A two-step pH-dependent liquid–liquid extraction combined with HPLC-fluorescence method for the determination of 10-hydroxycamptothecin in mouse liver tissue

Figures & data

Figure 1.  Chemical equilibrium reaction between the lactone (a) and carboxylate (b) forms of CPTs (R=H, CPT; R=OH, HCPT).

Figure 2.  Representative chromatograms of: (A) a blank mouse liver tissue homogenate sample without the first LLE step; (B) a blank mouse liver tissue homogenate sample using the two-step LLE; (C) a blank mouse liver tissue homogenate spiked with HCPT (20 ng/mL) and I.S. (CPT 200 ng/mL); (D) a mouse liver tissue homogenate sample at 4 h after oral administration of HCPT at a dose of 80 mg/kg and spiked with I.S. (CPT 200 ng/mL).

Figure 3.  A representative calibration curve of HCPT in liver tissue homogenate samples.

Table 1.  Accuracy and precision for the analysis of HCPT in mouse liver tissue homogenates.

Nominal, C (ng/mL)	Measured, C (ng/mL)	(RSD %)	Relative error (%)	Accuracy (%)
Inter-day (n = 30)
2.5	2.47	8.14	−0.90	98.8
10	10.22	1.01	2.29	102.2
80	80.14	3.02	0.18	100.18
Intra-day (n = 18)
2.5	2.42	10.02	−3.30	96.8
10	9.87	4.94	−1.30	98.7
80	79.98	4.68	−0.02	99.98

Table 2.  Stability of HCPT in liver tissue homogenate samples (n = 6).

Experimental condition		Nominal, C (ng/mL)	Measured, C (ng/mL) ± SD	Relative error (%)
Liver tissue homogenate QC samples	2 h at RT	2.5	2.56 ± 0.17	2.4
		10	10.01 ± 0.06	0.09
		80	80.13 ± 2.41	0.17
	Autosampler 24 h	2.5	2.63 ± 0.12	5.5
		10	10.51 ± 0.87	5.1
		80	78.34 ± 2.28	−2.08
	30 days storage at −80°C	2.5	2.65 ± 0.19	6.01
		10	9.92 ± 0.03	−0.77
		80	78.54 ± 1.21	−1.82
	3 freeze-thaw cycles	2.5	2.64 ± 0.11	5.92
		10	10.67 ± 0.05	6.79
		80	78.63 ± 2.57	−1.71
Standard solutions	4 h at RT	2.5	2.54 ± 0.11	1.6
		10	10.11 ± 0.23	1.1
		80	80.09 ± 2.17	0.11
	30 days at 4°C	2.5	2.45 ± 0.13	−1.93
		10	10.21 ± 0.05	2.1
		80	79.54 ± 3.38	−0.58

Table 3.  The pharmacokinetic parameters of HCPT after oral administration of 80 mg/kg of HCPT.

Parameters	HCPT
Cmax (ng/mL)	69.40 ± 6.07
Tmax (h)	1.00
T1/2 (h)	4.73 ± 0.73
MRT (h)	6.25 ± 1.17
AUC0–24 (ng·h/mL)	318.60 ± 43.02
AUC0–∞ (ng·h/mL)	331.12 ± 50.53

Figure 4.  Mean concentration-time profiles of HCPT in mouse liver tissue homogenate samples from four subjects each receiving a single dose of 80 mg/kg HCPT.