ABSTRACT
Objective To use high-performance liquid chromatography-time of flight mass spectrometry (HPLC-TOF/ MS) for examining the steroidal saponins in rat urine after oral administration of Paris polyphylla extract, so as to lay a foundation for studying the metabolism of steroidal saponins in vivo. Methods SD rats were administered with an oral dose of 1 6 g Paris polyphylla extracts/kg body weight. The urine samples were collected 24 h after administration by oral gavage. The sample analysis was carried out on a reverse phase MG-C18 column (3. 0 mm× 100 3. 0μm) using a gradient mobile phase system of acetonitrile-water containing 0. 1% formic acid. TOF/MS was applied for qualitative analysis under positive and negative ion modes. The steroidal saponins in rat urine were identified by using the accurate molecular weight obtained by TOF/ MS and formula database. Results A total of 20 steroidal saponins were identified in the rat urine, and two pairs of isomers were deduced through their fragment ions and standards: polyphyllin VI and pennogenin-3-0-α-L-rhamnopyranosyl(l →4)-α-L-rhamnopyranosyl (1 → 3)-[-α--rhamnopyranosyl (1 → 2)]-β-D-glucopyranoside; and gracillin and pennogenin-3-O-α-L-rhamnopyranosyKl → 4)-α-L-rhamnopyranosyl (1 → 4)-β-D-glucopyranoside. Conclusion The established analysis method is accurate, reliable for identifying steroidal saponins in vivo, which paves a way for further pharmacokinetics and pharmacodynamics study of Paris polyphylla.
ABSTRACT
Objective To introduce an accelerated solvent extraction (ASE) -HPLC-TOF/MS method for simultaneous determination of Polyphyllin I, Polyphyllin II, Polyphyllin VI, Polyphyllin VII, Dioscin and Gracillin in Paris Polyphylla. Methods The extraction was performed by ASE under the following conditions: extracting solvent 70% ethanol; extracting temperature 120°C; pressure 1. 17 MPa (1 700 psi); and static time 6 min. The separation was carried out on a MGC18 column (3.0 mm× 100 mm, 3. 0 μm). Elution was done with a linear gradient mobile phase system consisting of 0. 1% formic acid (V/V) water and acetonitrile. The flow rate was set at 0. 8 ml/min, the sample injection volume was 3 μl, the column temperature was kept constant at 25°C, nebulizer gas pressure was 275. 8 kPa (40 psi), drying gas flow rate was 10 L/min, and gas temperature was 350°C. Results The calibration curves for Polyphyllin I, Polyphyllin II, Polyphyllin VI, Polyphyllin VII, Dioscin and Gracillin were linear within the range of 0. 500 0-50. 00 μg/ml (r = 0. 999 8), 0. 422 3-42. 23 μg/ml (r = 0. 999 7), 0. 612 0-61. 20 μg/ml (r = 0. 999 9), 0. 714 0-71. 40 μg/ml (r = 0. 999 5), 0. 448 0-44. 80 μg/ml (r = 0. 999 9) and 0. 436 0-43.60 μg/ml (r = 0. 9997), respectively; and the average recoveries (n=6) were 98. 9%, 98. 0%, 102. 5%, 101. 9%, 103. 1% and 97. 9%, respectively. Conclusion The introduced method is solvent-saving and time-saving; it can also be used for high-throughput analysis and determination of steroidal saponins in Paris Polyphylla.