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.

Hepatic Uptake Mechanism of Ophiopogonin D Mediated by Organic Anion Transporting Polypeptides.

BACKGROUND AND OBJECTIVES: Ophiopogonin D (OPD) is one of the main active ingredients of SMI (Shenmai injection) which is widely used in clinical practice in China. Our previous study indicated  that OPD might be transported from blood into liver mediated by organic anion transporting polypeptides (OATPs/oatps). This study aims to explore the hepatic uptake mechanism of OPD in rat and human. METHODS: Rosuvastatin (a competitive inhibitor of oatp1b2, oatp1a1, and oatp1a4), glycyrrhizic acid (a specific inhibitor of oatp1b2), digoxin (a specific inhibitor of oatp1a4), bromosulfophthalein (BSP), and ibuprofen (a specific inhibitor of oatp1a1) were used to study the uptake of OPD in rat hepatocytes. Furthermore, the uptake of OPD in human OATP1B1*1a-HEK293T cells was also investigated, and rosuvastatin, BSP, rifampin, and glycyrrhizic acid were all used as the competitive inhibitor of OATP1B1. RESULTS: OPD can be taken in rat primary hepatocytes with K m (Michaelis Menten constant) of 8.10 µM and V max (maximum velocity) of 54.39 nmol/min/mg protein. The uptake of OPD in rat hepatocytes was inhibited significantly by rosuvastatin and glycyrrhizic acid. However, digoxin, BSP, and ibuprofen had no effect on the uptake of OPD in rat hepatocytes. OPD can also be transported by OATP1B1*1a-HEK293T cells with K m of 5.50 µΜ and V max of 29.07 nmol/min/mg protein. Compared with rosuvastatin, OPD has a higher affinity with OATP1B1 and can be transported faster in unit time. Rosuvastatin, BSP, rifampin, and glycyrrhizic acid all exhibited a certain extent inhibitory effect on the transport of OPD in OATP1B1*1a-HEK293T cells. CONCLUSIONS: Overall, this study indicates OATP1B1 in human and oatp1b2 in rats might participate in the hepatic uptake of OPD.

Quantitative determination of gracillin by HPLC-MS/MS after oral administration and its application to a pharmacokinetic study.

A sensitive and credible high performance liquid chromatography hyphenated to mass spectrometry (HPLC-MS/MS) was established to quantify the concentration of gracillin in rat plasma. The plasma samples were subjected to a direct protein precipitation process with acetonitrile as a precipitant in a single-step. Ginsenoside Rb1 was selected as an internal standard (IS). The chromatographic separation of analyte and IS were carried out on an Inersil ODS-3 C18 column (250×4.6mm, 5µm) with a binary solvent system containing acetonitrile and 0.1% formic acid in water at a flow rate of 1mLmin(-1) under a gradient elution mode. Mass spectrometric detection was performed on a triple quadrupole tandem mass spectrometer by the multiple reaction monitoring (MRM) mode to examine the precursor-to-daughter ion transitions of 1110.3→948.2 for IS and 886.1→739.9 for gracillin, respectively, in a positive electrospray ionization mode. The calibration curve showed a promising linearity over a concentration range of 0.065-800ngmL(-1) with a better regression coefficient of r(2)=0.9960. The intra- and inter-day precisions (as relative standard deviation) of the assay at three quality control levels were all less than 3.48%, while the intra- and inter-day accuracies (as relative error) ranged from -8.43% to 9.74%, whose data were within the acceptable limits. The mean extraction recoveries of analyte from rat plasma were all more than 74.11%, and no notable matrix effect was observed. Stability experiments revealed that gracillin remained stable throughout the analytical procedure under various stored conditions. The above validated method was successfully used to investigate the pharmacokinetic behaviors of gracillin orally administrated to rats at three proportion doses. The pharmacokinetic analysis would pave the way for understanding the pharmacological actions and provide a meaningful foundation for further development and application in preclinical and clinical use of gracillin in the near future.

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.

Liquid chromatography tandem mass spectrometry in study of the pharmacokinetics of six steroidal saponins in rats.

A rapid, simple and sensitive high-performance liquid chromatography tandem mass spectrometry method was developed and validated for simultaneous determination of six main steroidal saponins in Paris polyphylla in rat plasma. Ginsenoside Rg3 was selected as the internal standard (IS). Plasma samples were pretreated with protein precipitation, and the separation was achieved on a reverse phase Agilent poroshell120 EC-C18 column using a gradient mobile phase system of acetonitrile-water containing 0.1% formic acid. The triple quadruple mass spectrometer was set in negative electrospray ionization mode and multiple reaction monitoring (MRM) was used for six steroidal saponins quantification. The precursors to produce ion transitions monitored for polyphyllin I, polyphyllin II, polyphyllin VI, polyphyllin VII, dioscin, gracillin and IS were m/z 899.5>853.4, 1059.5>1013.5, 783.4>737.4, 1075.5>1029.5, 913.5>867.4, 929.5>883.4 and 819.5>783.4, respectively. The intra- and inter-day precisions (RSD%) were less than 13% and the average extraction recoveries ranged from 85% to 97.0% for each analyte. Six steroidal saponins were proved to be stable during sample storage, preparation and analytical procedures. The established method was employed for simultaneous quantification and successfully used for the first time for the pharmacokinetics evaluation of the six main compounds after intragastric administration of P. polyphylla extract in Sprague-Dawley rats.

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).

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.

Determination of ruscogenin in crude Chinese medicines and biological samples by immunoassay.

Ruscogenin is a major bioactive steroidal aglycone found in the Chinese medicine, Ophiopogon japonicus. We have developed a quantitative determination of ruscogenin with an indirect enzyme-linked immunosorbent (ELISA) assay using polyclonal antibodies against ruscogenin conjugated with bovine serum albumin. This assay was highly sensitive, and it had considerably less cross-reactivity to diosgenin and sarsasapogenin, but high cross-reactivity to ruscogenin glycosides. The assay was successfully used for the measurement of ruscogenin concentrations in crude Chinese medicines and in biological samples from a pharmacokinetics study of ruscogenin.

Ovine metabolism of lithogenic sapogenins. Synthesis of [2,2,4,4-(2)h(4)]sarsasapogenone, [2,2,4,4-(2)h(4)]sarsasapogenin, and [2,2,4,4-(2)h(4)]episarsasapogenin and evaluation of deuterium retention in a sheep-dosing trial.

The suitability of [2,2,4,4-(2)H(4)]sarsasapogenone (1b), [2,2,4,4-(2)H(4)]sarsasapogenin (2b), and [2,2,4,4-(2)H(4)]episarsasapogenin (3b) as isotopically labeled dosing substrates to determine the levels of free and conjugated sapogenins present in feces from sheep grazing saponin-containing plants implicated in the development of ovine heptagenous photosentization diseases was investigated. A 1:4 mixture of [2,2,4,4-(2)H(4)]sarsasapogenin (2b) and [2,2,4,4-(2)H(4)]episarsasapogenin (3b), obtained by reduction of [2,2,4,4-(2)H(4)]sarsasapogenone (1b), was found to retain 94% of incorporated deuterium, when dosed to one sheep. The recovery of the dosed mixture of genins 2b and 3b was calculated to be 85%. Considerable loss of deuterium and a lower recovery of genin material were observed when [2,2,4,4-(2)H(4)]sarsasapogenone (1b) was dosed.

Ovine metabolism of saponins: evaluation of a method for estimating the ovine uptake of steroidal saponins from Narthecium ossifragum.

A sheep was dosed three times per day over six consecutive days with 70 g Narthecium ossifragum, and once on the seventh day with 70 g N. ossifragum. Additionally, it was dosed once on days 1-7 with 20 mg of [20,23,23-2H3]sarsasapogenin. After 7 days, the sheep was killed and GC-MS analysis of the free and conjugated sapogenin content in bile, urine, rumen, duodenum, jejunum, colon and rectum samples collected from the sheep, faecal samples collected on days 4-7, and dosed plant material was performed. The N. ossifragum contained mainly sarsasapogenin and smilagenin. Only neglible levels of deuterium-labelled sarsasapogenins were detected in the samples from the animal. Ingested saponins were quickly hydrolysed in the rumen to free sapogenins and, in part, epimerized at C-3 to afford episapogenins. The absorption of free sapogenins appeared to occur in the jejunum. The concentration of sapogenins in faeces reached a plateau 108 h after dosing started.

Plant glycosides in a liposomal drug-delivery system.

Plant glycosides were incorporated into the liposomal surface to study their sugar-specific uptake by various tissues. Two steroid glycosides, namely floribundasaponin D, with rhamnose as terminal sugar, and gracillin, with glucose and rhamnose as end sugars, were selected for the purpose. 125I-human IgG encapsulated liposomes composed of egg lecithin (phosphatidylcholine), cholesterol, dicetyl phosphate (optional) and either floribundasaponin D or gracillin, when injected into the tail vein of rat, showed significantly higher uptake in the rat liver than in appropriate controls. Whereas the uptake of floribundasaponin D liposomes was observed to be non-specific, the increased uptake of the gracillin liposomes, as judged from the inhibition studies with asppropriate sugars, was specific for glucose, although the receptor was unable to distinguish between the alpha and beta anomers ('anomerically blind'). The liver-perfusion studies showed that the uptake of gracillin liposomes was mostly by non-parenchymal cells.