A precise and reliable reversed-phase high-performance liquid chromatographic method with ultraviolet detection was developed and validated to determine cinitapride in human plasma. After liquid-liquid extraction, chromatographic separation was achieved on a Nucleosil C18 (25 cm × 4.6 mm, 5
Cinitapride (Figure
Chemical structure of cinitapride.
Several analytical methods have been reported for the determination of cinitapride in biological samples, pure and pharmaceutical dosage forms using various methods. A survey of literature revealed UV spectrophotometric methods [
The present study reports analysis of cinitapride in human plasma using the Nucleosil C18 column following a liquid-liquid extraction of the sample. The purpose of this study was to develop a rapid, economical, precise, and accurate RP-HPLC method for the determination of cinitapride in human plasma.
Cinitapride was provided by Zydus research laboratories limited (Ahmedabad, India). All analytical grade ammonium acetate, triethyl amine, HPLC grade methanol, and acetonitrile were purchased from Merck (Mumbai, India) while high purity water was prepared using a Milli-Q water purification system (Mumbai, India). Isolated human plasma obtained from Lion’s blood bank (Guntur, India) and stored in a freezer.
HPLC experiments were performed on a Shimadzu HPLC system (Shimadzu, Japan) equipped with Nucleosil C18 (25 cm × 4.6 mm, 5
A stock solution was prepared by dissolving accurately weighed 100 mg of cinitapride in 100 mL of HPLC-grade methanol to yield a final concentration of 1 mg/mL of the drug. The stock solution (1000
Five hundred microliters of each standard solution (drug spiked human plasma) was pipetted into a series of polypropylene tubes and vortexed briefly. Three milliliters of
The developed method was validated as per US FDA guidelines [
The selectivity of the proposed method was examined by analyzing the human blank plasma for endogenous interference. The absence of interfering peaks at the same retention time of cinitapride was considered as evidence for selectivity of the method.
Linearity was studied by spiking blank plasma samples with appropriate volume of stock solutions of cinitapride yielding 1 to 35 ng/mL. Calibration data were generated by injecting extracted solutions of the drug into the HPLC system.
For precision and accuracy studies, samples were prepared at three concentration levels: low (LQC), medium (MQC), and high (HQC) quality controls, corresponding to 3, 15, and 35 ng/mL of cinitapride, respectively, with six replicates each. The intraday and interday precision were determined by analyzing the prepared samples on the same and three different days, respectively. Precision was characterized by the percent coefficient of variation (%CV) whereas accuracy was expressed as a percent recovery of the drug.
Recovery of cinitapride was evaluated by comparing the detector response of cinitapride in three quality control samples (LQC, MQC, and HQC) with the response of the same concentration in methanolic solutions.
The limit of detection (LOD) and quantification (LOQ) were calculated from a calibration curve constructed using solutions containing a cinitapride in the range of detection limit.
Six aliquots each of the three quality control samples were kept at room temperature (25 ± 5°C) after spiking into human plasma. After completion of 6 hrs, the samples were extracted and analyzed against the concentration of a freshly prepared one. Percent changes (bias) for cinitapride concentration for spiked samples over stability testing period of 6 hrs at room temperature was determined and compared to nominal values.
During HPLC method development, different options were examined to optimize detection parameters, chromatography conditions, and sample extraction. The UV detector was used for the estimation of cinitapride at 260 nm to maximize the signal of the drug and minimize the signal of plasma interferents.
The compositions of mobile phase were optimized through several trials to achieve good resolution and symmetric peak shape for the drug. Optimization of HPLC conditions performed on chromatographic parameters including retention time, column efficiency (HETP) of the various compositions, and velocity of mobile phase. Efficiency values (
A typical chromatogram of blank plasma.
A typical chromatogram of cinitapride in human plasma.
System suitability data.
Drug | USP plate count | Tailing factor ( |
Retention time (min) ( |
Peak area ( |
||
---|---|---|---|---|---|---|
Cinitapride 15 ng/mL | 5602 | 1.05 | Mean ± SD | %RSD | Mean ± SD | %RSD |
3.25 ± 0.01 | 0.28 | 493 ± 13 | 2.6 |
Selectivity was evaluated by extracting different blank plasma samples. The absence of interfering peaks at the retention time of cinitapride was considered as evidence for selectivity of the method. A chromatogram obtained from the blank plasma preparations indicates that there is no any interference of plasma components at the retention time of the drug. Chromatograms of both blank plasma and cinitapride in plasma are given in Figures
The peak areas of calibration standards were proportional to the amount of cinitapride over the concentration range of 1 to 35 ng/mL. The linearity of a calibration curve was proved by high correlation coefficient (
Six replicates of three quality control samples were prepared for recovery determination, and the peak areas obtained were compared to the peak areas obtained from extracted samples of the same concentration levels. The percent recovery ranged from 86.6% to 99.93% (Table
Recovery study.
S. no. | LQC response | MQC response | HQC response | |||
---|---|---|---|---|---|---|
Extracted | Unextracted | Extracted | Unextracted | Extracted | Unextracted | |
1 | 110 | 116 | 459 | 543 | 1086 | 1170 |
2 | 94 | 119 | 496 | 549 | 1069 | 1176 |
3 | 116 | 120 | 483 | 551 | 1091 | 1182 |
4 | 89 | 114 | 492 | 542 | 1035 | 1172 |
5 | 92 | 109 | 469 | 549 | 1092 | 1169 |
6 | 100 | 116 | 491 | 551 | 1110 | 1183 |
Mean | 100.16 | 115.66 | 481.67 | 547.50 | 1080.50 | 1175.33 |
SD | 10.74 | 3.93 | 14.66 | 3.99 | 25.87 | 6.05 |
% CV | 10.72 | 3.40 | 3.04 | 0.73 | 2.39 | 0.51 |
% recovery | 86.60 | 87.98 | 91.93 |
LQC, 3 ng/mL; MQC, 15 ng/mL; HQC, 35 ng/mL of cinitapride.
The precision of the developed method was evaluated as intraday and interday precision. They were examined by analyzing six replicates of three quality control samples on the same day and three consecutive days. The percent coefficient of variation of both intraday and interday precision was ≤7.1% (Table
Precision and accuracy of cinitapride.
Concentration (ng/mL) | Accuracy (%nominal) ( |
Precision (%CV) | |
---|---|---|---|
Interday ( |
Intraday ( |
||
3 | 86.04 | 5.57 | 7.10 |
15 | 90.40 | 4.68 | 3.04 |
35 | 93.88 | 2.03 | 4.35 |
The LOD and LOQ determined for cinitapride were 0.1192 and 0.3612 ng/mL, respectively. The values obtained were satisfactory. All the results for validation parameters are summarized in Table
Validation parameters.
Parameters | Results |
---|---|
Selectivity | Pass |
System suitability | Pass |
Linearity (ng/mL) | 1–35 |
Range (ng/mL) | 0.1–35 |
LOD (ng/mL) | 0.1192 |
LOQ (ng/mL) | 0.3612 |
Accuracy and precision | Pass |
Short-term stock stability | Pass |
Long-term stock stability | Pass |
Long-term plasma stability | Pass |
Bench-top stability | Pass |
Bench-top, short-term stability, long-term stability, and freeze-thaw cycle for cinitapride were investigated at different quality control samples. The results revealed that cinitapride was stable in plasma for at least 6 hrs at room temperature and 35 days at 2–8°C. It was approved that repeated freezing and thawing (three cycles) of plasma samples spiked with cinitapride at all levels did not influence their stability. In addition, dilution integrity confirmed that dilution of the samples with analyte-free control human plasma did not affect the stability. The findings obtained from all these stability studies are shown in Table
Stability studies.
Stability | Spiked final concentration (g/mL) | Mean ± SD | %CV | %nominal | %RE |
---|---|---|---|---|---|
BT | 3 | 2.79 ± 0.10 | 3.47 | 93.05 | −6.95 |
15 | 14.52 ± 0.22 | 1.51 | 96.79 | −3.20 | |
35 | 33.83 ± 0.49 | 1.45 | 96.65 | −3.35 | |
ST | 35 | 32.82 ± 0.68 | 3.61 | 93.78 | −6.22 |
LT | 35 | 33.22 | 2.52 | 94.90 | −5.10 |
FT | 3 | 2.78 ± 0.08 | 3.09 | 2.78 | −7.22 |
10 | 9.59 ± 0.25 | 2.63 | 95.90 | −4.10 | |
25 | 24.46 ± 0.18 | 0.73 | 97.84 | −2.16 | |
DI | 14 | 13.55 ± 0.28 | 2.04 | 96.80 | −3.20 |
7 | 6.82 ± 0.11 | 1.65 | 97.38 | −2.62 |
BT: bench-top stability, 6 hrs; ST: short-term stability, 6 hrs; LT: long-term stability, 35 days; FT: freeze-thaw stability, 3 cycles; DI: dilution integrity.
Optimization of RP-HPLC conditions and extraction of cinitapride from human blood plasma by liquid-liquid extraction have been performed and analyzed by the HPLC-UV method. The developed method was validated for its selectivity, linearity, precision, accuracy, and stability. The method can be used to analyze cinitapride in human blood plasma, so that the results obtained can be directly used to determine the bioavailability and its bioequivalence.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that there are no conflicts of interest regarding the publication of this article.
The authors thank the Southern Institute of Medical Sciences, College of Pharmacy, Mangaldas Nagar, A.P., India, for providing necessary facilities to carry out all research work.