Determination and Pharmacokinetics of Ibuprofen in Rabbit Plasma by GC-MS Method
Bilal Yilmaz1* and Vedat Akba2
1Department of Analytical Chemistry, Faculty of Pharmacy, Ataturk University, 25240, Erzurum, Turkey 2Criminal Police Laboratory, 25060, Erzurum, Turkey
*Corresponding Author E-mail: yilmazb@atauni.edu.tr
ABSTRACT:
This paper describes a gas chromatography-mass spectrometry (GC-MS) method for determination of ibuprofen in rabbit plasma. Ibuprofen and internal standard (IS) naproxen were extracted from plasma by using liquid-liquid extraction method. The samples were separated by GC on a DB-5MS analytical column and determined by a quadrupole mass spectrometer detector operated under selected ion monitoring mode (SIM). Excellent linearity was found between 0.05 and 5.0 μg/mL (r=0.998) for plasma samples. Intra-day and inter-day precisions expressed as the relative standard deviation (RSD) for the method were 3.29-5.14% and 4.26-5.84%, respectively. The mean recovery of ibuprofen samples was 92.03%. This assay was successfully applied to a pharmacokinetic study of ibuprofen in New Zealand white rabbits. As a result, the plasma half-life was 2.68 ± 0.920 h and the mean AUC0-12 h was 187.24 ± 22.42 μg/mL h. The maximum plasma concentration (Cmax) of 59.8 ± 7.416 µg/mL reached 2.0 ± 0.288 h after administration.
INTRODUCTION:
Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most commonly prescribed agents worldwide to treat a variety of pain-related conditions, including arthritis and other rheumatic diseases. In addition, epidemiological studies have shown that long-term use of NSAIDs reduces the risk of developing Alzheimer’s disease and delays its onset1-.3.
Ibuprofen, 2-(4-isobutylphenyl)propionic acid, is a non-steroidal anti-inflammatory, analgesic and antipyretic drug. It is extensively used in the treatment of acute and chronic pain and many rheumatic and musculoskeletar disorders4.
Several methods have been reported for determination of ibuprofen including high-performance liquid chromatography (HPLC)5-13, LC-MS-MS14 and capillary electrophoresis (CE)15-17 in biological samples.
In addition, no method is reported till date for determination of ibuprofen by GC-MS from rabbits which had been given ibuprofen. Therefore,
this report describes a simple and specific GC procedure with MS detection for determining ibuprofen in rabbit plasma..
The developed method was validated by using linearity, stability, precision, accuracy and sensitivity parameters according to International Conference on Harmonization (ICH) guidelines18
The advantages of present method include simple and single step extraction procedure using inexpensive chemicals and short run time. Also, this method was used to assay the ibuprofen in plasma samples obtained from three rabbits which had been given an oral tablet of Artril tablet (600 mg ibuprofen).
MATERIALS AND METHODS:
Ibuprofen and naproxen were obtained from Sigma (St. Louis, MO, USA). N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), ethylacetate, hexane and acetonitrile were purchased from Sigma-Aldrich (St. Louis, MO, USA). Artril tablet (600 mg ibuprofen) was obtained Eczacıbaşı Pharmaceutical Industry (Istanbul, Turkey).
GC-MS System:
Chromatographic analysis was carried out on an Agilent 6890N gas chromatography system equipped with 5973 series mass selective detector, 7673 series autosampler and chemstation (Agilent Technologies, Palo Alto, CA). HP-5 MS column with 0.25 μm film thickness (30 m × 0.25 mm I.D., USA) was used for separation. Splitless injection was used and the carrier gas was helium at a flow rate of 1 mL/min. The injector and detector temperatures were 250°C. The MS detector parameters were transfer line temperature 280°C, solvent delay 3 min and electron energy 70 eV.
Preparation of the Stock and Standard Solutions:
The stock solution of ibuprofen (1 mg/mL) was prepared and diluted with acetonitrile to give standard solutions of 0.05-5.0 μg/mL (0.05, 0.2, 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 μg/mL). Standard calibration samples were prepared daily by spiking 0.5 mL of drug-free rabbit plasma with 0.1 mL of appropriate ibuprofen standard solutions to achieve final concentrations of 0.05-5.0 μg/mL for plasma. The working solution of IS was prepared by dissolving in acetonitrile to obtain a concentration of 1.0 μg/mL. The quality control (QC) samples were separately prepared at the concentrations of 0.1, 1.5 and 4.5 μg/mL for plasma.
Derivatization and Sample Preparation:
MSTFA is an effective trimethylsilyl (TMS) donor. MSTFA reacts to replace labile hydrogens on a wide range of polar compounds with a TMS group and is used to prepare volatile and thermally stable derivatives for GC-MS19. To increase the performance of the gas chromatographic separation, ibuprofen and IS were derivatized using MSTFA (Figure 1). The hydroxy (-OH) groups were converted to the corresponding silyl (-O-TMS) groups.
Blood samples were collected into the tubes containing disodium EDTA and centrifuged at 4500 × g for 10 min. A 0.5 mL of the resultant plasma samples were spiked with 0.1 mL of ibuprofen, 0.1 mL of internal standard and 0.5 mL H3PO4 solutions were added. After vortex mixing for 5 s, 4 mL of ethylacetate and hexane was added (2:3, v/v), the mixture was vortexed for 30 s and then centrifuged at 3000×g for 3 min. The organic layer was transferred into another tube and evaporated to dryness at room temperature under nitrogen gas. The dry residue was dissolved in 100 µL of a mixture of acetonitrile and MSTFA (50:50, v/v). The mixture was vigorously shaken and then delayed at room temperature for 10 min. 1 µL sample was injected into the GC-MS system.
Rabbits:
The study was conducted in accordance with the Animal Ethical Guidelines for Investigations in Laboratory Animals and was approved by the Ethical Committee for Medical Experimental Research and Application Centre of Ataturk University. The rabbits are male which is 4.8-5.2 kg weight. The rabbits were housed with free access to food and water, except for the final 2 h before experiment. After a single oral administration of 600 mg of ibuprofen (Artril tablet), 1.5 mL of blood samples were collected from the marginal ear vein at 0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, 10.0, and 12 h time-points into EDTA collection tubes. The blood samples were centrifuged at 4000 rpm for 10 min and the plasma was taken and stored at -20 °C until analysis.
Pharmacokinetic Analysis:
The maximum plasma concentration (Cmax) and the time to reach maximum concentration (Tmax) were directly determined from the plasma concentration versus time curves. The area under the curve from 0 to t (AUC0-t) was calculated by the linear trapezoidal rule. The area under the curve from 0 h to infinity (AUC0-∞) was estimated by summing the area from 0 to t (AUC0-t) and t to infinity (AUCt-∞), where AUCt-∞ = Ct/Kel, with Ct defined as the last measured plasma concentration at time t, and kel the slope of the terminal portion of the ln(plasma concentration) versus time curve. The elimination half-life (t1/2) was calculated using the pharmacokinetic relationship t1/2 = ln(2)/kel20.
Method Development and Optimization:
Ibuprofen is a polar molecule so a capillary column coated with 5% phenyl and 95% dimethylpolysiloxane was used separation. The injection port and detector temperature was set to 250°C. Different temperature programs were investigated to give an optimum temperature program as follows; initial temperature was 150°C, held for 1 min, increased to 220°C at 20°C/min held for 1 min, and finally to 300°C at 10°C/min with a final hold of 1 min. The injector volume was 1 mL in splitless mode.
To confirm the complete derivatization of ibuprofen and IS each compound was derivatized and analyzed separately. After establishing the optimum reaction conditions, the compounds were mixed together and then derivatized in order to perform a simultaneous analysis.
The effects of time and temperature on the reaction were investigated. Therefore, ibuprofen and IS were dissolved in acetonitrile. To 100 µL of 2.0 μg/mL ibuprofen solution and 100 µL of MSTFA solution were added and reacted at room temperature, 50 and 75°C for 5, 10 and 20 min. The resulting samples were quantitated by GC-MS system. After standing for 10 min at room temperature, maximum peak areas were quantitated.
Specificity and Separation:
It was determined that liquid-phase extraction process was necessary at the sample preparation procedure. Several solvents (ethylacetate, hexane, dichloromethane, acetonitrile, butanol and chloroform) were tested for the extraction. Finally, ethylacetate and hexane mixture (2:3, v/v) proved to be the most efficient in extracting ibuprofen from rabbit plasma. Following this procedure, the samples were derivatizated with MSTFA.
The fragment ions (m/z 73 and 185) were used for quantitation of ibuprofen and IS in SIM mode. The retention times of ibuprofen-TMS and IS-TMS derivatives were 4.5 and 7.7 min, respectively and the total run time of analysis was 8 min. Representative chromatograms of (a) drug-free plasma, (b) the plasma spiked with ibuprofen (0.5 μg/mL) and IS (1.0 μg/mL), (c) the rabbit plasma obtained at 1 h after a single dose of 600 mg ibuprofen was given in Figures 2. There is no interference in the chromatogram of drug-free plasma.
Figure 1. MS spectra after deriatization of ibuprofen (a) and naproxen (IS) (b) with MSTFA.
Figure 2. Representative chromatograms of (a) drug-free plasma, (b) the rabbit plasma spiked with ibuprofen (0.5 μg/mL) and IS (1.0 μg/mL), (c) the rabbit plasma obtained at 1 h after a single oral dose of 600 mg ibuprofen.
Commonly prescribed drugs (carvedilol, nebivolol, atenolol, metoprolol, mexiletine, rofecoxib, medazepam, diazepam, disulfiram, estradiol valerate and medroxyprogesterone acetate) were analysed for possible interference. No interference was observed under the chromatographic conditions.
Calibration and Linearity:
Calibration curve was linear over the range 0.05-5.0 μg/mL for plasma The regression equation was as follows:
A = 0.2984C - 0.0264 (r = 0.998) for plasma, where A is the peak area ratio (Aibuprofen/AIS) and C is the concentration of ibuprofen (μg/mL).
Recovery:
As shown in the Table 1, the mean recovery of ibuprofen was 92.03% for plasma. The mean recovery of the internal standard was found to be 88.27% for plasma.
Table 1: Recovery of ibuprofen in plasma (n=6)
|
Sample
|
Concentration (µg/mL) |
Recovery %
|
RSD%
|
|
|
Added |
Found (Mean ± SD) |
|||
|
|
0.1 |
0.093 ± 0.003 |
93.0 |
3.22 |
|
Plasma |
1.5 |
1.36 ± 0.048 |
90.7 |
3.53 |
|
|
4.5 |
4.16 ± 0.234 |
92.4 |
5.63 |
Precision and Accuracy:
The values of precision and accuracy of ibuprofen are summarized in Table 2. Intra-day and inter-day relative standard deviation (RSD) values were found within 5.14% and 5.84% for plasma. The results were determined analysing the samples spiked with ibuprofen at three different concentrations. Accuracy of the method expressed as relative error (RE) was below 12.00%.
Table 2: Intra-day and inter-day precision and accuracy of ibuprofen in plasma (n=6)
|
|
Concentration (µg/mL) |
RSD% |
RE% |
|
|
Sample |
Added |
Found (Mean ± SD) |
|
|
|
Plasma |
0.1 |
0.091 ± 0.003 |
3.29 |
9.00 |
|
Intra-day |
1.5 |
1.32 ± 0.051 |
3.86 |
12.00 |
|
|
4.5 |
4.03 ± 0.207 |
5.14 |
10.44 |
|
Inter-day |
0.1 |
0.094 ± 0.004 |
4.26 |
6.00 |
|
|
1.5 |
1.43 ± 0.062 |
4.33 |
4.67 |
|
|
4.5 |
4.21 ± 0.246 |
5.84 |
6.44 |
Sensitivity:
The limit of quantification values for each sample were accepted as the lowest concentration on the calibration curves for 0.05 μg/mL. Under the experimental conditions, the limit of detection value was 0.015 μg/mL for plasma with a signal to noise ratio 3.
Stability:
The derivative of ibuprofen-TMS was stable in this solvent for at least 48 h at 4°C. The stability of stock solutions of ibuprofen in acetonitrile was checked and proved to be stable for at least 1 week at 4°C. The stabilities of ibuprofen and IS in a biological fluid are affected by the chemical properties, the storage conditions and the matrix effects. The stability of ibuprofen under various conditions is described in Table 3. Under all conditions tested, ibuprofen was stable with detected concentrations of at least 86.27% for plasma samples of the initial concentration.
Table 3: Stability of ibuprofen in plasma (n=3)
|
Treatment
|
Recovery (Mean ± SD) |
||
|
Plasma concentration (µg/mL) |
|||
|
0.1 |
1.5 |
4.5 |
|
|
Three freeze-thaw cycles |
92.24 ± 3.124
|
88.14 ± 2.736
|
94.15 ± 2.064
|
|
Stored at RT for 24ha |
88.76 ± 2.635
|
92.47 ± 2.536
|
87.34 ± 2.567
|
|
Stored at -20 0C for 2 weeks |
87.46 ± 2.432
|
88.34 ± 2.962
|
88.63 ± 2.863
|
|
Stored at -20 0C for 4 weeks |
86.27 ± 2.862
|
91.03 ± 2.086
|
86.79 ± 2.062
|
a RT, room temperature
Pharmacokinetic Study:
To check the applicability of the method, the pharmacokinetic parameters of ibuprofen were investigated in three rabbits after a single oral administration of 600 mg of the drug. The concentration-time profile is shown in Figure 3. The main pharmacokinetic parameters of ibuprofen were calculated and summarized in Table 4.
Figure 3: Mean plasma ibuprofen concentration-time profile for three rabbits after a single oral dose of ibuprofen, 600 mg.
Table 4: Mean pharmacokinetic parameters of ibuprofen for three rabbits after oral administration of Artril tablet (600 mg)
|
Parameters |
(Mean ± SD) |
RSD% |
|
Maximum plasma concentration |
59.8 ± 7.416 |
12.40 |
|
Cmax (µg/mL) |
|
|
|
Time required for maximum plasma concentration (Tmax) |
2.0 ± 0.288
|
14.40
|
|
Area under curve |
187.24 ± 22.42 |
11.97 |
|
AUC(0-12 h) (µg/mL h) |
|
|
|
Area under curve at infinite time |
217.76 ± 37.14 |
17.06 |
|
AUC(0–∞) (µg/mL h) |
|
|
|
Plasma half life(T1/2) (h) |
2.68 ± 0.920 |
34.32 |
DISCUSSION:
Today, GC-MS is a powerful technique for highly specific and quantitative measurements of low levels of analytes in biological samples. As compared to HPLC, high-resolution capillary GC has been less frequently used20.
During method development, it became evident that ibuprofen and IS were very sensitive to matrix effects during the derivatization process in plasma and urine. Sample preparation techniques, such as liquid-liquid extraction was used in order to minimise matrix suppression effects.
GC-MS method sensitivity is not enough for the determination of ibuprofen in plasma. For this reason, MSTFA was chosen as a chromagenic derivatization reagent. In this study, the purpose of the derivatization reaction is the raise of sensitivity thus the possibility of working in low concentrations has been occurred.
When this method is applied to plasma samples, its sensitivity was found to be adequate for pharmacokinetic studies. The present method has the following advantages over the reported method6,7,11. CE methods are a little simpler and faster with respect to the deproteinization step and analysis time. But the reported detection limits of 1 µg/mL16 and 8 µg/mL17 are not sensitive enough for the pharmacokinetics studies of the drug in vivo.
Calibration curve of ibuprofen was linear over the concentration range of 0.5-40 µg/mL for plasma which is as good as or superior to that reported in other papers5,7,9,12,14,15,17.
Ibuprofen was extracted from plasma with a solid phase extraction procedure by Farrar et al.12. This method is also the most comprehensive method which can extract ibuprofen in a single extraction procedure. In this study, the recovery percentage of ibuprofen is high12,14,17, extraction processes do not take much time6-8; additionally, the retention time is short which is an advantage5,13-15.
Bonato et al.14 have reported LC method with tandem mass detection for the analysis of ibuprofen in plasma. The calibration curve of LC-MS-MS method was linear for ibuprofen in the range 0.12-190 μg/mL. Intra- and inter-day precision values were lower than 15%. The maximum recovery of ibuprofen was 73.9%. The LOQ of method was found 0.12 μg/mL. Detection using LC-MS-MS would be a more sensitive approach but is costly and not yet available for every laboratory.
In statistical comparison (p > 0.05) with other methods in the literature5,6,12,13,15 the proposed method has indicated high accuracy and recovery.
In conclusion, a simple and sensitive GC-MS method has been developed for the determination of ibuprofen in rabbit plasma. The method was completely validated by using sensitivity, stability, specificity, linearity, accuracy and precision parameters for determination of ibuprofen in plasma. Additional advantages of this method include small sample volume (0.5 mL), good extraction recovery from plasma and a readily available internal standard. Also, the extraction and derivatization procedures in this study were simple. Therefore, the method can be very useful and an alternate to performing pharmacokinetic studies in determination of ibuprofen for clinical use.
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Received on 28.12.2009 Modified on 17.02.2010
Accepted on 18.08.2010 © RJPT All right reserved
Research J. Pharm. and Tech. 4 (1): January 2011; Page 52-56