The role of mass spectrometry in drug metabolite analysis

 Mass spectrometry is an essential technique for performing drug metabolite analysis, which is characterized by high detection sensitivity, a wide range of molecular masses analyzed, and a large amount of molecular structure information provided. Moreover, mass spectrometry is often used in conjunction with other analytical methods, such as chromatography and electrophoresis, which further expands the application of mass spectrometry. For example, some researchers have used ultra-high pressure liquid chromatography-time-of-flight mass spectrometry for metabolite analysis of drugs in rats to investigate the substances that exert drug effects.

1. Mass spectrometry can be used for drug metabolite analysis

Drug metabolites are derived from the metabolic reactions of drugs, and the structure of metabolites is closely related to the type of metabolic reactions and the structural properties of precursor compounds. After the metabolic transformation of a drug in vivo, only some structural modifications are generally made on the basis of the parent drug. The parent drug structure naturally does not change much, so the metabolites and the parent drug often have similar mass spectrometry characteristic ions, according to which the metabolites can be identified and combined with other fragment characteristics to make reasonable inferences about their structures.

When performing drug metabolite analysis, it is also necessary to compare the electron bombardment MS spectrum of the metabolite with the synthetic MS spectrum. The chemical structure is ultimately determined with the help of NMR spectroscopy. Structural information on the parent molecule and metabolite molecules can also be obtained directly by multilevel mass spectrometry (MSN) when no preparation of pure metabolites is required, and the biological samples are only subjected to a simple pretreatment.

In analyzing drug metabolites, mass spectrometry has become the primary detection tool for drug metabolites because of its wide range of applications and high sensitivity. Since the background matrix of drug metabolites is more complex, direct mass spectrometry injection significantly influences the ionization efficiency of drug metabolites. Therefore, in drug metabolite analysis, mass spectrometry is often used with liquid chromatography, gas chromatography, and other chromatographic techniques with strong separation capacity.

2. UHPLC coupled with mass spectrometry for metabolite analysis of drugs in rats

The determination of blood components by mass spectrometry is fast, microscopic, and accurate, which can solve the problems of low content, multiple elements, difficult separation, and significant biological individual differences in the analysis of plasma components. The combination of ultra-performance liquid chromatography and mass spectrometry for drug metabolite analysis can identify the major chemical components of a drug as well as the drug prototype components in animals, providing a basis for finding the active chemical in a prescription. Medicilon Pharmacokinetics Lab has passed the GLP  certification by  NMPA, Following the guiding principles of ICH,  NMPA, and FDA. The lab offers in vivo and in vitro pharmacokinetic tests according to our client's needs and provides them with complete sets of pharmacokinetic evaluation and optimization services.

To identify the metabolites in plasma, urine, and feces after administration of Damp Heat Paralysis capsules to rats, some investigators used a column of Waters C18 (2.1 mm×50 mm, 1.7 μm) with a column temperature of 40°C. The mobile phase: 0.1% formic acid aqueous solution - 0.1% formic acid acetonitrile solution, gradient elution. The ultra-high pressure liquid chromatography-time-of-flight mass spectrometer was used with an electrospray ionization ion source and positive and negative ion mode detection[1].

The results showed that the UHPLC-MS method analyzed seven prototypical components of Damp Heat Paralysis capsules in blood, six prototypical components of Damp Heat Paralysis capsules in urine, and 11 prototypical components of Damp Heat Paralysis capsules in feces. It was found that the components measured in vivo may be the material basis for the efficacy of the tablets.

The metabolites of nobiletinin rats were also analyzed by ultra-performance liquid chromatography-triple quadrupole mass spectrometry (uplc-ms/ms)[2]. Seven metabolites of nobiletin were observed by comparing the total ion flow chromatograms of rat plasma, bile, and urine samples before and after drug administration. 

Further, by combining the chromatographic retention behaviors of the metabolites, and primary and secondary mass spectrometry information, three of them were presumed to be Ⅰ-phase metabolites, which were obtained by the removal of one and two methyl groups from nobiletin, respectively. The other four were supposed to be phase Ⅱ metabolites, which were generated by further sulfation and glucuronidation of phase I metabolites, respectively.   

Four Phase Ⅱ metabolites were reported among the above products for the first time. The results suggest an essential role of phase Ⅱ metabolism, especially glucuronidation, in the metabolic pathway of trichostatin. They indicate that the gene polymorphism of glucuronosyltransferase and the associated drug interactions causing potential changes in trichostatin activity and toxic side effects deserve further attention.

Performing metabolite analysis of drugs in animals can be of great use as it can provide vital information for further study of the metabolites of drugs and their mechanism of action.

[1] Analysis of metabolites in rats with oral damp-heat paralysis capsules [J].

[2] Analysis of metabolites of nobiletin in rats by ultra-performance liquid chromatography-mass spectrometry[J].