About Authors:
Emanual Michael Patelia*, Rakesh Thakur, Jayesh Patel
Department of Pharmaceutical analysis and chemistry (Gujarat technical university)
Department of Pharmacology (University of Bedfordshire)
ricky.emanual@gmail.com
Abstract:
To develop and validate liquid chromatography-tandem mass spectrometric method for the quantification of ciprofibrate from human plasma. Ciprofibrate and furosemide (IS) were extracted from human plasma using Oasis HLB 1cc 30 mg solid phase extraction cartridge. The chromatographic separation was performed on ACE C18, 50×4.6 mm, 5μ column. The mobile phase consisted of 0.001% ammonia in methanol-acetonitrile-water (70:20:10, v/v/v) was delivered at rate of 1 mL/min. Detection and quantitation were performed by a triple quadrupole equipped with electrospray ionization and multiple reaction monitoring in negative ionization mode (API 3200). The most intense [M-H]- transition for ciprofibrate at m/z 287.0→85.0 and for IS at m/z 328.9.0→204.9 were used for quantification. The developed method was successfully applied for bioequivalence study of ciprofibrate. The method was found to linear over the range of 25-30000 ng/mL (r>0.998). The lower limit of quantitation (LLOQ) was 25 ng/mL. The extraction recovery was above 90%. The accuracy was found to 101.26%-106.44%. The intra and inter-day precision expressed as % CV were 1.15% and 5.25%, respectively. The stability testing was also investigated and it was found that both drug and IS were quite stable. A simple, rapid, sensitive, accurate and precise LC-ESI/MS/MS method has been developed for the quantification of ciprofibrate from human plasma using SPE method. The method exhibited good linear response over the selected concentration range 25-30000 ng/mL. Selectivity and sensitivity were sufficient for detecting and quantifying ciprofibrate in human plasma. These features coupled with a short run time at 1.8 min compared to reported methods, facilitated a high analysis throughput, with the ability to quantify a larger number of clinical samples in a shorter time frame.
REFERENCE ID: PHARMATUTOR-ART-1942
Introduction:
Ciprofibrate is isobutyric acid used to reduce high levels of lipids such as triglycerides in the blood.
It is also used to reduce the likelihood of cardiovascular disease events [1-3]. Ciprofibrate is primarily indicated in conditions like Hypercholesterolaemia, Hyperlipidemia and Hypertriglyceridaemia. In patients with low HDL and high risk of atheromatous disease [4-6]. High-performance liquid chromatographies coupled with electrospray tandem mass spectrometry have developed for quantification of Ciprofibrate in human plasma for pharmacokinetic studies [7]. High-performance liquid chromatography has developed for determination of ciprofibrate in human plasma [8]. Stability-Indicating HPLC has developed for the determination of ciprofibrate in Bulk drug and Pharmaceutical dosage form [9]. High-performance liquid chromatography have developed for the determination of bezafibrate, ciprofibrate and fenofibric acid in human plasma [10]. Spectrophotometric method have developed for the determination of Ciprofibrate content in tablets [11]. Method has developed for achiral and chiral determination of Ciprofibrate and its glucuronide in human urine by capillary electrophoresis [12]. Densitometry and videodensitometric TLC method have developed method for the determination of bezafibrate and ciprofibrate in pharmaceutical formulations [13]. The method official is in British Pharmacopeia 2011 [14]. All reported methods have long run time. Hence, it felt necessary to develop and validate a rapid and selective method that can be successfully applied to a bioequivalence study. In the present paper we would like to present a simple and high-throughput protein precipitation method for quantification of Ciprofibrate using Glimipiride as an internal standard with LC-MS/MS detection. The application of this validated method in analyzing samples from a bioequivalence study involving Ciprofibrate is also presented.
Experimental conditions:
Chemicals and Reagents
The reference standard of Ciprofibrate was provided by accutest pharmaceuticals Ltd. (Mumbai, India). The reference standard Furosemide was obtained from Derivados Quimicos, India. Purity of both the standards was higher than 100.1 %. The Ciprofibrate API, were obtained from Derivados Quimicos, India. High-purity water was prepared in-house using a Milli-Q A10-gradient water purification system (Millipore, Bangalore, India). LC-grade methanol and acetonitrile were purchased from J.T. Baker Inc, and sigma, germany. (Phillipsburg, NJ, USA). Ortho-phosphoric Acid and Sodium hydroxide were procured from AR Grade, Rankem Fine Chemical Ltd., New Delhi, India. Ammonia was procured form SD fine chemicals Mumbai.Drug-free (blank) human plasma containing heparin was obtained by enrolling healthy volunteers and taking their consent before bleeding. The plasma thus obtained was stored at −20°C prior to use.
Calibration Curve and Quality Control Samples
Two separate stock solutions of Ciprofibrate were prepared for bulk spiking of calibration curve and quality control samples for the method validation exercise as well as the subject sample analysis. The stock solutions of Ciprofibrate and Furosemide were prepared in 0.001% ammonia in methanol-acetonitrile-water (70:20:10, v/v/v) at free base concentration of 100 μg/mL and 1000 μg/mL. Primary dilutions and working standard solutions were prepared from stock solutions using Methanol:Water (80:20 v/v) solvent mixture. These working standard solutions were used to prepare the calibration curve and quality control samples. Blank human plasma was screened prior to spiking to ensure that it was free of endogenous interference at retention times of Ciprofibrate and internal standard Furosemide. An eight-point standard curve and four quality control samples were prepared by spiking the blank plasma with an appropriate amount of Ciprofibrate. Calibration samples were made at concentrations of 500, 1000, 2000, 10000, 20000, 100000, 200000, 400000 and 600000 ng/mL, and quality control samples were made at concentrations of 1000, 2000, 10000, 20000, 100000, 200000, 400000 and 600000 ng/mL for Ciprofibrate.
Liquid Chromatography and Mass Spectrometric Conditions
The chromatographic separation was performed onACE C18 column with 50x4.6 mm i.d., 5 μ particle size. The mobile phase consisted of 0.001% ammonia in methanol:acetonitrile:water (70:20:10, %v/v/v) (pH: 6.5) was delivered at rate of 1.0 mL/min with Splitter. Detection and quantitation were performed by a triple quadrupole equipped with electrospray ionization and multiple reaction monitoring in negative ionization mode. The tuning was performed with ion-spray voltage -4500 V and temperature was 550 °C. The most intense [M-H]- transition for Ciprofibrate at m/z 287.0 85.0 and for IS at m/z 328.9 204.9 were used for quantification. The developed method was successfully applied for bioequivalence study of Ciprofibrate. The method was found to linear over the range of 25-30000 ng/mL (r≥0.992). The lower limit of quantitation (LLOQ) was 25 ng/mL. Selectivity and sensitivity were sufficient for detecting and quantifying Ciprofibrate in human plasma. These features coupled with a short run time compared to reported methods, facilitated a high analysis throughput, with the ability to quantify a larger number of clinical samples in a shorter time frame.
Figure 1: Product ion mass spectrum of Ciprofibrate
Figure 2: Product ion mass spectrum of glimepride.
The data acquisition was ascertained by Analyst 1.4.2 software. For quantification, the peak area ratios of the target ions of the analyte to those of the internal standard were compared with weighted (1/ x2) least squares calibration curves in which the peak area ratios of the calibration standards were plotted versus their concentrations.
Plasma Sample Preparation
300 μL of the sample (volunteer plasma) was transferred in 2 mL micro centrifuge tube, added 15 μL of IS working solution (10 μg/mL) mixed by vortexing and extracted using solid phase extraction.
685 μL 0.5% Ortho-phosphoric acid in water was added to each plasma sample preparation and vortexed for 1 min. The cartridges were conditioned with 1 mL of methanol (100%). The cartridges were equilibrated with 1 mL of water (100%). 1 mL plasma sample preparation were transferred to each cartridges (blank plasma, zero, calibration std. and quality control samples) on pre equilibrated cartridges and passed through plasma by applying vaccum of 5 mmHg pressure. Each sample cartridges were washed with 1 mL of water (100%). Each sample cartridges were washed with 1 mL 10 % methanol (10%). Drug and internal standard were eluted with 1 mL of elution solvent 0.001% v/v ammonia in methanol:acetonitrile:water (70:20:10% v/v/v) & transferred to vial for injection.
Validation:
A thorough and complete method validation of Ciprofibrate in human plasma was carried out following US FDA guidelines [14]. The method was validated for selectivity, sensitivity, matrix effect, linearity, precision and accuracy, recovery, dilution integrity, partial volume, reinjection reproducibility, and stability. Selectivity was performed by analyzing the human blank plasma samples from six different sources (or donors) with an additional haemolysed group and lipemic group to test for interference at the retention times of analytes. The assessment of matrix effect (coeluting, undetected endogenous matrix compounds that may influence the analyte ionization) constitutes an important and integral part of validation for quantitative LC-MS/MS method for supporting pharmacokinetics studies. It was performed by processing six different lots of plasma samples in quadruplet. LQC and HQC working solutions were spiked following extraction in duplicate for each lot. The % CV at each level was calculated by taking the mean value obtained by injecting the postextracted samples prepared in duplicate from each plasma lot, which should be less than ten.The intra-run (within a day, and inter-run (between days, accuracy was determined by replicate analysis of quality control samples (at LLOQ (lower limit of quantification), LQC (low quality control), MQC (medium quality control), HQC (high quality control), and ULOQ (upper limit of quantification) levels. The % CV should be less than 15% and accuracy (% RE) should be within 15% except LLOQ where it should be within 20%.Accuracy is defined as the percent relative error (% RE) and was calculated using the formula % RE = ((E −T)/T) 100, where E is the experimentally determined concentration and T is the theoretical concentration. Assay precision was calculated by using the formula % CV = (SD/M) (100), where M is the mean of the experimentally determined concentrations and SD is the standard deviation of M. The % change was calculated by using the formula % change = (S/F − 1) 100, where S is the mean concentration of stability samples and F is the mean concentration of freshly prepared samples. The extraction efficiencies of Ciprofibrate and Glimipiride were determined by analysis of six replicates at each quality control concentration level for Ciprofibrate and at one concentration for the internal standard Glimipiride. The percent recovery was evaluated by comparing the peak areas of extracted standards to the peak areas of unextracted standards (spiked into extracted matrix of same lot).The dilution integrity experiment was performed with an aim to validate the dilution test to be carried out on higher analyte concentrations above upper limit of quantification (ULOQ), which may be encountered during real subject sample analysis. Dilution integrity experiment was carried out at 1.7 times the ULOQ concentration. Six replicates each of 1/2 and 1/4 concentrations were prepared and their concentrations were calculated by applying the dilution factor 2 and 4 against the freshly prepared calibration curve. In real subject samples with insufficient plasma volume, the partial volume experiment was performed on medium quality control (MQC) concentration level to validate the method. Six replicates each of half and quarter volume of the total volume of plasma required for processing were prepared and their concentrations were calculated by applying the concentration factor 2 and 4 against the freshly prepared calibration curve.LQC and HQC samples were injected to check re-injection reproducibility, after which the system was turned off and then restarted after two hours. The same samples were then reinjected, and original values were compared with re-injected values with respect to % change, which should be less than 10%.As a part of the method validation, stability was evaluated in stock solutions and in plasma under different conditions, maintaining the same conditions that occurred during study samples handling and analysis. Stock solution stability was performed by comparing area response of the analyte and the internal standard in the stability sample, with the area response of sample prepared from fresh stock solution. Stability studies in plasma were performed at LQC and HQC concentration level using six replicates at each level. Analyte was considered stable if the % change is less than 15% as per US FDA guidelines [14].
The stability of the spiked human plasma samples stored at room temperature (bench top stability) was evaluated for 4 to 24 hrs. The stability of the spiked human plasma samples stored at −70°C in coolant (coolant stability) was evaluated for 24 h. The autosampler sample stability was evaluated by comparing the extracted plasma samples that were injected immediately (time 0 hrs), with the samples that were re-injected after storing in the autosampler at 5°C for 52 hrs. The reinjection reproducibility was evaluated by comparing the extracted plasma samples that were injected immediately (time 0 h), with the samples that were re-injected after storing in the refrigerator at 2–8°C for 19 h. The freeze-thaw stability was conducted by comparing the stability samples that had been frozen at −70°C and thawed three times, with freshly spiked quality control samples. Six aliquots each of LQC and HQC concentration levels were used for the freeze-thaw stability evaluation. For long-term stability evaluation, freshly prepared calibration curve and quality control samples were injected along with the stability samples. The concentrations obtained after 16, 39, 87, and 221 days intervals were compared with initial concentrations. The interaction of K2EDTA with analyte was determined by storing the analyte in K2EDTA coated vaccutainer used for plasma sample collection. The four sets of low and high QCs were prepared in the subject sample collection vaccutainer coated with K2EDTA and kept in deep freezer at –70 ± 5±C for about 48 hrs. After completion of 48 hrs the samples were thawed at room temperature, extracted along with precision and accuracy batch and compared with freshly prepared quality control samples. The % change in concentration between freshly prepared samples and anti-coagulant effect sample should be within ± 15%.
Application of Method:
The validated method has been successfully used to analyze Ciprofibrate concentrations in sixty human volunteers under fasting conditions after administration of a single tablet containing 120 mg Ciprofibrate as an oral dose. The study design was a randomized, two-period, two-sequence, two-treatment single-dose, open-label, bioequivalence study using BATCH NO.Y01L manufactured by Derivados Quimicos, as the reference formulation. The study was conducted according to current GCP guidelines and after signed consent of the volunteers. Before conducting the study, it was also approved by an authorized ethics committee. There were a total of 25 blood collection time-points including the predose sample, per period. The blood samples were collected in separate vaccutainers containing heparin as anticoagulant. The plasma from these samples was separated by centrifugation at 3500 rpm within the range of 2–8°C. The plasma samples thus obtained were stored at −70°C till analysis. Following analysis the pharmacokinetic parameters were computed using WinNonlin software version 5.2 and 90% confidence interval was computed using SAS software version 9.2.
Results and Discussion:
Method Development
During method development different options were evaluated to optimize detection parameters, chromatography, and sample extraction.
Mass Spectra
The LC-ESI-MS/MS with negative and multiple reactions monitoring mode provides a selective method for determination of ciprofibrate and IS. The negative ionization mode was chosen for ion product since there was presence of carboxylic group in the structure of ciprofibrate and IS. The ion source was set to 550oC to enhance the sensitivity. As the [M-H]- transitions at m/z 287.0 85.0 for ciprofibrate and m/z 328.9 204.9 for IS was most intense ones, thus were selected for the quantification. The optimized parameters- Declustering potential, Collision cell entrance potential and Collision cell exit potential for Ciprofibrate and IS were -22.0 V, -11.0 V, 1.70 V and -28.0 V, -16.0 V, -4.0 V, respectively.
Chromatography
The different concentration of acids like formic acid, acetic acid were tried from 0.001-0.1%, but it was found that as the concentration of acid increased, response of analyte decreased. The buffers like ammonium acetate and ammonium formate were also tried from 2mM-5mM for mobile phase and the improved chromatography was found but the intensity of analyte was not enough to quantify LLOQ. So, that next attempt was made using various proportion of methanol:acetonitrile:water like (60:30:10, %v/v/v), (70:20:10, %v/v/v), (80:10:10, %v/v/v). Good chromatography was obtained but still response was not enough. So, that ammonia was added from 0.001-0.05% and improved response was found at LLOQ level in presence of ammonia at all proportion because ammonia increases ionization of ciprofibrate. Finally, 0.001% ammonia in methanol:acetonitrile:water (70:20:10, %v/v/v) (pH: 6.5) was selected as mobile phase. ACE C18 column with 50x4.6 mm i.d., 5 μ particle size column was selected to reduce the run time. Low flow rate was selected to 1.0 mL/min to increase the efficiency of column and to reduce the usage of mobile phase.
Extraction
Protein precipitation method (PPT)
Various protein precipitating agents like acetonitrile, methanol and 5% Perchloric acid were attempted. But no satisfactory results were obtained. The interferences were observed from the biological matrix at the retention time of the analyte and internal standard (Ion Supression). Less recovery was also obtained for the analyte and internal standard. Hence, the protein precipitation method was not selected.
Liquid-liquid extraction (LLE)
The LLE method was carried out using various organic solvents like tertiary butyl methyl ether (TBME), diethyl ether, and ethyl acetate: dichloromethane (80:20, %v/v) and the combination of these solvents in different ratios had been attempted. The recovery was obtained for analyte from 60-80% at different proportion but it was not reproducible and it is also one of the time consuming method, so LLE was ruled out.
Solid phase extraction (SPE)
Oasis HLB 1cc 30mg cartridges were used for solid phase extraction. Cartridges were conditioned using 1.0 mL methanol and then 1.0 mL water. For washing, water and methanol in water (5, 10, and 15%) in varying proportions were attempted. Good result was obtained with water and methanol in water (10%). The analyte and internal standard eluted using mobile phase. The eluent was then directly transferred to LC-MS/MS vials for injection. Recovery obtained by this extraction technique was found good and no matrix effect was observed. Hence, this method was selected for present study.
Method Validation:
Selectivity and Sensitivity
Representative chromatograms obtained from blank plasma, plasma spiked with lower limit of quantification, and real subject sample for Ciprofibrate and Glimipiride are shown in Figure 3. The mean % interference observed at the retention time of analytes between eight different lots of human plasma including haemolysed and lipemic plasma containing heparin as an anticoagulant was calculated and the value was found to be 0.00% and 0.00% for Ciprofibrate and Glimipiride, respectively, which was within acceptance criteria. Six replicates of extracted samples at the LLOQ level in one of the plasma sample having least interference at the retention time of Ciprofibrate were prepared and analyzed. The % CV of the area ratios of these six replicates of samples was 2.65% for Ciprofibrate confirming that interference does not affect the quantification at the LLOQ level. Utilization of selected product ions for each compound enhanced mass spectrometric selectivity.
Figure 3: Representative chromatograms of Ciprofibrate (left) and Glimipiride (right) in human plasma. (A) Blank plasma, (B) LLOQ, and (C) Real subject sample.
The LLOQ for Ciprofibrate was 25 ng/mL. The intra-run precision and intra-run accuracy (% CV) of the LLOQ plasma samples containing Ciprofibrate were 0.68-4.46%, respectively. All the values obtained below 25 ng/mL for Ciprofibrate were excluded from statistical analysis as they were below the LLOQ values validated for Ciprofibrate.
Matrix Effect
The matrix effect was evaluated at low and high QC samples obtained from six different plasma lot including lipemic and haemolysed plasma. The mean % accuracy and precision (% CV) were 106.09 % & 107.52 % and 2.96 % & 0.62 %, respectively. The results are within the acceptance criteria of ± 15 %.
Linearity, Precision and Accuracy, and Recovery
The peak area ratios of calibration standards were proportional to the concentration of Ciprofibrate in each assay over the nominal concentration range of 25–30000 ng/mL. The calibration curves appeared linear and were well described by least-squares linear regression lines. As compared with the 1/x weighing factor, a weighing factor of 1/x2 properly achieved the homogeneity of variance and was chosen to achieve homogeneity of variance. The regression squares were greater than 0.998 for Ciprofibrate. The deviation of the back calculated values from the nominal standard concentrations was less than 15%. This validated linearity range justifies the concentration observed during real sample analysis.The inter-run precision and accuracy were determined by pooling all individual assay results of replicate quality control over three separate batch runs analyzed on three different days.
Table 1: Intrarun and interrun precision and accuracy (n = 6) of Ciprofibrate in human plasma.
|
Run |
Concentration added (ng/mL) |
Mean concentration found (ng/mL) |
% CV |
% RE |
|
Intra- |
25.000 |
51.777 |
4.46 |
1.16 |
|
75.000 |
1468.198 |
1.75 |
1.31 |
|
|
750.000 |
8668.107 |
1.15 |
8.12 |
|
|
25000.000 |
56823.853 |
0.68 |
4.08 |
|
|
Inter- |
25.000 |
52.777 |
4.26 |
2.16 |
|
75.000 |
1488.198 |
4.15 |
9.31 |
|
|
750.000 |
8568.107 |
2.15 |
3.12 |
|
|
25000.000 |
55423.853 |
1.68 |
6.08 |
CV: coefficient of variation; RE: relative error.
Six post extracted replicates (samples spiked in extracted matrix of same lot) at low, medium, middle, and high quality control concentration levels for Ciprofibrate were prepared for recovery determination, and the areas obtained were compared versus the areas obtained for extracted samples (shown in Table 2) of the same concentration levels from a precision and accuracy batch run on the same day.
Table 2: Recovery for Ciprofibrate and Glimipiride (n = 6).
|
Analytes |
Level |
A |
B |
% Recovery |
% CV |
|
Ciprofibrate |
LQC |
9985.17 |
8566.33 |
85.79 |
0.99 |
|
MQC |
85648.83 |
76047.00 |
88.79 |
1.26 |
|
|
HQC |
3017781.83 |
2666043.33 |
88.34 |
0.63 |
|
|
glimipiride |
MQC |
75982 |
67449 |
88.77 |
1.31 |
A: mean area of unextracted sample(n=6); B: mean area of extracted sample(n=6); mean recovery was found to be 87.64% for Ciprofibrate and 88.77 %for Glimipiride; CV: coefficient of variation.
Dilution Integrity and Partial Volume
The mean % accuracy and precision of two times diluted QC samples was 103.83% and 0.38%, respectively.
Reinjection Reproducibility and Stabilities
Reinjection reproducibility exercise was performed to check whether the instrument performance remains unchanged after hardware deactivation due to any instrument failure during real subject sample analysis. % Change was less than 10.25% for LQC and HQC level concentration; hence batch can be reinjected in case of instrument failure during real subject sample analysis. Also samples prepared were reinjected after 29 hours which shows % change less than 2.01 % for LQC and HQC level concentration; hence the batch can be reinjected after 29 hours in case of instrument failure during real subject sample analysis.Stock solution stability was performed to check stability of Ciprofibrate and Glimipiride in stock solutions prepared in .001% ammonia in methanol-acetonitrile-water (70:20:10, v/v/v) and stored at 2–8°C in a refrigerator. The freshly prepared stock solutions were compared with stock solutions prepared before 16 days. The % change for Ciprofibrate and Glimipiride was 1.79% and 1.26%, respectively, indicating that stock solutions were stable at least for 16 days.Bench-top, coolant and autosampler stability for Ciprofibrate was investigated at LQC and HQC levels. The results revealed that Ciprofibrate was stable in plasma for at least 17 h at room temperature, 26 h in a coolant at −70°C, and 34 h in an autosampler at 10°C. It was confirmed that repeated freezing and thawing (three cycles) of plasma samples spiked with Ciprofibrate at LQC and HQC levels did not affect their stability. The long-term stability results also indicated that Ciprofibrate was stable in matrix up to 11 days at a storage temperature of −70°C. The results obtained from all these stability studies are tabulated in Table 3.
Table 3: Stability results for Ciprofibrate(n = 6).
|
Stability |
Level |
A |
% CV |
B |
% CV |
% Change |
|
Autosampler (34 h, 10°C) |
LQC |
82.26 |
2.11 |
82.69 |
2.50 |
-0.13 |
|
HQC |
23079.83 |
1.91 |
23352.09 |
1.39 |
2.21 |
|
|
Bench top (17 h at room temp.) |
LQC |
77.57 |
3.85 |
76.99 |
7.25 |
-0.74 |
|
HQC |
27511.61 |
1.50 |
27075.31 |
0.25 |
-1.59 |
|
|
Drug interaction with K2 EDTA (-70± 5°C, 48 hrs) |
LQC |
82.76 |
2.51 |
82.67 |
1.79 |
-0.11 |
|
HQC |
23009.83 |
1.01 |
23484.23 |
0.99 |
2.06 |
|
|
Reinjection (29 h, 2–8°C) |
LQC |
82.76 |
2.51 |
82.49 |
2.00 |
-0.33 |
|
HQC |
23009.83 |
1.01 |
23472.09 |
1.59 |
2.01 |
|
|
3rd freeze-thaw cycle (−70°C) |
LQC |
83.09 |
0.99 |
79.44 |
0.93 |
-4.40 |
|
HQC |
24038.26 |
0.59 |
24043.84 |
1.17 |
0.02 |
|
|
Long term (11 days, −70°C) |
LQC |
75.45 |
2.24 |
78.32 |
4.02 |
3.80 |
|
HQC |
25372.69 |
0.94 |
23713.97 |
1.01 |
-6.54 |
A: mean value of comparison samples (original concentrations before storage) concentrations (ng/mL); B: mean value of stability samples (measured concentration after storage) concentrations (ng/mL); CV: coefficient of variation; h: hours, temp: temperature.
Application:
The validated method has been successfully used to quantify Ciprofibrate concentrations in sixty human volunteers, under fasting conditions after administration of a single tablet containing 100 mg Ciprofibrate as an oral dose. The study was carried out after approval from an independent ethics committee and after obtaining signed approval from the volunteers.
The pharmacokinetic parameters evaluated were Cmax (maximum observed drug concentration during the study), Tmax(time to observe maximum drug concentration), and T1/2 (terminal half-life as determined by quotient 0.693/Kel, where elimination half life = 38-86 hr). The mean Cmax that was observed for Ciprofibrate in case of both test and reference formulations was 23.89 and 24.12 μg/mL, respectively, peak plasma concentrations attained within 1-4 hr. The 90% confidence intervals of the ratios of means Cmax, AUC0-72 all fell within the acceptance range of 80%–125%.
Conclusion:
The developed LC-MS/MS assay for Ciprofibrate is rapid, selective, and suitable for routine measurement of subject samples. The method is linear over the concentration studied i.e. 25.00 ng/ml to 30000.00 ng/ml of Ciprofibrate, which is sufficient enough to give data for calculation of the required pharmacokinetic data and establish bioequivalence. The other major advantage of this validated method is the run time of 1.5 minutes which allows the quantitation of over 240 plasma samples per day.
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