Determination of metabolites

Related definitions

After drugs enter the body, they generally undergo physical changes and chemical changes. Physical changes refer to the binding reaction between drugs and biological macromolecules such as plasma proteins; chemical changes refer to metabolic reactions in the body.

Metabolic reactions in the body are also called biotransformations, which are divided into one-phase metabolism and two-phase metabolism. Phase one metabolism refers to the oxidation reaction, reduction reaction, and hydrolysis reaction of the drug in the body. Phase two metabolism refers to the conjugation reaction of drugs in the body, including: glucuronic acid conjugation, sulfation, methylation, acetylation, amino acid conjugation, glutathione conjugation, etc. The MetID team of Medicilon is composed of experienced scientists. We provide fast and reliable in vivo and in vitro MetID and reactive metabolite capture services. We also support new drug screening and domestic and oversees IND filings. Since the establishment of MetID team, Medicilon has successfully completed multiple different types of research projects for clients, including challenging peptide MetID research.

The analysis and research of drugs and their metabolites in vivo will provide scientific basis for the relationship between drug concentration, drug efficacy and toxicity, as well as the study of drug action mechanism and pharmacokinetics. Therefore, the progress in recent years has been remarkable and has become An important branch of drug research and formed a new discipline.

Bioavailability: The rate and extent to which a drug in a dosage form is absorbed into the bloodstream.

Bioequivalence: When different preparations of a drug are given the same dose under the same test conditions, there is no significant statistical difference in the main kinetic parameters that reflect the rate and extent of absorption.

Study the significance of metabolite determination

2.1 Measuring the distribution of drugs and their metabolites in blood tissues and organs is an important indicator to measure the effectiveness and safety of drugs;

2.2 Determine the changes in concentrations of drugs and metabolites over time to provide reliable pharmacokinetic data;

2.3 Studying the metabolic pathways of drugs, the pharmacological activity of metabolites, and the speed of generation and elimination is of certain significance for rational use of drugs, avoiding drug toxicity, and finding new drugs.

2.4 Conducive to the preservation of biological samples. After biological samples such as blood are collected, plasma esterase may continue to hydrolyze the ester drugs in the sample, and other enzymes may also continue to produce metabolic reactions on the sample drugs. Therefore, enzyme inhibitors are often added. .

2.5 It is conducive to the selection of analytical methods for the in vivo metabolic reactions of most drugs. However, changes in certain groups on the molecular structure of the drug make the chemical structure of the metabolites very similar to that of the parent drug, resulting in no major differences in some physical properties and spectra. Analysis and immunoassay methods often face interference from metabolites. When you want to determine the content of drugs and metabolites in biological samples, chromatography is the appropriate choice. Because of its separation and analysis functions, it can avoid interference from metabolites or directly Determination of metabolites.

2.6 It is helpful to select methods for sample separation and extraction. After drugs are metabolized in the body, the metabolites generated generally increase in polarity and enhance water solubility. Therefore, when separating and extracting drugs from biological samples, the polarity and balance between them are often used. Depending on the difference in dissociation behavior, choose an appropriate pH buffer and solvent system to separate them.

Situations in which metabolites in the body need to be measured

3.1 When conducting phase 1 clinical pharmacokinetic trials of drugs,

3.1.1 If active metabolites (pharmacological and toxicological) are known and the concentration is high enough, it can be determined

a) The study is about its pharmacokinetics, and metabolites need to be measured

b) Only conduct bioequivalence experiments, and metabolites do not need to be measured when the original drug can be measured.

3.1.2 If there is an active metabolite known, but the concentration is low and difficult to measure, it does not need to be measured.

3.1.3 If the metabolites will affect the metabolism, distribution and excretion of the drug, the measurement of the metabolites should be considered when studying its pharmacokinetic characteristics.

However, it is difficult to judge whether a metabolite is active in the human body, because its activity is supported by its preclinical data. At this time, you can refer to the March 2005 version of "Technical Guiding Principles for Clinical Pharmacokinetic Studies of Chemical Drugs" and FDA's "Bioavailability and Bioequivalence Studies for Orally Administered Drug Products - General Considerations" (2003)

3.2 When detecting the concentration of metabolites in biological samples,

3.2.1 When metabolites can affect the safety and effectiveness of drugs:

Pharmacokinetic studies: Simultaneous determination of concentrations of parent drug and metabolites,

Equivalence test: The concentrations of the parent drug and metabolites are measured simultaneously, and the parent drug is used as the main judgment indicator when evaluating equivalence.

3.2.2 When the activity of the main metabolite is unclear:

Pharmacokinetic studies: determination of parent drug and metabolite concentrations,

Equivalence test: Determination of parent drug concentration.

3.2.3 When the prodrug concentration is very low and the metabolite is the main form in the body:

Pharmacokinetic studies: determination of parent drug and metabolite concentrations,

Equivalence evaluation: Determine the concentration of metabolites.

3.2.4 Prodrugs:

Pharmacokinetic studies: determination of parent drug (if available) and metabolite concentrations,

Equivalence test: Determination of metabolite concentration.

Biological samples commonly used for metabolite determination

Biological samples commonly used for metabolite determination include blood samples (plasma, serum and whole blood), urine samples, and saliva.

Collection and storage of commonly used biological samples: plasma - add anticoagulant to whole blood, centrifuge, and take the supernatant; serum - centrifuge the whole blood, take the supernatant. After blood collection, plasma or serum must be separated by centrifugation as soon as possible and stored frozen below -20°C. Urine sample - collect urine samples within a certain period of time after taking the medicine, record the volume, mix evenly and take a certain amount, measure the concentration of the drug in the urine, and calculate the cumulative amount of the drug in the urine within a certain period of time. Saliva - After gargling for 15 minutes, collect the mixed saliva that naturally flows out of the mouth or flows out after stirring in the mouth with the tongue, and centrifuge to take the supernatant. If the sample cannot be measured in time after collection, it can be refrigerated at 4°C. If it is left for a long time, it needs to be frozen and stored at -20°C ~ -80°C.

Methods for determination of metabolites

The determination of metabolites is one of the most classic and very effective methods in drug metabolism research.

Since the drug will eventually be excreted from the body through one or more metabolites associated with the parent nucleus, the isolation, identification and measurement of metabolites can provide a certain understanding of the drug's metabolic pathway. In recent years, high-speed, efficient and highly sensitive analytical methods such as HPLC, GC-MS, LC-MS and GC-MS-MS, LC-MS-MS, LC-NMR and LC-MS-NMR have been used for the analysis of metabolites. Analysis and determination.

5.1.HPLC method

Since the content of drugs and metabolites in body fluids is extremely small and mixed with a large number of endogenous components, only reliable analysis methods can ensure accurate analysis results. Gas chromatography-mass spectrometry (GC-MS) is a powerful tool for analyzing trace and even ultra-trace compounds and is widely used in this field. However, many drugs and metabolites lack volatility and thermal stability, which limits its application. In recent years, high-performance liquid chromatography (HPLC) has been characterized by its wide application range, high separation efficiency, easy recovery of components, and speed and simplicity. Along with the development and application of highly sensitive and selective detectors, it has been used in many analyses. The method has come from behind and has become one of the indispensable means for analyzing drug metabolites in the body.

Because the content of drugs and metabolites in the body is very small, it is difficult to collect enough samples to measure the four major spectra, namely infrared, ultraviolet, nuclear magnetic resonance and mass spectrometry. Therefore, using HPLC as a tool to identify unknown substances has become one of the difficult problems that need to be solved. Although LC-MS is effective, it cannot be widely used because the instrument is expensive. At present, empirically inferred compounds are still commonly used. In order to remove the interference of endogenous components and ensure the column efficiency and life of the chromatographic column, it is extremely important to pretreat biological samples.

5.1.1 Separation

Different pretreatments are performed for the different biological sample types selected. For example, blood samples (including serum and plasma) can be loaded directly for solid phase extraction. However, if the drug binds to protein, the extraction recovery rate will be reduced. Therefore, the protein in the metabolites must be removed to release the bound drug to determine the total concentration. . Obtain a "cleaner" extract, reduce emulsification, and eliminate interference with the measurement. For saliva samples, centrifugal precipitation is mainly used to remove mucin, and the supernatant is taken to determine the drug concentration. Determination of conjugates in urine often requires acid hydrolysis or enzymatic hydrolysis to hydrolyze the conjugates.

Commonly used protein removal methods:

Protein precipitation method - formation of insoluble salts or salting out and dehydration. Commonly used protein precipitating agents include organic solvents (acetonitrile, methanol, ethanol, acetone, etc.), ammonium carbonate salts, inorganic acids (trichloroacetic acid, perchloric acid, etc.) and heavy metal salts (mercury salts, tungstates, copper salts, etc.) . It is most widely used because biological samples treated with organic solvents or acids are compatible with reversed-phase HPLC analysis.

Tissue enzyme digestion method - proteolytic enzymes, such as trypsin, pepsin, etc.

In recent years, methods for filtering proteins using microporous membranes have been reported, which have the advantage of being simple and fast, but can only measure free compounds. Using acidic and alkaline ion exchange resins to remove proteins that coexist with alkaline and acidic compounds can complete the purification and enrichment processes at the same time, so it is often used.

5.1.2 Extraction

There are usually two methods for extracting analytes from biological samples: liquid phase extraction and solid phase extraction, which are especially necessary when analyzing ultra-trace compounds.

Liquid phase extraction is also called solvent extraction. Commonly used organic solvents include: n-alcohol, dichloromethane, chloroform, ethyl acetate, diethyl ether, benzene, n-hexane, etc. and their mixed solutions. Sometimes a small amount of methanol or ethanol can be added to reduce emulsification. In order to effectively extract acidic and alkaline drugs, it is usually necessary to adjust the pH of the sample. Alkaline drugs are generally adjusted to 12 units higher than their pKa, and acidic drugs are adjusted to 12 units lower than their pKa, so that the drugs are in a non-dissociated state. Easily extracted. When extracting highly ionized compounds (such as quaternary ammonium salts or sulfonates) and amphoteric compounds (such as amino acids), ion pairs must be added for trial production. Acidic compounds generally use quaternary ammonium salts or tertiary ammonium salts, and alkaline compounds use alkyl or aryl sulfonates to form ion pairs to improve organic solvents. Since organic solvents are generally toxic and flammable, evaporation of the solvent may cause analytes to evaporate. Loss and sometimes emulsification phenomenon, etc. Therefore, a simpler and more effective extraction method has become the goal sought.

Among them, solid-phase extraction method shows broad prospects. Classic solid phase extraction agents include alumina, activated carbon and diatomaceous earth. Since alumina can selectively adsorb o-phenolic hydroxyl compounds, it is still commonly used today. It is usually adsorbed in a weakly alkaline medium (around pH 8), and the impurities are washed away with water and then the analyte is eluted with acid.

Factors affecting liquid-liquid extraction: pH value of aqueous phase, extraction solvent and ionic strength, etc.

1) Aqueous phase pH

Alkaline drugs: pH 2 to 3 units higher than the pKa of the drug; acidic substances: < pKa 2 to 3 units

2) Extraction solvent

The selection of the extraction solvent must consider not only the selectivity of the extraction, but also the convenience of operation. While satisfying the extraction efficiency, select a solvent with as little polarity as possible to achieve a suitable recovery rate and reduce interfering substances to lowest.

Generally, the selection can be based on the principle of similar miscibility, and a solvent with a low boiling point can be selected.

When the properties of the drugs in the sample are unknown, ether and chloroform can be used as extraction solvents for acidic and alkaline drugs respectively.

3) Ionic strength

Adding neutral salts, such as NaCl, to the water phase can increase the ionic strength, causing water molecules in the solution to strongly associate with inorganic ions, resulting in fewer water molecules associated with the drug, making the drug solubility in the water phase smaller, and thus Conducive to organic solvent extraction.

4) The volume of organic phase and aqueous phase is 1:1 or 2:1

Factors affecting liquid-solid extraction:

Separation and recovery rate are important indicators reflecting extraction efficiency. The main factors affecting extraction rate are:

1) Eluent flow rate - if the flow rate is too fast, the resolution will decrease, the sample will be lost, the recovery rate will be low, and the reproducibility will be poor.

2) Sample loading capacity - the effective loading capacity depends on the capacity factor of the analyte, the amount of stationary phase and the sample concentration. Overload, resulting in sample loss.

Select pretreatment and detection methods based on the drug's pKa value, lipophilicity, solubility, distribution coefficient, etc.

A) Drugs with lipophilic properties are extracted with solvents at appropriate pH values

B) Methods of selective precipitation of proteins, solid phase extraction, ion pair extraction or extraction after derivatization for drugs with strong polarity or hydrophilicity, etc.

C) GC assay for drug selection with volatile properties

D) Select analytical detection methods with spectral or electrochemical characteristics

E) The extraction and concentration technology should be selected based on the stability of the drug: avoid using strong acid or strong alkaline solvents for drugs that are unstable to acids and bases, avoid high-temperature evaporation of solvents for drugs that are unstable to heat, and avoid exposure to light for drugs that are unstable to light.

The selection of sample processing steps and analysis methods is shown in the figure below

 

5.2 Other methods for measuring metabolites

Since drugs are ultimately excreted through one or more metabolites associated with the parent nucleus, isolation and identification of metabolites can provide a certain understanding of the drug's metabolic pathway. Special analysis methods such as GC-MS, HPLC-MS, GC-MS-MS, LC-MS-MS, LC-NMR and LC-MS-NMR analysis methods can also be used at this time. They have high speed, high efficiency and high sensitivity. characteristics and absolute specificity. And the structure of metabolites can be identified while detecting extremely small amounts of metabolites.

Absorbance ratio, photodiode matrix detection, evaporative light scattering detection, or the application of dual detectors (UV, fluorescence) are also some special methods. In the absence of the above techniques, the peak shape and retention time of each chromatographic peak should be carefully compared.