G Model

ARTICLE IN PRESS

CHROMA-356266; No. of Pages 8

Journal of Chromatography A, xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Simultaneous determination of Eperisone Hydrochloride and Paracetamol in mouse plasma by high performance liquid chromatography-photodiode array detector Marcello Locatelli a,b,∗ , Roberta Cifelli a , Cristina Di Legge a , Renato Carmine Barbacane c , Nicola Costa d , Massimo Fresta d,e , Christian Celia a,e,f , Carlo Capolupo g , Luisa Di Marzio a a

University “G. d’Annunzio” Chieti-Pescara, Department of Pharmacy, via dei Vestini 31, 66100 Chieti, Italy Interuniversity Consortium of Structural and Systems Biology, Viale Medaglie d’oro 305, 00136 Roma, Italy c University “G. d’Annunzio” Chieti-Pescara, Immunology Division, Department of Experimental and Clinical Science, via dei Vestini 31, 66100 Chieti, Italy d University of Catanzaro “Magna Graecia”, Department of Health Sciences, Viale “S. Venuta”, 88100 Catanzaro, Italy e University of Catanzaro “Magna Græcia”, Inter-regional Research Center for Food Safety & Health, Viale “S. Venuta”, 88100 Catanzaro, Italy f Houston Methodist Research Institute, Department of Nanomedicine, Houston, TX 77030, USA g Unità Operativa di Farmacia Ospedaliera, Presidio Ospedaliero Soveria Mannelli, Viale R. Rubbettino, 88049 Soveria Mannelli (CZ), Italy b

a r t i c l e

i n f o

Article history: Received 10 December 2014 Received in revised form 2 February 2015 Accepted 3 February 2015 Available online xxx Keywords: HPLC-PDA Method development Eperisone Hydrochloride Paracetamol Plasma Sample preparation

a b s t r a c t This paper reports the validation of a quantitative high performance liquid chromatography-photodiode array (HPLC-PDA) method for the simultaneous analysis, in mouse plasma, of Eperisone Hydrochloride and Paracetamol by protein precipitation using zinc sulphate–methanol–acetonitrile. The analytes were resolved on a Gemini C18 column (4.6 mm × 250 mm; 5 ␮m particle size) using a gradient elution mode with a run time of 15 min, comprising re-equilibration, at 60 ◦ C (±1 ◦ C). The method was validated over the concentration range from 0.5 to 25 ␮g/mL for Eperisone Hydrochloride and Paracetamol, in mouse plasma. Ciprofloxacin was used as Internal Standard. Results from assay validations show that the method is selective, sensitive and robust. The limit of quantification of the method was 0.5 ␮g/mL for Eperisone Hydrochloride and Paracetamol, and matrix-matched standard curves showed a good linearity, up to 25 ␮g/mL with correlation coefficients (r2 ) ≥ 0.9891. In the entire analytical range the intra and inter-day precision (RSD%) values were ≤1.15% and ≤1.46% for Eperisone Hydrochloride, and ≤0.35% and ≤1.65% for Paracetamol. For both analytes the intra and inter-day trueness (bias%) values ranged, respectively, from −5.33% to 4.00% and from −11.4% to −4.00%. The method was successfully tested in pharmacokinetic studies after oral administration in mouse. Furthermore, the application of this method results in a significant reduction in terms of animal number, dosage, and improvement in speed, rate of analysis, and quality of pharmacokinetic parameters related to serial blood sampling. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Biological fluids such as serum, plasma and urine are very complex matrices that could vary over several orders of magnitude in components concentration. The use of high performance extraction procedures and separation techniques are essential for the

∗ Corresponding author at: Analytical and Bioanalytical Chemistry, University “G. d’Annunzio” Chieti-Pescara, Department of Pharmacy, Italy. Tel.: +39 0871 3554590; fax: +39 0871 3554911. E-mail address: [email protected] (M. Locatelli).

correct quali-quantitative determination of specific compounds at low concentration levels. Several papers have been published on this topic [1–3], reporting instrument configurations [3–5], extraction procedures [6], multi-components analyses [7–9], and also using chemometric approach [10]. Often, different active principle associations are used for disease treatment, therefore analytical procedures must be developed and validated in order to maintain (or improve) sensitivity and selectivity, respect to the target compounds. Recently, Eperisone Hydrochloride (or (2RS)-1-(4-ethylphenyl)2-methyl-3-(piperidinyl)-1-propanone hydrochloride, Fig. 1a), a potent new generation antispasmodic agent [11–13] was used in

http://dx.doi.org/10.1016/j.chroma.2015.02.008 0021-9673/© 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: M. Locatelli, et al., Simultaneous determination of Eperisone Hydrochloride and Paracetamol in mouse plasma by high performance liquid chromatography-photodiode array detector, J. Chromatogr. A (2015), http://dx.doi.org/10.1016/j.chroma.2015.02.008

G Model CHROMA-356266; No. of Pages 8

ARTICLE IN PRESS M. Locatelli et al. / J. Chromatogr. A xxx (2015) xxx–xxx

2

Millipore Milli-Q Plus water treatment system (Millipore Bedford Corp., Bedford, MA, USA). The Eperisone Hydrochloride and Paracetamol combination was customized as reported in Supplementary material, Section S.5. 2.2. Experimental animals, drug administration, and serial sampling collection and storage

Fig. 1. Chemical structures of Eperisone Hydrochloride (a), and Paracetamol (b).

association with Paracetamol (or N-(4-hydroxyphenyl)acetamide, Fig. 1b), a centrally and peripherally acting non-opioid analgesic and antipyretic agent, for the treatment of moderate to severe pain. Eperisone Hydrochloride shows low bioavailability after oral administration and broad first-pass metabolism [14,15], which leads to low plasma concentration. In order to evaluate biological fluid concentration, a sensitive method for the determination of Eperisone in plasma is required. Several papers have reported the determination in pharmaceuticals and biological fluids using classic instrument configuration, such as High Performance Liquid Chromatography (HPLC) [16], but also hyphenated ones, such as Gas Chromatography–Mass Spectrometry (GC–MS) [17,18], and High Performance Liquid Chromatography–Mass Spectrometry (HPLC–MS) [19–21]. On the other hand, Paracetamol (acetaminophen) is quickly absorbed from the gastrointestinal tract and is primarily metabolized through conjugation with glucuronic and sulphuric acid in order to form glucuronide (Paracetamol-glucuronide, PG) and sulphate (Paracetamol-sulphate, PS) derivatives, respectively, and consequently excreted in the urine [22]. Several papers were recently published also for Paracetamol, reporting its determination (and/or its metabolite profiles) in different matrices [23–25], its characterization [26], and its selective extraction using magnetic molecularly imprinted polymer (m-MIPs) for solid-phase extraction and sample clean up [27]. Furthermore, only one paper is present in the literature that reports the simultaneous determination of these two drugs in pharmaceuticals [28], but no work considers their simultaneous determination in biological fluids, such as plasma. In continuation of our studies on method validation for drugs, metabolites, impurities, and particularly, quantitative analyses of drug-associations for clinical purposes [29–34], we report herein a new high performance liquid chromatography-photodiode array (HPLC-PDA) method for the simultaneous determination of Eperisone Hydrochloride and Paracetamol in mouse plasma, and their quantitative evaluation samples after single oral dose of 0.5 mg/kg (Eperisone Hydrochloride) and 5 mg/kg (Paracetamol) of commercially available formulation.

2. Experimental 2.1. Chemicals and reagents Eperisone Hydrochloride (>99% purity index) was purchased from Santa Cruz Biotechnology (Dallas, USA), while Paracetamol (98% purity index) was supplied by Sigma-Aldrich (Milan, Italy). Ciprofloxacin (IS, >98% purity index), ammonium acetate (>98% purity index), and acetic acid (to obtain ammonium acetate buffer at pH = 3) were obtained by Fluka Chemie (Buchs, Switzerland). Methanol and acetonitrile (AcN) (HPLC-grade) were purchased from Carlo Erba Reagenti (Milan, Italy). Water is produced by

All experimental animals (C57BL/6JOlaHsd mice, 4 weeks old), supplied by Harlan Laboratories S.r.l. (Udine, Italy), were housed in individually metabolic cages, and all procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with Institutional Animal Ethics Committee (IAEC) of University “Magna Graecia” of Catanzaro, Department of Health Sciences (as reported in Supplementary Material, Section S.6). For pharmacokinetic studies anaesthetized animals received orally a drug equivalent dosage of 0.5 mg/kg of Eperisone Hydrochloride and 5 mg/kg of Paracetamol, respectively according to the following therapeutic protocol: group 1 (untreated mice or control, n = 6); group 2 (0.5 mg/kg Eperisone Hydrochloride, n = 6); group 3 (5 mg/kg Paracetamol, n = 6); group 4 (0.5/5 mg/kg Eperisone Hydrochloride/Paracetamol, n = 6). The different formulations were dissolved in mineral water and administered in bolus (1 mL). The control group received single oral dose of mineral water for pharmacokinetic comparison. Serial blood samples (200 ␮L) were drawn from the retro-orbital plexus at 30 min, 1 h, 4 h, 8 h, 16 h and 24 h after oral administration, collected in heparinized polythene tubes, centrifuged for 10 min at 4000 rpm at 4 ◦ C. Plasma was separated and stored at −20 ◦ C until further analysis. 2.3. Plasma sample preparation A 90 ␮L aliquot of mouse blank plasma was mixed with a 5 ␮L aliquot of analytes working solutions and 5 ␮L aliquot of Internal Standards working solution (at final concentration of 25 ␮g/mL) and vortexed for 1 min (10% of matrix modification in calibration and QC samples preparation, 5% of matrix modification in real samples analyses). For protein precipitation, 50 ␮L of ZnSO4 solution (5%, p:v) in water:methanol:acetonitrile were added, stirred, and centrifuged at 12,000 × g for 10 min. Then the supernatant, after a filtration on Phenex-PTFE (4 mm, 0.45 ␮m) syringe filters (Phenomenex, Torrance, CA, USA), was transferred into vials and 20 ␮L of samples were injected into the HPLC-PDA system. 2.4. Apparatus and chromatographic condition HPLC analyses were performed on a waters liquid chromatograph 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, USA). Empower v.2 Software (Waters Spa, Milford, MA, USA) was used for data acquisition and elaboration. A Gemini C18 packing column (4.6 mm × 250 mm, 5 ␮m particle size; Phenomenex, Torrance, CA, USA) was employed for the separation, protected by a disposable Security Guard column (4.0 mm × 3.0 mm, 5 ␮m particle size; Phenomenex, Torrance, CA, USA) and the column was thermostated at 60 ◦ C (±1 ◦ C) using a Jetstream2 Plus column oven. For quantitative analyses, selective detection was performed at 259, 245, and 279 nm for Eperisone Hydrochloride, Paracetamol, and Ciprofloxacin (IS), respectively (see Supplementary material, Section S.1 for analytes and Internal Standard UV/vis spectra).

Please cite this article in press as: M. Locatelli, et al., Simultaneous determination of Eperisone Hydrochloride and Paracetamol in mouse plasma by high performance liquid chromatography-photodiode array detector, J. Chromatogr. A (2015), http://dx.doi.org/10.1016/j.chroma.2015.02.008

G Model CHROMA-356266; No. of Pages 8

ARTICLE IN PRESS M. Locatelli et al. / J. Chromatogr. A xxx (2015) xxx–xxx

Table 1 Chromatographic gradient elution. Time (min)

% Aa

% Bb

% Cc

0 8 9 15

79 35 79 79

9 25 9 9

12 40 12 12

a b c

Ammonium acetate buffer (10 mM, pH 3). AcN. Methanol.

3

Stability of the two analytes was evaluated by comparing the corrected area (analyte/IS area) of the QC samples with those obtained for samples subjected to stability tests. During the longterm stability studies samples were stored frozen at −20 ◦ C and +4 ◦ C for 30 days; during short-time stability samples were kept at room conditions for 24 h. Furthermore stability after three freeze–thaw cycles, and stability study of analytes in the stock solutions for 30 days were also assessed.

3. Results and discussion Gradient elution mode was performed using a ternary solvent system composed by ammonium acetate buffer (10 mM, pH 3), AcN, and methanol at 1.2 mL/min flow rate, as reported in Table 1. 2.5. Stock solution, calibration and quality control samples The three chemical standards stock solutions were made at the concentration of 1 mg/mL in a final volume of 10 mL of mobile phase. Combined working solutions of mixed standards at the concentrations ranging from 5 to 250 ␮g/mL were obtained by dilution of a mixed solution at 500 ␮g/mL in volumetric flasks containing the mobile phase. Finally the eight calibration standards were obtained as previously reported in mouse plasma sample preparation Section 2.3, and injected into the HPLC-PDA system. 2.6. Method validation In order to demonstrate the suitability of the developed analytical method, validation was carried out according to International Guidelines [35–37]. In this way, Limit of Detection (LOD), Limit of Quantification (LOQ), linearity, intra- and inter-day trueness and precision, selectivity, recovery, and stability were tested for each analyte in mouse plasma. The LOQ of the method was defined as the concentration of the lowest standard in mouse plasma on the calibration curve for which (a) the analyte peak is identifiable and discrete, (b) the analyte response is at least ten 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 mouse plasma samples fortified with known concentrations. Precision and trueness studies were carried out at the LOQ and at three Quality Control (QC) concentration levels by injecting six individual preparations of the analytes in mouse plasma and calculating the RSD% and Bias% of the back-calculated concentrations. Calibration curves in mouse plasma were calculated by analysing six-times these eight non-zero concentration standards prepared in freshly spiked plasma. Concentrations of the QCs and unknown samples were calculated by interpolating their analyte peak area/Internal Standard area ratio on the calibration curve, and quantitative analyses for Eperisone Hydrochloride, Paracetamol, and Ciprofloxacin (IS) were performed at 259, 245, and 279 nm, respectively. Selectivity was tested by analysing, under optimized chromatographic conditions, blank mouse plasma samples from different sources, and by comparing them with spiked mouse plasma samples at a concentration close to the LOQ. The method efficiency (recovery) was measured by the comparison of peak areas ratios obtained from samples spiked before and samples spiked after extraction procedure (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.

3.1. Optimization of extraction procedure An important step in the determination of different compounds is the procedure employed to obtain representative samples with maximum recovery of all analytes. This can often be a problem due to the difference in physiochemical properties between analytes. Due to the fact that the cited analytes are different (an antispasmodic and an analgesic/antipyretic agent), the first step was to remove the large protein whilst maintaining good recoveries and avoiding degradation and/or chemical modification for all analytes (and IS). Several assay were tested, such as precipitation with organic solvent (methanol, acetonitrile) [38,39], also acidified with phosphoric acid, trichloroacetic acid (TCA) in water, or by liquid–liquid extraction using diethyl ether: dichloromethane [40]. These procedures allow good recovery for Internal Standard (approx. 85%), but very low values for one (or both) analytes (approx. 20–40%). Solid phase extraction (SPE) was also tested in order to reduce the solvent consumption, maximize the analytes recovery, and to optimize the sample clean-up procedure. Several SPE cartridges were considered, such as Oasis HLB (Waters Spa, Milford, MA, USA) [41], Strata-X (Phenomenex, Torrance, CA, USA), and Sep-Pak C18 (Waters Spa, Milford, MA, USA) in different extraction solvent system, differing in organic modifier percentage, buffer solution, and pH (i.e. methanol, water, and phosphate buffer (pH 7, 10 mM)). All these procedures allow good recovery for Paracetamol (approx. 90–95%) and/or Internal Standard (approx. 85%), but low values were obtained for Eperisone Hydrochloride, probably as a result of stronger interactions with the stationary phase. Following our previously published research [9], MicroExtraction by Packed Sorbent (MEPS) procedure for multianalytes analysis was also tested. In particular, the needle, fitted with a barrel insert and needle assembly (BIN) containing a C18 and a silica sorbent (SGE Analytical Science, Australia), were tested starting from general conditions applied to plasma matrix for cartridge conditioning, sample extraction, and elution according to that reported in literature [42]. Also in this case, as previously reported for SPE extraction, using MEPS (both C18 and silica) it is possible to obtain a quantitative recovery for Paracetamol (approx. 99%), and better recovery for Internal Standard (approx. 90–93%), even if the recovery value for Eperisone Hydrochloride remains lower (approx. 60%). These values can be applied for the quantitative analysis of Paracetamol in biological fluids. Conversely, Eperisone Hydrochloride shows the low drug bioavailability and increased metabolism after oral administration [14,15], thus shows low drug bioavailability and the need to develop more accurate procedures to quantify the drug into biological samples. For this reason, another approach, based on protein precipitation, was applied using a ZnSO4 (5%, p:v) solution in water:methanol:acetonitrile (5:3:2, v:v:v), in order to retain small molecules in solution and to remove large proteins, followed by stirring and then centrifugation at 12,000 × g for 8 min, following a recently reported procedure [43,44]. The use of sample: protein precipitation solvent volume ratio of 1:0.5 not only allows for a low

Please cite this article in press as: M. Locatelli, et al., Simultaneous determination of Eperisone Hydrochloride and Paracetamol in mouse plasma by high performance liquid chromatography-photodiode array detector, J. Chromatogr. A (2015), http://dx.doi.org/10.1016/j.chroma.2015.02.008

G Model CHROMA-356266; No. of Pages 8

ARTICLE IN PRESS M. Locatelli et al. / J. Chromatogr. A xxx (2015) xxx–xxx

4

dilution factor, but obtains a higher recovery for both analytes and Internal Standard. As reported by Mollica [43], and Carlucci [44], this solution does not generate problems in the analysis phase for which the supernatant obtained following centrifugation is directly injected into the system, even if is generally good practice use at least the same solvent system used for chromatographic analysis. 3.2. HPLC separation and method development Chromatographic conditions were optimized over several trials to achieve good resolution, increased analyte signal, to minimize the run time, and to avoid the presence of interferences. Several isocratic and gradient mobile phases using different acids and buffers were examined in order to obtain the separation conditions of the cited analytes and Internal Standards. The chromatographic behavior of the analytes was investigated employing several HPLC columns including Luna C18 (250 mm × 4.6 mm, 5 ␮m particle size, Phenomenex, Torrance, CA, USA); Hypersil® BDS C18 (250 mm × 4.6 mm, 5 ␮m particle size, Thermo Fisher Scientific, Waltham, MA, USA), HyperClone C18 (Phenomenex, Torrance, CA, USA), and finally a Gemini C18 column (250 mm × 4.6 mm, 5 ␮m particle size, Phenomenex, Torrance, CA, USA) in mobile phases, differing in organic modifier, buffer solution, and pH, i.e. (a) 20 mM sodium dihydrogen phosphate buffer (pH = 3) and methanol [45], (b) water and acetonitrile–water (80:20, v:v), and finally a ternary solvent system composed by ammonium acetate buffer (10 mM, pH 3), AcN, and methanol using a flow rate of 1.2 mL/min, respectively. Isocratic conditions under different organic modifier type and percentages were first tested in order to avoid assay transferability problems in the future. Unfortunately, these conditions could not be applied because of overlapping, interfering peaks present in the matrix with the analyte signals, that could warrant the use of a more selective detector, such as Mass Spectrometer (after a deep matrix effects evaluation), or novel flow derivative spectrophotometric approach in order to extract only the signal of interest [5]. The last solvent system was chosen to allow the mobile phase polarity to benefit the retention of the analytes, to resolve the analytes and interfering peaks, and to maintain an acceptable overall run time. With optimized conditions a robust baseline analyte separation was achieved in 8 min, followed by re-equilibration time. Under these conditions, the analyte retention times were 7.66(±0.18), 4.10(±0.06), and 6.11(±0.10), for Eperisone Hydrochloride, Paracetamol, and Ciprofloxacin (IS), respectively (see Supplementary material, Section S.2 for System Suitability Test (SST) separation). The within-assay precision (repeatability) was determined by performing six consecutive assays in the same day on QC samples spiked at three different analyte concentration levels, i.e. 1.5 (low level), 7.5 (medium level) and 17.5 (high level) ␮g/mL, which are within the range of the calibration curve. The QC samples were also analyzed on different days to assess the between-assay precision (intermediate precision). The trueness of the method was evaluated at the same analyte concentration levels by comparing the measured analyte concentrations of the QC samples with their nominal values. This data is summarized in Table 3. LOQ values are 0.5 ␮g/mL for Eperisone Hydrochloride and Paracetamol, and on the basis of the signal-to-noise ratio of the chromatograms, the LODs of the method could also be set at 0.1 ␮g/mL for Eperisone Hydrochloride and Paracetamol, as reported in Table 2. As illustrated in Fig. 2, selectivity and specificity of the method were tested on blank plasma samples extracted and analyzed by HPLC-PDA assays without any fortification (a), and after addition of merely the Internal Standards (b) or analytes plus Internal Standards (c). Under these conditions the analyte retention times

confirmed the retention times obtained by the real sample analyses and no interfering peaks are present. Carryover was not obvious in the biological matrices, for this reason, a blank plasma sample was analyzed after the analysis of plasma fortified at the upper limit of quantification (ULOQ) and no “memory effects” was observed. In addition, intra-matrix variability was assessed by analyses of six different lots of mice, and no interferences for the two analytes and Internal Standard were found. 3.3. Method validation 3.3.1. Linearity, accuracy, and LOQ Calibration curves were obtained plotting the corrected area (ratio analyte area/IS area) for each level versus the nominal concentration level corresponding to each standard solution. The linearity of the standard curves was assessed with the intercept, slope and determination coefficient and their variations in the range of 0.5–25 ␮g/mL for Eperisone Hydrochloride and Paracetamol. Eperisone Hydrochloride and Paracetamol calibration samples were prepared by diluting the working standard solution of these analytes in mouse plasma, and at least eight concentration levels were used. The calibration curves were linear over the range described with a least-squares linear-regression determination coefficient (r2 ) ≥ 0.9891, using a weighting factor of (1/x2 ). Calibration curves, obtained at corresponding analytes (and IS) maximum wavelengths were plotted using weighted linear least-squares regression analysis, as permitted by the method validation guidelines, stating that “standard curve fitting is determined by applying the simplest model that adequately describes the concentration–response relationship using appropriate weighting. . .” [35]. All calibration curve parameters are reported in Table 2. The precision and trueness were acceptable since RSD% and Bias% values were lower than 15%, as reported in Table 3. Limits of quantitation obtained were 0.5 ␮g/mL for Eperisone Hydrochloride and Paracetamol. 3.3.2. Selectivity and intra-matrix variability In the present study, selectivity was studied by analysing six plasma samples from different animals. As the ICH guideline requires [36], the studied blanks showed neither area values higher than 20% of the LOQ’s areas at the analyte retention times, nor higher than 5% of the IS area at its corresponding retention time. Representative chromatograms at 262 nm obtained from control plasma after extractions are shows in Fig. 2. The peak present near to the Paracetamol retention time represents interference, but does not affect the peak integration phase, as shown by the results obtained in the validation. Furthermore, its intensity is lower when the chromatograms are extrapolated at 245 nm (corresponding to maximum wavelength for Paracetamol), as reported in Supplementary material, Section S.3. 3.3.3. Recovery, stability, and assay parallelism assessment The recoveries were calculated for Eperisone Hydrochloride and Paracetamol at low, medium and high concentrations, the results are shown in Table 4. The method efficiency was measured by the comparison of the peak area ratios obtained from samples spiked before protein precipitation and samples spiked after protein precipitation processes (maximum recovery). It allows an overall recovery range from 81.8% to 94.6% for the cited analytes. No decreases in the measured concentrations or change of the chromatographic behavior due to degradation of the analyte were observed in stock solutions and spiked plasma samples, or extracts maintained at room temperature. Spiked plasma samples stored at −20 ◦ C, and at +4 ◦ C were stable for at least 1 month and after a

Please cite this article in press as: M. Locatelli, et al., Simultaneous determination of Eperisone Hydrochloride and Paracetamol in mouse plasma by high performance liquid chromatography-photodiode array detector, J. Chromatogr. A (2015), http://dx.doi.org/10.1016/j.chroma.2015.02.008

G Model

ARTICLE IN PRESS

CHROMA-356266; No. of Pages 8

M. Locatelli et al. / J. Chromatogr. A xxx (2015) xxx–xxx

5

Table 2 Mean linear calibration curve parameters obtained by linear least-squares regression analysis of six independent nine non-zero concentration point. Analyte

Linearity range (␮g/mL)

Slopea

Intercepta

Detemination coefficient (R2 )

Eperisone Hydrochloride Paracetamol

0.5–25 (0.1 ␮g/mL ) 0.5–25 (0.1 ␮g/mL b )

0.030 (±0.009) 0.023 (±0.009)

0.042 (±0.034) 0.005 (±0.008)

0.9891 0.9908

a b

b

Values at 95% confidence intervals on the mean of six independent calibration curves. In round bracket are reported LOD values obtained by signal-to-noise ratio = 3; slope and intercept are reported for calibration in ␮g/mL units.

Fig. 2. Chromatograms obtained after extraction and analyses of (a) blank mouse plasma, (b) blank mouse plasma spiked with 25 ␮g/mL of Internal Standard, and (c) blank mouse plasma spiked with 25 ␮g/mL of Internal Standard and 10 ␮g/mL of analytes. 20 ␮L injected.

minimum of three freeze-thaw cycles (see Supplementary material, Section S.4). A parallelism check was performed by analysing a high drug concentration plasma sample diluted 1:10 (v:v) with the pooled corresponding matrix used for preparation of standards and QC samples.

The results obtained indicate that plasma levels above the top calibration standard and up to 100 ␮g/mL can be measured upon dilution of the sample with accuracy (precision and trueness) comparable to those achieved for concentrations within the calibration range.

Table 3 Intra-day and inter-day precision (RSD%), trueness (bias%) of the analytical method obtained from the analysis of QC samples. Intra day Eperisone Hydrochloride

Inter day Paracetamol

a

Theoretical Mean back-calculateda BIAS% RSD%

−1.42 −5.33 −0.01

−1.44 −4.00 −0.14

Theoreticala Mean back-calculateda BIAS% RSD%

−7.37 −1.73 −0.09

6.88 −8.27 0.35

Theoreticala Mean back-calculateda BIAS% RSD%

17.2 −1.71 −1.15

−15.5 −11.4 −0.25

Eperisone Hydrochloride

Paracetamol

1.5 −1.42 −5.33 −0.09

−1.41 −6.00 −0.08

−7.37 −1.73 −0.46

−7.05 −6.00 −0.45

18.2 4.00 1.46

−16.6 −5.14 −1.65

7.5

17.5

Data are expressed as the mean values of six experiments (n = 6). a Concentration expressed as ␮g/mL unit.

Please cite this article in press as: M. Locatelli, et al., Simultaneous determination of Eperisone Hydrochloride and Paracetamol in mouse plasma by high performance liquid chromatography-photodiode array detector, J. Chromatogr. A (2015), http://dx.doi.org/10.1016/j.chroma.2015.02.008

G Model

ARTICLE IN PRESS

CHROMA-356266; No. of Pages 8

M. Locatelli et al. / J. Chromatogr. A xxx (2015) xxx–xxx

6

Table 4 Recovery values of the analytical method obtained from the analysis of QC samples. Eperisone Hydrochloride QC low concentration level Recovery Standard deviation RSD%

82.6 1.82 1.78

QC medium concentration level Recovery Standard deviation RSD%

81.8 5.85 5.96

QC high concentration level Recovery Standard deviation RSD%

92.1 4.18 4.17

Paracetamol

1.5 82.9 2.88 2.83 7.5 87.3 9.43 8.18 17.5 94.6 1.65 1.64

Data are expressed as the mean values of six experiments (n = 6).

3.4. Comparisons with existing methods The developed method presents several advantages in terms of overall analytical performances especially related to the fact that the just reported assays consider merely only Eperisone Hydrochloride [16–21] or Paracetamol [23–27] in pharmaceuticals and biological matrices, also using complex instrument configurations that require trained personnel. No paper reports the simultaneous determination, in biological matrices, of these drugs, even if their selectivity (and, generally) their sensitivity can be higher than the reported herein [21]. The validated assay permits the simultaneous determination of this drug association in mouse plasma sample after a simple, and rapid protein precipitation with LOQ from 4 to 10-folds (for Paracetamol and Eperisone Hydrochloride, respectively) lower than the reported for solid dosage form [28]. Recently another paper consider the drug association analysis using High Performance Thin Layer Chromatography (HPTLC), with comparable LOQs values, but still remains focused on tablet dosage form [46]. Another advantages of the reported assay is the use of Mass Spectrometry-compatible solvent system, that can allow its future application to a more complex instrument, such as HPLC–MS/MS, in order to further increase fundamentals analytical parameters (LOD, LOQ, selectivity). The developed method, unfortunately, require a gradient elution, and this fact can bring to some transferability problems related to a different void volume. In addition, the validated assay requires approx. 6 min for re-equilibration, and this fact bring to a complete run-time analysis of 15 min, and this can influence the high productivity (or high-throughput) when applied to clinical studies.

Fig. 3. Pharmacokinetic profile obtained for the analytes in mouse plasma for single drug administration and for drug association. Errors bars represents Standard Error of the Mean (SEM) values (n = 6).

3.5. Application to pharmacological study The developed method was tested for the quantification of Eperisone Hydrochloride and Paracetamol association in mouse plasma samples. Mouse plasma samples were submitted to protein precipitation, and HPLC analyses. Plasma concentrations vs time profiles were analyzed using the PKSolver software 2.0 version, an add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel [47] and pharmacokinetic parameters, including observed maximum concentration (Cmax ), time (tmax ), half-time (t1/2 ), area under the curve (AUC0−t ), and total area under the curve (AUC0-inf ) were calculated using non-compartmental analysis. The mean residence time (MRT) was determined based on AUMC/AUC, and reported in Table 5. Fig. 3 reports pharmacokinetic profiles after dosage of only Eperisone Hydrochloride (0.5 mg/kg), only Paracetamol (5 mg/kg), and the association of the two drugs (0.5/5 mg/kg Eperisone Hydrochloride/Paracetamol) administered in bolus, respectively. In Supplementary material, Section S.7 reports Table S.7.1 with concentration values and SEM for the tested molecules in plasma used in Fig. 3.

Table 5 Pharmacokinetic parameters for the tested Eperisone Hydrochloride and Paracetamol association dose. Single administration

t1/2 (h) tmax (h) Cmax (␮g/mL) AUC0−t (␮g/ml h) AUC0-inf obs (␮g/ml h) AUC0−t/0-inf obs (␮g/ml h2 ) AUMC0-inf obs MRT0-inf obs (h)

Association

Eperisone Hydrochloride

Paracetamol

Eperisone Hydrochloride

Paracetamol

2.83 4 1.89 11.7 12.1 0.966 75.1 6.20

3.43 1 2.11 13.4 13.9 0.964 73.4 5.30

2.62 4 2.49 14.5 14.9 0.975 83.4 5.61

3.40 1 2.42 15.4 15.9 0.969 85.5 5.39

Data were the mean values of six experiments (n = 6); parameters were calculated using PKSolver software version 2.0; AUC were calculated using non-compartmental analysis.

Please cite this article in press as: M. Locatelli, et al., Simultaneous determination of Eperisone Hydrochloride and Paracetamol in mouse plasma by high performance liquid chromatography-photodiode array detector, J. Chromatogr. A (2015), http://dx.doi.org/10.1016/j.chroma.2015.02.008

G Model CHROMA-356266; No. of Pages 8

ARTICLE IN PRESS M. Locatelli et al. / J. Chromatogr. A xxx (2015) xxx–xxx

4. Conclusions Determination and quantification of Eperisone Hydrochloride and Paracetamol using HPLC-PDA in plasma samples was successfully performed on a Gemini C18 column using a ternary solvent system composed of ammonium acetate buffer (10 mM, pH 3), ACN, and methanol as mobile phase at 1.2 mL/min flow rate. The analytical performance was validated and the method was successfully tested in the determination of these compounds in samples from mice undergoing a single oral dose of the tested analytes. In the explored range the method is accurate, selective, and sensitive enough to allow the analysis in mouse plasma samples after a simple protein precipitation step. Neither endogenous compounds nor other co-administered drugs showed interferences in terms of selectivity, and the analyses 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 Standards method. The results suggest that the reported methodology is a suitable tool for the efficient detection, identification, and quantification of these two analytes, and to evaluate the pharmacological dosages for the reported association. Acknowledgment The authors gratefully acknowledge the financial support given for this research from the University “G. d’Annunzio” of ChietiPescara. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma. 2015.02.008. References [1] M. Locatelli, Anthraquinones: analytical techniques as a novel tool to investigate on the triggering of biological targets, Curr. Drug Targets 12 (2011) 366–380. [2] M. Locatelli, L. Governatori, G. Carlucci, S. Genovese, A. Mollica, F. Epifano, Recent application of analytical methods to phase I and phase II drugs development: a review, Biomed. Chromatogr. 26 (2012) 283–300. [3] M. Locatelli, D. Melucci, G. Carlucci, C. Locatelli, Recent HPLC strategies to improve sensitivity and selectivity for the analysis of complex matrices, Instrum. Sci. Technol. 40 (2012) 112–137. [4] M. Locatelli, G. Carlucci, Advanced capillary electrophoresis techniques in the analytical quantification of drugs, metabolites and biomarkers in biological samples, Glob. J. Anal. Chem. 1 (2010) 244–261. [5] S. Zaza, S.M. Lucini, F. Sciascia, V. Ferrone, R. Cifelli, G. Carlucci, M. Locatelli, Recent advances in the separation and determination of impurities in pharmaceutical products, Instrum. Sci. Technol. 43 (2015) 182–196. [6] S. Genovese, F. Tammaro, L. Menghini, G. Carlucci, F. Epifano, M. Locatelli, Comparison of three different extraction methods and hplc determination of the anthraquinones aloe-emodine, emodine, rheine, chrysophanol and physcione in the bark of Rhamnus alpinus L. (Rhamnaceae), Phytochem. Anal. 21 (2010) 261–267. [7] M. Locatelli, S. Genovese, G. Carlucci, D. Kremer, M. Randic, F. Epifano, Development and application of high-performance liquid chromatography for the study of two new oxyprenylated-anthraquinones produced by Rhamnus species, J. Chromatogr. A 1225 (2012) 113–120. [8] M. Locatelli, F. De Lutiis, G. Carlucci, High performance liquid chromatography determination of prulifloxacin and five related impurities in pharmaceutical formulations, J. Pharm. Biomed. Anal. 78–79 (2013) 27–33. [9] M. Locatelli, V. Ferrone, R. Cifelli, R.C. Barbacane, G. Carlucci, Microextraction by packed sorbent and HPLC determination of seven non-steroidal antiinflammatory drugs in human plasma and urine, J. Chromatogr. A 1367 (2014) 1–8. [10] D. Melucci, S. Fedi, M. Locatelli, C. Locatelli, S. Montalbani, M. Cappelletti, Application of pyrolysis-gas chromatography–mass spectrometry and multivariate analysis to study bacteria and fungi in biofilms used for bioremediation, Curr. Drug Targets 14 (2013) 1023–1033. [11] Y. Kuroiwa, I. Sobue, Y. Tazaki, T. Nakanishi, E. Ohtomo, K. Itahara, Effects of E-0646 on cases of spasticity – a double blind comparison using tolperisone hydrochloride, Clin. Eval. 9 (1981) 391–419.

7

[12] S. Iwase, T. Mano, M. Saito, G. Ishida, Effect of a centrally-acting muscle relaxant, Eperisone Hydrochloride, on muscle sympathetic nerve activity in humans, Funct. Neurol. 7 (1992) 459–470. [13] M. Matsunaga, Y. Uemura, Y. Yonemoto, K. Kanai, H. Etoh, S. Tanaka, Y. Atsuta, Y. Nishizawa, Y. Yamanishi, Long-lasting muscle relaxant activity of Eperisone Hydrochloride after percutaneous administration in rats, Jpn. J. Pharmacol. 73 (1997) 215–220. [14] T. Fujita, T. Takamatsu, T. Hisamoto, J. Tsutsumi, K. Kinoshita, T. Kanai, Studies on the metabolic fate of 4 -ethyl-2-methyl-3piperidinopropiopenone hydrochloride (1): absorption, disposition and excretion in rats and guinea pigs, Pharmacometrics 21 (1981) 835–846. [15] K. Mihara, M. Matsumura, E. Yoshioka, K. Hanada, H. Nakasa, S. Ohmori, M. Kitada, H. Ogata, Intestinal first-pass metabolism of Eperisone in the rat, Pharm. Res. 18 (2001) 1131–1137. [16] Y. Owada, M. Takahashi, S. Iwasa, H. Ichiba, K. Sadamoto, T. Fukushima, Enantiomeric separation of tolperisone and eperisone by reversed-phase HPLC with cellulose tris(3-chloro-4-methylphenylcarbamate)-coated chiral column, Biomed. Chromatogr. 28 (2014) 102–105. [17] T. Takamatsu, K. Yamakazi, M. Kayano, Determination of eperisone in human plasma by gas chromatography–mass spectrometry, J. Chromatogr. 584 (1992) 261–266. [18] T. Saito, T. Yamagiwa, Y. Yui, S. Miyazaki, A. Nakamoto, A. Namera, S. Inokuchia, Monolithic spin-column extraction and GC–MS method for the assay of eperisone in human serum, J. Health Sci. 56 (2010) 598–605. [19] L. Ding, X. Wang, Z. Yang, Y. Chen, The use of HPLC/MS, GC/MS, NMR, UV and IR to identify a degradation product of Eperisone Hydrochloride in the tablets, J. Pharm. Biomed. Anal. 46 (2008) 282–289. [20] Y. Song, A. Shi, L. Song, Bioequivalence of Eperisone Hydrochloride tablets in healthy volunteers, Chin. Pharm. J. 35 (2000) 186–189. [21] X. Wei, L. Ding, J.-M. Gao, J. Li, S.-O. Zhang, J.-P. Shen, Y.-D. Zhang, Pharmacokinetics and bioequivalence of Eperisone Hydrochloride tablet in healthy subjects, Yaoxue Xuebao 39 (2004) 309–311. [22] A.J. Cumming, M.L. King, B.K. Martin, A kinetic study of drug elimination: the excretion of Paracetamol and its metabolites in man, Br. J. Pharmacol. Chemother. 29 (1967) 150–157. [23] A. Trettin, J. Jordan, D. Tsikas, LC–MS/MS analysis of uncommon Paracetamol metabolites derived through in vitro polymerization and nitration reactions in liquid nitrogen, J. Chromatogr. B 966 (2014) 171–178. [24] A.P. Dewani, P.G. Shelke, R.L. Bakal, S.S. Jaybhaye, A.V. Chandewar, S. Patra, Gradient HPLC-DAD determination of Paracetamol, phenylephrine hydrochloride, cetirizine in tablet formulation, Drug Res. 64 (2014) 251–256. [25] B. Mohammed Ishaq, K. Vanitha Prakash, G. Krishna Mohan, Development and validation of RP-HPLC method for simultaneous estimation of tapentadol and Paracetamol in bulk drug and its pharmaceutical dosage form, Res. J. Pharm. Technol. 7 (2014) 208–212. [26] P. Wiczling, W. Struck-Lewicka, T. Kubik, D. Siluk, M.J. Markuszewski, R. Kaliszan, The simultaneous determination of hydrophobicity and dissociation constant by liquid chromatography–mass spectrometry, J. Pharm. Biomed. Anal. 94 (2014) 180–187. [27] S. Azodi-Deilami, A.H. Najafabadi, E. Asadi, M. Abdouss, D. Kordestani, Magnetic molecularly imprinted polymer nanoparticles for the solid-phase extraction of Paracetamol from plasma samples, followed its determination by HPLC, Microchim. Acta 181 (2014) 1823–1832. [28] S.G. Khanage, P.B. Mohite, S. Jadhav, Development and validation of UV–vis spectrophotometric method for simultaneous determination of Eperisone and Paracetamol in solid dosage form, Adv. Pharm. Bull. 3 (2013) 447–451. [29] S. Persiani, E. Roda, L.C. Rovati, M. Locatelli, G. Giacovelli, A. Roda, Glucosamine oral bioavailability and plasma pharmacokinetics after increasing doses of cristalline glucosamine sulfate in man, Osteoarthr. Cartil. 13 (2005) 1041–1049. [30] A. Roda, L. Sabatini, A. Barbieri, M. Guardigli, M. Locatelli, F.S. Violante, L.C. Rovati, S. Persiani, Development and validation of a sensitive HPLC-ES-MS/MS method for the direct determination of glucosamine in human plasma, J. Chromatogr. B 844 (2006) 119–126. [31] S. Persiani, R. Rotini, G. Trisolino, L.C. Rovati, M. Locatelli, D. Paganini, D. Antonioli, A. Roda, Synovial and plasma glucosamine concentrations in osteoarthritic patients following oral crystalline glucosamine sulphate at therapeutic dose, Osteoarthr. Cartil. 7 (2007) 764–772. [32] E. Pastorini, M. Locatelli, P. Simoni, G. Roda, E. Roda, A. Roda, Development and validation of a HPLC-ESI-MS/MS method for the determination of 5aminosalicylic acid and its major metabolite N-acetyl-5-aminosalicylic acid in human plasma, J. Chromatogr. B 872 (2008) 99–106. [33] C. Parolini, S. Caligari, D. Gilio, S. Manzini, M. Busnelli, M. Montagnani, M. Locatelli, E. Diani, F. Giavarini, D. Caruso, E. Roda, A. Roda, C.R. Sirtori, G. Chiesa, Reduced biliary sterol output with no change in total faecal excretion in mice expressing a human apolipoprotein A-I variant, Liver Int. 32 (2012) 1363–1371. [34] C. Celia, E. Trapasso, M. Locatelli, M. Navarra, C.A. Ventura, J. Wolfram, M. Carafa, V.M. Morittu, D. Britti, L. Di Marzio, D. Paolino, Anticancer activity of liposomal bergamot essential oil (BEO) on human neuroblastoma cells, Colloid Surf. B 112 (2013) 548–553. [35] CDER and CVM Guidance for Industry, Bioanalytical Method Validation. Food and Drug Administration, May 2001, http://www.fda.gov\cder\ guidance\4252fnl.pdf [36] International Conference on Harmonisation of technical requirements for registration of pharmaceuticals for human use. Harmonised Tripartite Guideline: Validation of Analytical Procedures: Text and Methodology, ICH Q2(R1), 2005.

Please cite this article in press as: M. Locatelli, et al., Simultaneous determination of Eperisone Hydrochloride and Paracetamol in mouse plasma by high performance liquid chromatography-photodiode array detector, J. Chromatogr. A (2015), http://dx.doi.org/10.1016/j.chroma.2015.02.008

G Model CHROMA-356266; No. of Pages 8 8

ARTICLE IN PRESS M. Locatelli et al. / J. Chromatogr. A xxx (2015) xxx–xxx

[37] I. Taverniers, E. Van Bockstaele, M. De Loose, Trends in quality in the analytical laboratory. II. Analytical method validation and quality assurance, Trend Anal. Chem. 23 (2004) 535–552. [38] T. Gicquel, J. Aubert, S. Lepage, B. Fromenty, I. Morel, Quantitative analysis of acetaminophen and its primary metabolites in small plasma volumes by liquid chromatography–tandem mass spectrometry, J. Anal. Toxicol. 37 (2013) 110–116. [39] R.N. Rao, S. Satyanarayana Raju, Enantioselective separation and simultaneous determination of tolperisone and eperisone in rat plasma by LC–MS/MS, Chirality 25 (2013) 622–627. [40] H. Li, C. Zhang, J. Wang, Y. Jiang, J.P. Fawcett, J. Gu, Simultaneous quantitation of Paracetamol, caffeine, pseudoephedrine, chlorpheniramine and cloperastine in human plasma by liquid chromatography–tandem mass spectrometry, J. Pharm. Biomed. Anal. 51 (2010) 716–722. [41] M.P. Patel, S.A. Varnum, D. Gandla, M.J. Zdilla, C.J. Martoff, Stable tetramethyl-1,10-phenanthroline osmium(III) complex in neutral pH as a photoluminescence following electron-transfer reagent for the detection of acetaminophen in urine and pharmaceutical formulations, Anal. Methods 6 (2014) 5818–5829. [42] M. Abdel-Rehim, Recent advances in microextraction by packed sorbent for bioanalysis, J. Chromatogr. A 1217 (2010) 2569–2580.

[43] A. Mollica, F. Pinnen, R. Costante, M. Locatelli, A. Stefanucci, S. Pieretti, P. Davis, J. Lai, D. Rankin, F. Porreca, V.J. Hruby, Biological active analogues of the opioid peptide Biphalin: mixed ␣/␤3 -peptides, J. Med. Chem. 56 (2013) 3419–3423. [44] G. Carlucci, F. Selvaggi, S. Sulpizio, C. Bassi, M. Carlucci, R. Cotellese, V. Ferrone, P. Innocenti, M. Locatelli, Combined derivatization and highperformance liquid chromatography with fluorescence and ultraviolet detection for simultaneous analysis of Octreotide and Gabexate Mesylate metabolite in human pancreatic juice samples, Biomed. Chromatogr. (2015), http://dx.doi.org/10.1002/bmc.3373 (in press). [45] S.A. Helmy, H.M. El-Bedaiwy, Simultaneous determination of Paracetamol and methocarbamol in human plasma By HPLC using UV detection with time programming: application to pharmacokinetic study, Drug Res. 64 (2014) 363–367. [46] N. Uchadadiya, F. Mehta, P. Sanchaniya, HPTLC-densitometric analysis of Eperisone Hydrochloride and Paracetamol in their combined tablet dosage form, Chromatogr. Res. Int. 2013 (2013) (article ID 464796). [47] Y. Zhang, M. Huo, J. Zhou, S. Xie, PKSolver: an add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel, Comput. Methods Progr. Biomed. 99 (2010) 306–314.

Please cite this article in press as: M. Locatelli, et al., Simultaneous determination of Eperisone Hydrochloride and Paracetamol in mouse plasma by high performance liquid chromatography-photodiode array detector, J. Chromatogr. A (2015), http://dx.doi.org/10.1016/j.chroma.2015.02.008