Mohammed E. Abdel-Hamid1, Leyla H. Sharaf1*, Oludotun A. Phillips1, Elijah O. Kehinde2, Alice Babu2
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
2Department of Surgery, Faculty of Medicine, Health Sciences Centre, Kuwait University, Kuwait.
*Corresponding Author E-mail: ls20504@hsc.edu.kw
ABSTRACT:
This work aims to the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring ciprofloxacin (CIP) in rabbits serum following intra-gastric dosing and application of the developed method for pharmacokinetic studies of CIP released from six generic and one reference products. A positive electrospray tandem mass spectrometry coupled with liquid chromatography was used. Chromatographic analysis was done using of XTerra® RP C8 column and a mobile phase of acetonitrile/water/formic acid (80:20:0.025v/v/v). A simple protein precipitation method using acetonitrile was used for cleaning up the serum samples before LC/MS/MS analysis. Quantification was achieved using multiple reaction monitoring (MRM) at m/z 332.7>245.5 (CIP) and m/z 364.7>263.2 (rufloxacin, internal standard). The pharmacokinetics of the absorbed CIP fractions from the examined brands were calculated using PK-software for non-compartmental pharmacokinetics data analysis. The developed LC-MS/MS method showed a good linearity (r≥ 0.99), precision (RSD%<6.1%) and accuracy (Bias%<8.5%)over the CIP concentration range 0.5-6µg/ml in rabbits serum. Stability studies indicated that CIP serum samples were stable for at least 4 month sat -35oC.The calculated pharmacokinetic parameters were in the ranges: Cmax 1.1-6.0 µg/ml, Tmax 0.5-1.0 hr , AUC0-t5.3-16.0 µg.hr/ml and the % relative bioavailability was from 35.6 to 77.5%. The developed LC-MS/MS method was successful for measuring CIP levels in rabbits serum following intra-gastric dosing. The proposed route of drug administration permits an accurate drug placement and collection of serum samples. Pharmacokinetic studies revealed some differences of the absorption patterns of the generic and reference products which reflects variation of the antibacterial efficacy among CIP brands.
KEYWORDS: Ciprofloxacin; LC-MS/MS; rabbits serum; pharmacokinetics study
INTRODUCTION
Fluoroquinolones (FQs) are synthetic broad-spectrum antibacterial agents derived from nalidixic acid. FQs are essentially used for treating a variety of bacterial infections such as respiratory and urinary tract infections including prostatitis. The mode of action of FQs is postulated to be through inhibition of bacterial gyrase, an enzyme involved in DNA replication, recombination and repair. Generally, FQs are well tolerated, however, adverse drug reactions such as gastric and photo sensitivity reactions and risk of tendon rupture complications are often observed1,2. CIP (Figure 1) is frequently prescribed against several bacterial infections for treatment and prophylactic purposes.
In clinical practice in Kuwait, CIP is prescribed as a prophylactic antibiotic during trans-rectal ultrasound guided biopsy of the prostate (TRUSGBP) at the urological unit. Generally, a prophylactic oral dose of 500 mg, one day before and three days after biopsy, was prescribed to avoid bacterial infections3.Between year2001 and 2005, even though the recommended CIP dosage regimen was given, infectious complications after TRUSGBP rose from 1.6% to 13.6%3. The pathogenic bacteria responsible for about 85% of the infectious complications was due to CIP resistant Escherichia coli. Consequently from the year 2006to2011, the prophylactic dose was modified by inclusion of an additional IV dose of 500 mg of amikacin3.Following this dose modification, the infectious complication rates remained less than 2% annually. Although the problem was attributed to bacterial resistance to CIP3, it was also noted that many CIP generic products other than the branded product were clinically used.
This may suggest that the bioavailability and the therapeutic efficacy of the alternative generic products might also contribute to this problem. This work reports on the development of a rapid and specific LC-MS/MS method for measurement of CIP in rabbits serum and the utility of the method in evaluating the efficacy of selected generic brands in comparison to a reference brand of CIP.
CIPROFLOXACIN (MW= 331.4)
RUFLOXACIN (IS) (MW= 363.0)
Fig. 1 : Chemical structures of ciprofloxacin (CIP) and rufloxacin (IS)
MATERIALS AND METHODS:
Chemicals and reagents
Powders of ciprofloxacin (CIP) and rufloxacin (RUF) as an internal standard (IS) were kindly donated by local pharmaceutical companies and were used without further purification. Drug free rabbits serum samples were supplied by Prof. Elijah O. Kehinde, Department of Surgery, Faculty of Medicine, Kuwait University and were stored in PVC tubes at -35oC. All solvents were of HPLC grade and the reagents were of analytical grade. Milli-Q purified water (Millipore, Bedford, MA, USA) was used throughout this work.
Instruments and conditions
A triple quadrupole tandem mass spectrometer (Quattro LC, Micromass, UK), fitted with positive electrospray ionization probe was used. The system was coupled with Waters Alliance 2690 LC and Waters Auto-sampler (Waters Association, Milford, MA, USA).The tuning parameters(Capillary voltage, cone voltage and collision energy) were optimized at 3.5 kV, 25 V and 25 eV, respectively. The source and desolvation temperatures were adjusted at100oC and 250oC, respectively. Quantification was achieved by internal standard calibration using Multiple Reaction Monitoring (MRM) transitions at m/z 332.7 > 245.5 and m/z 364.7 > 263.2 for CIP and IS, respectively. System operation and data acquisition were controlled by MassLynx software. The chromatographic analyses were conducted using XTerra® RP C8 column (250 mm x4.6 mm ID, 5µm particle size) and a mobile phase of acetonitrile-water-formic acid in a ratio of 80:20:0.025 v/v/v. The flow rate of the mobile phase was 0.4 mlmin-1 and the run-time was five minutes.
Standard solutions and calibration curves
Stock solutions (1 mgml-1) of CIP and IS were separately prepared in methanol/water (80/20). An aliquot of CIP stock solution was diluted with water to give a working solution of 0.1 mgml-1. On the other hand, the IS stock solution was further diluted with acetonitrile to yield a working solution of 0.2 mgml-1. The calibration curves of CIP were prepared by spiking drug-free rabbits sera with CIP solution to give a concentration range0.5-6µgml-1. Similarly, quality control samples were prepared in rabbits serum at concentrations 2, 4, 6 µgml-1.
Assay procedure
In 1.5 ml-Eppendorf tube, a 100 µl aliquot of CIP-spiked serum sample was mixed with 20 µlaliquot of IS solution. An aliquot of 300 µl of acetonitrile was added as a protein precipitant. The mixture was mixed by vortex for 30 seconds and then centrifuged at 980 g for 10 min. The clear supernatant was transferred to a glass tube and the solvent was evaporated under N2 gas at 70oC. The residue was reconstituted in 80 µl of the mobile phase and a 20 µl aliquot of clear solution was injected into LC-MS/MS. A 100 µl aliquot of rabbits serum sample was similarly treated without adding the IS solution.
Method Validation:
Linearity: The linearity was established by calculating the means of slopes and correlation coefficients of at least 10 duplicate calibration curves over the concentration range 0.5-6 µg ml-1 of ciprofloxacin in serum using least squares linear regression analysis.
Limit of quantification (LLQ):
The lower limit of quantification (LLQ) was determined on the basis of the lowest concentration of ciprofloxacin in spiked rabbit serum samples that produces RSD% and %DEVs < 25%.
Precision and accuracy:
Precision and accuracy of the developed method were established by analyzing QC rabbits serum samples spiked with CIP at low (0.5µg ml-1) and high (6 µg ml-1) concentrations. The percentage relative standard deviation (RSD%) and percentage deviation from nominal concentrations (DEV%) were calculated in one day to assess the intra-day precision and accuracy, respectively. In addition, the inter-day precision and accuracy were also measured over a period of three weeks.
Recovery studies:
QC rabbit serum samples spiked with CIP at concentrations 2, 4, 6 µg ml-1 were analyzed by the described assay procedure. Recoveries of CIP were computed by comparing the found and added concentrations.
Stability studies:
The serum samples were stored at -35oC for ~ 4 months. The remaining concentrations of CIP expressed as log % drug remaining were calculated at monthly intervals and plotted versus time to establish the stability profiles. The values of the degradation rate constant (Kdeg) were determined from the negative slopes of the stability curves, whereas the degradation half-lives (t½) were determined from the formula 0.693/kdeg.
Intra-gastric administration of CIP brands in rabbits
Mature male New Zealand white (NZW) rabbits (>1 year old, weight>3 kg) were used. The animals were provided and cared for according to guidelines provided by the Kuwait University Animal Resources Centre, Health Sciences Centre, Kuwait University, Kuwait. The animal experimental protocol was approved by the Kuwait University Animal Ethics Committee. NZW rabbits were anaesthetized by IV administration of Ketamine HCl and Xylazine mixture at the appropriate dose. The stomach of the animal was exposed via amidline anterior abdominal wall incision. The calculated dose of each CIP brand (56 mg /kg in 5 ml water) was directly injected into the gastric cavity below the oesophagogastric junction under direct vision(Figure2).The wounds were closed and the animal was left to recover. Aliquots of ~2 ml of the venous blood were collected via ear veins at the time-intervals 0, 15 min, 30 min, 1 hr, 2 hr, 3 hr, and 6 hr in plain tubes. The serum layers were separated and stored at -35oC prior to analysis. A 100µl aliquot of the rabbit serum sample was similarly treated as above mentioned in the assay procedure. The concentration of CIP was automatically computed by the instrument quantifying program using weighing 1/x least squares linear regression analysis.
Fig. 2 : Intra-gastric dosing of CIP to NZW rabbits
Serum concentration-time curves
The measured serum concentrations of CIP at different time intervals were used to establish the serum concentration-time profiles. The pharmacokinetic parameters such as Cmax, Tmax, AUC0-t, AUC0-„,Ke and t1/2were determined from the curves using PK-software for non-compartmental pharmacokinetics data analysis (PK Solutions 2, Summit, Research Services).
RESULTS AND DISCUSSION:
Underoptimized mass spectrometer tuning parameters, CIP and RUF (IS) were detected at the molecular mass ions m/z 332.7 and 364.7, respectively. In daughter (MS/MS) scan mode, CIP and IS produced the daughter mass ions at m/z 245.5 and 263.2, respectively(Figure 3). The MRM transitions at m/z 332.7>245.5 for CIP and 364.7>263.2 for IS were selected for the quantification of CIP in rabbits serumusing internal standard calibration. A preliminary protein precipitation procedure using acetonitrile as a protein precipitant was used to clean up the rabbits serum samples from endogenous constituents before injection to mass spectrometer. This step was necessary to minimize the ion suppression and matrix effects in LC-MS/MS analyses. The composition of the mobile phase was examined to achieve good detection and resolution of the analytes at a short run-time. A mobile phase of acetonitrile/water/formic acid in a ratio 80/20/0.025 v/v/v was appropriate. The presence of formic acid improves the ionization and detection of analytes and the high percentage of organic modifier permits rapid elution of CIP and IS. The selected RP C8 column improves the peaks shape and helps the removal of trace amounts of serum constituents with subsequent elimination of the ion suppression effect.
Under the selected mass and chromatographic conditions, CIP and IS were rapidly eluted at retention time < 5 min. Figure 4 showed representative chromatograms of drug-free serum sample and a rabbits serum sample at 1 hour interval following animal dosing. Figure 4a displayed no interference of endogenous serum constituents at the MRM transitions of analytes, whereas Figure 4b exhibited a rabbits serum concentration of CIP at 3.16 µg ml-1.
a)
b)
Fig. 3. MS/MS scans of CIP (a) and IS (b)
a)
b)
Fig. 4. MRM chromatograms of drug-free rabbits serum sample (a) and rabbits serum sample at 1 hr interval following intra-gastric dosing of CIP brand (Ciflox®) (b).
The specificity of LC-MS/MS method was further proved by the absence of interferences of selected antibacterial agents such asrufloxacin, norfloxacin, gatifloxacin, nalidixic acid and linezolid. The linearity of LC-MS/MS was established by using internal standard calibration. As the utilization of isotope-labeled CIP as internal standard was not feasible, several compounds were tested. Rufloxacin(Figure 1) was found appropriate as the chemical structure and ionization patterns are relatively similar to CIP. Calibration curves in rabbits serum were constructed over the concentration range 0.5-6 µg ml-1 of CIP. Using
linear regression analysis, the average regression equation of five calibration curves was: y = -0.04 + 0.29 x using 1/x weighting with a correlation coefficient (r≥0.99), where y is the peak area ratio of CIP / IS and x is the concentration of CIP (µg ml-1). The lower limit of quantitation (LLQ) was 0.491 µg ml-1 (RSD%:4.07), whereas the lower limit of detection (LLD) was 0.204 µg ml-1(RSD%:14.71) (Table 1).The intra-day and inter-day precision and accuracy of LC-MS/MS method for determination of CIP at low, medium and high concentrations were appropriate, as indicated from RSD% values (Table2). Recovery studies of CIP from rabbits serum were in the range of 93.5-105.0 % (Table 2) indicating the appropriateness of the protein precipitation method for cleaning up the serum samples. Stability studies of refrigerated serum samples of CIP, indicated that the collected rabbits serum were stable at -35oC for at least 4 months. The degradation kinetics indicated that the degradation rate constant Kdeg and degradation half-life t1/2, were 0.033 month-1 and 21.0 months, respectively (Table 3).
Table 1: Lower limit of quantitation (LLQ) and lower limit of detection (LLD) for the determination of CIP in rabbits serum by LC-MS/MS
LLQ LLD
Nominal Found Bias% Nominal Found Bias%
Concentration mean Concentration mean
µg ml-1 µg ml-1 µg ml-1 µg ml-1
0.5 0.482 -3.6 0.2 0.20 0.0
0.517 +3.4 0.17 -15.0
0.510 +2.0 0.22 +10.0
0.469 -6.2 0.24 +20.0
0.479 -4.2 0.19 - 5.0
Mean ± SD 0.491 ± 0.02 0.204 ± 0.03
RSD%* 4.07 14.71
Bias%** -6.2 to +3.4 -15.0 to +20.0
*RSD%= [SD / Mean] x 100
** Bias%= [Found Concentration-Nominal Concentration] / [Nominal Concentration] x 100
Table 2: The intra-day and inter-day precision (RSD%), accuracy (DEV%) and recovery % for measurement of CIP in rabbits serum (n=5) by LC-MS/ MS
Nominal Found RSD%# DEV%* Recovery %
Concentration mean ± SD
µg ml-1 µg ml-1
Intra-day precision and accuracy
2.0 2.13 4.2 +3.0 to +6.5 105.0± 1.6
4.0 3.68 3.8 -7.0 to + 6.5 93.5± 0.5
6.0 6.19 1.1 +2.0 to +4.7 103.1± 1.1
Inter-day precision and accuracy
2.0 2.10 3.8 -2.5 to + 8.5 103.9± 4.2
4.0 3.80 3.9 +2.5 to 7.0 96.0±3.8
6.0 6.10 1.6 +2.3 to - 1.2 100.8±1.7
#RSD%= [SD / Mean] x 100
*DEV%= [Found Concentration-Nominal Concentration] / [Nominal Concentration] x 100
Table 3:Stability study of CIP in rabbits serum samples at -35 oC
Time (Month) Mean Found (n=5) % Remain* log%
Concentration, µg ml-1 Remain
0.0 6.0 100.0 2.0000
1.0 5.9 98.3 1.9926
2.0 5.8 96.7 1.9854
3.0 5.6 93.3 1.9698
4.0 5.4 90.0 1.9542
*% Remain = [Found Concentration at 1,2,3,4 months / Concentration at 0.0 time] x 100
The validated LC-MS/MS method was used to measure CIP concentrations in rabbits serum following intra-gastric dosing at a 56 mg of CIP brand/kg. This route ensures a complete placement of drug doses in the stomach and permits avoidance of possible drug losses as by oral route. Figure 5 showed the rabbits serum concentration-time curves for six generic and one reference brands of CIP following intra-gastric administration.
Fig. 5 :Rabbits serum concentration-time curves of seven brands of CIP products following intra-gastric dosing of NZW rabbits
The various pharmacokinetic parameters representing drug absorption such as Cmax, Tmax and AUC were calculated (Table 4). The computed parameters were in good agreement with the reported values 2. As shown in Table 4, the branded reference product (A) displayed Cmax,Tmax, AUC0-t and AUC0-„at 6µg/ml, 1 hr, 16 µg.hr/ml and 24.3 µg.hr/ml, respectively. The Cmax value of the generic brands (B, C, D, E, F and G) was in the range of 1.1-2.9 µg/ml and Tmax was between 0.5-1.0 hr. The AUC0-t values were in the range of 5.3 -12.4 µg.hr/ml, whereas the AUC0-„ values ranged from 7.7 to 21.7 µg.hr/ml. The pharmacokinetic parameters Ke and t½ representing drug elimination were in the ranges of 0.15-0.29 hr-1 and 2.43-4.73 hr, respectively.
Table 4: Pharmacokinetic (PK) parameters of CIP after a single intra-gastric dose (56 mg/kg) of CIP brands in NZW rabbits (n=3)
PK Parameter CIP Product
A B C D E F G
Mean (n=3)
Cmax (µg/ml) 6.0 2.9 1.1 1.3 1.5 1.6 1.3
Tmax (hr) 1.0 0.5 1.0 1.0 0.5 1.0 1.0
AUC0-t (µg.hr/ml) 16.0 12.4 5.3 5.5 6.7 6.5 5.7
AUC0-„ 24.3 21.7 8.9 7.7 13.9 10.1 9.7
MRT (hr) 4.8 6.5 5.9 5.6 8.0 5.2 6.0
Ke (1/hr) 0.29 0.18 0.23 0.18 0.15 0.27 0.21
t1/2 (hr) 2.43 3.81 3.08 3.79 4.73 2.62 3.26
Relative bioavailability %* -- 77.5 33.1 34.4 41.9 40.6 35.6
Relative bioavailability %** -- 89.3 36.6 31.7 57.2 41.6 39.9
A: Ciprofloxacin® ( Rambaxy, UK),B: Ciflox® (KSP, Kuwait),C: Ciprofar® (PPA, Egypt)
D: Ciprotab®(Fidson, India), E:Ciprofloxacin® (Reddy, USA),F: Ciprofloxacin® (EEPIA, Egypt),G: Ciprobay® (Bayer, Canada)
Percentages are made using AUC0-t *, AUC0-„ ** values
As reported in the literature, HPLC analyses using either ultraviolet or fluorescence detection were commonly used for measurement of CIP in biological samples4-10. Although the reported HPLC methods were accurate and precise, however, some of them were tedious, time consuming and required a complete resolution of CIP and IS. Recently, LC-MS/MSanalyses have been extensively used as alternatives of HPLC assays for measurement of quinolonesdue to marked sensitivity, selectivity and sample throughput11-15. In the this study, a rapid and specific positive electrospray LC-MS/MS method was developed for the determination of CIP in rabbits serum. The CIP concentrations were used to calculate the pharmacokinetic parameters representing the
rate and extent of drug absorption from CIP products. A newly proposed intra-gastric route of products administration was used to ensure a complete placement of CIP doses in the stomach and to avoid possible drug losses, however, the technique needs particular skill and experience. The pharmacokinetic studies indicated a percentage relative bioavailability for the generic products in the range of 33.1-77.5% based on AUC0-t and 31.7-89.3 % based on AUC0-„compared to reference product. These findings suggested that the antibacterial efficacy for the generic products was lower than the branded product. The results indicated that not only bacterial resistance to the antibiotic but also low product bioavailability should be considered. Moreover, the study shed light on the potential problems of the marketed substandard products, which are manufactured below acceptable quality control standards and therefore, may be dangerous to patient's health or ineffective for treatment or prophylaxis of bacterial infections16.
CONCLUSIONS:
A rapid and specific positive electrospray LC-MS/MS method was established for measurement of CIP concentrations in rabbits serum for pharmacokinetic studies. A newly intra-gastric dosing procedure of CIP products in rabbits was proposed. The reported study revealed that the regular use of substandard products of CIP can lead to therapeutic failure or drug resistance. Although the study was performed on animal models, and not on humans, it clearly showed that switching from abranded product to generic products may not be safe and it can lead to products efficacy deterioration. The newly described procedure of drug dosing together with LC-MS/MS method may be of interest for researchers in the areas of drug discovery and drug pharmacokinetic studies.
ACKNOWLEDGEMENT:
This study was supported by Kuwait University Research Grant MS 01/05.The excellent technical assistance of Ms. Alice Babu of dosing rabbits and collecting blood samples, was highly appreciated.
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Received on 08.02.2012 Modified on 23.02.2012
Accepted on 09.03.2012 © RJPT All right reserved
Research J. Pharm. and Tech. 5(5): May2012; Page 624-631