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.
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