Chromatography-mass spectrometry for the in vivo metabolite analysis of saponins

 Saponins are one of the main active ingredients of many herbal medicines such as ginseng, Polygala tenuifolia, Platycodon grandiflorum, licorice, rhizoma anemarrhenae, and radix bupleurum. Pharmacological studies have shown that saponins have biological activities such as antibacterial, antitumor, modulation of body metabolism and immunity, and treatment of cardiovascular diseases and diabetes mellitus. The in vivo metabolites of saponins were analyzed by chromatography-mass spectrometry to provide favorable evidence for the elucidation of the therapeutic mechanism of Chinese medicine.

Liquid chromatography-mass spectrometry (LC/MS) is a bioanalytical technique that combines the high separation performance of high-performance liquid chromatography HPLC with the high sensitivity and specificity of tandem mass spectrometry. It does not require complete chromatographic separation between analytes, and its multi-window detection capability allows the quantitative analysis of multiple components simultaneously. 

Medicilon’s Bioanalysis Department boasts a professional, scientific research team with analysis laboratories equipped with advanced instruments. In the context of information management, our experiments and researches are compliant with the standards of  FDA/NMPA GLP  and involve pharmacokinetics,  toxicokinetics,   pharmacodynamics, immunogenicity, and bioequivalence, to provide our clients with services, including selection and development, preclinical and clinical researches of micromolecule drugs, biological preparations, vaccines, and biomarkers.

Chinese herbal medicines and their prescriptions are complex in composition. HPLC coupled with UV or DAD detectors can only provide signals such as retention time and UV absorption for individual peaks, while the structural information available for unknown components is quite limited. The identification of chromatographic peaks must have controls, which are difficult to obtain for most of the chemical elements of TCM, and for the in vivo drug analysis of TCM, the general detection techniques are challenging to meet the requirements for the determination of blood concentration after drug administration.

The application of HPLC/MS can combine the advantages of the high separation efficiency of HPLC and the high sensitivity and specificity of tandem mass spectrometry and give the molecular weight information of the measured components. The structural information of the measured substances can also be derived through multi-stage tandem mass spectrometry analysis.

1,Liquid chromatography-tandem mass spectrometry for the analysis of pseudo ginsenoside metabolites in human blood

To establish a liquid chromatography-tandem mass spectrometry method for determining pseudo ginsenoside GQ concentration in human plasma. An appropriate amount of internal standard was added to the plasma samples, extracted with ethyl acetate, and then analyzed by Waters Xevo TQS LC-MS/MS. A Poroshell 120 EC C8 column (2.1 mm×50 mm, 2.7 μm) was used at 40 ℃ with methanol-10 mmol-L-1 ammonium acetate aqueous solution (80:20) as the mobile phase at a flow rate of 0.3 m L-min-1. The determination was performed in the negative ionization mode using multiple reaction ion monitoring (MRM) in scanning mode with an electrospray ionization source (ESI).

The linear range of the method was 2.500~5 000 ng-m L-1, the minimum limit of quantification was 2.500 ng-m L-1, the intra-day, and inter-day precision was less than 15%, the accuracy was between 85% and 115%, the extraction recovery was about 9%~11%, the matrix effect was about 66%~73%, and the stability investigation results were good. The pharmacokinetic tests showed that the peak time was two h, and the half-life was approximately ten h after static injection of pseudo ginsenoside GQ 120 mg-sub-1 once daily for 5 d. The main pharmacokinetic parameters were the same at d 1 and d 5 of the trial, and the calculated accumulation coefficients were RC max=0.964±0.099 and RAUC=0.965±0.181, both of which were close to 1, respectively.

This method applies to the human pharmacokinetic study of pseudo ginsenoside GQ. Under this dosing regimen, there was no significant accumulation of pseudo ginsenoside GQ in humans, and continuous dosing did not affect the human pharmacokinetic process of pseudo ginsenoside GQ.

2,LC-MS/MS for the analysis of metabolites of yarrow saponin in rat blood

High-performance liquid-phase tandem mass spectrometry (LC-MS/MS) was used to determine the content of maidenhair saponin G in rat plasma and to study its pharmacokinetic characteristics in rats. Methods A Phenomenex Luna C18 column (150 mm×2 mm, three μm) was used with acetonitrile-water (containing 0.1% formic acid) as the mobile phase at a flow rate of 0.2 mL-min~(-1), and ginsenoside Rg3 was used as the internal standard; rats were injected with maidenhair saponin G 0.25, 0.5 and 1 mg-kg-1 in the tail vein, and blood was collected at different time points after drug administration. 

The blood was collected at other time points after drug administration, and the blood concentrations were determined by LC-MS/MS method as described above. The pharmacokinetic parameters were fitted by DAS 3.0 software and a non-atrial model. 

The results showed good linearity between 0.01~1.0 μg-m L-1 yari saponin G and peak area, and the methodological investigations all followed the requirements; the plasma pharmacokinetic parameters of rats after intravenous administration were: t1/2=3.447±0.898 h, MRT0-∞=4.568±1.075 h, CL=0.858±0.171 L-h-1-kg, and the AUC and Cmax increased equivalently with the increase of the administered dose. The AUC and Cmax increased equiproportionally with increasing amounts, which was consistent with linear pharmacokinetics. This method is simple, sensitive, and accurate and is suitable for determining yarrow saponin G in rat plasma and its pharmacokinetic study.

Some researchers have also used HPLC-ESI-MS/MS for the qualitative and quantitative analysis of saponins in blood saponin injection. Other researchers used pressurized solution extraction (PLE) with the HPLC-DAD-MS technique to determine nine saponins and two poly ethynyl alcohols in ginseng leaves and ginseng, a rapid method for the detection of herbal medicines and help control the quality of ginseng.

Establishing a reliable analytical method is a precursor to performing in vivo metabolite analysis of drugs. With the development of modern chromatographic coupling techniques, separating and identifying multiple trace metabolites in vivo has become a continuous process. In particular, LC-MS sample pretreatment is simple and generally does not require hydrolysis or derivatization treatment. LC-MS technology avoids the complicated and tedious work of separating and purifying metabolites and allows the separation and identification of difficult-to-identify in vivo trace metabolites.