Abstract.A simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and applied for quantification of venlafaxine in human plasma. The plasma samples were extracted from plasma with anhydrous diethyl ether. The analyte was separated on a Inertsil ODS-3 column (50 mm×2.1 mm, 3 μm, Tokyo Japan) an isocratic mobile phase of methanol-10 mmol/L ammonium acetate (85:15,v/v) at a flow rate of 0.30 ml/min. Total chromatographic run time was 3.0 min. Quantitation was performed on a triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source and turbo ion spray interface and operating in multiple reaction monitoring (MRM) mode to detect parent→production transition for venlafaxine (m/z 278→57 ) and internal standards (m/z 455→165). The method was validated over the concentration range of 2.00~200 ng/mL for venlafaxine in human plasma, with a correlation coefficient of 0.99963. Both the intra-batch and inter-batch precision in six validation batches across three concentration levels (LLOQ, LQC, MQC, and HQC) were less than 14.4%, The accuracy determined at these levels was within -3.1% to 0.53%. This method was successfully applied in analysis of venlafaxine in the human plasma in a bioequivalence study in which 20 healthy volunteers underwent single-dose or multi-dose, two-period, two-sequence, and crossover design test.
Key words: Venlafaxine；Bioequivalence； LC-MS/MS, Plasma
1.Introduction
Venlafaxine {1-[2-(dimethylamino)-1-(4-methoxyphenyl) ethyl] cyclohexanol, VEN} has been widely used in the treatment of depression and a good choice in treating children with cataplexy and nightmares, symptoms in the narcolepsy [1]. It is one of the second generation antidepressants with a novel phenethylamine bicyclic structure which is different from the tricylic anti-depressant or monoamine oxidase inhibitor [2,3], and can selectively inhibit the re-uptake of norepinephrine and serotonin, with weak inhibition of dopamine re-uptake [4,5]. The mechanism underlying in the anti-depressant effects of venlafaxine in humans is believed to be associated with its potentiation of neurotransmitter activity in the central nervous system. VEN does not have high affinity for muscarinic, histaminergic or α1-adrenergic receptors in vitro [6]. In humans, it is metabolized in the liver by cytochrome P450 (CYP) enzymes to one major active metabolite (O-desmethylvenlafaxine) and the other two minor metabolites (N, O-didesmethylvenlafaxine and N-desmethylvenlafaxine)[7]. Several methods have been developed for the analysis of VEN in the human biological matrix, including gas chromatography(GC) and high performance liquid chromatography- ultraviolet ray(HPLC-UV) spectrometry or mass spectrometry, and recently, determination of VEN with LC-MS/MS has also been reported. Although the method is sensitive and efficient, but the total chromatographic run time is relatively long, which may not be applied in the routine subject analysis. In addition, these methods have a high on-column loading of the analytes at the ULOQ level, which may reduce the efficiency of the column and may affect the column life.
In the present study, we described a rapid, selective and sensitive method with LC-MS/MS in multiple reaction monitoring mode for the quantification of venlafaxine in human plasma using verapamil as an internal standard. The solid phase amounts of solvents or biological matrix and has a short turn ?around time for the alnalysis. The paper describes a simple and efficient method that was successfully applied in a pharmacokinetic study of venlafaxine extended release capsule formulation in 20 healthy Chinese male subjects under fed condition.
2.Materials and Methods
2.1Chemicals, materials and reagents
Venlafaxin hydrochloride extended release capsules (batch number:20080307, 75mg/pill) were supplied by Wansheng Pharmaceutical Company (Beijing, PR China), while standard reference capsules (batchnumber:0711210, 75mg/pill, BH20060182, J20060015) were produced by Ireland Wyeth Pharmaceuticals. Reference substance of venlafaxine(99.6%) was obtained from Wanshegn pharmaceutical Co. (Beijing, PR China). The reference substance of verapamil (99.5%) was obtained from Department of Pharmacochemistry, Shenyang Pharmaceutical University (Shenyang, PR China). Methanol (HPLC grade reagent) was obtained from Tedia Co., ammonium acetate(HPLC grade reagent) was obtained from Dikma Co.. Anhydrous ether (analytical grade reagent) was purchased from Yuwang Chemical Agent Factory (Shandong, PR China). Double distilled water was purified by Millipore Direct-Q (Millipore Corporation, Benford, MA, USA). Human blank plasma was supplied by Centre Blood Stantion (Shenyang, PR China).
2.2 Equipments
Analysis was performed on a system consisting of ACQUITY Ultra Performance LCTM (Waters Co., Milford, MA, USA) with a pump autosampler and an on-line degasser. Quantitative analysis was performed by using a triple quadrupole mass spectrometer equipped with an electrospray ionization interface(ESI) and operated in the multiple reaction monitoring(MRM). All the parameters were controlled by Masslynx 4.1(Waters Co., Milford, MA, USA) work station.
2.3 LC-MS/MS conditions
The testing samples were separated on an Inertsil ODS-3 column(50mm×2.1mm, 3μm, Tokyo Japan). The oven temperature was set at 40℃. The mobile phase consisted of methanol-10mmol/L ammonium acetate (85:15, v/v) and was eluted at a flow rate of 0.30mL/min. The injection volume was 5 μL.
The detection was acquired in the positive ion mode. Nitrogen was used as desolvation gas at a flow rate of 350 L/h. Desolvation temperature was set at 300℃. The ionization source worked at 110℃. Scan time was 0.10s, capillary and cone voltages were 1.2KV and 20V for ESI. Voltages were 19eV and 25eV respectively for venlafaxine and verapamil(inner substance). Quantitation was done using the MRM mode to monitor precursor→product ion transition of m/z 278→m/z 57 for venlafaxine, and m/z 455→m/z 165 for verapamil.
2.4 Preparation of standard stocks and quality control (QC) sample
The standard stock solution of venlafaxine was prepared by dissolving requisite amount of venlafaxine in CH3OH-H2O (10:90,v/v), and the working standards of venlafaxine were prepared from solution at ng/mL in CH3OH-H2O (10:90,v/v). The calibration standards (2.00, 5.00, 10.0, 20.0, 50.0, 100, 200ng/mL) were prepared daily by spiking 50μL of each working standard into human blank blood. QC samples of 4.00ng/mL (LQC), 25.0ng/mL (MQC) and 150ng/mL (HQC) were prepared by spiking, respectively. All the standard, intermediate and working stock solutions were prepared and stored at 4℃ until used. Drug free plasma and all spiked plasma samples were stored at -20℃.
2.5 Sample preparation
Samples from the -20℃ freezer thawed for 30-45min at room temperature. Aliquotsof plasma (200μL) were transferred into a 10mL glass test tube with a ground stopper, then 50μL of CH3OH-H2O solution (10:90,v/v), 50μL of inner substance solution of verapamil (204ng/mL) were added in order, and then the mixture was vortexed for 30 sec, then 200μL of 0.1mol/L NaOH solution was added, and vortexed for 30 sec again, extracted with 3mL of anhydrous diethyl ether by vortexing for 60 sec, and then centrifuged at 3500 rpm for 10 min. The supernatant was separated and dried at 40℃ under gentle stream of nitrogen. Residue was dissolved by 100 μl of CH3OH-H2O solution (85:15, v/v), and then vortexed for 30 sec and 5μl was injected into the LC-MS/MS system.
2.6 Bioequivalence study in healthy volunteers
The bioequivalence study of two venlafaxin hydrochloride extended release capsules was conducted in twenty Chinese healthy male subjects after they were informed on the purpose, protocol and risk involved in the study. All subjects gave written consent and the protocol was approved by the local ethics committee. In the study, theses volunteers weighing 68～78kg aged 21-25 years old. Health check for all subjects was done by general physical examination, ECG and laboratory tests such as hematology and urine examination. The study was conducted in accordance with the current Good Clinical Practices (GCP), International Conference on Harmonization (ICH) and FDA guidelines.
2.6.1 A randomized, single-dose, two-period, two-sequence and crossover design
Subjects were orally administered with a single dose of test and reference formulation with 250mL of water. Drinking was not allowed and supine position was restricted 2h post-dose. Standardized meals were provided as per schedule. All subjects were forbidden to smoke, drink and take any thing with caffeine during the study. 5mL of venous blood sample was collected in tubes containing heparin before administration (0h), and at 1.0, 2.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10.0, 12.0, 24.0, 48.0, 72.0 h after administration of drug. Plasma was separated by centrifugation at 5000 rpm for 15min, and then stored at -20℃ until analysis. Crossover trial was performed after 17 days.
2.6.2 A randomized, multi-dose, two-period, two-sequence, and crossover design
Subjects were orally administered with a single dose of test and reference formulation with 250mL of water everyday for 5 days. 3~5mL of blood was collected respectively before administration with a single dose drug at the 6, 7 and 8d, and blood samples were detected to get plateau concentration and make sure if homeostasis is attained. Blood samples were collected at 1.0, 2.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10.0, 12.0, 24.0, 48.0, 72.0 h in the 8d after administration of a single dose of drug. Plasma was separated by centrifugation at 5000 rpm for 15min, and then stored at -20℃ until analysis. Crossover trial was performed 17 days later.
3 Results and Discussion
3.1 Method development
3.1.1 Selection of the internal standard
In the present study, verapamil was selected as the internal standard, and the experimental results showed that verapamil and venlafaxine were similar in nature. Verapamil could be fully dissolved in solvents, without chemical reaction between them. The time of peaks was located close to each other and did not interact their sensitivities, and the results demonstrated that verapamil was an appropriate internal standard substance.
3.1.2 LC-MS/MS conditions
The chromatographic conditions were optimized through several trials to achieve good responses and symmetric peak shapes for both analyte and verapamil in a short run time. It was found that the mixture of methanol-10mmol/L ammonium acetate (85:15, v/v) was appropriate as the mobile phase. The Inertsil ODS-3 column (50 mm×2.1 mm, 3μm, Tokyo Japan) was faster and more effective than other traditionary columns. The total chromatographic elution was undertaken within 2.5 min at a flow rate of 0.30mL/min.
3.2 Method validation
3.2.1 Linearity and sensitivity
The peak area ratios of the calibration standards were proportional to the concentration of the drug in plasma ove the range tested. Calibration curves consisted of venlafaxine of six concentrations spiked in human plasma: 2.00, 5.00, 10.0, 20.0, 50.0, 100, 200ng/mL, and samples were prepared with the method described in “2.6”. The calibration curve of the six standards was Y=2.089×10-2X ± 1.662×10-3 (r=0.99962).
Six QC samples of venlafaxine at a concentration of 2.00ng/mL were prepared, and every bath was repeated three times, and then they were analyzed with the standard curve, then the concentrations which were calculated according to the standard curve at the same day were compared with the concentration of the prepared control samples, then the lower limit of quantification and day precision (RSD) were obtained.
3.2.2 Precision and accuracy
Venlafaxine of low, medium and high concentrations (4.00, 25.0, 150 ng/mL) quality control (QC) samples were prepared by the method described in “2.5". Six samples were prepared for each concentration, duplicate analysis of the same batch was performed according to the standard curve. The results were calculated according to the standard curve measured at the same day and compared with the prepared control samples. Experimental results indicate that human plasma venlafaxine analytical methods comply with the requirement of relevant standards.
3.2.3 Recovery
QC samples of venlafaxine of low, medium and high concentrations (4.00, 25.0, 150 ng/mL) were prepared from 200μL of blank human plasma based on the method described in “2.4”. Five samples at each concentration were analyzed. Another 200μL of blank human plasma was treated by the same way, but without series of standard solutions and the internal standard. The residue added with the internal standard solution and corresponding series of standard solution of 50μL were vortex mixed, and dried by nitrogen at 40 ℃. The residue was dissolved in 100 mL mobile phase, and 5mL of sample was taken for analysis to obtain the corresponding peak area (Determination of the average of the three). Extract recoveries were calculated by peak area ratio of every concentration handled in two ways. In the method, the extract recoveries of venlafaxine at the concentration of 4.00, 25.0, 150 ng/mL were 92.4%, 86.4% and 91.6%, respectively, with an average of 90.1%.
3.3 Application of the assay
3.3.1 One-dose experiment
After the subjects were administrated with a single oral dose of HCl venlafaxine sustained release capsules of test and reference formulation.As the result, Tmax was 6.10±0.97h and 5.80±1.01h, Cmax was 60.99±35.81ng/mL and 63.88±38.19ng/mL, t1/2 was 11.69±3.48h and 11.90±4.42h, AUC0-t was 1292.3±1317.6ng?h/mL and 1251.1±1227.5ng?h/mL, AUC0-∞ was 1386.1±1429.4 ng?h/mL and 1351.5±1354.4ng?h/mL, respectively. Calculated with AUC0-t, the mean relative bioavailability of venlafaxine was (102.1±9.5)% and there was no significant difference between the test and reference formulation about the main pharmacokinetic parameters including AUC0-t, AUC0-∞, Cmax, Tmax and t1/2.
The results of variance analysis and two/one-sided t test showed that all inequivalence hypotheses of AUC0-t ,AUC0-∞ and Cmax were rejected(P