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Simultaneous quantification of nimesulide, phenylpropanolamine, caffeine and chlorpheniramine in rat plasma by RP–HPLC/PDA method and application to pharmacokinetic studies in healthy rat subjects
⁎Corresponding author. Tel.: +91 08702462662; fax: +91 870 2459547. pnr.nitw@gmail.com (Pothuraju Nageswara Rao)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Simultaneous determination of nimesulide, phenylpropanolamine, chlorpheniramine and caffeine in rat plasma by reversed-phase high performance liquid-chromatography (RP–HPLC) with photodiode array (PDA) detection method was developed and validated. Sample preparation based on a simple extraction procedure consisting of deproteination and extraction with methanol solution followed by volume make up with the aqueous component of the mobile phase obtained best recoveries of the analytes. The chromatographic conditions were optimized and the analytes were separated on XBridge™ C18 (3.5 μm, 4.6 × 150 mm) column in isocratic elution with the mobile phase composition of acetonitrile and 10 mM ammonium acetate buffer (pH 4.0, 0.1% formic acid) (18:82 v/v%) at the flow rate of 1 mL min−1 and the effluents were monitored in the wavelength range of 220–275 nm. The method was linear for all analytes over the following concentration (ng mL−1) ranges: nimesulide 250–4000; phenylpropanolamine 100–1500; chlorpheniramine 20–500; and caffeine 10–100. Acceptable precision, accuracy and recoveries were obtained for quality control (QC) samples at three concentrations (low QC, middle QC and high QC). The percentage of relative standard deviation (% RSD) of Inter and intra-run precision of all molecules was <15% and the percentage of accuracy was 100 ± 10. The analytes were more stable in rat plasma at different storage conditions. Finally the method was efficiently applied to pharmacokinetics study in rat plasma.
Keywords
Nimesulide
Phenylpropanolamine
Caffeine
Chlorpheniramine
Simultaneously
RP–HPLC
1 Introduction
Nimesulide (NMS) is [N-(4-Nitro-2-phenoxyphenyl) methane sulfonamide] a relatively cyclooxygenase-2 (COX-2) selective, non-steroidal anti-inflammatory drug (NSAID) with analgesic and antipyretic properties (Rainsford, 2006). Phenylpropanolamine (PPA) is [(1RS, 2SR)-2-amino-1-phenylpropan-1-ol] a sympathomimetic compound, which has been widely used as an over the counter (OTC) and prescription medication for cough, cold, and nasal decongestant (Flavahan, 2005). Chlorpheniramine (CPM) is [3-(4-chlorophenyl)-N, N-dimethyl-3-pyridin-2-yl-propan-1-amine] a first-generation alkylamine antihistamine used in the prevention of the symptoms of allergic conditions such as rhinitis and urticaria (Carlsson and Linqvist, 1969). Caffeine (CFN) [1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione 3,7-dihydro 1,3,7-trimethyl-1H-purine-2,6-dione] acts as a central nervous system stimulant, temporarily warding off drowsiness, mood elevator and restoring alertness (Nehlig et al., 1992). The combined dosage form of NMS, PPA, CPM and CFN is more effective in controlling common cold and severe allergic cases than individual drugs (Fig. 1). Diphenylpyraline (DPP) [4-benzhydryloxy-1-methyl-piperidine] was used as an internal standard (IS) for determination of all these drugs in spiked rat plasma samples. All the compounds are white, crystalline powders and polarity nature so easily soluble in polar solvents such as water, methanol, etc. Till to date no RP–HPLC method has been reported for the simultaneous determination of NMS, PPA, CPM and CFN in rat plasma. Several methods have been reported for the determination of NMS, PPA, CPM and CFN individually or combination with other drugs, such as HPLC with UV/PDA detection (Kumar et al., 2012; Nageswara Rao et al., 2005; Rolim et al., 2007 and Pavan Kumar et al., 2006) or fluorescence detection (Fu-Ming and Chen-Wen, 2005), Liquid Chromatography–Spectrophotometry (Hadad et al., 2007), Liquid chromatography–mass spectrometry (LC–MS) or Liquid chromatography-tandem mass spectrometry (LC–MS/MS) (Bao, 2006; Zou et al., 2008; Li et al., 2010 and Chen et al., 2004), Fourier transform-near infrared (FT–NIR) (Ajayakumar et al., 2012), Stripping Voltammetry (Furlanetto et al., 2000), Micellar Electrokinetic Capillary Chromatography (Dalmora et al., 2007), High performance thin layer chromatography (HPTLC) (Dhongle et al., 2011). The four analytes are highly different polarity nature and wide range of concentrations in plasma after an oral dose. This method is validated and has been successfully applied to pharmacokinetics study of compounds in six healthy Wistar rats.
2 Experimental conditions
2.1 Chemical and reagents
Nimesulide (NMS), phenylpropanolamine hydrochloride (PPA), chlorpheniramine maleate (CPM), caffeine (CFN) and diphenylpyraline (IS) (purity > 99% for all drugs) were gift samples from Shantha Biotechniques Ltd., Hyderabad, India. CureX tablets (containing 100 mg NMS, 12.5 mg PPA, 4 mg CPM and 30 mg CFN) were purchased from Mycure pharma Pvt. Ltd (Bangalore, India) (manufactured by Embiotic Laboratories (P) Ltd., Bangalore, India). HPLC grade acetonitrile and methanol (Sigma Aldrich, Mumbai, India) and analytical reagent grade (AR) ammonium acetate, acetic acid and formic acid (SD Fine Chemicals, Mumbai, India) (>99.5% purity) were used. Deionized water was prepared by using a Millipore synergy (Millipore, France).
2.2 Instrumentation
The HPLC system consisting of a quaternary LC-20AD pump, a SPD-M20A diode array detector, a SIL-20AC auto sampler, a DGU-20A5 degasser and CBM-20A communications bus module (all from Shimadzu, Kyoto, Japan) was used. The pH measurements were carried out by Elico, model LI 120, pH meter equipped with a combined glass–calomel electrode. The chromatographic and the integrated data were recorded using HP-Vectra (Hewlett Packard, Waldron, Germany) computer system using LC-Solution data acquiring software (Shimadzu, Kyoto, Japan). Separation and quantitation were made on an XBridge™ C18 column (150 × 4.6 mm I.d, 3.5 μm particle size) (Waters, Chromatographie technik GmbH, Germany).
2.3 Preparation standard and quality control (QC) solutions
All stock solutions of 1000 μg mL−1 of NMS, PPA, CPM and CFN were prepared independently with mobile phase. The solution was prepared by dissolving 100 mg of each drug in sufficient amount of mobile phase and the volume was completed to 100 mL volumetric flask with the same solvent. Similarly 1000 μg mL−1 of IS was prepared. From the stock solutions, working solutions (Table 1) were prepared by serial dilution of each sample. Three quality control samples of each drug were prepared in μg mL−1 such as 0.5, 1, 2 for NMS, 0.2, 0.6, 1 for PPA, 0.03, 0.12, 0.18 for CPM and 0.015, 0.025, 0.04 for CFN, respectively. This was presented in Table 1. A 1.0 mg mL−1 stock solution of IS was used to prepare a 200 ng mL−1 working IS solution. All the stock and working standard solutions were stored at 4 °C and brought to room temperature (20 °C) before use.
| Concentration (ng mL−1) | NMS | PPA | CPM | CFN |
|---|---|---|---|---|
| C1 | 250 | 100 | 20 | 10 |
| C2 (QC 1) | 500 | 200 | 30 | 15 |
| C3 | 750 | 400 | 60 | 20 |
| C4 (QC 2) | 1000 | 600 | 120 | 25 |
| C5 | 1500 | 800 | 150 | 40 |
| C6(QC 3) | 2000 | 1000 | 380 | 70 |
| C7 | 4000 | 1500 | 500 | 100 |
| Concentration ranges | 250–4000 | 100–1500 | 20–500 | 10–100 |
2.4 Sample preparation
Protein precipitation extraction method was used for preparation of plasma sample from the rat blood. The spiking 900 μL of blank plasma sample was transferred into a 10 mL centrifuge tube. To the above centrifuge tube 50 μL standard solutions, 50 μL of IS (4 μg mL−1) and 2 mL of methanol were added (final concentration of IS was made to 200 ng mL−1), then vortexed for 10 min and centrifuged at 4000 rpm for 20 min from that 100 μL of the supernatant solution was carefully transferred into vial and injected into HPLC system. The unique sample was also prepared for invitro analysis by addition of 15 μL of NMS (100 μg mL−1); 15 μL of PPA (100 μg mL−1); 10 μL of CPM (15 μg mL−1); 10 μL of CFN (15 μg mL−1); 50 μL of IS (12 μg mL−1) and 2 mL of methanol to 900 μL of blank plasma in centrifuge tube. The final concentration was made to 500 ng mL−1 of NMS and PPA, 50 ng mL−1 of CPM and CFN, 200 ng mL−1 of IS. This solution was vortexed and sonicated as above and supernatant solution was injected into HPLC system. The QC samples at three concentrations (low QC, middle QC and high QC) of each drug were prepared in a similar fashion.
2.5 Method validation
The method was validated for selectivity, matrix effect, precision, accuracy, linearity, sensitivity, recovery and stability according to the US Food and Drug Administration (FDA) guidelines for the validation of bioanalytical method (FDA, 2001). The selectivity of this method was investigated by analyzing six individual rat blank plasma samples. Each blank sample was tested for interference using the present analytical method and compared with spiked sample whose concentration of the analyte was at the Low Limit of Quantification (LLOQ). Calibration standard samples in rat plasma were prepared for three separate batches. Intra (same day) and inter (different days (10 days))-batch precision and accuracy were evaluated by measurement of reference in plasma in five replicates of QC samples at three different concentrations (low, medium and high QC) for three separate batches. The matrix effect was evaluated by adding known amounts of the analyte to pre-treated blank plasma samples, immediately before injection (Taylor, 2005). The peak areas obtained from these assays were compared to those of the standard solutions and the corresponding peak area ratios were calculated. Recovery was determined by comparing the detector response of the pre-extracted spiked sample with those spiked post-extraction onto a blank matrix because the analytes and IS have the same matrix. The small variations of flow rate (±0.2 mL min−1), volume of acetonitrile in mobile phase (±5 vol%), concentration of buffer (±5 mM) and pH (±1)) were studied for robustness test. The stabilities of quality control samples were tested under different storage conditions; i.e. three freeze–thaw cycles, room temperature (at 20 °C) for 24 h for short term stability and re-injection after one month (at −20 °C) for long term stability.
2.6 Animals and plasma sample preparation for pharmacokinetic study
In vivo drug release was investigated in male Wistar rats. Six Wistar rats (190–210 g) were procured from Mahavir enterprises, Hyderabad, India. The use of animals was approved by the ‘Institutional Animal Ethical Committee’ (169/99/CPCSEA, University College of Pharmaceutical Sciences, Warangal, India); throughout the experimental period, the animals were housed under standard condition in cages at room temperature (20 ± 2) °C; relative humidity (60–70)% exposed to 12/12 h light/dark cycle. They were fed with standard laboratory diet supplied by Rayans biotechnologies Pvt Ltd., Hyderabad, India. Food and water was allowed ad libitum during the experiment. The study was performed in three groups (18 Wistar rats were divided into 3 groups (3 × 6 rats), again these three groups were subdivided into 3 groups (3 × 2 rats). The samples were collected in different time intervals using subdivided groups. During the time of sample collection 2 rats were used for 12 h, then 2 rats were used for 12–24 h and remaining 2 rats were used for 24–36 h after injection of drugs. First two groups received a single oral dose of 25 mg kg−1 of standard and test solutions (combination of pure drugs) and third group received a single oral dose of CureX tablet. Serial blood samples were collected into the processed test tube at variable time-points (0, 0.2, 0.5, 1, 2, 3, 5, 6, 12, 24, 36 h for PPA; 0, 0.4, 0.6, 0.8, 1.2, 2, 3, 4, 5, 6, 12, 18 h for NMS; 0, 0.2, 0.6, 1, 1.5, 3, 3.5, 5, 7.5, 10, 12, 24 h for CPM; 0, 0.3, 0.6, 1, 1.5, 3, 6, 10, 15, 19, 24 h for CFN, respectively) into heparinized collection tubes. Whole blood sample was centrifuged for 20 min at 4000 rpm for assortment of rat plasma and stored at −20 °C. The stored plasma samples were allowed to thaw at room temperature (20 °C) before processing. An aliquot (900 μL) was pipetted into a 10 mL polypropylene tube, and 100 μL of IS (2 μg mL−1) and 2 mL of methanol were added (final concentration of IS was made to 200 ng mL−1). The mixture was vortex mixed briefly, and centrifuged at 4000 rpm for 20 min after standing for 5 min at room temperature; that 100 μL of the supernatant solution was carefully transferred into vial and injected into HPLC system. Statistical analysis was performed on Microsoft Excel 2003 and pharmacokinetics parameters were calculated on “Ramkin” pharmacokinetic software based on noncompartment model.
3 Results and discussion
To obtain the best chromatographic conditions, different columns, mobile phases with different pH and type of organic solvent were tested to provide sufficient selectivity and sensitivity in short separation time. The four analytes and IS had different retention properties because of their different polarities (Fig. 1). The best chromatographic conditions took place on XBridge™ C18 column with mobile phase consisting of 10 mM ammonium acetate (pH 4.0, 0.1% formic acid): acetonitrile (82:18% v/v) at flow rate 1 mL min−1 and the eluate were monitored in the wavelength range of 220–275 nm (Fig. 2). The influence of organic solvent concentration was carefully studied, increasing organic solvent concentration to improve peak shape and decrease the run time. Different pH of buffer was used for the separation of all analytes. Finally, pH 4.0 was chosen as the optimum value for better peak shape and reasonable run time. Other pH conditions, PPA and CPM were more retained on the column (the retention time of PPA and CPM were 21.76 and 23.82 minutes, respectively) and solvent consumption was also more. Diphenylpyraline was used as internal standard (IS) applied for determination of these drugs owing to retention characters that not affected seriously by change of pH, so it eluted with reasonable resolution and absorbance characters that show high absorbance at the chosen wave length (234 nm) that increases the sensitivity of the method. The proposed method succeeds in the determination of the studied mixture in spiked plasma sample and also in real rat plasma sample.
3.1 Method validation
The described method was validated in terms of linearity, sensitivity, recovery, specificity, stability, precision and accuracy according to FDA guidelines (FDA, 2001).
3.2 Specificity and sensitivity
The specificity of the extraction and chromatographic method tested the ability of the method to differentiate and quantitate the analyte in the presence of other endogenous constituents in the sample and to detect potential interferences. No interfering peaks were observed at the retention times of the analytes. The LLOQ was found at 250 ng mL−1 for NMS, 100 ng mL−1 for PPA, 20 ng mL−1 for CPM and 10 ng mL−1 for CFN. Fig. 2 shows RP–HPLC standard chromatogram of NMS, PPA, CPM, CFN and IS; the blank rat plasma sample and the blank plasma sample spiked with NMS, PPA, CPM, CFN and IS.
3.3 Linearity, accuracy and precision
All calibration curves were linear within the concentration ranges tested (Table 1). Typical equations of calibration curves and correlation coefficients were as follows: NMS: y = 280.37C + 157.84, r = 0.9982; PPA: y = 135.88C – 142.04, r = 0.9990; CPM: y = 125.72C – 68.596, r = 0.9973; CFN: y = 227.87C + 191.24, r = 0.9986. The calibration curves were linear. Results for accuracy and precision at the LLOQ for five replicates were (92.87 ± 3.0)% for NMS, (91.3 ± 7.2)% for PPA, (93.87 ± 6.8)% for CPM and (92.71 ± 5.9)% for CFN. The relative standard deviation (RSD) of precision values for intra-batch and inter-batch was <15% of each QC level. The accuracy deviation values for intra-batch and inter-batch were all within (100 ± 10)% of the actual values at each QC level. This was shown in Table 2, which revealed that the precision and accuracy of mixture at three QC concentrations were within the acceptable limits.
| Analyte | Intra batch (n = 5 replicates on same day) | Interbatch (n = 5 replicates on 10 different days) | |||||
|---|---|---|---|---|---|---|---|
| Concentration added (μg mL−1) | Concentration found (mean ± SD, μg mL−1) | Accuracy (%) | Precision (%RSD) | Concentration found (mean ± SD, μg mL−1) | Accuracy (%) | Precision (%RSD) | |
| NMS | 0.5 | 0.463 ± 0.015 | 92.61 | 5.3 | 0.481 ± 0.014 | 96.20 | 5.7 |
| 1 | 0.927 ± 0.147 | 92.70 | 6.4 | 0.987 ± 0.139 | 98.71 | 6.7 | |
| 2 | 1.951 ± 0.263 | 97.55 | 5.1 | 1.934 ± 0.301 | 96.70 | 4.9 | |
| PPA | 0.2 | 0.201 ± 0.012 | 100.50 | 4.7 | 0.203 ± 0.018 | 101.50 | 4.8 |
| 0.6 | 0.598 ± 0.077 | 99.67 | 5.9 | 0.589 ± 0.032 | 98.17 | 5.9 | |
| 1 | 1.039 ± 0.093 | 103.90 | 3.4 | 0.986 ± 0.014 | 98.61 | 2.8 | |
| CPM | 0.03 | 0.029 ± 0.003 | 96.67 | 2.4 | 0.029 ± 0.004 | 96.67 | 4.3 |
| 0.12 | 0.118 ± 0.010 | 98.33 | 6.3 | 0.119 ± 0.013 | 99.17 | 6.2 | |
| 0.18 | 0.188 ± 0.022 | 104.44 | 3.9 | 0.181 ± 0.017 | 100.56 | 4.8 | |
| CFN | 0.015 | 0.014 ± 0.001 | 93.33 | 6.1 | 0.014 ± 0.001 | 93.33 | 6.1 |
| 0.025 | 0.024 ± 0.002 | 96.00 | 4.6 | 0.024 ± 0.003 | 96.00 | 5.5 | |
| 0.040 | 0.041 ± 0.005 | 102.50 | 4.8 | 0.041 ± 0.004 | 102.5 | 4.5 | |
3.4 Recovery and matrix effect
Recovery was determined by comparing the detector response of the pre-extracted spiked sample with those spiked post-extraction onto a blank matrix because the analytes and IS have the same matrix. The recoveries of all analytes of QC concentrations were in the range of (80 ± 15)% in plasma. The recoveries for each analyte were shown in Table 3. The inter-subject variability of matrix effect at every concentration level should be <15% for acceptable performance (Taylor, 2005). The matrix effect could be considered the same for pre and post-extraction spiked samples. The matrix effect was shown in Table 3. For all the analytes the matrix effects were in the range of (90 ± 10)%. The results obtained were good, since the sample/standard peak area ratios of matrix effect were always higher than 86.7% (Table 3).
| Analyte | Concentration (μg mL−1) | Recovery (%) (mean ± S.D) | Matrix effect, sample/standard peak area ratio (%) |
|---|---|---|---|
| NMS | 0.5 | 79.61 ± 5.1 | 89.7 |
| 1 | 82.23 ± 5.5 | 92.8 | |
| 2 | 80.17 ± 4.9 | 86.7 | |
| PPA | 0.2 | 78.56 ± 4.1 | 95.4 |
| 0.6 | 87.03 ± 4.3 | 96.7 | |
| 1 | 81.51 ± 4.5 | 95.1 | |
| CPM | 0.03 | 85.72 ± 6.1 | 94.7 |
| 0.12 | 90.54 ± 6.3 | 92.6 | |
| 0.18 | 89.41 ± 3.8 | 93.2 | |
| CFN | 0.015 | 91.03 ± 2.3 | 94.5 |
| 0.025 | 90.47 ± 1.9 | 91.8 | |
| 0.04 | 89.56 ± 4.4 | 95.3 | |
3.5 Robustness
According to the ICH guidelines (ICH, 1996) the robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, deliberate variations in method parameters and provides an indication of its reliability during normal usage. Three different types of method parameters exist: basic, internal and external parameters. The robustness study was limited to investigating the influence of basic and internal parameters. External parameters, such as different laboratories, analysts and instruments, were not included in the study (This study comes under ruggedness study, which was not done). The internal parameters (Flow rate, percentage of acetonitrile, buffer concentration and pH) were studied and shown in Table 4. The small variation of flow rate (±0.2 mL min−1), volume of acetonitrile in mobile phase (±5 vol%), and concentration of buffer (±5 mM) and pH (±1) were tested. The experiments were run randomly with a plasma sample spiked with 0.5 μg mL−1 of NMS, 0.5 μg mL−1 for PPA, 0.05 μg mL−1 for CPM and 0.03 μg mL−1 for CFN and IS respectively. The selected responses (tailing factor, resolution and retention time) were observed (Table 4) and the results indicated that the different combinations of significant parameters do not drastically affect responses, so that the developed method was considered to be robust.
| Parameter | Variation | Resolution (Rs) | Tailing factor (Tf) | Retention time (tR) | |||
|---|---|---|---|---|---|---|---|
| Between CFN and NMS | Between CPM and PPA | Tf of NMS | Tf of PPA | tR of CPM | tR of CFN | ||
| Flow rate (mL min−1) | 0.8 | 5.82 | 3.58 | 1.11 | 1.37 | 13.71 | 7.05 |
| 1 | 4.15 | 3.17 | 1.02 | 1.11 | 14.32 | 7.16 | |
| 1.2 | 4.01 | 2.59 | 1.23 | 1.29 | 14.95 | 8.60 | |
| Variation in pH | 3 | 4.00 | 3.96 | 1.27 | 1.25 | 14.88 | 6.47 |
| 4.0 | 4.15 | 3.17 | 1.02 | 1.11 | 14.32 | 7.16 | |
| 5 | 5.81 | 3.86 | 1.19 | 1.24 | 18.66 | 9.51 | |
| Concentration of buffer (mM) | 5 | 6.12 | 5.02 | 1.10 | 1.26 | 19.52 | 8.67 |
| 10 | 4.15 | 3.17 | 1.02 | 1.11 | 14.32 | 7.16 | |
| 15 | 5.39 | 4.99 | 1.09 | 1.27 | 15.46 | 7.52 | |
| Volume of acetonitrile (volume%) | 13 | 4.86 | 3.11 | 1.19 | 1.20 | 16.81 | 9.06 |
| 18 | 4.15 | 3.17 | 1.02 | 1.11 | 14.32 | 7.16 | |
| 23 | 5.27 | 4.34 | 1.17 | 1.29 | 13.91 | 6.88 | |
3.6 Stability
The result of the stability validation was presented in Table 5. The results revealed the final concentration of the drugs in each quality control samples at stability conditions i.e. three freeze–thaw cycles, room temperature for 24 h and re-injection after 30 days at −20 °C. The standard deviation (SD) value (n = 5) of final concentration of all drugs after storing the samples in all the stability conditions was found to be <15%. The accuracy of stored samples was found to be nearly equivalent to 100%. Hence, it can be inferred that all the drugs were stable in rat plasma.
| Analyte | Concentration (μg mL−1) | Remaining percentagea (mean ± SD) | ||
|---|---|---|---|---|
| Three freeze–thaw cycles | Room temperature for 24 h | Re-injection after 30 days at −20 °C | ||
| NMS | 0.5 | 98.14 ± 2.1 | 97.11 ± 3.0 | 98.01 ± 3.7 |
| 1 | 99.25 ± 3.4 | 97.98 ± 5.9 | 97.55 ± 4.8 | |
| 2 | 97.54 ± 4.7 | 96.89 ± 4.7 | 98.14 ± 7.6 | |
| PPA | 0.2 | 100.19 ± 1.8 | 100.27 ± 2.4 | 98.25 ± 4.9 |
| 0.6 | 101.32 ± 1.1 | 100.48 ± 5.8 | 96.52 ± 6.8 | |
| 1 | 99.78 ± 2.4 | 98.85 ± 2.8 | 97.14 ± 5.7 | |
| CPM | 0.03 | 96.48 ± 5.6 | 95.82 ± 5.9 | 99.81 ± 6.8 |
| 0.12 | 98.85 ± 4.3 | 97.15 ± 6.7 | 101.21 ± 7.4 | |
| 0.18 | 97.52 ± 3.6 | 96.14 ± 2.8 | 98.47 ± 5.8 | |
| CFN | 0.015 | 99.0 ± 0.9 | 95.86 ± 8.7 | 102.85 ± 4.4 |
| 0.025 | 98.74 ± 1.5 | 99.29 ± 5.8 | 96.17 ± 2.6 | |
| 0.04 | 98.29 ± 3.8 | 97.65 ± 5.1 | 100.26 ± 4.1 | |
3.7 Application to pharmacokinetic study
The developed method was applied to quantify four analytes concentration in pharmacokinetic study carried out on rats. The mean plasma concentrations versus time profile following a single oral administration of each analyte to 3 rats was presented in Fig. 3. The important pharmacokinetic parameters (Maximum plasma concentration (Cmax), Time required to reach maximum plasma concentration (Tmax), Plasma half life (t1/2), Area under curve at 24 h (AUC0→∞) and Mean residence time (MRT)) were calculated and presented in Table 6. Therefore, the terminal phase of analytes in the study was well characterized and the analytical assay was able to detect low concentrations at the end of the plasma concentration time profile. The present developed HPLC assay method could be successfully applied to the determination of all analytes in several pharmacokinetic studies conducted in any institution.
| Parameters | Tmax (h) | Cmax (ng mL−1) | AUC0-∞ (ng × h mL−1) | T1/2 (h) | MRT (h) |
|---|---|---|---|---|---|
| NMS | 0.8 ± 0.02 | 3591 ± 101.20 | 23940 ± 682.8 | 6.3 ± 1.3 | 8.3 ± 1.9 |
| PPA | 3.0 ± 0.07 | 450 ± 18.71 | 461 ± 68.34 | 13.2 ± 2.5 | 15.9 ± 3.7 |
| CPM | 1.5 ± 0.11 | 177 ± 13.82 | 375 ± 29.45 | 7.8 ± 1.4 | 8.6 ± 1.8 |
| CFN | 0.9 ± 0.03 | 98 ± 7.93 | 840 ± 43.08 | 6.5 ± 0.9 | 7.6 ± 2.1 |
4 Conclusions
A rapid and sensitive RP–HPLC method has been developed for the simultaneous quantitation of NMS, PPA, CPM and CFN in rat plasma. The method involves simple sample preparation and a short run allowing high sample throughput. The method was found to be selective and sensitive enough to extract and quantify these four drugs in small volume of rat plasma. The method was found to be linear (correlation coefficient (r) >0.9970 for all drugs). The accuracy, precision, specificity robustness and stability were found to be within the acceptable limits according to FDA and ICH (for robustness study) guidelines. The drug was stable in rat plasma and also applied to adequate sensitivity for use in the pharmacokinetic studies. In summary it can be suitable for use in all laboratories equipped with sophisticated or unsophisticated instruments.
Acknowledgments
The authors thank the Directors, National Institute of Technology, Warangal and Indian Institute of Chemical Technology, Hyderabad, for providing research facilities and encouragement. Mr. Thippani Ramesh thanks MHRD, Govt of India for providing financial assistance.
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