifies that all materials are uniquely identified. The library can now be used to identify new unknown substances by comparing their spectra with those of the library and determining the hit quality for each substance in the library. If this hit quality is less than the threshold for one substance and greater than the threshold for all other substances, the unknown substance is identified.
Identified liquids can be measured either by flow measurement in the sample compartment (as shown in Figure 1) or with a fiber optic immersion probe. In any case, the lower NIR absorbances (compared to mid-IR) allow for much longer sample path lengths (i.e. 1 - 10mm). Because of this difference in length, the measurement paths in the sample compartment become more advantageous, as it allows the use of generic low-cost glass tubes instead of precision cells, reducing the cost and time of measurements.
QUANTITATIVE ANALYSIS OF ACTIVE INGREDIENTS
Another important part of qualitative/quantitative analysis in the pharmaceutical industry is the quantitative analysis of concentrated active ingredients. This type of analysis often requires extensive laboratory testing of proof prints of specimens that break down during testing. In contrast, Fourier-NIR provides a time-saving and non-destructive way to perform quantitative analysis of concentrates in mixtures of powders or liquids, as well as in already manufactured pharmaceutical tablets and capsules.
EFFICIENT SAMPLING
A key factor in the success of FT-NIR for quantitative analysis is the choice of sampling method, often a combination of automated and manual sampling. Bruker facilities manufacture sampling accessories specifically for the needs of the pharmaceutical industry. For example, an autosampler (Fig. 5) can be installed in the sample compartment of any Bruker FT-NIR spectrometer.
This accessory features a customizable sample disc that can hold up to 30 samples. The user handles the tablet slots and the movement of the disc by the OPUS software or a user-defined macro and/or communication with a centralized distributed control system within the manufacturing plant.
EXAMPLES OF ACTIVE INGREDIENT ANALYSIS
An example of a quantitative analysis of an active ingredient concentrate in a finished pharmaceutical Fourier-NIR is the determination of the concentration of acetylsalicylic acid (ASA) in aspirin tablets. To perform this analysis, the least squares (LSM) method was used to process the spectra obtained from aspirin tablets with a known ASA concentration. The ASA concentration in the samples ranged from 85% to 90%. In addition to ASA, the tablets contained two types of starch in the range of 0%-10%.
To set up an LSM model for this multi-component system, with a total resolution of 8cm-1 44 spectra were registered. The optimal range for ACK was determined using the OPUS-Quant/2 software package (peer-to-peer validation). The root-mean-square error was 0.35%, and the discrepancy R 2 - 93.8%. This error was within the limits specified by the customer. A graph of true and calculated concentrations is shown in Figure 6.
SAMPLE THROUGH PACKAGING
In addition, determination of the concentration of the active ingredient of aspirin tablets through the plastic materials of the transparent packaging was demonstrated using a fiber optic diffuse reflectance sensor, as shown in Figure 7. In the resulting spectra, convex ranges from the polymeric material of the transparent packaging appeared, but two separate regions (6070-5900cm-1 and 4730-4580cm -1 ) containing peaks from aspirin are still visible and were used to create a calibration model.
A graph of true and found concentrations is shown in Figure 8). The root-mean-square error was 0.46%, and the discrepancy R 2 - 91.30%, these values are again within the limits specified by the customer. The spectra obtained in this example are shown in Figure 9.
BENEFITS OF INCREASED RESOLUTION
IN SPECTRAL ANALYSIS
Until recently, most of the published results in NIR spectroscopy have been obtained with low resolution dispersive instruments, which have a spectral resolution between 6 and 10 nm (from 15 cm-1 to 25 cm -1 , at 2000 nm). The advent of Fourier-NIR spectrometers has led to significant advances in high-resolution capabilities (better than 2 cm-1 ) NIR spectroscopy.
NIR spectra are usually characterized by high absorbance, which does not require high resolution. At that time there were often situations when the desired calibration model from low resolution spectra could not be created. In addition, the high resolution directly affects the wavelength accuracy of the instrument and, consequently, the stability of the results and the "transportability" of the calibration models.
In an experiment, to demonstrate the value of increasing resolution in spectral analysis, the NIR spectra of 5 tablets were measured with various low concentrations of the active ingredient. The spectra were measured at a resolution of 8 cm-1 and 2 cm -1 , after which an identification model for tablets was created using OPUS. With a resolution of 2 cm-1 , the model could only distinguish between placebo and tablets with active ingredients, while at a higher resolution of 8 cm-1 , all concentrations are clearly distinguishable.
Figure 10a shows the spectra and plot obtained for the first two principal components of the measurements at 8 cm-1 . Figure 10b shows the spectra and plot obtained for the first two main components of measurements at 2 cm-1 . The 5 areas in the last graph indicate that the higher resolution model clearly distinguishes the 5 concentration levels of the active ingredient.
COVER THICKNESS DETERMINATION
Fourier-NIR spectroscopy has also been successfully used to determine the layer thickness on pharmaceutical tablets. Several tests were carried out in this study, including experiments with non-linear relationships between the measure of light absorption and layer thickness, the similarity of the composition of the core and coating of the material, and the lack of sufficient calibration samples for standard calibration of the LSM. Peak at 7184 cm-1 , which differentiates the core material from the coating material, was identified when high resolution NIR spectra (2 cm-1, 0.4 nm at 7184 cm-1 ) on the Fourier - NIR spectrometer IFS-28/N from Bruker (see Figure 11).
Studies show that the layer thickness can be modeled as a polynomial approximation of the peak region of this sample peak (see Fig. 12), while the least squares calibration of the same data is not possible due to the lack of a sufficient number of calibration samples. Also, this calibration has been successfully used for a number of tablets, but is unacceptable for fiber optic measurements of diffuse reflection, due to insufficient penetration of the fiber into the core.
TRANSFER CALIBRATION
Developing a stable and operationally reliable calibration model is a very time-consuming, resource-intensive job that involves preparing and analyzing a large number of samples using the standard method, and then analyzing them using the Fourier-NIR method. Thus, it is important that a calibration model be developed that can be used over time, and for which it does not matter what kind of instrument is used, the type of sources, detectors, sensors, etc.
In addition, several factors affect the transfer of a calibration from one instrument to another. This includes, for example, the wavelength and photometric accuracy of various instruments. Therefore, for all calibration models that are transferred from one instrument to another, it is necessary to remeasure at least the original set of calibrations (or the complete set of calibrations) on the new instrument in order to determine the correction factors that will allow the model to work on the new instrument.
Sometimes this leads to difficulties in transferring the calibration model, and sometimes, in the case of rare or changing calibration samples, such a transfer is not possible at all.
Usually, the difficulty in transferring the calibration model is caused by the wavelength accuracy on these two instruments. The absence of a stable wavelength axis is a factor that greatly limits the possibility of transferring the calibration model among dispersive instruments. Therefore, Brooker's product line of Fourier-NIR high-resolution instrument spectrometers has the great advantage of using the wavelength axis as a calibration method.
For this, a narrow region in the spectrum of atmospheric water vapor with a known constant wavelength is considered, which is used as a wavelength standard. This allows Fourier - NIR spectrometers (manufactured by Bruker Optik GmbH, Germany) to provide much higher wavelength accuracy than any dispersive instrument. As a result, direct transfer of calibration from one Fourier-NIR instrument to another is possible. The advantage of this feature in avoiding costly recalibration while saving time, money and effort cannot be underestimated.
One such example of transferring a calibration model for quantifying alcohol content in spirits is shown in Table 1. Calibration was performed on an IFS-28/N Brooker spectrometer with immersion probe A, and was subsequently transferred to a Brooker Vector 22/N spectrometer with immersion probe B. After transmission, comparison R 2 and standard deviation errors showed the success of the forward transfer of the calibration. Additional tests have shown the success of direct transfer of other calibration models from instrument to instrument, as well as direct transfer of models on the same instrument, after replacing all major components of the system, including the NIR source, HeNe laser, detector, sensors and electronics.
CONFORMITY TEST
Often it is necessary to determine the conformity of the final product to a certain standard. This is easy to do on Bruker spectrometers, using the Compliance Test . For several selected samples of each substance, a number of spectra are measured, which will be checked against the spectra determined independently by a standard method. For each substance, an average spectrum is generated along with the standard deviation spectrum. New samples of the substance are then analyzed, their spectra compared with the stored average spectrum, and whether the new spectrum is within acceptable limits defined by the standard deviation spectrum and a customer-adjustable factor. A typical compliance test report is shown in Figure 13.
MIXTURE ANALYSIS
In many pharmaceutical processes it is often necessary to analyze the process of mixing two or more components. Mixture analysis plays an important role in mixing powders where samples tend to be heterogeneous. The optimal ratio in the mixture determines the final product. The mixing process must be checked in real time using Fourier-NIR spectroscopy. Spectra are taken from the correct reference mixtures, and then the average spectrum and the standard deviation spectrum are calculated. After that, spectra are taken during mixing, processed and compared with the average spectrum. The mixing process is stopped if the resulting spectrum falls outside a user-defined threshold for the average spectrum of the desired mixture.
CONCLUSION
Fourier spectroscopy - NIR is a fast, easy to use and reliable tool for quality assurance and quality control in the pharmaceutical industry. The enhanced performance of the Fourier Transform technology enables more difficult studies and allows calibration to be transferred directly. In addition, among consumers in the pharmaceutical industry, methods such as raw material identification and quality control, determination of the concentration of the active ingredient, end product conformity test, and mixture analysis in products are common.
