Process of using clinoptilolite zeolite nanocomposite in the extraction of triptorelin acetate using magnetic solid phase

Abstract

The process of using clinoptilolite zeolite nanocomposite in the extraction of triptorelin acetate drug using magnetic solid phase is in the field of laboratory and medical technology. This invention is to solve the technical problem of low concentration of triptorelin acetate drug in vitro for measurement, which low concentration cannot be measured with HPLC (high performance liquid chromatography) and measurement with LC-Mass is also expensive. The basis of the proposed solution is the use of clinoptilolite zeolite nanocomposite in the extraction of the drug triptorelin acetate using a magnetic solid phase, which, based on the magnetic solid phase extraction (MSPE) method, has the power to concentrate drug solutions containing the peptide triptorelin acetate and allows for the accurate measurement of the drug triptorelin acetate in vitro. This method is used in pharmaceutical companies and laboratories in the extraction of the drug triptorelin acetate using a magnetic solid phase.

Description

Description of the invention Title of the invention Process of using clinoptilolite zeolite nanocomposite in the extraction of triptorelin acetate using magnetic solid phase Technical background of the relevant invention The technical field of the invention is the measurement and evaluation of the concentration level of the drug triptorelin acetate in an in vitro environment. Triptorelin acetate is a synthetic analogue of gonadotropin-releasing hormone (GnRH) that is used with long-term action to help treat prostate cancer. In addition to helping treat prostate cancer, this hormone has a wide range of uses in helping treat uterine endometriosis, female infertility, uterine fibroids, and precocious puberty. The technical field of this invention can be considered laboratory and medical. Technical problem and statement of invention objectives In order to measure the drug triptorelin acetate in vitro (outside the body), where the drug concentration is very low and it is not possible to measure it with the help of HPLC (high-performance liquid chromatography) and measurement with LC-Mass is also expensive, for this reason, the magnetic solid phase extraction (MSPE) method was used to concentrate the sample, which has features such as repeatability, selectivity, high enrichment factor, high speed, simplicity, and no need for filtration and centrifugation during the extraction process. Also, the reason for using magnetic zeolite as an inorganic nanosorbent instead of an organic nanosorbent is due to its unique magnetic properties, low cell toxicity, easy chemical modification of the surface, and easy control of interparticle interactions in solutions and structures. In fact, the purpose of this invention is to provide a method for measuring the drug triptorelin acetate in vitro using a clinoptilolite zeolite nanocomposite that is very accurate and low cost. A description of the state of the prior art and the history of developments related to the claimed invention. 1- Introduction to the drug Triptorelin acetate Triptorelin acetate is a synthetic analogue of gonadotropin-releasing hormone (GnRH) that has long-acting effects and was approved by the FDA in 1985 to help treat prostate cancer. It is widely used to help treat prostate cancer, endometriosis, female infertility, uterine fibroids, and precocious puberty. The structure of triptorelin acetate is shown in Figure 2. Triptorelin acetate is a gonadorelin analog with similar properties. This synthetic peptide with a molecular mass of 1311 g / mol inhibits steroid production from the ovaries and testes, resulting in decreased testosterone (in men) and estrogen (in women). Triptorelin acetate is used to help treat prostate cancer, estrogen-dependent breast cancer, and other diseases affected by sex hormones by reducing levels of sex hormones in both sexes. It is available as the base, acetate, diacetate, or pamoate. Triptorelin acetate is rapidly absorbed following subcutaneous injection, with peak plasma concentrations occurring within 40 minutes of a single dose. Its biological half-life is approximately 7.5 hours, although longer half-lives have been reported in patients with prostate cancer and shorter half-lives have been observed in some healthy individuals. Also, patients with prostate cancer are required to take this drug for one year or more, and due to the short biological half-life of this drug, repeated daily injections are required. Due to the problems caused by daily injections, slow-release injectable systems of triptorelin acetate have been developed and produced. 2- Triptorelin acetate injectable slow-release system (microspheres and locally formed systems) Controlled drug delivery systems are systems by which drugs reach their target site at a specific rate and duration of action, so that they have the desired effects. The most important issue in these systems is maintaining this desired rate. The basic formulation of this technique consists of a drug and a carrier, usually a polymer, which allows the drug to be released at the desired site over a period of time and at a controlled rate. To design a controlled drug delivery system, factors such as the minimum therapeutic level of the drug used, the rate of drug elimination from the body, the kinetics and mechanism of drug release from the system, and the physical and chemical limitations on the system must be considered. Nowadays, studies on the controlled release of drugs and other bioactive agents from delivery systems have attracted the attention of researchers all over the world. Perhaps the most appropriate definition of controlled release is the following: "Controlled release is a method that delivers chemical or biological active substances to the desired target at the required speed and duration". Maintaining the desired release rate and drug concentration level in the body is the main goal of these methods. Among the types of drug delivery systems, implantable systems can be mentioned. 1-2- New generation of implantable injectable drug delivery systems: Since the 1990s, various subcutaneous or intramuscular controlled drug delivery systems based on biodegradable polymers have been developed in the form of implants or microparticles. Implants are generally prepared by mixing the drug, polymer, and additives, and melt extrusion, melt compression molding, or injection molding are used to obtain the desired shape and size. There are many problems in these processes, including: high process temperature, inhomogeneity of the system, the need for surgery, etc. In addition, the preparation of implants, especially biodegradable microparticles, is complex and costly, and requires control of formulation variables and multi-step processes. As a result, in order to avoid the need for surgery to implant large implantable systems, biocompatible and biodegradable polymer particles were used for controlled drug delivery. Biodegradable polymers used in these systems are of natural or synthetic origin and are degraded in the body by enzymatic, non-enzymatic, or both. These polymers are degraded into biocompatible and non-toxic byproducts that are then eliminated from the body through normal metabolic pathways. Polymers used in these systems must have other characteristics such as drug compatibility, desirable mechanical properties, and ease of processing. 2-2- New generation of injectable drug delivery systems: Injectable drug delivery systems have made significant progress over the past few years. This attention is due to the advantages that these systems have, such as simple application, effective local drug delivery, long-term drug delivery, reduced drug dosage (along with reduced side effects common to most drug delivery systems), and increased patient comfort and acceptance. In early studies, drug delivery systems such as emulsions, liposomes, biodegradable microspheres, and micelles were studied. Although these formulations have had great success in specific applications, they still have drawbacks that require further research and study. In contrast, semi-solid injectable systems that form in situ have been further developed due to their special advantages. These implantable systems are made of biodegradable polymers that can be injected into the body with a syringe and solidify upon injection to form a semi-solid reservoir. There are two or more in situ formed injectable products on the market, including Atridox® and Eligard®, both of which are based on the Atrigel technology. This technology uses the PLGA polymer dissolved in N-methyl-2-pyrrolidone, a water-miscible solvent. The drug powder is dissolved in a polymer solution containing an organic solvent or dispersed as particles in this solution before use. In situ-forming injectable systems are classified into four categories based on the mechanism of polymer solidification within the body: thermoplastic pastes, in situ crosslinking systems, solidification of organic gels at the injection site, and in situ polymer deposition. One strategy used to produce in situ-forming systems is the polymer precipitation strategy, which was first used by Dunn et al. in 1990. Polymer precipitation can occur due to solvent removal and changes in temperature or pH. In this system, a biodegradable and water-insoluble polymer such as poly(L,D-lactic), poly(L,D-lactic-co-glycolide), and poly(L,D-lactic-co-3-caprolactone) is mixed with a biocompatible organic solvent containing the drug, and after mixing, a solution or suspension is formed. When the resulting formulation is injected into the body, the water-soluble organic solvent disperses and water penetrates into the organic phase. This process leads to phase separation, precipitation of the polymer, and the formation of a solid polymer reservoir at the injection site. This method was developed by Atrix Laboratories and is named Atrigel technology. Solvents used in this method include (N-methyl-2-pyrrolidone NMP), propylene glycol, acetone, dimethyl sulfoxide (DMSO), tetrahydrofuran, 2-pyrrolidone, and triacetin, but the best preferred solvents are NMP and DMSO due to their pharmaceutical properties, while NMP is also FDA-approved at the permitted concentration. The maximum plasma drug concentration for the injectable slow-release system of triptorelin acetate with doses of 3.75 mg (one month), 11.25 mg (three months), 22.50 mg (six months) is 28.43, 38.50, 11.44 micrograms per milliliter (ppm), respectively. Also, in measurements performed in in vitro (outside the body) or in vivo (inside the body) environments, since the minimum drug concentration is much lower than these values, it is necessary to prepare the sample before measurement. 3- Sample preparation In general, all the steps that are performed to introduce a sample to one of the analytical systems are called sample preparation. Since the matrix of most analytes is such that it is not possible to directly inject the sample into the device for analyte analysis, sample preparation has become one of the most important analytical processes. The purpose of this step is to perform one or a combination of the processes of purification, extraction, or preconcentration. Preconcentration of the analyte is to improve sensitivity, amplify the signal, and reduce the measurement limit. Purification is also to eliminate or minimize the effect of other chemical species in the sample matrix, which increases selectivity. This step usually takes up a large part of the test time. Choosing the appropriate sample preparation method is very effective in qualitative and quantitative determination of the analyte. Thus, sample preparation has a significant impact on the quality of the subsequent analysis steps.Nowadays, the development of sample preparation methods is focused on reducing time, using very small amounts of sample, reproducibility, no sample wastage, compliance with green chemistry, and method automation. 1.3- Liquid-liquid extraction (LLE) Liquid-liquid extraction is one of the oldest extraction methods and is based on the transfer of the analyte from an aqueous sample to a water-immiscible solvent (such as carbon tetrachloride, diethyl ether, chloroform, toluene, dichloromethane, chlorobenzene, etc.). This method is time-consuming and tedious and requires the use of large amounts of sample and toxic organic solvents, which in addition to being expensive, leads to environmental pollution. Low reproducibility, emulsion formation, and low concentration factor are other disadvantages of this method. 2.3- Capillary Electrophoresis Extraction (CEE) In this method, sample preparation is based on the difference in migration speed in an electric field, such as electrophoresis, and the basis of separation and extraction in methods based on the difference in migration speed is based on kinetic equilibria. One of the most important limitations of this method is its inherent low sensitivity. 3-3- Electromembrane extraction (EME) Unlike most methods based on liquid phase microextraction (LPME), in which the extraction of analytes is based on the phenomenon of passive diffusion and mass transfer, electromembrane extraction is based on electrokinetic migration. The main advantage of this method is the reduced extraction time, especially when dealing with small volumes of sample. In this method, one electrode is placed in the sample solution and the other electrode is placed in the acceptor solution inside the hollow fiber. The pH of the sample solution is adjusted so that the desired analytes are ionized. A certain voltage is applied to the electrodes based on the nature of the analyte, and this potential difference applied across the membrane acts as a driving force. For cations, the cathode is placed in the acceptor solution and the anode is in the sample solution. For the extraction of anions, the potential polarity is reversed. Finally, the acceptor aqueous solution is collected and used for analysis by any analytical technique.One of the disadvantages of this method is that the instability of analytes at high voltages leads to redox reactions in the electrodes and electrolysis. On the other hand, the formation of air bubbles on the surface of the hollow fiber and the surface of the electrodes, which is caused by performing extraction at high voltages, leads to a decrease in the mass transfer rate and a decrease in repeatability. 4.3- Solid Phase Extraction (SPE) This method was first developed in the 1950s. In solid phase extraction, a liquid sample is passed through a suitable solid phase and then the analytes adsorbed on the solid phase are washed away with a suitable organic solvent. In solid phase extraction (SPE) as a selective method, there are various types of adsorbents depending on the type of analyte and sample matrix. These include conventional reversed phase adsorbents such as C8-C18, normal phase adsorbents such as silica and alumina, ion exchange adsorbents, combined adsorbents (such as ion exchange + reversed phase) and functionalized resins based on styrene-divinylbenzene polymers. Among all the available adsorbents, silica adsorbents, bonded silica, polymers, carbon-based adsorbents and molecularly imprinted adsorbents are the most widely used in solid phase extraction (SPE). Solid-phase extraction (SPE) is considered a suitable alternative to liquid-liquid extraction (LLE) because solid-phase extraction (SPE) does not require a large amount of organic solvent and the extraction time of the analyte in this method is shorter than that of liquid-liquid extraction (LLE). In addition, this method uses a small cartridge for analyte extraction, which is relatively inexpensive and can be recycled for other experiments. Also, the entire process of solid-phase extraction (SPE) can be automated, unlike liquid-liquid extraction (LLE). These advantages have led to many efforts to improve and develop new approaches to the method, use various adsorbents to improve the selectivity of the adsorbent towards the analyte and even selectivity towards the analyte, increase the capacity, physical and chemical resistance of the adsorbent. The solid phase extraction (SPE) method has advantages such as repeatability, selectivity and high enrichment factor, but on the other hand it faces limitations such as time-consuming, difficult and also the need to use large amounts of toxic and environmentally destructive organic solvents. Considering the limitations mentioned in solid phase extraction (SPE), the magnetic solid phase extraction (MSPE) method, which is derived from solid phase extraction (SPE), was first proposed and used in 1999. The advantages of this method over conventional solid phase extraction (SPE) include high speed, simplicity and no need for filtration and centrifugation during the extraction process, because the magnetic zeolite nano-sorbent containing the absorbed analyte (triptorelin acetate) is separated by applying an external magnetic field and washed with a suitable solvent of water / acetonitrile in a ratio of 50:50 and finally the magnetic zeolite nano-sorbent is reused. 5.3- Magnetic solid phase extraction (MSPE) Due to the aforementioned limitations of solid phase extraction (SPE), the magnetic solid phase extraction (MSPE) method, which is derived from solid phase extraction (SPE), was first proposed and used in 1999. The advantages of this method over conventional solid phase extraction (SPE) include high speed, simplicity, and the absence of the need for filtration and centrifugation during the extraction process, because the nanosorbent containing the adsorbed analyte is separated by applying an external magnetic field and washed with a suitable solvent, and finally the nanosorbent is reused. Also, one of the nanosorbents used in the magnetic solid phase extraction method is magnetic zeolite. 4- Introduction to magnetic zeolite Zeolites are crystalline aluminosilicates with pores and channels the size of molecules that are used in industry as ion exchange resins, molecular sieves, adsorbents and catalysts. The body of zeolites is generally made of silicon, aluminum and oxygen, and cations, water and other molecules are located in the pores. The empirical formula of zeolite is shown as the following formula, where M represents the exchangeable cation with a valence of n. M is usually an ion from the alkali and alkaline earth metal group, although metals, nonmetals and other inorganic cations may also neutralize the negative charge of Al in the structure. Mx / n[(AlO2)x(SiO2)]. wH2O Zeolites with a characteristic silica structure and micropores are used as hosts and protectors for nanoparticles. The interaction between magnetic nanoparticles, which are synthesized as compounds of Fe, Ni, Co, metal oxides (Fe3O4, Fe2O3), ferrites (MFe2O4) and metal alloys (FePt, CoPt), with zeolites leads to the expansion of their applications in magnetic, electronic, sensor and adsorbent fields. In general, magnetic microcylinders containing a magnetic core and a silica shell have attracted much attention due to their unique magnetic properties, low cell toxicity, easy chemical modification of the surface and easy control of interparticle interactions in solutions and structures. These coatings are stable in aqueous environments with minimal pH, but lose their stability under alkaline conditions. 5- Tools and methods for measuring triptorelin acetate in laboratory conditions and in-vitro environment The most common methods used to measure triptorelin acetate are chromatography-based methods such as high-performance liquid chromatography (HPLC) equipped with ultraviolet and fluorescence detectors and liquid chromatography coupled to mass spectrometry (LC-MS). 1-5- HPLC method One of the methods for measuring pharmaceuticals, including triptorelin acetate, is liquid chromatography (LC). This method has advantages that have made it the most widely used method for measuring pharmaceuticals compared to other methods. This method can measure very thermally unstable, non-volatile, highly polar or high molecular weight drugs that cannot be measured by gas chromatography (GC). Liquid chromatography (LC) can be performed at normal temperatures, so it does not damage the structure of the drug. Today, researchers are trying to provide simple, effective and inexpensive methods that can be used in a variety of products and for compounds with diverse structures, and also overcome the disadvantages of routine methods for analyzing these compounds. Among the disadvantages of common methods for analyzing these compounds, we can mention high cost, time-consuming and the use of large amounts of toxic solvents. 2.5- LC-MS method Due to the increasing use of protein drugs and their instability during storage and when placed inside the body, increasing the stability of this group of substances is essential. Therefore, in recent years, attempts have been made to increase the stability of these compounds by forming drug complexes, and the LC-MS method has been used to measure the drug triptorelin acetate. Considering the advantages and disadvantages mentioned above of extraction methods such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE), in this invention, the magnetic solid-phase extraction (MSPE) method was used in the measurement of triptorelin acetate in vitro due to the following advantages. Providing a solution to an existing technical problem along with an accurate, sufficient, and integrated description of the invention In this invention, the magnetic solid phase extraction (MSPE) method was used. As shown in Figure 1, in this method, the nano-sorbent (magnetic zeolite) is dispersed in the sample solution and the analyte (triptorelin acetate) is adsorbed on the surface of the nano-sorbent (magnetic zeolite) while being agitated. The nano-sorbent (magnetic zeolite) along with the adsorbed analyte (triptorelin acetate) is separated from the sample solution using an external magnetic field. The analyte (triptorelin acetate) is washed from the surface of the nano-sorbent (magnetic zeolite) by a suitable washing solvent (water / acetonitrile in a ratio of 50:50). The external magnetic field is again used to separate the nano-sorbent (magnetic zeolite) from the washing solvent (water / acetonitrile in a ratio of 50:50) containing the analyte (triptorelin acetate), which is finally analyzed by a suitable instrumental method such as HPLC. It is measured. This invention resulted in a method with a high concentration factor of 100, which is obtained by dividing the volume of the initial absorption solution (200 cc) by the volume of the washing solvent (2 cc), as well as a low detection limit of 0.07 micrograms per milliliter (PPm) and much more desirable sensitivity. Finally, the optimized method was used to analyze controlled release drug samples. Magnetic zeolite nanosorbent has been used as an efficient adsorbent for the extraction and concentration of triptorelin acetate drug in the treatment of prostate cancer. In addition, the factors affecting the recovery of this drug have been optimized and the proposed method has been evaluated with the figures of merit and finally the application of the proposed method for the measurement of triptorelin acetate drug in controlled release samples has been investigated. The chemicals used in the different laboratory stages of this invention are as follows: Chemical formula Chemical Na3K3(Al6Si30O72)24H2O Zeolite Clinoptilolite FeCl2.4H2O Iron (II) chloride tetrahydrate FeCl3.6H2O Iron (Ш) chloride hexahydrate C2HF3O2 Trifluoroacetic acid C2H3N Acetonitrile - buffer Sodium acetate C5H9NO N-methyl-2-pyrrolidone HCL Hydrochloric acid [C3H4O2]x[C2H2O2]y Polymer poly(lactide-glycolide) NaOH Sodium hydroxide NH3 Ammonia C64H82N18O13 Triptorelin acetate Table 1: Chemicals used in various laboratory stages of this invention Preparation of magnetic clinoptilolite zeolite nanocomposite 1- To prepare magnetic clinoptilolite zeolite nanoparticles, first pour 0.5 grams of ground zeolite into an Erlenmeyer flask, add 50 ml of distilled water, and sonicate for 0.5 hours. Then add one gram of ferric chloride II and two grams of ferric chloride III to the solution and sonicate for 15 minutes. Then bring the solution to 50 degrees Celsius, add 3 ml of ammonia dropwise, and place on a stirrer for half an hour until homogenized. Then filter the solution several times, wash with distilled water until the solvent is removed, and finally pour it into a plate and place it in an oven for 24 hours at 90 degrees Celsius to dry. 2- To ensure correct preparation, the resulting nanoparticles are characterized using various methods such as X-ray diffraction (Figure 3), dynamic light scattering (DLS) (Figure 4), Fourier transform infrared spectrometer (Figure 5), and the absorption of the analyte (triptorelin acetate) by magnetic zeolite nanosorbent is examined using an HPLC device. 3- After completing the steps of preparing the magnetic zeolite nanosorbent and characterizing it using the various methods above, the efficiency of the prepared magnetic zeolite nanosorbent was used to extract the drug triptorelin acetate in controlled release samples. Magnetic solid phase extraction (MSPE) is performed with a solution containing magnetic zeolite nanosorbent and analyte solution (triptorelin acetate). To improve the adsorption process, the solution is stirred in a vortex mixer at room temperature and then the magnetic zeolite nanosorbent is separated using an external magnet. After the desorption step, the analyte concentration (triptorelin acetate) is measured by HPLC. In this invention, the parameters affecting the extraction efficiency such as the amount of magnetic zeolite nano-adsorbent, extraction time, temperature, pH, type and volume of desorption solvent (water and acetonitrile in a ratio of 50:50) have been optimized, then the figures of merit of the optimized method, including linear range, limit of detection (LOD), repeatability (0.75%), extraction efficiency and concentration factor have been calculated. The devices used in the different laboratory stages of this invention are as follows in the table below. High-performance liquid chromatography (HPLC) measuring instruments KNAUER model 5050, made in Germany METTLER TOLEDO model SEVENEASY digital pH meter, made in Germany KERN digital balance model 770, made in Germany X-ray diffraction model 5000 D, KV40, MA 40, made in Germany SIEMENS RETSCH BALL MILL model 100 PM USP4 device for dissolution testing Fourier transform infrared spectrometer (FTIR) model EQUINOX, made in Germany VEGA3 scanning electron microscope TESCAN (CZECH REPUBLIC) Alloy magnet... Linear Lab shaker model Hs501, IKA Labortechnick Table 2: Devices used in various laboratory stages of this invention Drug absorption stage of triptorelin acetate with magnetic clinoptilolite zeolite nanocomposite In order to adsorb the drug to the magnetic zeolite nano-adsorbent particles, 25 ml of a 5 ppm triptorelin acetate drug solution was added to the magnetic zeolite nano-adsorbent prepared in a 100 cc beaker. Then, using 1 N sodium hydroxide, the pH was adjusted to 6.8 and the beaker was placed in a shaker incubator at 25 °C to adsorb the drug triptorelin acetate to the magnetic zeolite nano-adsorbent. After a specified period of 30 minutes, the magnetic zeolite nano-adsorbent particles were separated from the solution using a strong magnet, the supernatant was removed as the adsorption solution, and after passing through a 0.22 micron syringe filter, the amount of triptorelin acetate in it was analyzed with an HPLC device. Parameter Amount of absorbent (grams) Volume of absorption solution (ml) pH of absorption solution Absorption time (minutes) Optimized 0.1 25 6.8 30 Table 3: Optimal parameters for the absorption of triptorelin acetate using magnetic zeolite nanosorbent After the absorption stage and determination of the concentration of the solution on the adsorbent using HPLC, it was observed that the concentration of the drug triptorelin acetate in the absorption supernatant was very low (for most samples, the concentration was less than 0.5 ppm), which indicates the success of the adsorption operation. Among the parameters examined in the absorption stage of the drug triptorelin acetate to the magnetic zeolite nanosorbent, it was seen that the most influential characteristic of the drug triptorelin acetate solution was that it significantly affected the adsorption of drug triptorelin acetate particles to the magnetic zeolite nanosorbent, such that in an acidic environment, the drug triptorelin acetate had a greater tendency to be present in the solution and less was adsorbed to the magnetic zeolite nanosorbent, but at pH about 6.8, the highest percentage of adsorption was observed, such that the concentration of the drug triptorelin acetate in the magnetic zeolite nanosorbent solution could not be measured (less than 0.5 ppm), meaning that all the drug triptorelin acetate was adsorbed to the magnetic zeolite nanosorbent and the solution was free of the drug triptorelin acetate. Desorption stage of triptorelin acetate drug from magnetic zeolite nanosorbent In order to desorb the drug particles of triptorelin acetate from the magnetic zeolite nano-adsorbent, a solution in which the drug triptorelin acetate is more soluble should be used. In order to desorb, 2 cc of the washing solution (water: acetonitrile 50:50) was poured onto the magnetic zeolite nano-adsorbent containing the drug triptorelin acetate and after covering the beaker lid, it was placed in a shaker incubator at a temperature of 37 degrees Celsius. It should be noted that using trifluoroacetic acid (TFA), the pH was adjusted to the range of 2 and then it was placed in a shaker incubator to desorb the drug triptorelin acetate from the magnetic zeolite nano-adsorbent into the solution. After 15 minutes, the magnetic zeolite nano-adsorbent particles were separated from the solution using a strong magnet and the supernatant was removed and after passing through a 0.22 micron filter, the amount of drug triptorelin acetate in it was analyzed with an HPLC device. Parameter Amount of adsorbent Volume of initial absorption solution Desorption temperature Desorption solution pH of absorption solution Optimized desorption time 0.2 g 200 ml 37°C 2 cc 50 / 50 solution of water and acetonitrile 2 15 minutes Table 4: Optimal parameters for the adsorption of triptorelin acetate using magnetic zeolite nanosorbent Finally, after optimizing all the adsorption and desorption parameters (Tables 3 and 4) and achieving a recovery of over 90% and a concentration factor of 100, the release rate of triptorelin acetate was measured using a USP4 device, examined and analyzed with an HPLC device, and we were able to achieve acceptable release within a period of 40 days. Drug release stage from the in situ formed system of triptorelin acetate by dynamic continuous flow method in USP Apparatus 4 The purpose of this section is to simulate the laboratory environment to the in vivo environment and compare the effect of the dissolution method (in terms of the dissolution device used) on the drug release profile of triptorelin acetate from the in situ formed system. In-situ injection system prepared For this purpose, 0.2 g of the polymer, drug, solvent (NMP) formulation was injected into a vial containing 10 ml of acetate buffer. The prepared formulation contained 59% solvent (198.24 mg), 39% polymer (131.04 mg), and 2% drug (6.72 mg). USP Apparatus 4 Continuous Flow Device The static release method with USP Apparatus 2 is suitable for a wide range of dosage forms, especially for oral and solid dosage forms. Because it acts more like the stomach and gastrointestinal tract environment, while the continuous flow apparatus USP Apparatus 4 is usually used in pharmaceutical research for various controlled release dosage forms. USP 4 seems to be the most suitable apparatus for in vitro release testing of controlled injectable products, especially for micro-travels, because it most closely resembles the subcutaneous environment of the body and the interstitial fluid in the body moves at a slow rate. USP Apparatus 4 uses the flow-through principle, where the release medium is pumped through a designed cell containing glass beads that holds the sample. Considering that determining the mode and rate of drug release is very crucial in investigating the performance of pharmaceutical formulations, this simulation is proposed in order to achieve the real release rate in vivo. Continuous flow The continuous flow release method is used to help simulate in vivo conditions in in vitro release experiments. A continuous flow device consists of a flow cell containing glass beads that holds the sample, a filter on top of the flow cell to prevent particles from passing through the sample cell, a syringe pump to move the release medium through the flow cell, and a water bath to maintain the medium at 37°C. The device may be configured as a closed-loop (Figure 3) or open-loop (Figure 4) configuration. In the closed configuration, the medium circulates continuously, while in the open configuration, it is connected to a sample collector and the medium passes through the cell only once. These flow cells can be used to study the kinetics of drug release from in situ formed implants (ISFI) to evaluate performance under more realistic conditions. In the USP device, there are 4 continuous flow glass chambers in the shape of a cylinder with an internal diameter of 22.4 mm, which are the sample locations. Then, the syringe was connected to a syringe pump through silicone hoses, and the two ends of the chamber were connected to each other through silicone hoses, and with the help of the syringe pump, the flow rate was adjusted at a rate of 4 mm / min in a closed loop from the 10 ml syringe into the cell and was set to automatic mode to establish a continuous flow into the cell by slowly injecting the buffer. In order to establish body temperature conditions, the flow cell and the buffer tank were floated in a glass water bath at 37 °C. Sampling of the buffer for the release test and determination of the amount of triptorelin acetate released was carried out at certain intervals and after leaving the chamber. Laboratory studies and experimental results In order to investigate the release of triptorelin acetate in the continuous flow method, the system was injected to determine the effect of the continuous flow method. The release of the system was carried out in the cell of the continuous flow device with acetate buffer at pH=6.8 and temperature of 37°C and at a flow rate of 4 ml / min. As can be seen in Figure 5, in the release diagram for continuous flow, the dormancy period after the initial explosive release was long and the secondary explosive release started later. The reason for this can be attributed to the circulation of the release medium and the lack of accumulation of acidic environment caused by the degradation around the system. In fact, the fluid movement and constant washing of the environment around the system delayed the degradation of the system for several days and the refreshing of the environment around the system was effective in the release process. In Figure 6, the graph of changes in the pH of the release medium at specific time intervals confirms that the pH decrease in the continuous flow method occurred on days 17 and 20, which is consistent with the secondary explosive release at this time. Explanation of shapes, maps and diagrams Figure 1. Magnetic solid phase extraction (MSPE) method Figure 2. Drug structure of triptorelin acetate Figure 3. XRD image of magnetic zeolite nanoparticles. Figure 4. Results of the effective diameter of nanoparticles prepared with 8 repetitions along with the particle size distribution graph. Figure 5. FTIR chromatogram of magnetic zeolite nanoparticles. Figure 6. Schematic representation of a closed-loop continuous flow device. Figure 7. Schematic representation of an open-loop continuous flow device. Figure 8. Diagram of drug release from the system over time under different conditions. Figure 9. Graph of pH changes in the release environment around the system over time under different conditions. Figure 10. Calibration curve of triptorelin acetate. Figure 11. In vitro dissolution test method in the laboratory A clear and precise statement of the advantages of the claimed invention over prior inventions. 1- Simplicity of the process 2- High performance speed 3-High enrichment factor 4- Process repeatability 5- Process selectivity 6- Using zeolite based on magnetic nanoparticles with high absorption capacity and time saving. 7-Easy extraction method for the drug triptorelin acetate. 8- Practical method for the absorption of triptorelin acetate by magnetic zeolite nano-adsorbent 9-Low price Description of at least one implementation method for implementing the invention In laboratories of research centers and pharmaceutical industries, 10 ml of the release medium samples collected from the USP4 device that evaluates the release of the slow-release drug product formed in the site of triptorelin acetate or microspheres containing triptorelin acetate are transferred into the test beaker and 100 mg of magnetic zeolite nanosorbent powder packaged in 100 gram packages are added to it. According to the pH of the release medium, the final pH of the prepared suspension is adjusted to 6.8 with the help of a pH meter and placed in a shaker incubator with low speed for 30 minutes so that the drug absorption of triptorelin acetate on the magnetic zeolite nanosorbent is carried out well. Then, using a strong magnet placed near the test beaker and tilting the test beaker, the supernatant solution is removed and the magnetic zeolite nanosorbent will remain in the beaker.Then, two milliliters of the desorption solution of water and acetonitrile in the ratio of (50:50) is added to the test beaker containing the magnetic zeolite nanosorbent containing the drug triptorelin acetate and with the help of a pH meter and trifluoroacetic acid, the pH is adjusted to the range of 2. The suspension is placed in a shaker incubator at a gentle speed for 15 minutes to completely extract the drug triptorelin acetate. Then, a strong magnet is placed next to the beaker and the supernatant solution is separated, which is ready to be injected into the HPLC device after passing through a 0.22 micron filter. Explicit mention of the industrial application of the invention This method has industrial applications in pharmaceutical companies, laboratories, and research works for the use of clinoptilolite zeolite nanocomposite in the extraction of triptorelin acetate drug using a magnetic solid phase.

Claims

Claims: Claim 1) What is claimed is a process for manufacturing magnetic nanoparticles of zeolite, which, based on the magnetic solid phase method (MSPE), has the power to concentrate pharmaceutical solutions containing the peptide triptorelin acetate and allows for the accurate measurement of the drug triptorelin acetate in vitro. Claim 2) According to claim 1. Magnetic nanoparticles can be made of various types of zeolite such as clinoptilolite, chabazite, mordantite or mixtures thereof. Claim 3) According to claim 1. The size of the magnetic nanoparticles is below 100 nanometers. Claim 4) According to claim 1. They are magnetized using metal compounds including iron, nickel, cobalt, metal oxides (Fe3O4, Fe2O3), ferrites (MFe2O4), metal alloys (FePt, CoPt), and zeolite nanoparticles. Claim 5) According to claim 1. The conditions for adsorption of triptorelin acetate drug onto one hundred milligrams of zeolite magnetic nanoparticles are 25-200 ml of a drug solution containing triptorelin acetate, adjusting the pH to 6.8 at a temperature of 25 degrees Celsius, and gentle mixing for about thirty minutes. Claim 6) According to claim 1. The conditions for the desorption of triptorelin acetate from two hundred milligrams of zeolite magnetic nanoparticles are to use a maximum of two milliliters of a solution of water and acetonitrile solvent in a ratio of 50:50 at a temperature of 37 degrees Celsius at a pH of 2 and gentle stirring.

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