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

Abstract

A new and specific HPLC–DAD method for the direct determination of Prulifloxacin and its active metabolite, Ulifloxacin, in human plasma has been developed. Plasma samples were analysed after a simple solid phase extraction (SPE) clean-up using a new HILIC stationary phase based high-performance liquid chromatography (HPLC) column and an ammonium acetate buffer (5 mM, pH 5.8)/acetonitrile (both with 1% Et₃N, v/v) mobile phase in isocratic elution mode, with Danofloxacin as the internal standard. Detection was performed using DAD from 200 to 500 nm and quantitative analyses were carried out at 278 nm. The LOQ of the method was 1 μg/mL of the cited analytes and the calibration curve showed a good linearity up to 25 μg/mL. For both analytes the precision (RSD%) and the trueness (bias%) of the method fulfil with International Guidelines. The method was applied for stability studies, at three QC concentration levels, in human plasma samples stored at different temperature of + 25, + 4 and −20 °C in order to evaluate plasma stability profiles.

• Extraction procedure
• HPLC–DAD
• Prulifloxacin
• stability profile
• Ulifloxacin

Introduction

Prulifloxacin, according to IUPAC nomenclature, is a thiazetoquinolone 6-fluoro-1-methyl-7-[4-[(5-methyl-2-oxo-1,3-dioxol-4-yl)methyl]-1-piperazinyl]-4-oxo-1H,4H-[1,3]thiazeto [3,2-a]quinoline-3-carboxylic acid, an anti-bacterial agent pro-drug of the quinolone carboxylic acid Ulifloxacin (Figure 1), characterized by a potent, and broad-spectrum anti-bacterial activity.

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

Prulifloxacin contains a quinolone skeleton with a four-member ring in the 1, 2 positions including a sulphur atom to increase anti-bacterial activity and an oxodioxolenylmethyl group in the 7-piperazine ring to improve oral absorption. This pro-drug was generally well tolerated in clinical trials, with a comparable tolerability profile to Ciprofloxacin and showed remarkable activity at 600 mg once daily for 10 days in patients with acute exacerbation of chronic bronchitis or other complicated lower urinary tract infections1.

In vitro anti-microbial activity studies were performed using Ulifloxacin against a large variety of clinical Gram-negative bacteria including community and nosocomial isolates (Escherichia coli, Klebsiella spp., Proteus, Providencia, Morganella spp., Moraxella catarrhalis and Haemophilus spp.) and Gram-positive bacteria (Staphylococcus aureus, Enterococcus spp. and Italian community isolates of Streptococcus pneumonie) bacteria2^,3.

Prulifloxacin pharmaceutical behaviour (pharmacokinetic and pharmacodynamic) and the use of this agent on daily basis (key factor in successfully infection treatment4) account for considering this pro-drug as an interesting antibacterial option for the treatment of acute exacerbations of chronic bronchitis5. Prulifloxacin also shows an important effect against a worldwide collection of gastroenteritis producing pathogens, including those causing traveller's diarrhoea6^,7.

After oral administration, Prulifloxacin is absorbed in the upper small intestine and then metabolised to Ulifloxacin by esterases, mainly paraoxonase, partly in the intestinal membrane and mostly in the portal blood and in the liver (first pass or pre-systemic metabolism)2^,3^,8.

In previously reported methods, analysis of merely Ulifloxacin was prevalently performed by HPLC with several type of detector, to improve the overall analytical performance, in particular sensibility and selectivity9. Tougou and co-workers8 used ethylchloroformate for the derivatization of Ulifloxacin into ethoxycarbonyl ulifloxacin to give a reasonable retention time on the column. Nakashima and co-workers10 used liquid–liquid extraction for sample preparation, while Carmignani and co-workers11 used multi-step extraction and solid phase extraction followed by liquid–liquid extraction and derivatization. Several papers report high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for the determination of Ulifloxacin in plasma12^,13.

Some published works also report an HPLC method with fluorescence detection (FLD)14. The assay employed solid phase extraction (SPE) involving multi-step purification and nitrogen evaporation and 0.5 mL plasma was needed.

In literature are present works that consider the determination of Prulifloxacin active metabolite by the use of spectroscopic analysis15^,16.

Other papers report the determination of Prulifloxacin active metabolite with different detection methods, such as traditional UV/Vis17 coupled also with a very powerful separation technique18 such as Capillary Zone Electrophoresis19, Fluorescence20^,21 and Chemiluminescence's22^,23.

Following our research, activity focused on drug analyses and characterization24–29, we report in the present study the development of a simple, rapid and sensitive HPLC method suitable for routine analysis of Prulifloxacin and its related active metabolite in human plasma that can be applied to stability testing and pharmacological trials.

Materials and methods

Chemical and reagents

Prulifloxacin and Danofloxacin (>99% purity index) were purchased from Sigma-Aldrich (Milan, Italy), while Ulifloxacin (>98% purity index) purchased from Suzhou Bichal Biological Technology Co. Ltd. (Jiangsu, China).

Acetonitrile (HPLC-grade) was purchased from Carlo Erba Reagents (Milan, Italy). Water is produced by Millipore Milli-Q Plus water treatment system (Millipore Bedford Corp., Bedford, MA). Ammonium acetate (>99% purity index) was from Fluka Chemika-BioChemik (Buchs, Switzerland) and acetic acid (glacial) was from Carlo Erba Reagents (Milan, Italy).

Samples preparation and solid phase extraction

A 450 μL aliquot of human plasma was mixed with a 25 μL aliquot of analytes working solutions and 25 μL aliquot of Danofloxacin (IS, at final concentration of 20 μg/mL) and vortexed for 1 min.

The sample was then diluted 1:4 (v:v) with buffer and vortexed for 1 min and transferred into the SPE cartridge for extraction procedure. The SPE cartridge (Oasis HLB, Waters Spa, Milford, MA, 100 mg/1 mL) preconditioned with 1 mL of acetonitrile and 1 mL of ammonium acetate buffer (5 mM, pH 5.8). Wash step was carried out with 1 mL of ammonium acetate buffer (5 mM, pH 5.8) and the analytes and Internal Standard elution was obtained using 3 mL of acetonitrile.

The eluate was evaporated to complete dryness under a stream of nitrogen and then the residue was dissolved in 250 μL of mobile phase, and 20 μL was injected into the HPLC system for the analysis.

Isocratic chromatographic condition

HPLC analyses were performed on a Waters liquid chromatography equipped with a model 600 solvent pump and a 2996 photodiode array detector. Mobile phase was directly on-line degassed by using Degassex, mod. DG-4400 (Phenomenex, Torrance, CA). Empower v.2 Software (Waters Spa, Milford, MA) was used for data acquisition and elaboration.

An HILIC packing column (Luna HILIC, 4.6 × 250 mm, 5 μm; Phenomenex, Torrance, CA) was employed for the separation and the column was thermostated at 25 ± 1 °C using a Jetstream2 Plus column oven.

For quantitative analyses, selective detection was performed at 278 nm. Isocratic elution mode was performed using a mobile phase containing a 15/85 ammonium acetate buffer (5 mM, pH 5.8)/acetonitrile (both with 1% Et₃N, v/v) at 1 mL/min flow rate.

All the sample solutions were previously centrifuged and 20 μL of the supernatant, after a filtration on Phenex-PTFE (4 mm, 0.45 μm) syringe filters (Phenomenex, Torrance, CA), was injected into the HPLC–DAD system.

Calibration, linearity and quality control samples

The two chemical standards stock solutions were made at the concentration of 1 mg/mL in a final volume of 10 mL of methanol. Combined working solutions of mixed standards at the concentrations of 20, 25, 40, 50, 80, 200, 250, 400 and 500 μg/mL were obtained by dilution of a mixed solution at 500 μg/mL in volumetric flasks containing the mobile phase. Finally the nine calibration standards were obtained as previously reported in sample preparation section, and injected into the HPLC–DAD system. Calibration curves were calculated by analysing these nine non-zero concentration standards prepared in freshly spiked plasma in sestuplicate. All quantitative analyses were performed at 278 nm. Concentrations of the QCs and unknown samples were calculated by interpolating their analyte peak area/internal standard area ratio on the calibration curve.

Limit of detection (LOD) and limit of quantification (LOQ)

The LOQ of the method was defined according to International Guidelines30–32 as the concentration of the lowest standard on the calibration curve for which (a) the analyte peak is identifiable and discrete, (b) the analyte response is at least 10 times the response of the blank sample and (c) the analyte response is reproducible with a precision less than 20% and trueness better of 80–120%. The LOD was estimated at a signal-to-noise ratio of 3:1 by injecting a series of samples with known concentrations.

Precision and trueness studies were carried out at the LOQ and at three QC concentration levels by injecting six individual preparations of the analytes and calculating the RSD% and bias% of the back-calculated concentrations.

Recovery

The method efficiency was measured by the comparison of peak areas ratios obtained from samples spiked before SPE processes and samples spiked after SPE processes (maximum recovery). The back-calculated concentrations were used to calculate the better extraction procedure leading to the maximum recovery for the cited analytes, minimizing solvent and time consumptions.

Results and discussion

Optimisation of solid phase extraction

An important step in the determination of pro-drug and related active metabolite is the procedure employed to obtain a representative samples with a maximum recovery for all analytes. This problem is especially related to the different physicochemical properties often present between the parent drug and its active metabolite24–29.

SPE has been a usual and generally adopted pre-treatment method for biological samples, suitable for most compounds, with a large variety of physicochemical properties and very effective in the removing the protein, salt and fat of the biological matrix. This technique was commonly used especially because it allows a lower solvent consumption and higher reproducibility for the extracted samples, if Internal Standard assay was adopted.

For these reasons, the extraction procedure, employed in this work, uses a simple SPE based on C18 based stationary phase, followed by a pre-concentration step, in order to obtain a better LOQ values.

Preconditioning step was carried out with 1 mL of acetonitrile and 1 mL of ammonium acetate buffer (5 mM, pH 5.8). Wash step was carried out with 1 mL of ammonium acetate buffer (5 mM, pH 5.8) to obtain neutral conditions, avoiding chemical degradation and Internal Standard loss, while the analytes and Internal Standard elution were obtained using 3 mL of acetonitrile.

HPLC isocratic separation

Several gradient and isocratic mobile phases using different acids and buffers were examined in order to obtain the separation conditions of the cited analytes and Internal Standard.

The chromatographic behaviour of the analytes was investigated employing several HPLC columns including Symmetry C18 (150 × 3.9 mm, 5 μm, Waters Spa, Milan, Italy); Zorbax SB-Aq (150 × 4.6 mm, 5 μm, Agilent Technologies, USA) and finally a Luna HILIC, column (4.6 × 250 mm, 5 μm; Phenomenex, Torrance, CA) in mobile phases, differing in organic modifier, buffer solution and pH, i.e. (a) methanol–water (both containing 0.5% formic acid, v/v)13, (b) acetonitrile–0.5% triethylamine buffer (pH 3.0)14 and (c) ammonium acetate buffer (5 mM, pH 5.8) and acetonitrile (12:88, v/v), respectively.

Isocratic conditions under different organic modifier type and percentages were firstly tested in order to avoid assay transferability problems in the future.

The Luna HILIC column, where the silica surface is covered with cross-linked diol groups to increase polar selectivity in hydrophilic conditions, was chosen to perform further experiments because it resulted in a better separation especially respect to peak symmetry, resolution and total analysis time using isocratic elution with mobile phase system consisting of ammonium acetate buffer (5 mM, pH 5.8, solvent A) and acetonitrile (solvent B). The choices of the column and the mobile phase were also addressed by the eventually future applications of this assay in HPLC–MS system, so that it would be possible to obtain lower LOD and LOQ values, very important parameters especially when a method was applied to biological matrices analyses. Carryover was not obvious in the biological matrices, for this reason, a blank plasma sample was analysed after the analysis of plasma fortified at the upper limit of quantification (ULOQ) and no “memory effects” was observed.

Figure 2 shows the chromatographic separation of standard mixture containing the two analytes and Internal Standard at 278 nm (20 μg/mL, 20 μL injected). A robust baseline analytes separation was achieved in 15 min. Under these conditions, as reported in Table 1, the analytes retention times were 4.72 ( ± 0.14), 6.42 ( ± 0.20) and 11.1 ( ± 0.53) min for Prulifloxacin, Ulifloxacin and Danofloxacin (IS) (n = 180, calibration and QC analyses), respectively.

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.

HPLC method development

Calibration curves, obtained at 278 nm and for six independent measures, were plotted using linear least-squares regression analysis. All calibration curves were linear over the concentration range tested of 1–25 μg/mL (LOD 0.75 μg/mL) with the determination coefficients r² of 0.9846 and 0.9903 for Ulifloxacin and Prulifloxacin, respectively.

The within-assay precision (repeatability) of the method was determined by performing six consecutive assays in the same day on QC samples spiked at three different anthraquinones concentration levels, i.e. 1.5 (low level), 5 (medium level) and 15 (high level) μg/mL, which are within the range of the calibration curve. The QC samples were also analysed in different days to assess the between-assay precision (intermediate precision) of the method.

The trueness of the method was evaluated at the same analytes concentration levels by comparing the measured analytes concentrations of the QC samples with their nominal values. These data are summarized in Table 2.

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.

As previously reported, the LOQ of the method was defined according to the Guidance for Industry on the validation of bio-analytical methods30–32.

Following these criteria, the LOQ values for each analytes are 1 μg/mL. On the basis of the signal-to-noise ratio of the chromatograms, the LOD of the method could also be set at 0.75 μg/mL.

As reported in Figure 3, selectivity and specificity of the method were tested on blank plasma samples extracted and analysed by HPLC–DAD assays without any fortification (a), and after addition of merely Internal Standard (b) or analytes plus Internal Standard (c). Under these conditions the analytes retention times confirmed the retention times obtained by the real samples analyses and no interfering peaks are presents.

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).

The method efficiency, as reported in Recovery section, was measured by the comparison of the peak areas ratios obtained from samples spiked before SPE processes and samples spiked after SPE processes (maximum recovery). It allows an overall recovery ranged from 90.1% to 107% for the cited analytes, as described in Table 3.

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.

During the analysis period and under the storage conditions, no decreases in the measured analytes and Internal Standard concentration or change of their chromatographic behaviour due to chemical and/or photochemical degradation were observed in the stock solutions and extracts samples.

Application to plasma sample

The developed method was applied to investigate the Prulifloxacin stability in human plasma samples stored under different temperature conditions. In this investigation, three different QC concentration levels in triplicate were used (1.5, 5 and 15 μg/mL) and the stability was followed for 6 h at defined times (15, 30, 60, 120, 240 and 360 min).

The different samples were submitted to SPE procedure and HPLC analyses at corresponding times and the quantitative results, reported in Table 4, were obtained plotting the total amount of remaining parent compound Prulifloxacin (expressed as μg/mL) versus time (expressed as minutes), as reported in Figure 4.

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.

As reported by Raju et al.33, the difference on the prodrug-active metabolite amount reported in Table 4 can be probably due to other degradation products that are not detected with the developed method.

Conclusions

The new HPLC–DAD method for the simultaneous determination of Prulifloxacin and its active metabolite Ulifloxacin in human plasma fulfils the acceptance criteria established for bio-analytical assays when they are applied in pharmaceutical analysis. In the explored range the method is accurate (precision and trueness), selective and sensitive enough to allow the analysis in human plasma after a simple SPE.

The analysis can be carried out by means of a relatively simple procedure, with a reduction of analytical variability and sample handling time, especially related to the use of Internal Standard method and an isocratic HPLC method that reduce the assay transferability problems.

The results suggest that the reported analytical methodology is a suitable tool for the efficient detection, identification and quantification of these two analytes, to evaluate their stability in other matrices and for clinical studies.

Declaration of interest

The authors report no declarations of interest.

Financial support to this study from the University “G. d'Annunzio” of Chieti-Pescara.