Quantification of DOX bioavailability in biological samples of mice by sensitive and precise HPLC assay

Figures & data

Figure 1. HPLC chromatograms of DOX in different samples. (A) DOX solution; (B) DOX in plasma sample; (C) DOX in heart sample; (D) DOX in liver sample; (E) DOX in spleen sample; (F) DOX in lung sample; (G) DOX in kidney sample; (H) DOX in tumor sample.

Table 1. Calibration curve of DOX in plasma and different tissues.

Sample	Linear range (µg/mL)	Equation	R
Plasma	3.03–40	A = 3297.2C + 3097.6	0.9977
Heart	2.22–30	A = 3472.4C + 2222.7	0.9990
Liver	2.22–30	A = 307.81C − 472.1	0.9976
Spleen	2.22–30	A = 352.03C − 590.95	0.9984
Lung	2.22–30	A = 125.95C + 326.95	0.9984
Kidney	2.22–30	A = 306.66C − 460.02	0.9986
Tumor	2.22–30	A = 3140.9C + 1141.6	0.9997

A is the peak areas of DOX, C is the concentrations of DOX.

Table 2. Accuracy and precision of the method for determination of DOX contents.

		Intra-day (n = 5)		Inter-day (n = 5)
Sample	C (µg/mL)	Accuracy (mean ± SD)	Precision (RSD %)	Accuracy (mean ± SD)	Precision (RSD %)
Plasma	3.03	2.88 ± 0.01	0.35	2.88 ± 0.01	0.35
	18.18	19.08 ± 0.07	0.37	19.07 ± 0.07	0.37
	36.36	35.44 ± 0.06	0.17	35.48 ± 0.07	0.20
Heart	2.22	1.88 ± 0.04	2.13	2.23 ± 0.03	1.35
	13.33	13.65 ± 0.23	1.69	13.66 ± 0.20	1.46
	26.67	26.44 ± 0.16	0.61	26.66 ± 0.21	0.79
Liver	2.22	2.18 ± 0.11	5.05	2.23 ± 0.16	7.17
	13.33	13.11 ± 0.16	1.22	13.14 ± 0.06	0.46
	26.67	26.9 ± 0.16	0.60	26.9 ± 0.23	0.86
Spleen	2.22	1.97 ± 0.08	4.06	2 ± 0.07	3.50
	13.33	12.84 ± 0.08	0.62	12.80 ± 0.06	0.47
	26.67	26.25 ± 0.07	0.27	26.35 ± 0.09	0.34
Lung	2.22	2.61 ± 0.06	2.30	2.58 ± 0.06	2.33
	13.33	12.77 ± 0.03	0.23	12.78 ± 0.04	0.31
	26.67	26.46 ± 0.32	1.21	26.51 ± 0.22	0.83
Kidney	2.22	1.89 ± 0.13	6.88	2.01 ± 0.03	1.50
	13.33	12.8 ± 0.02	0.16	13.33 ± 0.05	0.38
	26.67	27 ± 0.32	1.19	26.68 ± 0.32	1.20
Tumor	2.22	1.96 ± 0.03	1.53	1.89 ± 0.04	2.15
	13.33	12.86 ± 0.16	1.24	12.87 ± 0.23	1.79
	26.67	26.43 ± 0.17	0.64	26.35 ± 0.21	0.80

Table 3. Recovery of the method for determination of DOX contents.

Sample	Added (µg/mL)	Recovery (%)	Mean ± SD	RSD (%)
Plasma	3.03	95.71	99.15 ± 3.26	3.29
	18.18	102.20
	36.36	99.55
Heart	2.22	98.65			99.43 ± 0.75	0.75
	13.33	100.15
	26.67	99.48
Liver	2.22	97.30	99.01 ± 1.67	1.69
	13.33	99.10
	26.67	100.64
Spleen	2.22	93.24			96.42 ± 2.87	2.98
	13.33	97.22
	26.67	98.80
Lung	2.22	104.50	99.85 ± 4.32	4.33
	13.33	95.95
	26.67	99.10
Kidney	2.22	99.55			99.31 ± 2.21	2.23
	13.33	96.99
	26.67	101.39
Tumor	2.22	98.32	97.81 ± 2.17	2.22
	13.33	95.43
	26.67	99.67

Figure 2. Mean DOX concentration–time curves in plasma after intravenous administration of free DOX, DOX/P(HB-HO) NPs and DOX/FA-PEG-P(HB-HO) NPs to tumor-bearing mice (n = 3) ((A) 0–48 h; (B) 0–12 h).

Figure 3. Biodistribution of free DOX, DOX/P(HB-HO) NPs and DOX/FA-PEG-P(HB-HO) NPs in blood, heart, liver, spleen, lung, kidney, and tumor at different times ((A) 4 h, (B) 24 h, (C) 48 h) after intravenous administration to tumor-bearing mice (n = 3). *p < 0.05 compared with free DOX; **p < 0.01 compared with free DOX.

Table 4. Main pharmacokinetic parameters of DOX formulations after i.v. administration.

	free DOX	DOX/P(HB-HO) NPs	DOX/FA-PEG-P (HB-HO) NPs
t1/2/h	7.23 ± 1.22	19.26 ± 4.68*	25.08 ± 2.97**
Cmax/µg mL^−1	20.12 ± 4.29	20.66 ± 5.46	22.78 ± 5.49
Tmax/h	0.25 ± 0.03	1.00 ± 0.01**	1.00 ± 0.08**
AUC0–t/µg·mL^−1·h	64.62 ± 15.98	99.25 ± 19.66	132.36 ± 25.38*
MRT/h	5.61 ± 1.23	15.99 ± 4.63*	25.14 ± 7.04**

t_1/2, terminal half-life; C_max, maximum concentration; T_max, time of maximum concentration; AUC_0–t, area under plasma concentration versus time curve (0–t); MRT, mean retention time.

*p < 0.05 compared with free DOX; **p < 0.01 compared with free DOX.