HPLC method with fluorescence detection for the determination of ligustilide in rat plasma and its pharmacokinetics

Figures & data

Figure 1. Typical chromatograms of LIG from rat plasma: (A) blank plasma sample; (B) blank plasma sample spiked with LIG; (C) plasma sample obtained at 1 h from rat after an i.v. administration of LIG (12.5 mg/kg); (D) standard solution of LIG spiked with IS. Peak 1, podophyllotoxin (internal standard); Peak 2, LIG.

Table 1. Precision and accuracy data for LIG in rat plasma (n = 5).

	Accuracy (%)		Precision (%)
Nominal concentration (ng/mL)	Within-batch	Between-batch	Within-batch	Between-batch
4.88	88.93 ± 0.21	90.37 ± 0.25	4.83	5.72
39	96.21 ± 1.19	96.13 ± 1.82	3.17	4.86
156	98.76 ± 4.96	98.67 ± 3.65	3.22	2.37
1250	99.35 ± 33.41	99.52 ± 27.12	2.69	2.18
5000	98.33 ± 198.13	97.95 ± 223.80	4.03	4.57

Table 2. Extraction recoveries of LIG from rat plasma (n = 5).

Nominal concentration (ng/mL)	Recovery (%) Mean ± SD	RSD (%)
4.88	89.07 ± 0.10	2.37
39	96.13 ± 0.81	2.13
156	97.97 ± 4.71	3.08
1250	99.71 ± 44.12	3.54
5000	99.50 ± 183.09	3.68

Table 3. Summary of stability studies of LIG in rat plasma under various storage conditions (n = 5).

Storage conditions	Nominal concentration (ng/mL)	Mean measured concentration (ng/mL)	Mean recoveries (%)	RSD (%)
Non-processed samples stored room temperature for 6 h	4.88	4.43	91.37 ± 0.12	2.61
	39.00	37.53	96.24 ± 0.72	1.92
	156.00	155.55	99.71 ± 3.20	2.06
	1250.00	1245.38	99.63 ± 27.15	2.18
	5000.00	4957.00	99.14 ± 145.74	2.94
80 °C for 4 weeks (Three freeze/thaw cycles)	4.88	4.37	90.15 ± 0.17	3.83
	39.00	37.53	96.23 ± 1.14	3.05
	156.00	153.61	98.47 ± 4.76	3.10
	1250.00	1240.13	99.21 ± 49.98	4.03
	5000.00	4946.00	98.92 ± 155.80	3.15
Processed samples stored on the rack of autosampler for 24 h (25 °C)	4.88	4.40	90.68 ± 0.11	2.51
	39.00	37.63	96.48 ± 0.93	2.46
	156.00	153.88	98.64 ± 4.20	2.73
	1250.00	1239.88	99.19 ± 27.90	2.25
	5000.00	4942.50	98.85 ± 151.24	3.06
Long-term stability (Non-processed samples stored −80 °C for 30 days)	4.88	4.43	91.36 ± 0.14	3.17
	39.00	38.31	98.22 ± 1.64	4.28
	156.00	152.46	97.73 ± 5.67	3.72
	1250.00	1243.38	99.47 ± 41.53	3.34
	5000.00	4937.50	98.75 ± 194.04	3.93

Figure 2. Plasma concentration--time curves of LIG in rats after oral and intravenous administration (n = 6). (A): intravenous administration of 12.5, 25 and 50 mg/kg, b.w. LIG; (B): oral administration of 12.5, 25 and 50 mg/kg, b.w. LIG.

Figure 3. Relationship between Cmax and AUC vs. dose in rats receiving single 12.5, 25 and 50 mg/kg, b.w. intravenous dose of LIG.

Table 4. Pharmacokinetic parameters of LIG in rats after oral and intravenous administration at three different doses (n = 6, mean ± SD).

		Intravenous doses (mg/kg, mean ± SD)			Oral doses (mg/kg, mean ± SD)
Parameters	Units	12.5	25	50	12.5	25	50
Cmax	μg/L	172.27 ± 7.08	582.84 ± 4.75	1048.72 ± 10.94	123.94 ± 6.17	353.99 ± 2.01	683.33 ± 6.22
Tmax	h	0.08	0.08	0.08	0.25	0.25	0.25
AUC(0∼∞)^a	μg/L*h	235.74 ± 2.85	493.52 ± 12.72	866.35 ± 17.43	176.1 ± 3.58	336.9 ± 2.73	744.58 ± 7.69
AUC(0∼t)	μg/L*h	230.78 ± 2.59	492.52 ± 12.90	836.14 ± 13.51	171.94 ± 8.69	336.17 ± 2.82	738.05 ± 13.67
MRT(0∼∞)	h	4.39 ± 0.73	2.93 ± 0.04	4.18 ± 0.63	4.02 ± 1.18	2.40 ± 0.11	2.39 ± 0.13
Cl	L/h/kg	53.03 ± 0.64	50.69 ± 1.31	57.74 ± 1.16	71.01 ± 1.44	74.21 ± 0.60	67.16 ± 6.92
λn	h^−1	0.16 ± 0.02	0.23 ± 0.01	0.11 ± 0.02	0.12 ± 0.01	0.21 ± 0.01	0.14 ± 0.02
t1/2	H	4.72 ± 1.78	3.02 ± 0.17	6.90 ± 1.49	5.60 ± 0.32	3.33 ± 0.07	5.23 ± 1.12
V	L/kg	360.04 ± 13.18	220.85 ± 17.76	572.62 ± 12.72	576.84 ± 34.02	356.80 ± 9.87	508.02 ± 20.97
F (%)					71.36	68.26	75.44

^aDose-normalized (based on 12.5 mg/kg) values were compared when statistical analysis was performed.