In drug design, the metabolite structure of some drugs or prodrugs that produce actual pharmacological activity in vivo is usually unstable under laboratory conditions, so during method development, method validation and sample analysis of bioanalytical analysis, keep Structural stability of drugs and their metabolites is a crucial step in the correctness of method stability data. The factors that usually lead to compound instability can be mainly divided into two categories: physical factors and chemical factors.
Physical factors
1. Photosensitivity
For photolabile drugs, they are commonly found in molecules with unsaturated carbon-carbon double bond structures and some molecules with heterocyclic double bonds. The former may undergo E/Z isomerization, typically vitamin A, and the latter One is carbonyl, nitroaromatic and aryl chloride groups, which can undergo dechlorination reactions, and are representative of drugs such as lomefloxacin and fluoroquinolone. Therefore, for light-sensitive samples, it is necessary to use a brown collection tube in each step of sample collection and processing, wrap tinfoil around the sampling tube, or more commonly operate under a yellow light of a specific wavelength.
2. Thermally stable
Since some drugs are in the matrix of biological samples, under the action of enzymatic decomposition or other factors, the degradation rate of drugs and other adverse chemical reactions will be accelerated with the increase of temperature. The best solution for such drugs is The samples should be frozen and stored immediately after collection. At the same time, they should be stored under wet ice conditions when operating on the test bench. In addition, long-term cryopreservation stability samples should be established to monitor the degradation of drugs, and samples should be stored at a suitable temperature, such as When storage at -20°C is unstable, you can choose to store at -80°C.
3. PH
The normal physiological pH of plasma and urine matrix is around 7.4, but it will increase to 8.8 with time. Since specific enzymes in biological matrix need to work within a certain pH range, so for chemical and Enzyme-labile compounds are very important for pH control. Commonly used pH regulators include various concentrations of formic acid, acetic acid, phosphoric acid, and citric acid.
Typical compounds that are pH-sensitive and labile include esters (lactones), acid compounds such as amides (lactams), and metabolites that readily form acyl glucuronic acids.
(1) For example, the representative statins of lactones will hydrolyze the lactone ring into β-hydroxystatin acid in a high pH environment such as physiological pH. Therefore, citric acid is added during sample collection to control the pH in the matrix. This transformation can be inhibited by an acidic environment.
(2) Generally containing carboxyl compounds (salicylic acid, ibuprofen and telmisartan and other compounds) are combined with glucuronic acid under the catalysis of uridine diphosphate glucosyltransferase in vivo to form acyl glucuronide (AG) ), and acyl glucuronide is considered to be a reactive metabolite, which is prone to nucleophilic substitution and can covalently bind to proteins and other macromolecules, which can easily cause serious adverse reactions. In the testing guidelines, AG is listed as a class of metabolites that require further safety assessment during method development. AG is easily hydrolyzed into glucuronic acid and parent drug under physiological pH conditions. In addition, this metabolite can also undergo intramolecular and intermolecular acyl transfer. Once hydrolyzed, the concentration of AG will be underestimated and the parent drug will be overestimated. Therefore, it is very important to prevent the hydrolysis of AG. It is usually necessary to add citric acid or phosphoric acid in time to adjust the pH to an appropriate acidity (PH2~4) when the sample is collected, and pre-treatment at low temperature to inhibit decomposition and molecular acyl transfer.
4. Enzymes
Specific enzymes in biological substrates are important factors leading to the instability of compounds. Among them, esterases are the most common enzymes. Esterases can be divided into cholylesterases, carboxylesterases, serine esterases, and arylesterases. Enzymes are present in different concentrations in whole blood, plasma, serum and tissue samples, but these enzymes are abundant in rodents and less in humans, including butyrylcholinesterase, acetylcholinesterase, albumin Esterase and paraoxonase are present in the human body.
Therefore, when encountering enzymatically unstable compounds,
• Appropriate enzyme inhibitors can be selected, usually those with low toxicity are preferred, eg paraoxon is highly neurotoxic.
·Choose blood collection tubes with appropriate anticoagulants. For example, sodium fluoride has a strong enzyme inhibitory effect, which can effectively inhibit the activities of phosphatase, acetylcholinesterase and butyrylcholinesterase.
Another simple and easy method is to directly collect the sample into the sampling tube of the organic solvent, because the organic solvent can directly and quickly denature the proteins in the matrix and the enzymes that affect the stability.
Chemical factor
1. Oxidation
This instability usually exists in compounds containing anthraquinone or phenolic structures, such as mitoxantrone, rifampicin, and levodopa. The most commonly used antioxidants are vitamin C and sodium metabisulfite, which can be very stable. effect.
For lipophilic compounds like vitamin A and vitamin E, the most commonly used antioxidant is 2,6-di-tert-butyl-p-cresol (BHT).
Another class of compounds that are easily oxidized are thiols, which are also prone to form disulfide bonds between molecules to form dimers. In order to stabilize these compounds, methacrylate derivatization reagents can be added to derivatize them. , can play a good role in stability.
2. N-oxide interconversion
Aliphatic and aromatic amines and drug molecules of aliphatic amines adjacent to the aromatic ring are often susceptible to nitric oxide metabolites formed by enzymatic oxidation in liver microsomals, such as clozapine, imipramine, and procainamide Wait. Due to the strong pharmacological and toxicological activities of N-oxidative metabolites, drug concentration monitoring together with the parent drug is often required in biological analysis. At the same time, N-oxides are a class of unstable compounds in biological samples. These compounds are usually unstable before they have been prepared from whole blood into plasma. If N-oxides are soluble in plasma and the original drug is not For distribution in plasma, it is best to measure the two compounds separately and for stability assessment.
Because the chemical properties of N-oxidation metabolites are relatively active, when the pH is changed to stabilize the parent drug, the metabolites are unstable. Therefore, when handling samples of such compounds, care must be taken in the collection and analysis stages, and the selection of appropriate PH, storage temperature and stabilizer and other conditions, such as avoiding the use of oxidants, using neutral PH or weak acid and weak base conditions, avoiding the use of high temperature during nitrogen blowing, etc.
In addition, N-oxides are thermally unstable, and conversion to parent drugs is common at high ion source temperatures, so ESI sources are recommended, and stable isotope-labeled internal standards (SIL-IS) are strongly recommended for these compounds. Rather than using analog internal standards, to reduce the adverse effects of in-source transformation.
3. Transformation of Chiral Compounds
All chiral compounds will interconvert under certain conditions, so you can choose to add buffer reagents such as phosphate or citrate to change their pH or store samples at -80°C to stabilize them.
