Stability study of Prulifloxacin and Ulifloxacin in human plasma by HPLC–DAD

Figures & data

Figure 1. Prulifloxacin, Ulifloxacin and Danofloxacin (IS) chemical structures.

Figure 2. Separation of standards solution mixture containing Prulifloxacin, Ulifloxacin and Danofloxacin (IS) (plasma sample, 20 μg/mL concentration level, 20 μL injected).

Table 1. Chromatographic parameters.

Analyte	Retention time (tR, min)*	Retention factor (k)	Selectivity (α)†	Relative retention (trR)†
Prulifloxacin	4.72 ± 0.14	1.25	0.61	0.74
Danofloxacin (IS)	6.42 ± 0.20	2.06	1.00	1.00
Ulifloxacin	11.1 ± 0.53	4.29	2.08	1.73

*Reported values are expressed as mean ± standard deviations. Chromatographic parameters were based on void retention time corresponding to 2.10 min.

†Selectivity factors and relative retention were calculated against Danofloxacin (IS); n = 180.

Table 2. Within-assay and between-assay precision (RSD%), trueness (bias%) of the analytical method obtained from the analysis of QC samples.

	Prulifloxacin		Ulifloxacin
	Within	Between	Within	Between
Theoretical*	1.5
Mean back-calculated*	1.42	1.60	1.48	1.61
RSD%	13.02	0.59	7.82	8.30
Bias%	−4.94	9.03	−1.51	7.08
Theoretical*	5
Mean back-calculated*	4.54	4.84	4.56	4.87
RSD%	0.34	5.65	6.37	7.65
Bias%	−11.45	−3.20	−8.81	−2.65
Theoretical*	15
Mean back-calculated*	14.37	15.49	14.50	15.64
RSD%	11.34	1.60	5.58	2.93
Bias%	−5.96	4.67	−3.34	4.28

Data are expressed as the mean values of six experiments.

*Concentration expressed as μg/mL unit.

Figure 3. Chromatograms obtained for Prulifloxacin and Ulifloxacin analyses: (a) human plasma used for the preparation of plasma calibration standards and QC samples, (b) human plasma sample fortified only with Danofloxacin (IS, at 20 μg/mL) and (c) a 25 μg/mL Prulifloxacin and Ulifloxacin plasma calibration sample with Danofloxacin (IS, at 20 μg/mL).

Table 3. Recovery values of the analytical method obtained from the analysis of QC samples.

	Prulifloxacin	Ulifloxacin
QC low concentration level	1.5
Recovery	93.4	99.0
Standard deviation	6.62	5.44
RSD%	7.07	5.4
QC medium concentration level	5
Recovery	90.1	104
Standard deviation	9.20	4.82
RSD%	10.2	4.63
QC high concentration level	15
Recovery	93.0	107
Standard deviation	6.30	1.50
RSD%	6.77	1.40

Data are expressed as the mean values of six experiments.

Figure 4. Stability curves at three concentration levels and three different storage temperatures for Prulifloxacin.

Table 4. Stability data at three concentration levels and three different storage temperatures for Prulifloxacin. Ulifloxacin was followed as principal metabolite and its concentration levels were reported.

		Temperature  = 25 °C ( ± 1 °C)		Temperature  = 4 °C ( ± 1 °C)		Temperature  = −20 °C ( ± 1 °C)
		Prulifloxacin	Ulifloxacin	Prulifloxacin	Ulifloxacin	Prulifloxacin	Ulifloxacin
QC low concentration level (1.5 μg/mL)	T 0	1.48 ± 0.19	ND	1.48 ± 0.19	ND	1.48 ± 0.19	ND
	T 15	1.32 ± 0.17	ND	1.39 ± 0.17	ND	1.10 ± 0.14	ND
	T 30	1.09 ± 0.14	ND	1.53 ± 0.19	ND	1.33 ± 0.17	ND
	T 60	1.10 ± 0.14	ND	1.25 ± 0.16	ND	1.37 ± 0.17	ND
	T 120	1.13 ± 0.14	ND	1.23 ± 0.15	ND	1.10 ± 0.14	ND
	T 240	1.16 ± 0.14	ND	1.29 ± 0.16	ND	1.36 ± 0.17	ND
	T 360	1.13 ± 0.14	ND	1.22 ± 0.15	ND	1.19 ± 0.15	ND
	QC medium concentration level (5 μg/mL)	T 0	5.23 ± 0.65	ND	5.23 ± 0.65	ND	5.23 ± 0.65	ND
T 15		5.14 ± 0.64	ND	4.50 ± 0.56	ND	4.49 ± 0.56	ND
T 30		4.20 ± 0.52	1.01 ± 0.13	2.57 ± 0.32	2.13 ± 0.27	2.96 ± 0.37	1.88 ± 0.24
T 60		3.24 ± 0.40	1.53 ± 0.19	2.44 ± 0.30	2.34 ± 0.29	2.64 ± 0.33	1.99 ± 0.25
T 120		3.15 ± 0.39	1.08 ± 0.14	2.57 ± 0.32	2.13 ± 0.27	2.60 ± 0.32	2.08 ± 0.26
T 240		2.61 ± 0.33	1.53 ± 0.19	2.29 ± 0.29	1.93 ± 0.24	2.46 ± 0.31	2.16 ± 0.27
T 360		2.52 ± 0.31	1.52 ± 0.19	2.17 ± 0.27	1.88 ± 0.23	2.03 ± 0.25	2.27 ± 0.28
QC high concentration level (15 μg/mL)		T 0	14.90 ± 1.86	ND	14.90 ± 1.86	ND	14.90 ± 1.86	ND
	T 15	12.04 ± 1.50	2.73 ± 0.34	12.56 ± 1.57	2.84 ± 0.35	11.18 ± 1.40	4.43 ± 0.55
	T 30	11.66 ± 1.46	2.85 ± 0.36	12.28 ± 1.53	2.85 ± 0.36	11.10 ± 1.39	3.06 ± 0.38
	T 60	11.61 ± 1.45	2.83 ± 0.35	12.29 ± 1.54	2.25 ± 0.28	11.01 ± 1.38	3.76 ± 0.47
	T 120	10.97 ± 1.37	3.05 ± 0.38	12.10 ± 1.51	2.18 ± 0.27	10.31 ± 1.29	3.72 ± 0.47
	T 240	11.54 ± 1.44	3.08 ± 0.39	12.19 ± 1.52	1.96 ± 0.25	9.79 ± 1.22	4.72 ± 0.59
	T 360	10.75 ± 1.34	3.21 ± 0.40	12.03 ± 1.50	2.34 ± 0.29	8.46 ± 1.06	5.95 ± 0.74

Data are expressed as the mean back-calculated values ± SEM (standard error of the mean) of three independently experiments. ND = not detected.