Materials and reagents
The ELX, RFX, and loperamide hydrochloride (IS) were collected from the local manufacturing pharmaceutical industry (Hyderabad, India). Deionized water used for sample preparation was obtained from Milli Q water purification system purchased from Millipore (Bangalore, India). HPLC gradient grade acetonitrile (ACN) was purchased from Merck (Mumbai, India). HPLC grade orthophosphoric acid (OPA), triethylamine (TEA) were obtained from Sigma Aldrich (Mumbai, India). In the present study, drug-free rat plasma was acquired from Bio needs Laboratory (Bangalore, India), and it was stored at − 20 °C until analysis. The centrifuge type 2-16P (Sigma, Zurich, Switzerland), nylon syringe filters 0.22 m (Millipore, India), and heparinized tubes were used to collect blood samples.
Preparation of buffer solution
1 mL Triethyl amine in 1 L water and the pH was adjusted to 2.5 with orthophosphoric acid.
Apparatus and chromatographic conditions
An alliance e2695 HPLC system (Waters Corp., Milford, MA, USA) with a quaternary pump, in-line degasser, auto-injector, column compartment, and PDA detector (model-2998) was used for HPLC analysis. Empower 2 software was used to collect chromatographic data. All separation was achieved on Agilent Symmetry C8 column (5 µm, 250 mm × 4.6 mm) at room temperature. Isocratic elution with HPLC grade acetonitrile and 7 mM TEA (pH 2.5) (40:60, v/v) at a flow rate of 1 mL min−1, an injection volume of 10 µL, and detection at 283 nm was used to determine ELX and RFX. A filter paper of 0.45 µm was used to filter all the solutions and solvents (Millipore, India).
Preparation of standard stock and working solutions
Standard stock solutions were prepared by accurately weighing 10 mg of ELX and 20 mg of RFX and dissolving them individually in 100 mL of HPLC grade ACN. Stock solutions were further diluted to prepare the working solutions. The IS working solution (1.5 ng mL−1) was made by diluting the IS stock solution with the same diluent. All the solutions were stored at 4 °C in a volumetric flask and warmed to room temperature just before use.
Preparation of plasma CC standards and QC samples
Calibration curve (CC) standards of the analytes were made by spiking a suitable volume of working solutions into blank plasma and final concentrations were obtained as 10.0, 25.0, 50.0, 75.0, 100.0, 125.0, 150.0, and 200.0 ng mL−1 for ELX; and 20.0, 50.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0 ng mL−1 for RFX. The quality control samples for both ELX and RFX were prepared at three different concentration levels in blank plasma. In case of ELX, the lower quality control concentration (LQC) was prepared to be 50.0 ng mL−1, the medium (MQC) and high quality control concentration (HQC) were prepared to be 100.0 ng mL−1 and 150.0 ng mL−1 respectively. Whereas in the case of RFX the LQC was 100.0 ng mL−1, the MQC was 200.0 ng mL−1 and HQC was 300 ng mL−1. All spiked samples were stored in a deep freezer at – 80 °C.
Sample preparation
To extract analytes from the plasma, we have employed the liquid–liquid extraction method [21,22,23]. A plasma sample of 100 µL spiked with analytes of 600 µL and IS of 500 µL was added to an Eppendorf tube of 5 mL and the mixing process has been carried out for 1 min using a vortex. 800 µL of diluent was added to the samples and vortexed. After adding 1000 µL of ACN to the tube and vortex for 10 min, the mixture was centrifuged at 4500 rpm for 20 min at 10 °C. The supernatant solution was taken into another clean vial and dried under a nitrogen environment at 25 °C. The residue was dissolved in 100 µL of mobile phase and, of which, 10 µL aliquot was injected into HPLC system.
Method validation
The US FDA bioanalytical method validation guidelines were used to validate the specificity, sensitivity, linearity, accuracy, precision, matrix effect, extraction recovery, and stability of the analytical method [24].
Specificity
To determine the specificity, six different samples of rat plasma and blank plasma spiked with target analytes, ELX and RFX were taken and retention times were analyzed. By analyzing the retention times of analytes and IS, no evidence of interfering substances was found under developed chromatographic conditions.
Sensitivity
The LLOQC samples were prepared and processed in six replicates to measure sensitivity, precision, and accuracy at the LLOQ level. The precision and accuracy should be within the acceptable limits of ≤ 20% and 80–120%, respectively.
Carry-over effects
Carry-over was tested by injecting a blank sample of plasma immediately after the analysis of the upper limit of quantification, with acceptability criteria of 20% of the response of the analytes at the lower limit of quantification and 5% of the IS peak response.
Linearity and LLOQ
To plot calibration curves, the ratio of peak areas of analytes to the IS was plotted against the concentrations of the analytes over the range from 5 to 200 ng mL−1 for ELX; and from 10 to 400 ng mL−1 for RFX. The concentration of internal standard was maintained at 1.5 ng mL−1. The linearity was assessed using linear regression analysis. The LLOQ is defined as the lowest concentration of analytes in the calibration curve. The precision and accuracy were found to be within the acceptance criteria of ≤ 20% and 80–120%, respectively.
Accuracy and precision
The determination of the accuracy and precision of this method was done by analyzing six replicates at four different QC levels: 5.0 (LLOQ), 50.0 (LQC), 100.0 (MQC), and 150.0 ng mL−1 (HQC) for ELX and 10.0 (LLOQ), 100.0 (LQC), 200.0 (MQC) and 300.0 ng mL−1 (HQC) for RFX. The precision and accuracy of the method were determined through the percentage of coefficient of variation (% CV) and percentage recovery.
Extraction recovery
Recovery of analytes was determined by comparing with the peak areas of analytes extracted from six replicates (n = 6) of QC samples at LQC, MQC, and HQC levels to the equivalent areas of un-extracted analytes at the same concentrations.
Matrix effect
The matrix effect was measured by comparing the peak area of analytes spiked to blank plasma extracted samples to the clean standard solutions. Matrix effect calculated using six different sources of plasma lots at the levels of LQC and HQC. No noticeable matrix effect was observed on this method.
Stability
The stability was assessed for LQC and HQC concentrations of ELX and RFX in rat plasma, using different storage and handling conditions of samples. The benchtop stability was tested at room temperature for 6 h. The QC samples were kept in auto-sampler vials at 10 °C for 24 h. For long-term stability, samples were stored in a deep freezer at – 80 °C for 28 days. Freeze–thaw stability was investigated through three freeze–thaw cycles from – 80 °C to room temperature.
Dilution integrity
The effect of dilution of samples on the accuracy and precision of the method was investigated by testing the dilution integrity. To investigate the dilution integrity at a twofold dilution and the spiked samples were prepared above the ULOQ and tested in six replicates. The precision and accuracy should be within the acceptance criteria of ≤ 20% and 85–115%, respectively.
Application to a pharmacokinetic study
Six healthy rats weighing approximately 200 ± 20 g were used in a pharmacokinetic study. An oral dose of ELX at 0.416 mg/kg and RFX at 0.832 mg/kg was given to each rat. The rats were fasted for 12 h before administering the drugs and had access to water during the experiment. The blood samples were collected at 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0 h after the administration of drugs. Blood samples were taken in heparin-containing tubes and centrifuged at 4500 rpm for 10 min at 8 °C. The plasma samples were then refrigerated at – 80 °C until they were analyzed. WinNonlin software was used to determine pharmacokinetic parameters.