Methods
Instruments and reagents
The analysis was performed on an Agilent 1200 HPLC. Other instruments involved were a vortex mixer, electronic balance, centrifuge (Xiangyi H2050R) and nitrogen blowing apparatus. The following reagents were used: acetonitrile (Thermo Fisher, HPLC grade), sodium dihydrogen phosphate (Sigma-Aldrich, spectrum pure), triethylamine (Thermo Fisher), phosphoric acid (National Pharmaceutical Group, analytical grade), venlafaxine standard (China Food and Drug Testing Institute), mexiletine hydrochloride (China Food and Drug Testing Institute) and O-desmethylvenlafaxine (Belling, Shanghai). Blank plasma samples were collected from our group’s healthy members (figure 1).
Figure 1Flowchart of the study.
Chromatographic conditions
Chromatographic separation was achieved on an Agilent Eclipse XDB-C18 (4.6×150 mm, 5 µm) at 30°C. The mobile phase consisted of water containing sodium dihydrogen phosphate (0.05 mol/L) and acetonitrile (72:28). The flow rate was set at 0.5 mL/min. The fluorescence excitation wavelength was 276 nm, and the emission wavelength was 598 nm. The injection volume was 10 µL.
Preparation of solutions
To prepare 0.05 (mol/L) sodium dihydrogen phosphate solution, NaH2PO4•H2O (3.44893 g) was dissolved in water (500 mL) plus triethylamine (10 µL), and phosphoric acid was used to adjust pH to 2.90. The standard stock solution of venlafaxine hydrochloride was prepared as follows: venlafaxine hydrochloride (2.33 mg) was accurately weighed into a 10 mL brown volumetric flask, dissolved with 50% methanol–water to achieve the stock solution (206 µg/mL). The standard stock solution of O-desmethylvenlafaxine (212 µg/mL) and the stock solution of internal standard (mexiletine, 193.6 µg/mL) were prepared as described above. The stock solution of internal standard was diluted to internal standard solution (50 µg/mL) with 50% methanol–water before use. The standard solutions containing venlafaxine and O-desmethylvenlafaxine were prepared as follows: appropriate volume of standard stock solutions of venlafaxine and O-desmethylvenlafaxine was mixed, then diluted with 50% methanol–water to prepare the standard solutions containing venlafaxine and O-desmethylvenlafaxine with concentrations of 25, 18.725, 12.5, 5, 2.5, 1.25, 0.5 and 0.25 µg/mL, respectively. All the stock and standard solutions were stored at 4°C before use.
Treatment of plasma samples
The plasma sample (500 µL) and 30 µL internal standard solution (mexiletine, 50 µg/mL) were added to a 5 mL stoppered centrifuge tube. The solution was mixed by vortex-mixing at 1500 rpm for 3 min, extracted with ether (3 mL) by vortex-mixing at 1500 rpm for 3 min. After centrifugation at 3000 rpm for 15 min, the supernatant was transferred to a new tube and evaporated to dryness under a stream of nitrogen at 30°C. The residue was re-dissolved in mobile phase (100 µL) by vortex-mixing for 10 s, and 10 µL supernatant was injected into HPLC.
Specificity
The specificity of the method was determined by injecting the plasma samples prepared from blank plasma and blank plasma spiked with internal standard, venlafaxine and O-desmethylvenlafaxine. The samples were processed by following the same operations in the Treatment of plasma samples section. The effects of blank plasma on the determination of drugs were observed by chromatogram. Then, the plasma chromatograms of the patients taking venlafaxine were examined.
Many patients in our hospital took venlafaxine in combination with benzodiazepines. Accordingly, in this study, we investigated the effects of alprazolam, clonazepam and lorazepam on the determination of venlafaxine. About 2 mg of alprazolam, clonazepam and lorazepam were respectively weighed and dissolved in 10 mL volumetric flasks with 50% methanol–water. The samples were directly injected into HPLC.
Drawing of calibration curves
The plasma samples containing venlafaxine and O-desmethylvenlafaxine were prepared by spiking each of above standard solutions (20 µL), blank plasma (500 µL) and internal standard (30 µL) at final concentrations of 1000, 750, 500, 200, 100, 50, 20 and 10 ng/mL. According to the operations in the Preparation of solutions section, the plasma samples were processed and then injected into HPLC. The chromatograms were recorded. The calibration curves were performed using a weighted (1/c2) least-squares linear regression method by measuring the peak-area ratio of the analytes to the internal standard. The abscissa X represented the plasma concentration of analytes and ordinate Y represented the peak-area ratio of the analytes to the internal standard.
Extraction recovery
The extraction recoveries of QC (quality control) samples were investigated at three different concentrations. According to the operations in the Preparation of solutions section, the QC samples, containing venlafaxine and O-desmethylvenlafaxine at concentrations of 20, 200 and 750 ng/mL, were prepared by spiking standard solutions and blank plasma (500 µL). After an injection into HPLC, the peak-area of venlafaxine and O-desmethylvenlafaxine was determined and recorded as A1. Each sample at different concentrations was parallelly measured five times. Also, an appropriate amount of the standard solution containing venlafaxine and O-desmethylvenlafaxine was blown dry under a nitrogen stream at 40°C, reconstituted by adding 100 µL mobile phase, then injected into HPLC. The peak-area ratio of the analytes to the internal standard was recorded as A2. The extraction recoveries were calculated by the values of A1/A2.
Method recovery
The QC samples containing venlafaxine and O-desmethylvenlafaxine at three different concentrations of 20, 200 and 750 ng/mL were prepared according to the methods of ‘drawing of standard curves’. The postoperations were processed as described in the Preparation of solutions section. Each of the QC samples was parallelly measured five times; the peak areas of the analytes and the internal standard were recorded as As and Ai, respectively. The analytes’ concentrations were calculated by substituting R=As/Ai into the standard curve equation, and then the recoveries were determined by the ratio of the measured concentrations to the prepared concentrations.
Precision
The QC samples were prepared as described above. Each of the QC samples was parallelly determined five times. Intraday precision was examined on the same day; the interday precision was determined on three consecutive days. The concentrations of the QC samples were calculated in accordance with the calibration curves. The relative SDs (RSDs) of intraday and interday precision were calculated on the basis of the menstruated results of the QC samples.
Stability and repeatability
The QC samples containing venlafaxine and O-desmethylvenlafaxine at three concentrations of 20, 200 and 750 ng/mL (n=5) were prepared as described in the Preparation of solutions section and stored at room temperature. The samples were determined after being placed for 0, 24 and 48 hours. The QC samples were stored at −20°C, determined at 0, 45 and 90 days. The chromatograms were recorded to investigate the stability of the stock solution.
Limit of quantitation (LOQ)
The plasma samples spiked with different concentrations of venlafaxine and O-desmethylvenlafaxine were prepared without internal standard and processed according to the pretreatment of blood samples. The limit of quantitation was defined by the signal-to-noise ratio (S/N=10).