Using HPLC and capillary. Study of pharmacokinetics and bioavailability - journal of pharmacokinetics and pharmacodynamics High performance liquid chromatography
As a manuscript
MELNIKOV Igor Olegovich
DEVELOPMENT OF MICRO METHODS FOR ANALYSIS OF AMINO ACIDS,
SHORT PEPTIDES AND OLIGONUCLEOTIDES
USING RP HPLC AND CAPILLARY
ELECTROPHORESIS
Specialty: 02.00.02 – Analytical chemistry
Dissertations for the degree of candidate of chemical sciences
MOSCOW 2006 The dissertation work was completed at the Department of Analytical Chemistry of the Moscow State Academy of Fine Chemical Technology named after.
M.V. Lomonosov and in the group of analytical protein chemistry of the Institute of Bioorganic Chemistry named after. Academicians M.M. Shemyakin and Yu.A. Ovchinnikov RAS.
Scientific director:
Candidate of Chemical Sciences, Associate Professor Glubokov Yuri Mikhailovich
Official opponents:
Doctor of Chemical Sciences, Professor Makarov Nikolay Vasilievich Candidate of Chemical Sciences Kirillova Yulia Gennadievna
Leading organization:
Institute of Biochemical Physics named after. N.M. Emanuel RAS
The defense will take place on December 20, 2006 at 12:00 at a meeting of the dissertation council D 212.120.05 at the Moscow State Academy of Fine Chemical Technology named after. M.V. Lomonosov at the address: 119571, Moscow, Vernadsky Avenue, 86, room. M-119.
The dissertation can be found in the library of the Moscow State Academy of Fine Chemical Technology named after. M.V. Lomonosov.
Scientific secretary of the dissertation council, candidate of chemical sciences Yu.A. Efimova Relevance work. Currently, clinical research is increasingly focused on the use of instrumental microanalytic methods for diagnosing the most common and dangerous socially significant diseases. A significant part of these methods do not fully and do not always provide timely and high-quality diagnosis, which often does not meet modern requirements for monitoring the biochemical status of a person.
Free amino acids and short peptides that are part of physiological fluids have important functional significance. In some cases, they can act as molecular markers of certain diseases.
Changes in their concentration are often associated with metabolic disorders, which indicate the development of a particular disease. Most existing methods for amino acid analysis are either not sensitive and selective enough for their identification, or require derivatization of amino acids, which significantly complicates the process of their determination. The problem of simple and cost-effective analysis of free genetically encoded amino acids has not yet been fully resolved. At the same time, the clinical analysis of amino acids requires their pre- or post-column modification for highly sensitive and selective determination. Changes in the content of molecular markers in physiological fluids may also be associated with the patient’s genetic predisposition to a particular disease. Hence the need to conduct a comparative analysis of DNA fragments in order to increase the reliability of determining the cause of the disease under study and more effective its therapy.
Meanwhile, the imported equipment necessary for amino acid and nucleotide analysis is, as a rule, expensive and inaccessible to most clinical laboratories. The situation is further aggravated by the fact that many devices are highly specialized for each type of disease, as a result of which there is multiple duplication of diagnostic methods, both in equipment and in methodology.
This sharply increases the cost of clinical studies and complicates the comparison. The author expresses gratitude to the head of the group of analytical protein chemistry at the Institute of Bioorganic Chemistry of the Russian Academy of Sciences, senior researcher, candidate of chemical sciences. I.V. Nazimov for constant help, attention and discussion of the results.
interpretation of the obtained analysis results. Thus, the development of new methods that expand the possibilities of using publicly available analytical equipment of domestic production for highly sensitive, rapid and reliable determination of free and modified amino acids, short peptides and oligonucleotides both for the structural analysis of biopolymers and their fragments, and for clinical diagnostic purposes is an urgent scientific task .
Goal of the work. Purpose dissertation work is the development of a complex of instrumental highly sensitive micromethods for the analysis of free and modified amino acids, short peptides and oligonucleotides using domestic instrumental base.
Scientific novelty.
1. Methods have been developed for the determination of unmodified genetically encoded α-amino acids using capillary zone electrophoresis and micellar electrokinetic chromatography with direct UV photometric and refractometric detection methods.
2. Methods for the joint determination of low-molecular aminothiols in blood plasma using reverse-phase high-performance liquid chromatography and capillary zone electrophoresis with fluorimetric and direct UV photometric detection methods have been developed and applied in clinical practice.
3. A method has been developed for determining fragments of the mutant gene for venous thrombosis based on the analysis of allele-specific polymerase chain reaction products using capillary gel electrophoresis with fluorimetric detection.
Practical significance . The developed method of amino acid analysis makes it possible to determine microquantities of genetically encoded amino acids without their preliminary derivatization, which significantly simplifies the existing analysis scheme. A set of methods for analyzing molecular markers of vascular accidents (homocysteine, cysteine, glutathione), as well as fragments of the mutant gene for venous thrombosis, has been developed and proposed for practical use. As a result of the work carried out, it was possible to demonstrate the possibility of effective use of domestic equipment for biochemical analysis of both protein and nucleic components on the same device for capillary electrophoresis. Determination of sulfur-containing amino acids and peptides in blood plasma using the method developed in this work was used to assess the risk factor of dozens of patients with reliably established infarction and pre-infarction conditions. The results of the work carried out were used in the creation and testing in practice of biomedical analysis of a universal, economical automated complex of instruments and methods for molecular diagnostics of some socially significant diseases (heart attacks, strokes, thrombosis), developed at the Institute of Analytical Instrumentation of the Russian Academy of Sciences.
Submitted for defense:
methods for determining genetically encoded amino acids in an aqueous solution without their preliminary derivatization using capillary zone electrophoresis and micellar electrokinetic chromatography;
optimized conditions for the derivatization of low molecular weight plasma aminothiols using fluorogenic reagents (monobromobimane and 5-iodoacetamidofluorescein);
method for analyzing low molecular weight aminothiols in blood plasma using RP HPLC and capillary electrophoresis;
results of determination of homocysteine content in blood plasma by RP HPLC and capillary zone electrophoresis methods;
a method for determining the mutant gene for venous thrombosis using capillary gel electrophoresis in linear poly-N,N'dimethylacrylamide.
Approbation of work. Main results works were presented at the 8th All-Russian Symposium on Molecular Liquid Chromatography and Capillary Electrophoresis (October 15-19, 2001, Moscow, Russia), the 3rd International Symposium on Separation Methods in Biosciences (May 13-18, 2003, Moscow, Russia, ), International Conference of Undergraduate and Postgraduate Students in Basic Sciences "Lomonosov-2005" (Section of Chemistry. April 12-15, 2005, Moscow, Russia), 2nd Scientific and Practical Conference "Current Problems of Medical Biotechnology" (September 12-14 2005, Anapa, Russia), 3rd Congress of the Society of Biotechnologists of Russia named after. Yu.A. Ovchinnikov (October 25-27, 2005, Moscow, Russia), 1st conference of young scientists at MITHT. M.V. Lomonosov (October 13-14, 2005, Moscow, Russia), International Congress on Analytical Sciences ICAS-2006 (June 25-30, 2006, Moscow, Russia), 31st International Congress of the Federation of European Biochemical Societies (24-29 June, Istanbul, Turkey), 26th International Symposium on Separations of Proteins, Peptides and Polynucleotides (October 16-20, 2006, Innsbruck, Austria).
Publications. Based on the dissertation materials, 12 works were published in the form of articles and abstracts.
Dissertation structure. The dissertation consists of an introduction, literature review, experimental part, discussion of results, conclusions and a list of cited literature.
The dissertation material is presented on 147 pages, contains 19 tables and 42 figures. The list of literary sources consists of 187 titles.
In the introduction the rationale for the topic is given, the goals of the research and the provisions submitted for defense are formulated, its scientific novelty and practical significance are noted. Data are provided on the testing and publication of the research results, as well as on the structure and scope of the dissertation.
1st chapter. General information about existing methods for analyzing the compounds under study is provided. Chromatographic methods and a number of generally accepted identification methods are described in more detail. Methods for determining low molecular weight aminothiols in blood plasma, as well as DNA fragments, are considered. A comparison of existing methods for the analysis of amino acids, short peptides and oligonucleotides is carried out and the relevance of further research in this area is substantiated.
Chapter 2. Data is provided on materials, reagents, methods of preparing the solutions used and performing the work.
Chapter 3. Results and discussion.
The content of free amino acids (AA) in physiological fluids is a diagnostic parameter for a number of diseases. The performed research is aimed at developing a technique that allows them to quickly and reliably separate and identify them. The analysis of mixtures of such AA was carried out using capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography. The refractive index of AA by the CZE method using mixtures of AA by the CZE method with refractometric detection length 90 cm, effective length 75 cm.
A) Buffer: 60 mM sodium borate, pH 11.0. Voltage: 20 kV. Current: 93 µA. Temperature: 21.0 °C of the optimal composition of the background electrical sample Injection time: 7.0 s (electrokinetic, voltage during sample injection - 5 kV). Introduced troll. For this purpose, the amount of AA was tested - 0.1 ng; alanine, tyrosine – 0.2 ng.
4 – Proline; 5 – Alanine; 6 – Tyrosine; 7 – Serin; – Aspartic acid; 9 – Methionine.
CAPS buffer systems, as well as their various combinations using the example of a model mixture of 8 AA with radicals of different nature and properties and the most different pKa values of the amino group. An example of the separation of such a mixture using a borate supporting electrolyte is shown in Fig. 1.
The optimal background electrolyte for the separation of free AA by this method is a solution containing 60 mM borate buffer (pH 11.0).
Using it, the influence of various factors (voltage, current, internal diameter and effective length of the capillary, method of sample introduction) on the separation efficiency was studied and it was shown that under experimental conditions it is not possible to completely separate a mixture of all encoded AAs. It follows from the experiment that ACs for which the difference in migration time exceeds 1 min are acceptablely separated. For this reason, the classical CZE method cannot separate and identify the co-presence of glycine, alanine, valine, leucine, isoleucine, histidine, phenylalanine and tyrosine, as well as aspartic, glutamic acids and cysteine. The selectivity coefficient for them is very low and close to 1. Arginine, lysine, proline, serine, aspartic acid and methionine are easily isolated and identified, i.e. AKs having a migration time of 5.5-10.0, 15 and 19.5-20.8 minutes. The selectivity coefficient for this group of AKs is in the range of 1.1 – 1.3. When using a phosphate supporting electrolyte (pH 11.4), a similar overall separation pattern was observed, but with poorer peak resolution. The studies performed show that classical CZE with a refractometric termination allows, in the best case, complete separation and identification of no more than 8 AA in an aqueous solution. In this case, the content of individual AA from the indicated non-separable ones in such a mixture should not exceed two.
The efficiency of AA separation is noticeably improved by adding methanol to background electrolytes with pH 7. When using 150 mM phosphate buffer (pH 2.0) with the addition of 30% vol. methanol, it was possible to separate 16 out of 20 genetically encoded AAs (Fig. 2). Unfortunately, it takes considerable time to completely separate this mixture.
Capillary: internal diameter 75 µm, total length – 65 cm, effective length – 50 cm.
Temperature: 28.0 oC. Sample injection time: 1.5 s (vacuum).
Identification: 1 - lysine, 2 - arginine, 3 - histidine, 4 - glycine, 5 - alanine, 6 - valine, 7 - isoleucine, 8 - leucine, 9 - serine, 10 - threonine, 11 - methionine, 12 - phenylalanine, 13 – glutamic acid, 14 – proline, 15 – aspartic acid, 16 – tyrosine.
Since the applied classical CZE at pH 7 did not provide the required quality of separation, it was decided to apply the MEKC method with direct UV detection to the analysis of free AAs. Sodium dodecyl sulfate (SDS) was added to the buffer solutions as a detergent. During the work, various concentrations of the components of the background electrolyte were used. The best results were obtained using a background electrolyte containing 133 mM boric acid, 33 mM sodium borate and 100 mM SDS, pH 9.5. The use of an electrolyte of the specified composition significantly increased the number of analyzed AA while simultaneously determining them at pH values of the background electrolyte lying in the main region. In Fig. Figure 3 shows an electropherogram of a mixture of 14 AA.
Rice. 3. Separation of a mixture of 14 free AAs by micellar electrokinetic chromatography with direct UV detection Capillary: internal diameter 50 μm, total length 122 cm, effective length 35 cm. Buffer:
133 mM boric acid, 33 mM sodium tetraborate (pH 9.5) 100 mM sodium dodecyl sulfate. Voltage: 20 kV. Current: 48 µA. Temperature: 27.5 oC. Sample injection time: 3.0 s (vacuum).
The administered amounts of AA were 5.0 ng. Alanine, valine, isoleucine and glycine – 7.0 ng.
Identification: 1 – Valine, 2 – Alanine, 3 – Isoleucine, 4 – Glycine, 5 – Serine, 6 – Threonine, 7 – Tyrosine, 8 – Histidine, 9 – Phenylalanine, 10 – Arginine, 11 – Lysine, 12 – Cysteine, 13 – Methionine, 14 – Glutamic acid. * - System peaks.
The obtained values of migration times are noteworthy.
Despite the smaller effective capillary length, they are, as a rule, higher than in the case of classical CZE, which is in fairly good agreement with the nature of the MEKC. This may also be related to the magnitude of the applied voltage per unit length of the capillary. The difference in migration time required for the separation of AC is significantly less than in the case of CZE. It is enough for it to be 0.5 minutes.
A more detailed study was carried out on the conditions for the separation of 14 genetically encoded AAs, usually present in the blood plasma of healthy donors in a free state. The effect of pH and additions of various organic solvents to the background electrolyte on the degree of peak resolution was studied. From the experiment it follows that to achieve complete separation of the mixture of 14 AA, the pH value must lie in the range of 9.0-10.0. At pH values outside the specified range, the necessary resolution of AK is not provided. Obviously, at pH 9 this is due to the difference between pKa (AA) values, and at pH 10 it is due to the partial decomposition of DDSNAC conjugates. The effect of organic solvent additions was studied using methanol, 2-propanol and acetonitrile. The data obtained show that the addition of any of the organic solvents leads to a significant change in migration times and selectivity coefficients. The nature of the change is determined by the nature and concentration of the additive. Methanol and acetonitrile do not improve the separation of the studied AA, which is apparently due to their low ability to form mixed AA-SDS-R conjugates, where R is an organic solvent molecule. The addition of 3-5% 2-propanol significantly improves the degree of resolution of the components, with a relatively small increase in the migration time of AA. With an increase in the concentration of 2-propanol, a noticeable increase in the migration time of AA is observed, which leads to a decrease in the rapidity of determination. Specially conducted studies have shown that the best separation of AA in the presence of an effective amount of an organic solvent (2-propanol) occurs if the background electrolyte contains 50 mM boric acid, 33 mM sodium borate and 50 mM SDS. In Fig. Figure 4 shows an electropherogram of a mixture of 14 AA.
The data presented indicate the effective separation of 13 out of 14 genetically encoded AAs. The total separation time does not exceed min. The migration time Sr is 0.03. Slightly larger Sr values, close to 0.06-0.08, are observed for alanine, valine and histidine.
Rice. 4. Separation of a mixture of 14 AA by MEKC with direct UV detection Capillary: internal diameter 75 μm, total length 61 cm, effective length 41 cm.
Buffer: 33 mM sodium tetraborate, 100 mM boric acid, 50 mM SDS, 5% 2-propanol, pH=10.2. Voltage: 25 kV. Current: 65 µA. Temperature: 29.5 oC. Sample injection time: 1.5 s (vacuum). The administered amounts of AA were 0.5 ng.
Identification: 1 -Lysine; 2 - Proline; 3 - Phenylalanine; 4 - Alanine; 5 - Valine; 6 - Glycine;
7 - Histidine; 8 - Tyrosine; 9 – Leucine + Isoleucine; 10 - Serin; 11 - Threonine; 12 - Glutamic acid; 13 - Cysteine.
The achieved level of resolution made it possible to conduct studies on the quantitative determination of the considered AAs. An analysis of a model mixture of 14 AA of known composition was carried out. Studies have shown that the MEKC method with direct UV detection using a borate buffer solution containing 3-5% 2-propanol makes it possible to quantify 14-16 genetically encoded AAs with an error (Sr) of 6-8% in less than 30 minutes. The accuracy of the results obtained was additionally carried out using the “entered-found” method (Table 1). Verification of the correctness of determining the content of genetically encoded AAs using the MEKC method (mo(AA) = 0.50 ng; entered - 1.00 ng) Analysis of homocysteine and other low molecular weight aminothiols in plasma blood Homocysteine (Hcy) and its accompanying aminothiols (AT) (encoded amino acid - cysteine (Cys), tripeptide glutathione (GSH)) in the blood plasma are molecular markers of dysfunction of the cardiovascular system. The main goal of this study was to develop a method for determining homocysteine content as a reliable risk factor for such diseases.
Quantitative analysis of low molecular weight aminothiols in blood plasma includes the reduction of disulfide bonds, deproteinization of blood plasma, modification of aminothiols with appropriate reagents, separation and identification of modified aminothiols by RP HPLC or CE with one or another detection method. In this work, oxidized and protein-bound aminothiols were reduced with triphenylphosphine. To remove heavy metal cations, an EDTA additive was used. The proposed reduction technique provided rapid (15 min) and complete reduction (96%) of disulfides and the release of sulfhydryl groups at room temperature. The yields of reduction reactions were determined using a standard procedure for measuring the content of free AT. High molecular weight plasma proteins were precipitated with 5-sulfosalicylic acid.
Its concentration was optimized during the study, which avoided loss of sensitivity due to dilution during the neutralization stage.
Modification of free sulfhydryls was carried out with monobromobimane (mBrB) or 5-iodoacetamido-fluorescein (5-IAF). The required pH value was maintained using diethanolamine (pK a = 8.9) and sodium orthophosphate, depending on the reagent used for AT modification. The use of diethanolamine made it possible to directly control the formation of target products by mass spectrometry. To identify AT derivatives, photometric and fluorimetric detection methods were used. The derivatives were characterized by absorption, fluorimetric and mass (MALDI-TOF, ESI) spectroscopy. Photometric and fluorimetric detection was carried out at wavelengths selected according to the absorption spectra (Hcy-MB - 234 nm) and fluorescence spectra of Hcy derivatives (390 nm (excitation) and 478 nm (fluorescence)). From the fluorescence spectrum of the Hcy-AF conjugate it follows that during fluorimetric detection, the optimal wavelength for excitation is 462 nm, and for fluorescence, 504 nm.
To unify the methods for determining and verify the fundamental possibility of using the same detector to identify both monobimane and fluorescein derivatives, the efficiency of excitation at a wavelength of 390 nm was studied. The intensity of the resulting fluorescence maximum, and, as a consequence, the detection sensitivity was an order of magnitude lower than when using radiation at a wavelength of 462 nm for excitation.
Individual monobromobimane (MB) and acetamidofluorescein (AF) AT derivatives, as well as their model mixtures, were analyzed. The individual monobromobimane derivatives Cys, Hcy and GSH eluted with retention times (min) of 6.01 ±0.19, 10.76 ±0.17 and 11.89 ±0.11, respectively (Fig. 5)1.
The same retention time is maintained when using mixtures of aminothiols of known composition. The experimental data obtained made it possible to calculate the yield of the modification reaction. It was no less than 97%, which is in good agreement with the known data, but obtained under more stringent sample preparation conditions. The resulting derivatives were isolated from the mixture and characterized by mass spectrometry.
Fluorescein derivatives Cys, Hcy and GSH were eluted with retention times (min) of 8.49 ± 0.12; 10.46 ±0.15 and 12.96 ±0.14, respectively (Fig. 6).
Chromatographic analysis was carried out on a domestic high-performance liquid chromatograph “MiliChrom A-02” (EkoNova, Novosibirsk) Fig. 5. RP HPLC of a model mixture of aminothiols modified with monobromobimane. Cys-MB-50.0 µM, Hcy-MB-25.0 µM, GS-MB-25.0 µM.
Fluorimetric detection (exc = 390 nm, exp = 478 nm) Fig. 6. RP HPLC of a model mixture of aminothiols modified with 5-iodoacetamidofluorescein. Cys-AF-100.0 µM, Hcy-AF-150.0 µM, GS-AFµM. Fluorimetric detection (abs = 390 nm, esp = 478 nm) When using 5-IAF as a label, better resolution of the peaks of thiol-fluorescent conjugates is achieved compared to thiol-monobimane conjugates. The yield of the AT modification reaction 5-IAF, determined experimentally by reducing the intensity of the fluorescent label peak, was 95%. All the resulting derivatives were isolated from the mixture and characterized by mass spectrometry.
The data obtained served as the basis for the development of a method for the quantitative determination of homocysteine. To construct a calibration curve, samples of mixtures with its content from 2.5 to 100 μM were used. The selected interval includes the range of physiological concentrations of Hcy (5-50 μM). mBrB was used as a label. The resulting calibration dependence of the chromatographic peak area on the homocysteine content in the mixture is linear throughout the studied concentration range and is described by the equation:
The relative standard deviation of the determination results by peak area does not exceed 0.083, and by recovery time – 0.026. The detection limit of fluorimetric detection of MB derivatives is 1 μM (Fig. 7).
Rice. 7. Change in the area of the chromatographic peak depending on the concentration of Hcy. The effectiveness of the method is confirmed by comparing chromatograms obtained for individual substances and for blood plasma samples prepared using the proposed method. The performed studies made it possible to develop a method for the quantitative determination of homocysteine, cysteine and glutathione in blood plasma and successfully use it for routine analysis of the above-mentioned antibodies in the form of their MB derivatives (Fig. 8). When developing the method, additional control was carried out using the “entered-found” method (Table 2).
Rice. 8. RP HPLC of blood plasma from a healthy donor. Cys-MB-192.4 µM, HcyMB-10.1 µM, GS-MB-15.7 µM. Fluorimetric detection Determination of Hcy in the form of MB derivatives using microcolumn HPLC Using the developed method, more than 50 samples of blood plasma from healthy patients and those suffering from cardiovascular diseases of varying severity were analyzed. For healthy patients, the average content (μM) of AT in blood plasma obtained from a vein on an empty stomach in the morning was:
for Hcy 12.75 ±3.21, for GSH 9.53 ±1.17 and for Cys 206.34 ±24.61. The obtained concentration values fall within the range of reference values given in the literature. For patients suffering from cardiovascular diseases, the found concentration of Hcy in the blood plasma depended on the severity of the disease. The results correlate with the clinical condition of the patients.
The possibility of analyzing AT using such a cheaper and more common detection method as photometric was investigated. The experiment showed that it provides sensitivity that allows one to determine pathologically high levels of AT in blood plasma. When using photometric detection, it is preferable to use 5-IAF as a label because it resolves the peaks to the baseline (Fig. 9), allowing for quantitation.
Rice. 9. RP HPLC of model mixtures of aminothiols modified with monobromobimane (a) and 5-iodoacetamidofluorescein (b). A) Cys-MB-50.0 µM, HcyMB-25.0 µM, GS-MB-25.0 µM. B) Cys-AF-50.0 µM, Hcy-AF-75.0 µM, GS-AF- 25.0 µM. Photometric detection (a = 234 nm, b = 250 and 300 nm) Thus, the work performed made it possible to optimize the sample preparation stage, carrying it out under “mild conditions” with a guaranteed quantitative yield of the analyzed component. On its basis, a method has been developed that allows one to quickly and reliably determine the content of low-molecular-weight antibodies in the blood plasma of healthy and sick patients. The measurement error does not exceed 8.5%. The lower limit of the range of measured concentrations (2.5 μM) indicates the fundamental possibility of using this technique to determine the reduced content of Hcy in blood plasma. The detection limit of the described method is 1 µM. The developed method was tested on real samples of patient blood plasma and can be used for routine use.
Determination of homocysteine and other low molecular weight aminothiols in plasma Fig. 10. CZE of the AT model mixture, has a migration time of 6.18 ±0.16; cysteine modified mBrB Hcy-MB-700.0 µM, Cys-MB-300.0 µM 6.83 ± 0.20 and glutathione - 8.54 ± 0.17 min, respectively (Fig. 10). Compared to GS-MB- 700.0 µM. (absorb = 234 nm).
Capillary: internal diameter 50 µm, full HPLC CZE method used, allowing length 82 cm, effective length 62 cm.
Buffer: 50 mM sodium tetraborate pH=11.0. reduce the time of AT analysis by 2-3 minutes.
Voltage: 25 kV. Current: 58 µA.
(vacuum) direct UV detection is not enough to determine small amounts of Hcy in blood plasma. At a homocysten content of 10 µM, the signal/background ratio is 2.5-3, and the relative standard deviation is in the range of 0.3-0.5. This method is applicable to control the content of Hcy in the blood plasma of patients with pathologically high levels (25 µM). The relative standard deviation when determining these concentrations is 0.12 for MB-Cys, 0.18 for MB-Hcy and 0.17 for MB-GS.
Capillary zonal electrophoresis with fluorimetric detection was carried out on a capillary ion analyzer “Nanofor 02” (INP RAS, St. Petersburg) Analysis of Hcy in the form of MV derivatives using RP HPLC with fluorimetric and CZE with photometric detection (n = 5; P = 0.95 ) Model mixtures of AF derivatives were studied by the CZE method with direct photometric (Fig. 11) and fluorimetric (Fig. 12) detection (Table 3). Photometric detection was performed at a wavelength of 492 nm, which corresponds to the excitation wavelength of 5-IAF. For fluorimetric detection, the excitation wavelength was 473 nm and the emission wavelength was 514 nm. It has been established that the use of 5-IAP makes it possible to increase the sensitivity of the determination of Hcy by the CZE method with fluorimetric detection.
Absorbance, 492 nm Fig. Fig. 11. CZE of a model mixture of AT, mo- Fig. 12. CZE of a model mixture of ATs modified with 5-iodoacetamido-modified 5-iodoacetamidofluorescein with direct UV fluorescein with fluorimetric Hcy-AF-100.0 µM, Cys-AF-300.0 µM, GS-AF-Hcy-AF-15, 0 µM, Cys-AF-15.0 µM, GS-AFµM. (abs = 473 nm, exp = 514 nm) 700.0 µM. (absorb = 492 nm).
Capillary: internal diameter 50 µm, total Capillary: internal diameter 50 µm, total length 68 cm, effective length 53 cm. length 65 cm, effective length 57 cm.
Buffer: 25 mM sodium tetraborate, 25 mM phosphate Buffer: 25 mM sodium tetraborate, 25 mM sodium phosphate pH = 11.2. Voltage: 20 kV. Current strength: sodium pH = 11.2. Voltage: 20 kV. Current strength:
22 µA. Temperature: 25.0 oC. The input time is 18 µA. Temperature: 25.0 oC. Sample injection time: 5 s (electrokinetic, voltage at: 5 s (electrokinetic, voltage at) The developed methods for determining the homocysteine content in blood plasma using RP HPLC and capillary electrophoresis have been introduced into the practice of biomedical analysis by JSC Medical Technologies, Ltd.
using capillary gel electrophoresis. Changes in the content of molecular markers in physiological fluids may also be due to the patient’s genetic predisposition to a specific disease. Hence the need to conduct a comparative analysis of DNA fragments in order to increase the reliability of determining the cause of the disease under study and more effective its therapy.
In this work, we investigated the possibility of using available domestic CE equipment to determine mutations in DNA molecules using the example of fragments of the mutant gene for venous thrombosis, using allele-specific PCR. Nucleotides were separated by capillary gel electrophoresis (CGE) using unmodified quartz glass capillaries with a diameter of 25 to 100 μm. Linear poly-N,N-dimethylacrylamide (pDMA) of domestic production was used as a separation matrix. The choice of this polymer is related to the possibility of its use in the chip version of the CGE. pDMA was synthesized at Synthol JSC from dimethylacrylamide monomer by radical polymerization. The chain length was controlled by changing the temperature or adding radical scavengers. Polymers containing 5-8% pDMA monomer were used. 0.1 M TBE (0.1 M TRIS, 0.1 M boric acid, and 2.5 mM EDTA; pH = 8.3) and 0.1 M TAPS (N-tris(hydroxymethyl)methyl) were used as background electrolytes. 3-aminopropanesulfonic acid; pH = 8.3) All studied polymers had a low level of native fluorescence (0.5 AU). Polymers prepared using 0.1M TAPS allow up to 5 separations without refilling the capillary with gel, while those containing TBE allowed no more than 3. These polymers provide resolution comparable to their corresponding Western counterparts, but are also more affordable .
Studies of a mixture of fluorescently labeled polynucleotides with lengths of 5-15 nucleotides. respectively, with a content of 10-9 M of each of them, it was possible to determine the optimal polymer composition for separating oligonucleotides with a chain length of up to 100 nt. (Fig. 13). This is a pDMA-based gel containing 6% monomer, 7M urea and 0.1M TAPS.
Rice. 13. Separation of a mixture of polynucleotides with lengths Capillary: internal diameter - 50 µm, total length - 45 cm, effective length - 38.5 cm. Polymer: 6% pDMA monomer, 0.1M TAPS, 7M urea. Working electrolyte: 0.1M TAPS. Voltage:
10 kV. Current: 4.3 µA. Temperature: 25.0 oC. Sample injection time is 10 s (electrokinetic, sample collection voltage is 10 kV).
Optimization of separation conditions (voltage, current, effective length and capillary diameter) made it possible to achieve separation to a baseline of oligonucleotides with a total length of less than 100 nt. and a difference in length of 1 n.o. (Fig. 14.).
Rice. 14. Separation of a mixture of polynucleotides with a difference in length of 1 bp.
Capillary: internal diameter - 50 µm, total length - 65 cm, effective length - 57.5 cm. Polymer: 6% pDMA monomer, 0.1M TAPS, 7M urea. Working electrolyte: 0.1M TAPS. Voltage:
11 kV. Current: 5.1 µA. Temperature: 25.0 oC. Sample injection time is 10 s (electrokinetic, sample collection voltage is 10 kV).
The data obtained served as the basis for the development of a system for rapid and effective diagnosis of the mutant gene for venous thrombosis (Leiden gene of factor V of the human blood coagulation system).
To prove the possibility of joint determination of wild- and mutant-type products (difference 5 bp), the following PCRs were performed: 1.
Wild type DNA (no mutation) + wild type primer; 2. Wild type DNA (without mutation) + FV Leiden primer; 3. DNA with heterozygous mutation FV Leiden + primer FV Leiden; 4. FV Leiden primer without DNA. The reaction products, after treatment with formamide and dilution with water, were analyzed by CGE with fluorimetric detection. Analysis of samples 1 and 3 showed the presence of the product in the mixture after PCR, and samples 2 and 4 showed its absence. To confirm this pattern, we analyzed a mixture of mutant primer/mutant product and wild-type primer/wild-type product samples in a 1:1 ratio (Fig. 15).
Rice. 15. Joint determination of mutant and non-mutant PCR products Capillary: internal diameter – 50 µm, total length – 45 cm, effective length – 38.5 cm. Polymer 6% pDMA monomer, 0.1 M TAPS, 7 M urea. Working electrolyte: 0.1M TAPS. Voltage: 12 kV.
Current: 6.5 µA. Temperature: 25.0 oC. Sample collection time: 10 s (electrokinetic, sample collection voltage – 10 kV).
The data obtained show the possibility of joint determination of allele-specific PCR products of the FV Leiden mutation and the wild type. The proposed approach, namely the selection and synthesis of allele-specific primers of different lengths and a common counter primer, followed by analysis by CGE with fluorimetric detection, can be extended to diagnose other genetically determined diseases. It should also be noted that it is possible to analyze oligonucleotides using standard domestic equipment used for the analysis of amino acids and short peptides.
1. Methods have been developed for the analysis of genetically encoded amino acids without their preliminary derivatization using micellar electrokinetic chromatography and capillary zone electrophoresis with refractometric and direct UV detection. The influence of the composition and pH value of the background electrolyte, as well as the addition of organic solvents, on the separation efficiency was studied.
2. Using the method of micellar electrokinetic chromatography with direct UV detection, a quantitative determination of a model mixture of 14 genetically encoded free amino acids was carried out.
3. A method has been developed for rapid and reliable determination of the content of low molecular weight aminothiols in the blood plasma of healthy and sick patients using reversed-phase HPLC with fluorimetric detection. The fundamental possibility of using this technique to determine low levels of homocysteine in blood plasma is shown. The developed method was tested on real samples of patient blood plasma.
4. The possibility of determining pathologically high concentrations of homocysteine in blood plasma by capillary zone electrophoresis with photometric detection has been demonstrated. The developed method was tested on real samples of patient blood plasma. The possibility of using 5-iodoacetamidofluorescein as an absorbing and fluorogenic label was studied.
5. A method has been developed for the selective determination of fragments of the mutant gene for venous thrombosis (FV Leiden mutation) by capillary gel electrophoresis with fluorimetric detection. The possibility of analyzing nucleotides with a chain length of up to 100 nucleotides has been demonstrated. and with a difference in length of 1 n.o.
The main materials of the dissertation are presented in the following works:
1. Melnikov I.O., Nazimov I.V., Stukacheva E.A., Glubokov Yu.M. Determination of the content of homocysteine and other low molecular weight aminothiols in blood plasma. // J. Anal. Chem. -2006. -T. 61. -No. 11. -S. 1185-1191.
2. Melnikov I.O., Glubokov Yu.M., Nazimov I.V. Capillary electrophoresis of free amino acids with refractometric and direct UV detection. // Inf.-anal. bulletin "Scientific notes of MITHT." M.:
MITHT. -2004. Vol. 11. -S. 54-57.
3. Melnikov I.O., Glubokov Yu.M., Nazimov I.V. Analysis of low molecular weight aminothiols by capillary electrophoresis and RP HPLC with fluorescent and direct UV detection. // J. “Modern high-tech technologies.” M.: Academy of Natural Sciences, -2005. -No. 3. -P.40-41.
4. Melnikov I.O., Nazimov I.V., Stukacheva E.A., Glubokov Yu.M. HPLC and HPCE determination of homocysteine as a criterion of vascular diseases risk factor. // FEBS J. -2006. -V. 273. -S.1. -P. 256.
5. Melnikov I.O., Nazimov I.V., Lobazov A.F., Popkovich G.V. Capillary electrophoresis of unmodified amino acids. // Abstract. report 8th All-Russian Symposium on Molecular Liquid Chromatography and Capillary Electrophoresis, Moscow, October 2001, P. 23.
6. Melnikov I.O., Nazimov I.V., Lobazov A.F., Popkovich G.B. Capillary Electrophoresis Of Coded Nonmodified Amino Acids With Refractometric Detection. // 3rd International Symposium on Separations in BioSciences, Moscow, May 2003, P. 263.
7. Melnikov I.O., Stukacheva E.A., Glubokov Yu.M., Nazimov I.V. Determination of homocysteine and other low molecular weight aminothiols in blood plasma by CEF and RP HPLC methods. // Materials of the International Conference of Students and Postgraduate Students in Basic Sciences “Lomonosov-2005”.
M.: MSU, 2005. Chemistry section. -T. 1. -S. 31.
8. Nazimov I.V., Melnikov I.O., Glubokov Yu.M., Sivoplyasova E.A. Instrumental micromethods for determining homocysteine as a risk factor for cardiovascular diseases. // Materials of the 2nd scientific and practical conference “Current problems of medical biotechnology”, Anapa, September 2005, -S. 15-16.
9. Melnikov I.O., Nazimov I.V., Sivoplyasova E.A., Glubokov Yu.M. Micromethods for the quantitative determination of homocysteine in blood plasma. // Materials of the 3rd Congress of the Society of Biotechnologists of Russia named after. Yu.A. Ovchinnikova, Moscow, October 2005, -S. 61.
10. Melnikov I.O., Sivoplyasova E.A., Glubokov Yu.M., Nazimov I.V. Determination of risk factors for cardiovascular diseases using chromatographic analysis methods. // Materials of the 1st conference of young scientists MITHT im. M.V. Lomonosov, Moscow, October 2005, -S. 31.
11. Melnikov I.O., Nazimov I.V., Stukacheva E.A., Glubokov Yu.M. Separation and identification of blood low molecular weight aminothiols by reversed phase high performance liquid chromatography and capillary electrophoresis. // International congress on analytical sciences ICAS-2006, Moscow, June 2006, -P. 204.
12. Melnikov I.O., Nazimov I.V., Patrushev L.I., Alekseev Ya.I., Glubokov Yu.M., Mosina A.G. Determination of human leiden gene mutation by high performance capillary gel electrophoresis. // 26th International Symposium on the Separations of Proteins, Peptides and Polynucleotides, LMP, Innsbruck, Austria, October 2006, -P. 24.
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Transcript
1 NOVOSIBIRSK STATE UNIVERSITY Faculty of Natural Sciences Department of Analytical Chemistry Course work Prediction of retention volumes and UV spectra of peptides in reverse phase HPLC Completed by: Kuchkina A.Yu., gr. 147 Scientific supervisor: Ph.D. Azarova I.N. Novosibirsk-2005
2 CONTENTS 1. Introduction Literature review 2.1. Peptides. Amino acids of HPLC peptides Prediction of retention volumes and UV spectra of peptides in RP HPLC Experimental part Results and their discussion 4.1. Monitoring the stability of the chromatographic system Calculation of peptide retention volumes Linear “composition retention” model Nonlinear “composition retention” model Calculation of UV spectra of peptides Conclusions Literature Appendix
3 1. INTRODUCTION Identification of proteins functioning in living cells based on known information about the structure of the genome, proteomics, is considered one of the most important tasks of modern molecular biology. This problem has arisen after the complete deciphering of the genomes of some organisms in the past few years. It turned out that genomes encode many more proteins than the body produces them. Identification of the protein of interest in the sum of all proteins is a complex methodological task that, as a rule, cannot be solved by a direct method. One of the approaches to solving this kind of problem, called peptidomics, is that the sum of proteins is hydrolyzed with a specific protease, and the sum of the resulting peptides is separated by capillary electrophoresis or high-performance liquid chromatography (HPLC). The ultimate goal is to detect a peptide (peptides) of a known structure, which is (are) a priori a fragment of a protein encoded by the genome of the organism under study. If such a peptide(s) is detected in a sample, it is concluded that the protein is being produced by the body. Methodological problems that arise when solving problems of this kind can be divided into 2 groups. The first problem is separating a mixture, usually consisting of several thousand peptides. The second is determination of the structure of the isolated individual peptides. The separation problem is solved by fractionating the primary mixture followed by separating the separated fractions into individual components. The second problem is solved mainly by mass spectrometric methods, which ultimately make it possible to determine the molecular masses of individual components (peptides). If a peptide has a fairly “unique” structure (a set of amino acids) - these usually include long (more than amino acid residues) peptides - then its molecular weight will also be “unique”, and the probability of erroneous identification becomes negligible. Since the peptide separation procedure is aimed at isolating a peptide with a known set of amino acids, the question arises: is it possible to predict the time of release of such a peptide from the HPLC column, knowing its amino acid composition? This approach was first demonstrated in the early 80s. The purpose of this study was to develop a methodology for predicting the retention volumes of peptides on a Milichrom A-02 chromatograph in reverse-phase HPLC (RP HPLC) mode using a mobile phase suitable for direct injection into the mass spectrometer. 3
4 2. LITERATURE REVIEW 2.1. Peptides. Amino acids Peptides are biopolymers built from α-amino acid residues connected to each other by peptide bonds (-NH-CO-). The general formula of a peptide can be presented as follows: NH 2 CH CO NH CH CO... NH CH R 1 R 2 Rn There is no clear boundary between proteins and peptides; as a rule, peptides include biopolymers containing no more than 100 amino acid residues. Amino acids are any organic acids whose molecules include an amino group; this name often means α-amino acids, because they have important biological significance. The molecules of most natural amino acids that make up proteins have the general formula H 2 NCH R As can be seen from the structural formula, amino acids (with the exception of proline) differ from each other only in the structure of the side chain R. Amino acids are amphoteric compounds, since their molecules contain both an acidic carboxyl group and a basic amino group. In a strongly acidic environment, the carboxyl group is predominantly undissociated, and the amino group is protonated. In a strongly alkaline environment, the amino group is in the form of a free base, and the carboxyl group is in the form of a carboxylate anion. In the crystalline state, amino acids exist in the form of a zwitterion, where a proton from the carboxyl group is transferred to the amino group. Only 20 amino acids are involved in the biosynthesis of natural peptides and proteins. Amino acids and their properties are given in table 1. Table 1. Amino acids and their properties. Amino acid name Code Structure p a - -NH 2 -R Glycine G H 2.35 9.78 Alanine A H 3 C 2.35 9.87 Valine V H 3 C CH CH 3 2.29 9.74 4
5 Name of amino acid Code Structure p a - -NH 2 -R Leucine L H 3 C CH CH 3 2.33 9.74 Isoleucine I H 3 C * CH CH 3 2.32 9.76 H 2 C Proline P HC NH 1.95 10.64 Phenylalanine F 2.20 9.31 Tryptophan W N H CH NH 2 2.46 9.41 Tyrosine Y HO CH NH 2 2.20 9.21 10.46 Serine S HO 2.19 9.21 Threonine T HO * CH CH 3 2.09 9.10 Cysteine C HS 1.92 10.70 8.37 Aspartic acid D HOOC 1.99 9.90 3.36 Glutamic acid E HOOC 2.10 10.47 4.07 Asparagine N H 2 N C O 2.14 8.72 Glutamine Q H 2 N C O 2.17 9.13 Lysine K H 2 N 2.16 9.06 10.54 Arginine R H 2 N C NH 1.82 8.99 12.48 NH Histidine H N NH 1, 80 9.33 6.04 Methionine M H 3 C S 2.13 9.28 5
6 Depending on the nature of the side chains, amino acids are divided into two groups: amino acids with non-polar (hydrophobic) aliphatic or aromatic R-groups; amino acids with polar (hydrophilic) R-groups. The first group includes 8 amino acids: alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan. Seven amino acids contain groups in the side chain that can carry a negative or positive charge: aspartic and glutamic acids are negatively charged at pH 7.0; lysine, arginine, histidine are basic amino acids whose side chains can be positively charged; under alkaline conditions, the tyrosine and cysteine side groups of HPLC peptides can be negatively charged. The separation of mixtures of peptides is a very difficult task. Currently, RP HPLC is the most important method for separating peptides. Peptides are polar substances, which prevents their interaction with the hydrophobic surface of the stationary phase and leads to interaction with residual silanol groups. In order to reduce the interaction with silanol groups, strong acids or salts are added to the eluent. To reduce the polarity of the peptides, chromatography should be performed at low pH values (below 3). If these principles are followed, HPLC proves to be a very flexible method for separating peptides. This is due to the fact that this method is characterized by a high separation speed, reproducibility of results and the ability to analyze microgram amounts of substances. Another advantage of RP HPLC is the ability to collect fractions in a small volume of volatile solvents, which simplifies further mass spectrometric analysis. As a rule, 0.1% trifluoroacetic and formic acids are used as volatile solvents. Trifluoroacetic acid is transparent in the UV region up to 205 nm, which is a great advantage when chromatography of complex mixtures. In addition, the peptides are highly soluble in trifluoroacetic acid. Elution of peptides from reverse phases is usually carried out in a gradient mode with the addition of an organic modifier. The most common organic modifier is acetonitrile. It is transparent in the UV region up to 200 nm and has good selectivity. 6
7 Another factor driving the widespread use of RP HPLC for the analysis of peptides is the ability to predict their chromatographic behavior Prediction of retention volumes and UV spectra of peptides in RP HPLC The idea that the chromatographic behavior of peptides containing less than 25 amino acid residues can be predicted by knowing them amino acid composition, first stated by J. Meek. The retention of peptides in the reverse phase is determined by the hydrophobicity of the amino acid residues included in their composition. Amino acids with aromatic or large aliphatic chains are the major contributors to retention. Based on the above, Meek proposed calculating the retention volumes of peptides in the reverse phase as the sum of the contributions of the amino acid residues that make up the peptide. He proposed a linear (additive) “composition retention” model: V R = V 0 + Z N* + Z C* + Z i (1) where V R peptide retention volume; v 0 is the free volume of the column; Z N* and Z C* are respectively the contributions of free -NH 2 and - or modified terminal groups of the peptide; Z i is the contribution of the i-th amino acid (retention constant). Meek chromatographed 25 peptides under gradient RP HPLC conditions and, solving the inverse problem, calculated the amino acid retention constants. The correlation coefficients of the dependence V R (calc.)=f(v R (exp.)) were 0.997 (at ph 2.1) and 0.999 (at ph 7.4). Despite the high correlation coefficients, this model contradicts the physical meaning, since the amino acid retention constants obtained in this way do not reflect real differences in their hydrophobicity and some of the contributions have negative values. In addition, there are many facts according to which, with an increase in the number of amino acid residues in a peptide, the surface of its hydrophobic contact with the reverse phase, which determines retention, increases nonlinearly. The authors of the work also made the assumption that the retention volumes of peptides are calculated from the sum of the contributions of amino acid residues. To calculate the retention volumes of peptides, they proposed the following model: V R = A* ln (1+ Z j * n j) + B (2) 7
8 where Z j is the empirical retention parameter, which is calculated based on the hydrophobicity of amino acids; A and B constants; n j is the number of amino acid residues j in the peptide. This model provides a good correlation between calculated and experimental peptide retention volumes. The disadvantage of the model is that it is valid only for a specific chromatographic system (linear gradient with a given slope, fixed flow rate, mobile and stationary phases). Thus, the application of this model is very limited. Therefore, the authors of the work under review proposed another model based on the theory of gradient elution, which allows one to calculate the retention volumes of peptides for a wider range of chromatographic systems. Considering that the available area of hydrophobic contact of the peptide with the stationary phase varies in proportion to its molecular weight to the power of 2/3, the authors selected a function for calculating the retention volumes of peptides: V R = f (3) where a, b, c are constants depending on the properties of a particular chromatographic systems were determined experimentally from chromatography data of 15 peptides selected for this purpose. The correlation coefficient of the dependence V R (calc.)=f(v R (exp.)) was 0.98. A significant drawback that limits the use of this method is the need to calculate constants a, b and c for a specific chromatographic system from preliminary experiments with model peptides, which is a very labor-intensive procedure. The work calculated the retention volumes and UV spectra of peptides with a known amino acid sequence, having previously determined the contributions of amino acids to the above parameters. The values of these contributions were found experimentally from chromatograms of solutions of individual amino acids obtained by multi-wavelength photometric detection under the same conditions under which the peptides were chromatographed. The authors of the work proposed the following equation for calculating the retention volumes of peptides: V R = 209 [(Z i) + Z C*+N* + V 0 ] 1/3 990 (4) where Z i is the retention coefficient of amino acid “i”; V 0 free volume of the column; Z C*+N* is the total retention constant of the terminal groups of the peptide. When calculating the UV spectra of peptides, the spectrum of the peptide was considered as the sum of the spectra of all its structural elements. To test the proposed method there were 8
9, 30 peptides were chromatographed, and the correlation coefficient between the calculated and experimentally found retention volumes was 0.95. The proposed method for calculating the UV spectra of peptides also has satisfactory predictive power. In this work, acetonitrile and an aqueous solution of lithium perchlorate (0.2 M LiCLO M HClO 4), which is a non-volatile substance, were used as eluents, which makes subsequent mass spectrometric identification of peptides very difficult. Therefore, it seemed appropriate to us to develop a method using a mobile phase of the composition acetonitrile - trifluoroacetic acid, all components of which are volatile substances and do not interfere with mass spectrometric analysis. 9
10 3. EXPERIMENTAL HPLC was carried out on a Milichrome A-02 chromatograph on a 2x75 mm column with ProntoSIL C 18 AQ phase (Bischoff Analysentechnik und Geräte GmbH, Germany) under the following conditions: eluent A: 0.01 M trifluoroacetic acid; eluent B: acetonitrile; linear gradient: 40 min from 5 to 100% B; flow rate: 100 µl/min; column temperature: 40 C; detection: at 210, 220, 230, 240, 250, 260, 280 and 300 nm, τ = 0.18 s; sample volume: 4 µl. Solution for testing the chromatographic system: KBr - 0.2 mg/ml; uridine - 0.2 mg/ml; caffeine-1 mg/ml; m-nitroaniline - 0.1 mg/ml; o-nitroaniline-0.1 mg/ml; solvent 2% acetonitrile in water. All reagents with a mass fraction of the main substance of at least 98%. Amino acids (Serva, Germany), acetonitrile “Grade 0” (NPF “Kriochrome”, St. Petersburg), and anhydrous trifluoroacetic acid (ICN Biomedicals, USA) were used in the work. Peptide samples were kindly provided by V.V. Samukov (NPO “Vector”, Novosibirsk) and I.V. Nazimov (IBCh RAS, Moscow). The concentrations of amino acids in the chromatographed solutions were 0.2 1 mg/ml, peptides 0.1 2 mg/ml. Chromatograms were processed using the Multichrome program (Ampersend JSC, Moscow). To calculate VR, areas of chromatographic peaks when detected at 8 wavelengths (S λ) and graphical representation of peptide chromatograms, the “CHROM P” program (ZAO Institute of Chromatography “EcoNova”, Novosibirsk) was used. Linearization of the curve shown in Figure 1 was carried out using the Microsoft Excel program (Microsoft Corporation). 10
11 4. RESULTS AND THEIR DISCUSSION 4.1. Monitoring the stability of the chromatographic system The stability of the chromatographic system was monitored using a procedure regulated by the methodology. The test solution was chromatographed and 14 parameters were calculated from the resulting chromatograms, each of which controls a certain indicator of the chromatographic system: VR bromide ion free volume of the column; spectral ratio S 280 /S 250 of uridine; detector tuning accuracy in the range from 250 to 280 nm; spectral ratio S 260 /S 280 caffeine linear range of the detector; spectral ratio S 260 /S 230 m - nitroaniline suitability of eluent A. The o-nitroaniline peak was used to control: V R deviation of the gradient from a given shape, spectral ratios, detector tuning accuracy in the range from 210 to 300 nm, asymmetry of peak A 10% violation in column packing , S 210 sample dosing accuracy. Periodic measurements of the chromatographic and spectral parameters of the model solution allowed us to verify the reproducibility of the operation of the chromatographic system used. The testing results are shown in Table 2. Table 2. Results of testing the chromatographic system, n = 8. Substance Parameter 11 Average value of parameter S r (%) Bromide V R, µl 148 1.0 Uridine S 280 /S 250 0.53 1.3 Caffeine S 260 /S 280 0.73 0.6 m-nitroaniline S 260 /S 230 0.80 1.0 o-nitroaniline V R, µl,6 S 220 /S 210 1.71 0.5 S 230 /S 210 1.76 0.7 S 240 /S 210 1.11 0.6 S 260 /S 210 0.58 0.9 S 250 /S 210 0.40 1.4 S 280 /S 210 0.59 0.9 S 300 /S 210 0.32 1.2 A 10% 1.05 1.1 Output signal (peak area) S 210, e.p.p. µl 25.00 1.4
12 The obtained values of S r allow us to draw a conclusion about the stability of the described chromatographic system Calculation of peptide retention volumes The initial chromatographic data necessary for calculating the amino acid retention coefficients were obtained from the chromatograms of these amino acids and peptides GG and GGG under the conditions specified in section 3. Amino acid retention coefficients calculated using the equation: Z i = V Ri V 0 Z C*+N* (5) where V Ri is the retention volume of amino acid “i”; V 0 free volume of the column (retention volume Br -, in this chromatographic system it was 148 µl); Z C+N is the total retention constant of the terminal groups of the peptide. Z C+N was calculated using the equation: Z C*+N* = V R (G) V 0 ((6) where V R (G) is the retention volume of glycine, µL; V R (GGG) and v R (GG) are the retention volumes of GGG and GG peptides , µl The sum of the retention coefficients of the end groups [(-NH 2) + (-CONH 2)] was considered equal to the sum of the retention coefficients of the groups [(-NH 2) + (-)].Chromatographic data and calculated retention constants of the structural elements of the peptides are given in table 3. Table 3. Retention volumes of amino acids and peptides GG and GGG Retention constants of structural elements of peptides Code V R, μl Z i, μl Code V R, μl Z i, μl N P S V G M Q Y D I H L T F E W K GG C GGG A (C*+N*) end - 25 R
13 Linear model “composition retention” To predict the retention volumes of peptides within the framework of the linear model proposed in the work, we calculated the retention volumes of 28 peptides (Table 4) and compared the obtained data with the data found experimentally (Figure 1). Table 4. Experimentally found and calculated peptide retention volumes (linear model). Peptide V R (exp.), µl V R (calc.), µl 1 GG GGG AS GRGDS TKPR WAGGDASGE GL MY KPVGKKRRPVKVYP W-D-YGGDASGE W-D-AGGDA NCMLDY SYSMEHFRWG W-D-VGGDASGE YGGFLRKYPK RPPGFSPFR MEHFRWG RVYIHPF YGGFLRRIRP KLK YGGFM DRVYIHPF QATVGDINTERPGMLDFTGK ELYENKRPRRPYIL DRVYIHPFHL Y-D-AGFL RPKPQQFFGLM -NH PQQFFGLM-NH GIGAVLKVLTTGLPALISWIKRKRQQ-NH
14 V VR R(calc.), µl VR(exp.), R µl Fig. 1. Comparison of experimentally found and calculated peptide retention volumes. The calculation of VR values was carried out according to equation (1). As can be seen from the data obtained, the linear model gives satisfactory results only for relatively hydrophilic peptides; for hydrophobic peptides, the calculated values are noticeably higher than the experimental ones. Similar results were obtained in the work Nonlinear “composition retention” model Linearization of the curve shown in Fig. 1 gives the equation: V R = 173 [(n Z i) + V 0 ] 1/3 785 (7) A comparison of the calculated values and the experimentally found retention volumes for 28 peptides is shown in Table 5 and Fig.
15 V R (calc.), µl VR VR (exp.), µl V R Fig. 2. Comparison of experimentally found and calculated peptide retention volumes. The calculation of VR values was carried out according to equation (7). 15
16 Table 5. Experimentally found and calculated peptide retention volumes (nonlinear model). Peptide V R (exp.), µl V R (calc.), µl 1 GG GGG AS GRGDS TKPR WAGGDASGE GL MY KPVGKKRRPVKVYP W-D-YGGDASGE W-D-AGGDA NCMLDY SYSMEHFRWG W-D-VGGDASGE YGGFLRKYPK RPPGFSPFR MEHFRWG RVYIHPF YGGFLRRIRP KLK YGGFM DRVYIHPF QATVGDINTERPGMLDFTGK ELYENKRPRRPYIL DRVYIHPFHL Y-D-AGFL RPKPQQFFGLM -NH PQQFFGLM-NH GIGAVLKVLTTGLPALISWIKRKRQQ-NH The correlation coefficient of the dependence V R (calc.)=f(v R (exp.)) was 0.96. The Appendix shows experimental and calculated chromatograms of a model mixture of 11 peptides. 16
17 4.3. Calculation of UV spectra of peptides Specific spectral coefficients of structural elements of peptides were taken from the work, because In the chromatographic system we use, the correct calculation of spectral ratios is hampered by systemic peaks, as well as poor retention of hydrophilic amino acids and short peptides. The values of the specific spectral coefficients are given in Table 6. Table 6. Spectral coefficients of UV-absorbing structural elements of peptides as the area of chromatographic peaks at C = 1 mm and a sample volume of 4 μl, (e.o.p. μl). λ, nm W F Y H C M R Q N E D N*+C* PS where S C*+N* spectral coefficients of the sum of groups (-NH+ -) of the peptide, S PS absorption of the peptide bond. The spectral characteristics of peptides as the area of their chromatographic peaks at C = 1 mM and a sample volume of 4 μl were calculated using the equation: a λ peptide = a λ N*+C* + (m-1) a λ λ PS + a i (9) where m is total the number of amino acid residues in the peptide, a λ N*+C* is the conditional peak area of the terminal groups of the peptide, and λ PS is the specific absorption of the peptide bond at wavelength λ λ, and i is the spectral characteristics of amino acid residues for wavelength λ. The quantities included in equation (9) were obtained as follows. The specific spectral characteristics of the structural elements of peptides at a wavelength λ=210 nm (a 210 i) were calculated as the area of their chromatographic peaks for a solution with a concentration of 1 mM and a sample volume of 4 μl according to the equation: a 210 i = a 210 AA a 210 N*+C *, (10) where a 210 AA is the peak area of the amino acid; a 210 N*+C* is the conventional peak area of the terminal groups of the peptide. The value of a 210 N*+C* was taken equal to a 210 Leu, since NH 2 groups are the only Leu chromophores in the nm wavelength range. 17
18 The specific absorption of the peptide bond (a 210 PS) was calculated as the difference between the specific absorptions of the GGG and GG peptides: a 210 PS = a GGG a GG (11) Spectral characteristics for wavelength λ were calculated using the equation: a λ i = a 210 i (S λ /S 210), (12) where S λ /S 210 are the spectral ratios of amino acids and peptides GG and GGG, which are the ratios of peak areas at wavelengths λ to the peak area at λ = 210 nm. Table 7 shows a comparison of the calculated spectral ratios S λ /S 210 for 28 peptides with those obtained experimentally. The experimentally obtained and calculated spectral ratios of the peptides are in fairly good agreement with each other. In most cases, the differences are no more than 0.06. For some peptides (5, 9, 12, 16, 18, 22, 23, 26, 27), more noticeable differences in predicted and experimental spectral ratios are observed. The reasons for these differences require additional research. Table 7. Comparison of experimental and calculated values of spectral ratios of peptides. Peptide Value Spectral ratios S λ /S 210 for wavelengths λ(nm): Vych Exp Vych Exp Vych Exp Vych Exp Vych Exp Vych Exp Vych Exp Vych Exp
19 Peptide Value Spectral ratios S λ /S 210 for wavelengths λ(nm): Vych Exp Vych Exp Vych Exp Vych Exp Vych Exp Vych Exp Vych Exp Vych Exp Vych Exp Vych Exp Vych Exp Vych Exp Vych Exp Calc Exp Calc Exp
20 Peptide Value Spectral ratios S λ /S 210 for wavelengths λ(nm): Vt Exp V V Exp From the data obtained it is clear that the described method for calculating the retention volumes of peptides of known composition and their UV spectra under conditions of gradient RP HPLC has satisfactory predictive power and may be useful in studies involving the separation of mixtures of peptides. 20
21 5. CONCLUSIONS 1. A method has been developed that makes it possible to predict the retention volumes of peptides of known composition in the gradient RP HPLC mode on a Milichrome A-02 chromatograph using a volatile mobile phase suitable for direct introduction of isolated fractions into a mass spectrometer. 2. A method has been developed for calculating the optical absorption of peptides at wavelengths of 210, 220, 230, 240, 250, 260, 280 and 300 nm directly in the mobile phase used to separate peptides. 3. The developed methods were tested for 28 model peptides. The correlation coefficient of the calculated retention volumes with the corresponding experimental values was 0.96. The discrepancy between the calculated and experimentally obtained spectral ratios S λ / S 210 was no more than 0, REFERENCES 1. Reznikov V.A., Shteingarts V.D. Amino acids. Novosibirsk: NSU, S. Dawson R., Elliott D., Elliott W., Jones K. Handbook of a biochemist. Moscow: Mir, p. 3. Ovchinnikov Yu.A. Bioorganic chemistry. Moscow: Enlightenment, p. 4. High-performance liquid chromatography in biochemistry (edited by Henschen A.). Moscow: Mir, p. 5. Meek J.L. //Proc. Natl. Acad. Sci. USA. 1980, V. 77. No.3. P Sakamoto Y., Kawakami N., Sasagawa T. // J. Chromatogr V P Sasagawa T., Okuyama T., Teller D.C. // J. Chromatogr V P Browne C.A., Bennet H.P.J., Solomon S. // Anal. Biochem V.124. P Meek J.L., Rossetti Z.L. // J. Chromatogr V P Azarova I.N., Baram G.I., Goldberg E.L. // Bioorganic chemistry. In the press. 11. Mass concentration of UV absorbing substances. Methodology for performing measurements using high-performance liquid chromatography. FR Irkutsk
22 7. Appendix Experimental (A) and calculated (B) chromatograms of a mixture of 11 peptides. Peptide numbers in accordance with Table A 0.5 e.o.p nm B nm Volume, µl
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PEPTIDES, natural or synthetic. compounds, the molecules of which are built from a-amino acid residues connected to each other by peptide (amide) bonds C(O) NH. The molecule may also contain a non-amino acid component (for example, a carbohydrate residue). Based on the number of amino acid residues included in peptide molecules, di-peptides, tripeptides, tetrapeptides, etc. are distinguished. Peptides containing up to 10 amino acid residues are called. oligopeptides containing more than 10 amino acid residues polypeptides Pri polypeptides with a mol. m. more than 6 thousand names. proteins
Historical reference. For the first time, peptides were isolated from enzymatic protein hydrolysates. The term "peptides" was proposed by E. Fischer. The first synthetic peptide was obtained by T. Curtius in 1881. E. Fischer by 1905 developed the first general method for the synthesis of peptides and synthesized a number of oligopeptides. buildings. Creatures E. Fischer's students E. Abdergalden, G. Leike and M. Bergman contributed to the development of peptide chemistry. In 1932, M. Bergman and L. Zerwas used a benzyloxycarbonyl group (carbobenzoxy group) in the synthesis of peptides to protect the a-amino groups of amino acids, which marked a new stage in the development of peptide synthesis. The resulting N-protected amino acids (N-carbobenzoxyamino acids) were widely used to obtain various peptides, which were successfully used to study a number of key problems in the chemistry and biochemistry of these B-B, for example, to study the substrate specificity of proteolytic. enzymes. Natural peptides (glutathione, carnosine, etc.) were first synthesized using N-carbobenzoxyamino acids. An important achievement in this area developed in the beginning. 50s P. Vaughan et al. synthesis of peptides using the mixed anhydride method (methods for peptide synthesis are discussed in detail below). In 1953, V. Du Vigneault synthesized the first peptide hormone, oxytocin. Based on the concept of solid-phase peptide synthesis developed by P. Merrifield in 1963, automatic ones were created. peptide synthesizers. Methods for controlled enzymatic synthesis of peptides have received intensive development. The use of new methods made it possible to synthesize the hormone insulin, etc.
Successes of synthetic peptide chemistry was prepared by advances in the development of methods for the separation, purification and analysis of peptides, such as ion exchange chromatography, decomposition electrophoresis. media, gel filtration, high performance liquid chromatography (HPLC), immunochemical. analysis, etc. Methods for analyzing end groups and methods for stepwise cleavage of peptides have also received great development. In particular, automatic systems were created. amino acid analyzers and automatic devices for determining the primary structure of peptides - the so-called. sequencers.
Peptide nomenclature. Amino acid residue of peptides carrying free. a-amino group, called N-terminal, and the carrier is free. a-carboxyl group - C-terminal. Peptide name imagecomes from the name. the amino acid residues included in its composition, listed sequentially, starting from the N-terminal one. In this case, trivial names are used. amino acids, in which the ending “in” is replaced by “sil”; exclusion of the C-terminal residue, called which coincides with the name. the corresponding amino acid. All amino acid residues included in the peptides are numbered starting from the N-terminus. To record the primary structure of peptides (amino acid sequence), three-letter and one-letter designations for amino acid residues are widely used (for example, Ala Ser -Asp Phe -GIy alanyl-seryl-asparagyl-phenylalanyl-glycine).
Structure. The peptide bond has the properties of a partially double bond. This is manifested in a decrease in the length of this bond (0.132 nm) compared to the length of a simple C N bond (0.147 nm). The partially doubly-connected nature of the peptide bond makes it impossible to freely rotation of substituents around it. therefore, the peptide group is planar and usually has a trans configuration (f-la I). Thus, the backbone of the peptide chain is a series of rigid planes with a movable (“hinge”) joint in the place where the asymmetrical parts are located. C atoms (in phase I are indicated by an asterisk).
In peptide solutions, the preferential formation of certain conformers is observed. As the chain lengthens, ordered elements of the secondary structure (a-helix and b-structure) acquire more pronounced stability (similar to proteins). The formation of a secondary structure is especially characteristic of regular peptides, in particular polyamino acids.
Properties. Oligopeptides are similar in properties to amino acids; polypeptides are similar to proteins. Oligopeptides are usually crystalline. substances that decompose when heated. up to 200 300 0 C. They are well soluble. in water, dil. to-takh and alkalis, almost no sol. in org. r-retailers. Exception: Oligopeptides built from hydrophobic amino acid residues.
Oligopeptides have amphoteric properties and, depending on the acidity of the environment, can exist in the form of cations, anions or zwitterions. Basic absorption bands in the IR spectrum for the NH group are 3300 and 3080 cm -1, for the C=O group 1660 cm -1. In the UV spectrum, the absorption band of the peptide group is in the region of 180-230 nm. Isoelectric the point (pI) of peptides varies widely and depends on the composition of amino acid residues in the molecule. The pK a values of the peptides are approx. 3, for a -N H 2 approx. 8.
Chem. The properties of oligopeptides are determined by the functions they contain. groups, as well as features of the peptide bond. Their chem. transformation into means. are at least similar to the corresponding amino acid ratios. They give it to me. biuret reaction and ninhydrin reaction. Dipeptides and their derivatives (especially esters) cyclize readily to form diketopiperazines. Under the influence of 5.7 n.
hydrochloric acid peptides are hydrolyzed to amino acids within 24 hours at 105 0 C.
Synthesis. Chem. peptide synthesis involves creating a peptide bond between the COOH group of one amino acid and the NH 2 of another amino acid or peptide. In accordance with this, carboxyl and amine components of the peptide synthesis process are distinguished. To carry out targeted, controlled synthesis of peptides, it is necessary to preliminarily. temporary protection of all (or some) functions. groups that do not participate in the formation of a peptide bond, and also preliminarily. activation of one of the components of peptide synthesis. After completion of the synthesis, the protecting groups are removed. When obtaining biologically active peptides, a necessary condition is the prevention of racemization of amino acids at all stages of peptide synthesis.
Naib. important ways of forming a peptide bond when carrying out r-tion in r-re-methods activating. ethers, carbodiimide, mixed anhydrides and azide method.
The method of activated esters is based on pre- the formation of an ester derivative of the carboxyl component by introducing into it an alcohol residue containing a strong electron-withdrawing substituent. As a result, a highly reactive ester is formed, which is easily subject to aminolysis under the action of the amino component of peptide synthesis. As an activator esters in the synthesis of peptides, penta-fluoro-, pentachlor-, trichloro- and n-nitrophenyl and a number of other esters of protected amino acids and peptides are widely used.
The carbodiimide method of peptide bond formation involves the use of decomp. substituted carbodiimides. Dicyclohexyl-carbodiimide is especially widely used in the synthesis of peptides:
X and Y-resp. N- and C-protective groups With this condensing reagent, it is possible to synthesize peptides in aqueous media, since the rates of hydrolysis and aminolysis of the intermediately formed O-acyl isourea (II) differ significantly. Various compounds are also used in the synthesis of peptides. water-soluble carbodiimides (for example, N-dimethylaminopropyl-N"-ethylcarbodiimide).
The mixed anhydride method is based on pre-treatment. activation of the carboxylic component of peptide synthesis by the formation of a mixed anhydride with a carboxylic or inorg. who. Naib. alkyl esters of chloroformic (carbonic chloride) compounds are often used, especially ethyl and isobutyl ethers, for example:
B - tertiary amine
When synthesizing peptides using this method, mixed anhydrides of N-acyl amino acids and pivalic (trimethylacetic) acids are very effective. Thanks to the strong put. Due to the inductive effect of the tert-butyl group, the electrophilicity of the carboxyl atom C in the pivalin acid residue is significantly reduced, and this, along with the steric. obstacles, suppresses unwanted collateral formation of urethane and free. N-acylamino acids, edges are carried out according to the scheme:
In one variant of the mixed anhydride method, 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline is used as a condensing agent. This is the connection. easily forms an intermediate with the carboxyl component of peptide synthesis. mixed anhydride, which quickly enters into the condensation solution, and unwanted substances are completely eliminated. side distribution.
A special case of the mixed anhydride method is the symmetric method. anhydrides, in which amino acid anhydrides 2 O are used. Their use eliminates the possibility of disproportionation or improper aminolysis.
The azide synthesis method involves the activation of the carboxyl component by converting it into the azide of an N-substituted amino acid or peptide:
Due to the instability of azides, they are free. form from the solution, as a rule, is not isolated. If, instead of alkali metal nitrites, alkyl ethers of nitrogenous compounds (for example, tert-butyl nitrite) are used for the solution with hydrazide, then azide condensation can be carried out in org. r-ritele; the resulting HN 3 is bound with tertiary amines. Azide condensation is often complicated by unwanted complications. side reactions (converting hydrazide not into azide, but into amide; solutionhydrazide with azide, leading to the formation of 1,2-diacyl-hydrazine; intermittent formation of isocyanate, which as a result of the Curtius rearrangement can lead to a urea derivative or the corresponding urethane, etc.). The advantages of the azide method are a low degree of racemization, the possibility of using serine and threonine without protecting hydroxyl groups.
To transform protected peptides are converted to free peptides using special methods of deblocking, which are based on solutions that ensure the detachment of decomposition. protective groups that guarantee the preservation of all peptide bonds in the molecule. Examples of deblocking: removal of the benzyl oxycarbonyl group of the catalytic. hydrogenolysis at atm. pressure and room temperature, elimination of the tert-butyloxycarbonyl group by mild acidolysis, as well as hydrolytic. cleavage of the trifluoroacetyl group under the action of dilute. foundation solutions.
When synthesizing biologically active peptides, it is important that racemization does not occur; edges can occur as a result of the reversible elimination of H + from the a -atom C of an N-acyl amino acid or peptide. Racemization is promoted by bases and compounds, high temperatures and polar compounds. The decisive role is played by racemization, catalyzed by bases, which can occur through the so-called. azlactone mechanism or through enolization according to the following scheme:
Naib. important ways to avoid racemization: 1) extension of the peptide chain in the direction from the C-terminus to the N-terminususing N-protecting groups such as ROC(O). 2) Activation of N-protected peptide fragments with C-terminal proline or glycine residues. 3) Use of the azide method (in the absence of excess tertiary base and maintaining low temperatures in the reaction medium). 4) Application activ. amino acid esters, aminolysis of which proceeds through a transition state, stabilizer. hydrogen bridges (for example, esters formed with N-hydroxypiperidine and 8-hydroxyquinoline). 5) Using the carbodiimide method with N-hydroxy compound additives. or Lewis's office.
Along with the synthesis of peptides in solutions, the synthesis of peptides using insoluble carriers is important. It includes solid-phase peptide synthesis (Maryfield method, or method) and peptide synthesis using polymer reagents.
The strategy of solid-phase peptide synthesis involves temporary fixation of the synthesized peptide chain on an insoluble polymer carrier and is carried out according to the following scheme:
Thanks to this method, it was possible to replace very complex and labor-intensive procedures for separation and purification of intermediates. peptides by simple washing and filtering operations, as well as reducing the process of peptide synthesis to a standard sequence of periodically repeated procedures that can be easily automated. Merrifield's method made it possible to significantly speed up the process of peptide synthesis. Based on this methodology, various types of types automatic peptide synthesizers.
The connection is highly productive. solid-phase synthesis of peptides with the separating abilities of preparative HPLC provides access to a qualitatively new level of chemistry. synthesis of peptides, which, in turn, has a beneficial effect on the development of various. areas of biochemistry, they say. biology, genetic engineering, biotechnology, pharmacology and medicine.
The strategy for the synthesis of peptides using polymer reagents involves temporary binding to high molecular weight. carrier activated carboxyl component or condensing agent of peptide synthesis. The advantage of this method is that reagents attached to the polymer can be introduced in excess, and the separation of synthesized peptides from insoluble polymers is not difficult.
An example of such a synthesis is passing the amino component in a given sequence through several. columns, each of which contains a polymer bound
Summary
The review focuses on studies of pharmacokinetics and bioavailability when creating new original drugs with a peptide structure. Much attention is paid to methods for the quantitative determination of peptide compounds in biomaterials, the study of their pharmacokinetic characteristics, factors influencing the bioavailability of these substances, and some pharmacokinetic data on drugs with a peptide structure introduced into medical practice are also provided.
Keywords: pharmacokinetics, short peptides, bioavailability, excipients
Introduction
Anxiety disorders are mental disorders characterized by general persistent anxiety, pathological fear, tension and nervousness. Currently, the prevalence of diseases associated with anxiety disorders ranges from 13.6 to 28.8% in Western countries and is constantly increasing due to the high pace of life, environmental and social tension.
Due to the significant increase in diseases associated with anxiety and depressive disorders, the development and implementation of new anxiolytic drugs is relevant. Today, drugs that have such a pharmacological effect are represented mainly by a group of benzodiazepine compounds, which are characterized by fatigue, drowsiness, memory impairment, mental and physical drug dependence, and withdrawal syndrome, which reduces the quality of life of patients. One such anxiolytic, devoid of these side effects, is the drug afobazole. The above confirms the need to search for other highly effective drugs that are free of adverse reactions of benzodiazepines. Science pays great attention to endogenous peptides. To date, the important role of the endogenous neuropeptide cholecystokinin in the pathogenesis of anxiety disorders has been established. It is known that cholecystokinin, acting on CCK-B receptors located in the central nervous system, exhibits anxiogenic activity - induces panic attacks, interacts with the opiate system and thus can have an anti-analgesic effect. It is also possible that cholecystokinin plays a role in the pathogenesis of depression and schizophrenia.
Since endogenous neuropeptides have low enzymatic stability, are subject to hydrolysis in the gastrointestinal tract, and are active only after penetration through the BBB, there was a need to search for potential anxiolytics (cholecystokinin receptor antagonists) with a more compact and protected structure that are effective when administered systemically.
Based on the hypothesis developed by Gudasheva T.A. back in 1985, about the possibility of simulating the structure of a non-peptide prototype with a certain neurotropic activity, as well as the active fragment of the original peptide with similar activity, a new dipeptide anxiolytic GB-115 (N-phenyl-N-hexanoyl-L-glycyl-L amide) was synthesized -tryptophan) is a retroanalog of cholecystokinin-4. The pharmacological activity of the compound has been established: it has been experimentally proven that GB-115 exhibits anxiolytic, anti-alcohol, antidepressant and analgesic properties. When administered orally, GB-115 demonstrated its maximum anxiolytic activity at a dose of 0.1 mg/kg. The drug stops the anxiogenic reaction induced by ethanol withdrawal at a dose of 0.2 mg/kg, p.o. Maximum analgesic activity is manifested at a dose of 10 mg/kg, and an antidepressant effect at a dose of 0.025-0.05 mg/kg, i.p.
Conducting experimental pharmacokinetic studies of a drug is a necessary step for its further promotion into medical practice. Improving pharmacokinetic parameters allows the creation of an optimal dosage form that would be distinguished by the appropriate degree and rate of absorption, distribution characteristics, pathways of metabolism and excretion. An assessment of relative bioavailability allows one to make a choice in favor of a dosage form with the best pharmacokinetic parameters for the compound being studied.
Pharmacokinetics is a modern, rapidly developing science that studies the characteristics of drug penetration into the body, distribution, biotransformation and elimination. The study of these processes, including their quantitative assessment, is the main goal of pharmacokinetics.
Pharmacokinetic study of new pharmacologically active substances in experiment is a mandatory step in the research, development and implementation of them into medical practice. The effectiveness of the drug directly depends on the processes of absorption, distribution and excretion of drugs from the body.
Pharmacokinetic data make it possible to determine the route and method of administration, the site of penetration of the drug, the approximate dosage regimen, as well as the main routes of drug elimination.
Absorption, distribution, metabolism and excretion of a drug compound are interconnected processes. All of them are influenced by many factors: the rate of absorption depends on the dosage form of the drug, the concentration of the active substance, the pH of the medium in which the substance is dissolved, intestinal motility and the state of the absorption surface area. The indicators of distribution and biotransformation of a drug are influenced by gender, age, the somatic state of the patient’s body, as well as the state of the body’s enzymatic systems, which is often due to individual differences. Thus, the rate of metabolism of some psychotropic drugs can vary from 6 to 30 hours in different patients. The removal of metabolites from the body can be affected by concomitant diseases, as well as the influence of other drugs.
To assess the various pharmacokinetic processes of drugs in the body of animals and humans, the corresponding pharmacokinetic parameters are calculated, including bioavailability (F, %) - the part of the drug dose that reaches the systemic bloodstream after its extravascular administration.
It is important to note the conditions for conducting pharmacokinetic experiments in preclinical trials of new pharmacologically active compounds.
The studied pharmacological agents are considered to be the object of research, which in preclinical practice is carried out on healthy animals: rats, mice, rabbits, dogs, monkeys and others, the weight of which should not differ from the standard for each species by more than 10%.
The main types of biological material are blood serum plasma, whole blood, various organs and tissues, urine, feces.
The route of administration is determined by the form of the drug, recommended on the basis of pharmacokinetic studies for further pharmacological study. Methods of administration can be different: intravenous, intraperitoneal, intramuscular, subcutaneous, oral, etc. The drug is administered orally to animals using a pharyngeal or duodenal tube on an empty stomach to avoid interaction of the drug with food.
Administration can be repeated or single. With a single administration, it is necessary to study the pharmacokinetics of the active substance using at least three dose levels. This is necessary to verify the linearity of pharmacokinetics.
The duration of the experiment should correspond to a time 5 times longer than the half-life.
The number of animals per point (corresponding concentration value) must be at least 5 if only one sample is taken from each animal from the sample (in experiments on rats in the case of decapitation: one animal - one point).
One of the important stages of the pharmacokinetic and biopharmaceutical study of a new pharmacologically active compound is the study of its absolute and relative bioavailability (see section “Bioavailability of Drugs”).
- Analytical methods for the determination of peptides and their derivatives
There are various methods for the qualitative and quantitative determination of amino acids, peptides and their derivatives. And it is necessary to reasonably select the optimal method for analyzing a potential drug with a peptide structure. This will allow us to achieve sensitive analysis and obtain accurate and reproducible results that would show the pharmacokinetics of a particular compound.
Classification:
- Liquid chromatography methods:
Thin layer liquid chromatography
High performance liquid chromatography
- Gas chromatography
- Immunochemical methods of analysis
- Capillary electrophoresis
1.2 Chromatography of amino acids and peptides
Chromatography is a physicochemical method for separating the components of an analyzed mixture, based on the difference in their distribution coefficients between two phases: stationary and mobile. The most promising chromatography methods are: gas chromatography (GC) and high-performance liquid chromatography (HPLC) in combination with a mass spectrometric detector - GC-MS and HPLC-MS. These methods are developing at a rapid pace, which is associated with the growth of tasks that have arisen in recent years: proteomics, metabolomics, analysis of biofuels, determination of biomarkers of diseases, creation and quality control of medicines, quality control and food safety, as well as terrorism (determination of toxic substances, harmful substances and combatants) and rapid determination of the consequences of emergency situations.
1.2.1 Liquid chromatography methods
1.2.1.1. High performance liquid chromatography
HPLC is a physicochemical method for separating the components of a mixture of substances, based on their different distribution between two immiscible phases, one of which is mobile and the other immobile. Depending on the polarity of the mobile and stationary phases, HPLC is usually divided into normal phase (the stationary phase is more polar than the mobile) and reverse phase (the stationary phase is less polar than the mobile).
Reverse-phase HPLC is often used to separate amino acids and peptides due to the fact that most analytes are highly soluble in aqueous mobile phases and have limited solubility in most non-polar solvents. However, normal-phase HPLC is used for the chromatography of short-chain amino acid derivatives and peptides with low hydrophobicity, which are not retained by the stationary phase in reverse-phase HPLC. Reversed phase HPLC was the gold standard for peptide separation and purification prior to the application of mass spectrometry in this field. RP-HPLC has the following advantages over other methods of chromatographic analysis: reproducibility of results, high separation power, selectivity (the ability to differentiate peptides with a difference of one amino acid), sensitivity, high speed of execution, and the use of a small volume of volatile solvents.
The selectivity and quality of peptide analysis in reverse-phase HPLC depends on the correct choice of phases: mobile and stationary.
As a stationary phase, adsorbents are used, which are silica gel modified with various chlorosilane derivatives. This phase has high strength and indifference to organic solvents. The reverse phase is distinguished by the characteristics of the matrix - silica gel and the structure of the grafted radical, which differs in the composition and structure of the carbon fragment. When chromatography of peptides, the choice of reverse phase is determined by the size and hydrophobicity of the peptides: for peptides with a short chain, hydrophilic peptides, phases C8 (n-octyl) and C18 (n-octadecyl) are used, for large and hydrophobic ones - phase C3 (trimethyl- or dimethylpropyl), C4 (n-butyl), C6 (phenyl).
To correctly select the mobile phase, it is necessary to take into account the pH, composition and concentration of the organic solvent:
To reduce the polarity of the peptides and ensure better retention by the adsorbent, the pH of the eluent should be in the range of 2-3. Also, to increase the retention time of peptides, so-called modifiers or ion-pair reagents (counterions), which are capable of forming ion-pairs with positively charged peptide groups, are introduced into the mobile phase. The main ionic modifier in RP HPLC is trifluoroacetic acid. It is easily removed from eluates by evaporation, dissolves peptides well, and is UV transparent in the short wavelength region, which does not create additional peaks during detection. Formic acid is also used as a modifier and provides good separation, but its use is limited by strong absorption in the UV region.
The influence of the organic solvent on the elution ability of the mobile phase is very large. So, the elution strength of the solvent increases in the following order: water - methanol - acetonitrile - ethanol - dioxane - tetrahydrofuran - 2-propanol - 1-propanol. This sequence is due to a decrease in the polarity of organic substances in this series. Acetonitrile is most often used as an organic component of the mobile phase, since it is transparent in the UV region up to 200 nm, has a low viscosity, is highly volatile, which allows, if necessary, to easily remove it from the collected eluate fraction, and is characterized by good selectivity.
The separation of peptide compounds can be carried out under isocratic conditions, where the concentration of the organic solvent is constant, or by gradient elution, in which case the concentration of the organic solvent increases over time. The test substances elute in order of increasing hydrophobicity.
1.2.1.2. Methods for detecting peptides in high-performance liquid chromatography: UV detection, mass spectrometry.
To accurately carry out qualitative and quantitative analysis after the separation of medicinal substances by HPLC, it is necessary to use equipment for their detection, which in turn has the following requirements: detectors must have high sensitivity (good signal, no noise), speed, wide linear dynamic range, stability , lack of interaction with the mobile phase.
One of the most common detection methods in high-performance liquid chromatography is ultraviolet, which is explained by the high sensitivity of the analysis, simplicity, and affordability from an economic point of view. However, UV detection is a less sensitive method than mass spectrometry. UV detectors are represented by four main types today:
- with a fixed wavelength;
- with a monochromator that allows you to change wavelengths in its range;
- with an automatically tunable monochromator that allows multi-wavelength, multi-channel detection;
- diode-matrix detectors that allow obtaining full spectral information in a given range.
Due to the presence of some chromophores in the composition of amino acids, as well as the peptide bond itself, it has become possible to detect peptide compounds using UV radiation using one of the four types of equipment listed above.
Peptide compounds are capable of absorbing UV radiation in three areas:
Above 250 nm (λ=280 nm), which is due to the presence of aromatic amino acids in the analyzed compound - tryptophan (λ=278 nm), tyrosine (λ=275 nm) and phenylalanine.
At 210-250 nm, such a signal can be given by other amino acids with intra- and intermolecular hydrogen bonds in protein molecules.
At 190 nm, which is explained by the presence of peptide bonds.
However, detection of the compounds under study is not carried out at wavelengths below 210 nm due to the influence of solvents used in HPLC, which have their own absorption at wavelengths shorter than 210 nm, as well as due to the presence of impurities. Therefore, when detecting peptide substances, the wavelength range above 250 nm is often used. If the compounds do not contain chromophores that would absorb UV radiation in this region, then they resort to the derivatization method.
Derivatization is the chemical modification of an analyte to produce a derivative compound that has improved analytical properties. When working with HPLC-UV through derivatization, it is necessary to obtain a compound that is registered in the UV spectrum in a region convenient for the analysis of biological material. So in the work of Rudenko A.O. When determining the most important amino acids in complex biological matrices, a method of derivatization of 16 amino acids was used. O-phthalaldehyde was used as a derivatizing agent.
The mass spectrometric detection method consists of three stages: ionization, mass-to-charge separation, and subsequent detection using a mass analyzer. For the analysis of drug compounds, “soft” ionization techniques are used: electrospray ionization, as well as matrix-assisted laser desorption (MALDI). These methods represent a gentle ionization mode, which is especially important for thermally unstable biomolecules. However, these types of ionization are not sufficiently informative, so they often resort to tandem mass spectrometry (MS/MS), a method for recording fragments of analytes. To be more precise, this method consists of several stages: first, the analyzed compounds are softly ionized, pass through the first analyzer, then their energy is increased, due to which the molecules under study are fragmented and the second analyzer records the resulting mass spectrum.
For the quantitative determination of new drug compounds, the following types of mass analyzers are used:
Quadrupole (mass analyzer based on three quadrupoles), which is the “gold standard” in the study of new medicinal compounds;
Time-of-flight (TOF), when used, achieves lower sensitivity than when using triple quadrupole analyzers.
Ion cyclotron resonance and orbital ion trap, which are high-resolution mass analyzers and are so far rarely used due to the high cost and complexity of such devices.
The use of mass spectrometry detection in combination with HPLC has made it possible to achieve high rates of analysis, increase the detection limit of drug compounds, and significantly improve the stability and accuracy of studies.
- Thin layer chromatography
Today, TLC is used to a much lesser extent as more high-tech methods of peptide separation have become available, such as HPLC, liquid column chromatography, ion exchange chromatography, protein polyacrylamide gel electrophoresis, and capillary electrophoresis. However, TLC proved to be a quantitative, high-tech, relatively inexpensive and easily reproducible method in its time. Thin layer chromatography was popular in the 80s - amino acids were isolated from plants, animals and various biological fluids.
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