A validated UPLC-MS/MS method for quantitative determination of a potent neuroprotective agent Sarsasapogenin-AA13 in rat plasma: Application to pharmacokinetic studies.

Sarsasapogenin-AA13(AA13), a sarsasapogenin derivative, exhibited good neuroprotective and anti-inflammatory activities in vitro and therapeutic effects on learning and memory dysfunction in amyloid-ß-injected mice. A sensitive UPLC-MS/MS method was developed and validated to quantitatively determine AA13 in rat plasma and was further applied to evaluate the pharmacokinetic behaviour of AA13 in rats that were administered AA13 intravenously and orally. This method was validated to exhibit excellent linearity in the concentration range of 1-1000 ng/mL. The lower limit of quantification was 1 ng/mL for AA13 in rat plasma. Intra-day accuracy for AA13 was in the range of 90-114%, and inter-day accuracy was in the range of 97-103 %. The relative standard deviation of intra-day and inter-day assay was less than 15%. After a single oral administration of AA13 at the dose of 25 mg/kg, Cmax of AA13 was 1266.4 ± 316.1 ng/mL. AUC0-48 h was 6928.5 ± 1990.1 h·ng/mL, and t1/2 was 10.2 ± 0.8 h. Under intravenous administration of AA13 at a dosage of 250 µg/kg, AUC0-48 h was 785.7 ± 103.3 h⋅ng/mL, and t1/2 was 20.8 ± 7.2 h. Based on the results, oral bioavailability (F %) of AA13 in rats at 25 mg/kg was 8.82 %.

Development and validation of a UPLC-MS/MS method for determination of Sarsasapogenin-AA22 in rat plasma and its application to a pharmacokinetic study.

A sarsasapogenin derivative, sarsasapogenin-AA22 (AA22), with cyclobutylamine at the 3-hydroxyl position of sarsasapogenin, has great neuroprotective activity in PC12 cells and NO production inhibitory activity in RAW264.7 cell lines. A method was developed to determine AA22 in rat plasma which was further applied to evaluate the pharmacokinetics of AA22 after taking a single dose of AA22. Liquid chromatography tandem mass spectrometry was used in the method, while diosgenin was used as internal standard. A simple protein precipitation based on acetonitrile was utilized. A simple sample cleanup promoted the throughput of the method considerably. The method was validated over the range of 1-1000 ng/mL with a correlation coefficient > 0.99. The lower limit of quantification was 1 ng/mL for AA22 in plasma. Intra- and inter-day accuracies for AA22 were 92-111 and 100-103%, respectively, and the inter-day precision was <15%. After a single oral dose of 25 mg/kg of AA22, the mean peak plasma concentration of AA22 was 2114 ± 362 ng/mL at 6 h. The area under the plasma concentration-time curve was 196,098 ± 69,375 h ng/mL, and the elimination half-life was 8.7 ± 2.2 h.

Quantitative determination of sarsasapogenin in rat plasma using liquid chromatography-tandem mass spectrometry.

Sarsasapogenin, a natural compound from Chinese medical herb Anemarrhena asphodeloides Bge., has recently received a great deal of attention due to its various bioactivities. In this study, an easy and applicable liquid chromatography tandem mass spectrometry method for the quantification of sarsasapogenin in rat plasma was developed. Sample preparation was accomplished through a simple one-step protein precipitation procedure with methanol. Negative electrospray ionization was performed using multiple reactions monitoring (MRM) mode with transitions of m/z 417.4/273.2 for sarsasapogenin, and 415.2/271.4 for diosgenin (internal standard). The calibration curve was linear over the range of 0.5-500ng/mL (r=0.9994), with a lower limit of quantification at 0.5ng/mL. The RSD of intra- and inter-day precision was below 6.41%, and accuracy ranged from 87.60% to 99.20%. The RSD of matrix effect and recovery yield was within ±15% of nominal concentrations and sarsasapogenin was stable during stability tests. This validated method had been successfully applied to the preclinical pharmacokinetic studies of sarsasapogenin in rats. The half-life (t1/2) was (15.1±2.3), (16.1±3.0) and (15.4±3.9) h after single intragastric administration of 25, 50 and 100mg/kg sarsasapogenin, respectively. And it was found that, the area under the plasma concentration versus time curve (AUC0-72h) and the maximum plasma concentration (Cmax) were linearly related to dose.

Rapid determination of ruscogenin in rat plasma with application to pharmacokinetic study.

A sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine ruscogenin in rat plasma using midazolam as the internal standard (IS). Sample preparation was accomplished through a liquid-liquid extraction procedure with ethyl acetate to 0.2mL plasma sample. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1mm×50mm, 1.7µm) with the mobile phase of acetonitrile and 1% formic acid in water with gradient elution at a flow rate of 0.40mL/min. Ruscogenin and IS were eluted at 1.74 and 1.11min, respectively. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 431.2→287.0 for ruscogenin and m/z 326.2→291.1 for IS. The linearity of this method was found to be within the concentration range of 2-1000ng/mL with a lower limit of quantification of 2ng/mL. Only 2.0min was needed for an analytical run. The matrix effect was 92.4-107.3% for ruscogenin. The intra- and inter-day precision (RSD%) were less than 11.2% and accuracy (RE%) was within ±9.8%. The recovery ranged from 75.4% to 86.3%. Ruscogenin was sufficiently stable under all relevant analytical conditions. The method was also successfully applied to the pharmacokinetic study of ruscogenin in rats.

One-month toxicokinetic study of SHENMAI injection in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: 'SHENMAI' injection (SMI) has been widely used in cardioprotection and modulation of the immune system because of its great efficacy. SMI primarily comprises the saponins from Panax ginseng and Ophiopogon japonicas. The profiles of saponins in SMI during long-term toxicokinetics remain unclear. MiR-146a possesses excellent sensitivity as a bio-marker in the innate immunity modification effect of SMI. AIM OF THE STUDY: Is to monitor the exposure level of SMI during a one-month toxicokinetic experiment, an analytical method involving ESI-LC-MS/MS technology was developed to determine 20 (S)-protopanaxadiol-type ginsenoside (Rb1, Rb2, Rc, Rd), 20 (S)-protopanaxatriol-type ginsenoside (Rg1, Re, Rf), oleanolic acid-type ginsenoside (Ro), and ophiopogonin D in rats. The levels of AST, CK, ALT, SOD, GSH-pX, MDA, miR-146a, and ECG were measured to explore the effects of SMI in cardiologic function and immune activity. RESULTS: Results show that the levels of AST, CK, and MDA decreased upon the administration of SMI. The level of miR-146a increased upon the administration of SMI dosage. During the administration of SMI, increasing exposure levels of 20 (S)-protopanaxadiol-type ginsenosides were also observed. CONCLUSION: The 20 (S)-protopanaxadiol-type ginsenosides were considered potential PK/TK markers because of their high exposure levels that continuously increased. Oxidative stress was slightly alleviated during the toxicokinetic study. Based on the level of miR-146a, negatively regulated innate immunity was observed. The regulation became more serious with increasing exposure levels of 20 (S)-protopanaxadiol-type ginsenosides. Negatively regulated innate immunity could be induced by long-term administration of SMI (>0.4g/kg).

Determination of deltonin in rat plasma by using HPLC-MS/MS and the application of this method in pharmacokinetic studies.

Deltonin is a naturally occurring spirostanol glycoside from Dioscorea zingiberensis C.H. Wright, which is used in traditional Chinese medicine. It exerts strong cytotoxic effect on C26 cells, inhibits C26 derived-tumor growth, and prolongs the survival of tumor-bearing mice after its oral administration, indicating its potential for use as an anti-tumor drug. To investigate the pharmacokinetic profiles of deltonin, a rapid, sensitive, and simplified high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay was developed and validated for the determination of deltonin in rat plasma. After acetonitrile-mediated plasma protein precipitation, chromatographic separation of deltonin was achieved using a reversed phase Hypersil Gold column (150mm×2.1mm, 5µm), with gradient elution using 0.1% formic acid and acetonitrile. Thereafter, deltonin was quantified using MS/MS with electrospray ionization (ESI) in positive multiple reaction monitoring (MRM) mode. The flow rate of the mobile phase was 200µL/min, and the retention time was 9.03min for deltonin and 6.31min for the internal standard (IS: 20(S)-ginsenoside Rb1). The linear range of the calibration curve was 2-5000ng/mL (r(2)>0.99), and the limit of detection (LOD) was 0.46ng/mL. The intra- and inter-day accuracies ranged from -2.8% to 11.1% and precisions (RSD) were within 13.1%. Deltonin was found to be stable under short-term temperature conditions, post-preparative temperature conditions, and after 3 freeze-thaw cycles conditions. The validated method was successfully applied to a pharmacokinetic study in rats after oral administration of deltonin (50 and 100mg/kg). The pharmacokinetics is characterized by high apparent clearance (CL/F) and apparent volume of distribution (Vd/F).

Pennogenin tetraglycoside stimulates secretion-dependent activation of rat platelets: evidence for critical roles of adenosine diphosphate receptor signal pathways.

BACKGROUND: Total steroidal saponins extracted from the rhizome of Paris polyphylla Sm. var. yunnanensis (TSSPs) have been demonstrated to promote hemostasis in vivo and induce platelet aggregation in vitro. Pennogenin tetraglycoside (Tg) has been identified as one of the active ingredients in TSSPs and can induce rat platelet aggregation. OBJECTIVE: To investigate the functional role of Tg in platelets and the signaling pathway mechanisms which mediate Tg-induced platelet aggregation. METHODS AND RESULTS: Using scanning electron microscopy, the turbidimetric method and flow cytometry, we demonstrated that Tg induces shape change and concentration-dependently induces aggregation, dense granule secretion and a-granule secretion in rat platelets. The activation characteristics were comprehensively confirmed using transmission electron microscopy. Apyrase and antagonists of the platelet adenosine diphosphate (ADP) receptors, P2Y1 and P2Y12, completely inhibited Tg-induced platelet aggregation, which was not sensitive to indomethacin or SQ29548 inhibition. Furthermore, ADP receptor antagonists inhibited Tg-induced a-granule secretion, and blockade of the P2Y1 receptor prevented Tg-induced platelet shape changes. Tg-induced dense granule secretion was not affected by ADP receptor antagonists or various various pharmacological inhibitors of the intracellular effectors involved in dense granule secretion signaling pathways. CONCLUSION: We identified that Tg directly induces platelet activation and demonstrated that Tg-induced platelet activation depends on dense granule secretion of ADP, which in turn activates the P2Y1 and P2Y12 receptor signaling pathways.

A sensitive indirect competitive enzyme-linked immunosorbent assay for the detection of sarsasapogenin in rat plasma.

AIM: To generate a polyclonal antibody against sarsasapogenin and to develop an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) method for the pharmacokinetic study of Sarsasapogenin in rats. METHODS: The antigen of sarsasapogenin was produced using an active ester method and subsequently used for raising polyclonal antibodies in rabbits. The specificity and sensitivity of the antibody were measured by IC-ELISA. Using the ELISA method, sarsasapogenin levels were measured in the serum of rats after an oral dose of 100 mg/kg. RESULTS: Polyclonal antibodies raised against sarsasapogenin-bovine serum albumin were generated and showed a high reactivity to sarsasapogenin. The antibodies exhibited minor cross-reactivity to ruscogenin (23%), diosgenin (22%), 25 (R, S) ruscogenin l-O-[beta-D-glucopyranosyl (1-->2)][beta-D-xylopyranosyl (1-->3)]-beta-D-fucopyranoside (26%) and no cross-reactivity to diammonium glycyrrhizinate and notoginseng R1. The detection range of sarsasapogenin by this ELISA method was approximately 2.4-760 ng/mL. The recovery rates of 10 ng/mL, 100 ng/mL, and 500 ng/mL were in the range of 91.0%-96.2% for intra-assay and 89.0%-92.0% for inter-assay. The coefficients of variation (CV%) for intra- and inter-assays at the three different sarsasapogenin levels were 3.1%-8.3% (n=6) and 6.0%-14.1% (n=6), respectively. CONCLUSION: The IC-ELISA method is a sensitive test for the determination of sarsasapogenin concentration in rat plasma and for pharmacokinetic (PK) studies.

Quantitative determination of ophiopogonin d by liquid chromatography/electrospray ionization mass spectrometry and its pharmacokinetics in rat.

A sensitive and rapid liquid chromatography-mass spectrometric method for the determination of ophiopogonin D in rat plasma was developed and validated. Chromatographic separation was performed on a C (18) column using a step gradient program with the mobile phase of 0.5 mmol/L ammonium chloride solution and acetonitrile. Ophiopogonin D was quantified using an electrospray negative ionization mass spectrometry in the selected ion monitoring (SIM) mode using digoxin as an internal standard. Good linearity was obtained in the concentration range of 2.5 - 480.0 ng/mL ( R2 = 0.9984). The lower limit of quantification (LLOQ) and lower limit of detection (LLOD) were 2.5 ng/mL and 1.0 ng/mL, respectively. Both the intra- and inter-day precision was less than 8.9 % and the accuracy was within 97.5 - 107.3 %. The pharmacokinetic study of ophiopogonin D in rats was then defined using the method after intravenous dosing (77.0 microg/kg). The plasma concentration-time profile for ophiopogonin D was best fitted to an open two-compartment model with a clearance of 0.024 +/- 0.010 L/min/kg and a terminal half life of 17.29 +/- 1.70 min. A comparison of the pharmacokinetics of ophiopogonin D as a pure compound and as a component of 'SHENMAI' injection revealed a significantly smaller clearance of ophiopogonin D (0.007 +/- 0.002 L/min/kg) for the latter formulation, consistent with an inhibition by one or more other components in the formulation.

Simultaneous determination of ginsenoside Rg1, Re, Rd, Rb1 and ophiopogonin D in rat plasma by liquid chromatography/electrospray ionization mass spectrometric method and its application to pharmacokinetic study of 'SHENMAI' injection.

A sensitive and rapid liquid chromatography-mass spectrometric method for the simultaneous determination of ginsenoside Rg1, Re, Rd, Rb1 and ophiopogonin D in rat plasma was developed and validated. Chromatographic separation was performed on a C18 column using a step gradient program with the mobile phase of 0.5mmol/L ammonium chloride solution and acetonitrile. The analytes and I.S. were detected using an electrospray negative ionization mass spectrometry in the selected ion monitoring (SIM) mode. The method was linear over the investigated concentration range with a good correlation coefficient higher than 0.997. The lower limits of detection (LLOD) of these analytes were all lower than 2.0ng/mL. The intra- and inter-day precisions were all no more than 7.5% and accuracies were within the range of 97.5-107.0%. The validated method was successfully applied to investigate the pharmacokinetics of ginsenoside Rg1, Re, Rd, Rb1 and ophiopogonin D in rat after intravenous administration of 'SHENMAI' injection.