Materials and reagents
Five reference substances (furosemide, torasemide, azosemide, bumetanide, and etacrynic acid) were obtained from the National Institutes for Food and Drug Control (Beijing, China). The purity of each of the five reference substances was higher than 99%. Chromatographic grade methanol and acetonitrile were purchased from Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). AR grade of potassium dihydrogen phosphate (KH2PO4), triethylamine, and phosphoric acid were obtained from Sinopharm Chemical, Shanghai, China. Pharmaceutical formulations containing furosemide, torasemide, azosemide, bumetanide, or etacrynic acid were obtained commercially.
Apparatus and analytical conditions
Chromatographic separations were achieved on an Agilent HPLC 1260 system equipped with a diode array detector (G4212B), autosampler (G1329B), quaternary pump (G1311C), and column oven (G1316A). A digital workstation with ChemStation software Version C.01.10 served as both a controller and data manager for the overall system. A Shimadzu HC-C18 column (150 mm × 4.6 mm, 5 µm) was applied during the study under the following analytical conditions: the mobile phase was composed of acetonitrile with 0.05 mol/L KH2PO4 (pH adjusted to 4.0 using diluted phosphoric acid) (36: 64, v/v) at detection wavelengths of 278 nm, at a flow rate of 1.0 mL/min. The column temperature was retained at 30 °C, and a sample volume of 20 µL was injected by an automatic sampler.
Laboratory prepared mixture standard solution
Furosemide, torasemide, azosemide, bumetanide, and etacrynic acid references were weighed precisely to 25.0 mg each, and then the compounds were dissolved in the mobile phase in a 50 mL volumetric flasks. These stock standard solutions were stored at 4 °C and warmed to room temperature before use. Mixed working solutions of the five LDs were prepared, and the mobile phase was added to form a mixed reference solution with a concentration of approximately 50 µg/mL of the reference substance.
Method validation
The method was validated according to the International Conference of Harmonization (ICH) guidelines [30]. The following parameters were investigated: linearity, precision, stability, accuracy, limit of detection and robustness.
Linearity and range
The linearity and range of the method were evaluated with the standard solution of the five LDs at six different concentrations. The concentrations ranged from 2.5 to 150.0 µg/mL for the five LDs, respectively. The mixed test working standard solutions were prepared by appropriate dilution of the stock solutions with mobile phase to the required concentrations for plotting the calibration curves. A 20 μL aliquot of each working solution was injected in triplicate into the chromatographic system (n = 3). The standard curves of the five LDs were constructed from the different concentrations of the mixed solution. Chromatograms were recorded, and the standard calibration curve was generated with peak area as the Y-axis (Y) and the concentration (µg/mL) of each standard solution as the X-axis (X).
Precision
The mixed standard solution at the same concentration was continuously injected six times on the same day or on different days according to the chromatographic conditions described in Section analytical conditions. The relative standard deviation (RSD) values were evaluated by the chromatographic peak area of the five LDs.
Stability
The same mixed working standard solutions were injected into the HPLC at 0, 1, 2, 4, 6 and 8 h with the same mobile phase. The RSDs of the five peaks areas were determined.
Recovery
The accuracy of the assay method was calculated in triplicate by adding known amounts of the five LD reference substances to commercial sample solutions. Each solution was prepared at three concentrations (i.e., 40, 50, and 60 µg/mL), and each solution was injected into HPLC in triplicate. Then, the peak areas were recorded, and the average recovery, and RSD % of each LD were calculated.
Robustness
The robustness study was conducted by examining the samples on three different HPLC instruments: a Dionex U3000, an Agilent 1260 series, and a Shimadzu LC-20A. Three different models of chromatographic columns were used, including an Agilent Zorbax Extend C18, Shimadzu HC-C18 column, and Kromasil C18 (150 mm × 4.6 mm, 5 um). The samples were determined to have various pH values (3.8, 4.0, and 4.2), flow rates (0.98, 1.0, and 1.02 mL/min), flow volume (34: 66, 36: 64, and 38: 62) of the mobile phase, and column temperatures (29, 30, and 31 °C). The separation degree and % RSD of the five LDs were investigated.
Qualitative investigation
UV spectral similarity
The mixed standard solutions of five LDs were injected into HPLC under the above analytical conditions. The chromatogram maps and UV spectra were recorded, and the similarity of the original and first-order spectra of the five LDs was analyzed by the ChemStation software Version C.01.10.
Relative retention time
Under the analytical conditions described in sections analytical conditions and robustness, the same standard solutions of the five LD mixtures were tested and peak retention times were recorded to determine the relative retention time (RRT). The RRT is calculated by the following formulae (1):
$$ {\text{RRT }} = ({\text{t}}_{{\text{A}}} - {\text{t}}_{0} ) \, /({\text{t}}_{{\text{R}}} - {\text{t}}_{0} ) $$
(1)
where t0, tR and tA represent the retention times of urine pyrimidine, etacrynic acid, and analyte, respectively.
Quantitative analysis of multiple components by a single marker (QAMS)
The application of the QAMS method in the quality control of the five diuretic drugs was based on the relative correction factor (RCF) of each component, which is proportional to the detection signal in a certain concentration range. In this study, we selected etacrynic acid as the internal standard, and used Eq. (2) to calculate the RCF of the other diuretics.
$$ {\text{RCF}} = \frac{{{\text{RCF}}_{{\text{s}}} }}{{{\text{RCF}}_{{\text{i}}} }} = \frac{{{\text{A}}_{{\text{s}}} /{\text{C}}_{{\text{s}}} }}{{{\text{A}}_{{\text{i}}} /{\text{C}}_{{\text{i}}} }} $$
(2)
where As is the peak area of the internal standard (etacrynic acid), Cs is the concentration of the internal standard (etacrynic acid), Ai is the peak area of other investigated components i, and Ci is the concentration of other investigated component i in the sample solution.
From the Eq. (2), we can derive the Eq. (3)
$$ C_{i} = RCF \times C_{s} \times \frac{{A_{i} }}{{A_{s} }} $$
(3)
We can use Eq. (3) to calculate the concentration of each component of the sample solution. Additionally, the effects of the different HPLC systems, the pH of the mobile phase, the gradient elution scheme, the flow rate, the injection volume and the column temperature given in section robustness for the RCF were investigated.
Analysis of the five LDs in commercial injections and tablets
To determine the content of furosemide, bumetanide, and torasemide, commercially available injections of the 3 diuretics were prepared with the mobile phase. To determine the content of azosemide and etacrynic acid in conventional tablets, ten tablets were weighed, and disintegrated by shaking for 1 min with 10 mL water in a 100 mL volumetric flask. 40 mL acetonitrile was added. The samples were ultrasonically treated for 20 min, and diluted with purified water to a volume of 100 mL. Then, the samples of the 5 diuretics were injected into the HPLC according to the above described analytical method.