Investigation of preclinical pharmacokinetics of N-demethylsinomenine, a potential novel analgesic candidate, using an UPLC-MS/MS quantification method

Abstract

N- Demethylsinomenine (NDSM), the in vivo demethylated metabolite of sinomenine, has exhibited antinociceptive efficacy against various pain models and may become a novel drug candidate for pain management. However, no reported analytical method for quantification of N- Demethylsinomenine in a biological matrix is currently available, and the pharmacokinetic properties of N- Demethylsinomenine are unknown. In the present study, an ultra-high performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) method for quantification of N- Demethylsinomenine in rat plasma was developed and utilized to examine the preclinical pharmacokinetic profiles of N- Demethylsinomenine. The liquid-liquid extraction using ethyl acetate as the extractant was selected to treat rat plasma samples. The mixture of 25% aqueous phase (0.35% acetic acid-10 mM ammonium acetate buffer) and 75% organic phase (acetonitrile) was chosen as the mobile phases flowing on a ZORBAX C18 column to perform the chromatographic separation. After a 6-min rapid elution, NDSM and its internal standard (IS), metronidazole, were separated successfully. The ion pairs of 316/239 and 172/128 were captured for detecting N- Demethylsinomenine and IS, respectively, using multiple reaction monitoring (MRM) under a positive electrospray ionization (ESI) mode in this mass spectrometry analysis. The standard curve met linear requirements within the concentration range from 3 to 1000 ng/mL, and the lower limit of quantification (LLOQ) was 3 ng/mL. The method was evaluated regarding precision, accuracy, recovery, matrix effect, and stability, and all the results met the criteria presented in the guidelines for validation of biological analysis method. Then the pharmacokinetic profiles of N- Demethylsinomenine in rat plasma were characterized using this validated UPLC-MS/MS method. N- Demethylsinomenine exhibited the feature of linear pharmacokinetics after intravenous (i.v.) or intragastric (i.g.) administration in rats. After i. v. bolus at three dosage levels (0.5, 1, and 2 mg/kg), N- Demethylsinomenine showed the profiles of rapid elimination with mean half-life (T_1/2Z) of 1.55-1.73 h, and extensive tissue distribution with volume of distribution (V_Z) of 5.62-8.07 L/kg. After i. g. administration at three dosage levels (10, 20, and 40 mg/kg), N- Demethylsinomenine showed the consistent peak time (T_max) of 3 h and the mean absolute bioavailability of N- Demethylsinomenine was 30.46%. These pharmacokinetics findings will aid in future drug development decisions of N- Demethylsinomenine as a potential candidate for pain analgesia.

Keywords: N-demethylsinomenine; UPLC-MS/MS; bioavailability; pharmacokinetics; rats.

FIGURE 1

The chemical structure and full scan product ion mass spectra of N-demethylsinomenine (NDSM) (A) and the internal standard (IS) metronidazole (B) with monitoring at m/z 316→239 for NDSM and m/z 172→128 for IS.

FIGURE 2

Comparison of the effect of different sample preparation approaches on matrix effect and recovery for NDSM. (A), protein precipitation with acetonitrile showed high recovery rate (87.65% ± 19.10%), but matrix effect (4.68% ± 0.14%) did not meet the requirements; (B), salting-out assisted liquid-liquid extraction with ammonium acetate showed poor recovery rate (58.75% ± 5.30%) and matrix effect (26.45% ± 3.07%); (C), liquid-liquid extraction with ultimate concentration of 0.083M NaOH and ethyl acetate met the requirements for recovery rate (78.98% ± 2.74%) and matrix effect (93.43% ± 6.32%); (D), liquid-liquid extraction with ultimate concentration of 0.154M NaOH and ethyl acetate exhibited high recovery rate (88.32% ± 5.42%), but the matrix effect (66.56% ± 1.88%) was not as good as method (C); (E), solid-phase extraction (SPE) of plasma sample anticoagulated by heparin exhibited low recovery rate (19.84% ± 1.65%); (F), SPE of plasma sample anticoagulated by EDTA showed higher recovery rate (51.40% ± 14.80%) than method (E) but still did not meet the standard.

FIGURE 3

Chromatograms of NDSM (MRM 316/239) and IS (MRM 172/128) in rat plasma samples. (A) blank plasma sample; (B) blank plasma spiked with NDSM at the LLOQ level and IS; (C) plasma sample at 24 h after oral administration of NDSM (20 mg/kg). No interference peak in blank plasma samples was observed at the retention time of NDSM (4.5 min) and IS (4.8 min).

FIGURE 4

Mean plasma concentration profiles of NDSM after oral administration of 10 mg/kg (i.g. L), 20 mg/kg (i.g. M), and 40 mg/kg (i.g. H) and intravenous administration of 0.5 mg/kg (i.v. L), 1 mg/kg (i.v. M), and 2 mg/kg (i.v. H) (mean ± SD, n = 6). More details can be obtained in Table 4.

FIGURE 5

The relationship between half-life (T_1/2z) and dose of NDSM after single intravenous (A) and oral administration (B), and the relationship between the area under the plasma concentration-time curve (AUC) and dose of NDSM after single intravenous (C) and oral administration (D) (mean ± SD, n = 6). The word “ns” indicated no differences for the half-life among the three dose levels (p > 0.05). The regression equation of AUC from zero to infinite (AUC_0-∞) versus dose was Y = 451.8X - 80.54 (r ² = 0.9768, p = 0.0973) for intravenous administration, and Y = 157.6X - 820.6 (r ² = 0.9768, p = 0.0693) for oral administration. These results indicated that the half-life of NDSM was independent of the dosage and did not extend with the increase of the dosage, while AUC_0-∞ increased in proportion to the dose.