| The Benazepril hydrochloride and Lercanidipine hydrochloride tablets is a new compound hypotensor. In this paper, a sensitive and selective High Performance Liquid Chromatography tandem mass-spectrometric method (HPLC-MS/MS) for simultaneously determination of LER, BEN and benazeprilat in plasma was established and toxicokinetics studies of Benazepril hydrochloride and Lercanidipine hydrochloride tablets were carried on Beagle dogs and rats.Objectives 1. Establish a sensitive and selective HPLC-MS/MS method for simultaneously determination of LER, BEN and benazeprilat in plasma.2. Detect the accumulation of BEN, benazeprilat and LER in Beagle dogs and rats after multiple dosing of compound preparations comparing with single preparations.Methods 1. Plasma samples prepared by acetonitrile precipitated protein processing were injected into the HPLC-MS/MS. BEN, benazeprilat, LER, gliclazide and ubenimex (used as internal standard, I.S.) were separated on a Poroshell 120 ODS column SB-C18 (2.7-Micron,3.0×100 mm) at column temperature of 30 ℃ and flow rate of 0.5 mL/min by 0.1% formic acid -acetonitrile (A:B) with a gradient rate of 0～1.5 min. A:65%; 2 min, A:30%; 2～4.5 min, A:30%; 5 min, A:65%; 5～6.5 min, A:65%. Mass spectrometer was operated using electronic spray ionization (ESI) with positive ionization mode.The following ion transitions (m/z):425.3'351.1 for BEN,397.1'351.1 for benazeprilat,612.3'280.2 for LER,324.1'127.2 for gliclazide (I.S.) and 309.2'119.9 for ubenimex (I.S.) were selected for quantification in multiple reactions monitoring (MRM) mode.2. In this study, active pharmaceutical ingredients Lercanidipine hydrochloride and Benazepril hydrochloride were used with the ratio of 1:3. The dosages of administration were shown as Lercanidipine hydrochloride+Benazepril hydrochloride. Beagle dogs were randomly divided into five groups:low dose (L group), mid group (M group), high group (H group), LER group and BEN group. Each group was orally administrated with and 180 mg/kg/d once a day for 39 weeks. The blood samples were obtained on the first and 274th day after administration. Rats were divided into five groups:low dose (L group), mid group (M group), high group (H group), LER group and BEN group. Each group was orally administrated with mg/kg/d and 120 mg/kg/d once a day for 26 weeks. The blood samples were obtained on the first day and 183th day after administration. All blood samples were determined by HPLC-MS/MS. Toxicokinetics parameter calculations were carried out by DAS2.1.1.Results 1. Beagle dog method validation results:In the method of LC-MS/MS, the linear range of the calibration curve of BEN, benazeprilat and LER in plasma were 12～12000 ng/mL,12～12000 ng/mL and 0.6-600ng/mL, respectively. The LLOQ were 12, 12,0.6 ng/mL. The intra-day and inter-day precisions (RSD) of three samples were less than 15%. BEN, benazeprilat and LER were stable after storage at -80℃ for 162 days and storage at room temperature for 17 h after protein precipitation. The mean matrix effects of BEN, benazeprilat and the LLOQ were in the range of 94.5%～103.8%, 94.7%～104.4% and 97.55%～107.8%. The absolute recoveries were in range of 90.2%～96.8%,79%～82.7%and 87.5%-103.0%.2. Rats method validation results:In the method of LC-MS/MS, the linear range of the calibration curve of BEN in plasma was 0.8～800 ng/mL and the LLOQ was 0.8 ng/mL. The linear range of the calibration curve of benazeprilat in plasma by LC-MS/MS method was 12～12000 ng/mL and the LLOQ was 12 ng/mL. The linear range of the calibration curve of LER in plasma was 1.5～1350ng/mL and the LLOQ was 1.5 ng/mL. The intra-day and inter-day precisions (RSD) of three samples were less than 15%. BEN, benazeprilat and LER were stable after storage at -80℃ for 149 days.3. In L group, M group, H group, Ben group and LER group, the Beagle dogs loose stools, vomit, eat less and gingival hyperplasia. Other toxicity effects including the increases of ALT, TBIL and TG in serum levels, BIL positive and kidney tubules dilation were also existed. Comparing with single preparations, the compound preparations showed similar chronic toxicity, which the ma in toxic organs were liver and kidney.On the first day after administration, the main toxickinetics parameters of BEN in L group, M group and H group were as follow:AUC0-t were 4276.6±2636.3, 12343.6±3319.9 and 35643.0±7163.1 μg/L·h. The main toxickinetics parameters of benazeprilat in L group, M group and H group were as follow:AUC0-t were 22171.3±8448.2,70660.1±23362.9 and 409848.3±98893.7 μg/L·h. The main toxickinetics parameters of LER in L group, M group and H group were as follow:AUC0-t were 125.6±79.9,283.9± 162.3 and 662.2±319.3 μg/L·h.The main toxickinetics parameters of LER in LER group were as follow:AUC0-t was 369.1±298.1μg/L·h. The main toxickinetics parameters of BEN in Ben group were as follow:AUC0-t was 27031.9±9235.4 μg/L·h. The main toxickinetics parameters of benazeprilat in BEN group were as follow:AUC0-t was 284450.5±86289.2 μg/L·h.On the 274th day after administration, the main toxickinetics parameters of BEN in L group, M group and H group were as follow:AUC0-t were 3837.9±1944.7, 14498.2±7456.1 and 33241.2±9815.2 μg/L·h. The main toxickinetics parameters of benazeprilat in L group, M group and H group were as follow:AUC0-t were 10086.6±2432.0,60389.8±34442.9 and 280135.8±72457.3 μg/L·h. The main toxickinetics parameters of LER in L group, M group and H group were as follow:AUC0-t were 130.3±104.2,790.9±386.1,1853.7±1427.5 μg/L·h. The main toxickinetics parameters of LER in LER group were as follow:AUC0-1 was 1939.1±1649.4 μg/L·h. The main toxickinetics parameters of BEN in BEN group were as follow:AUC0-t was 30629.9±5465.9 μg/L·h. The main toxickinetics parameters of benazeprilat in BEN group were as follow:AUC0-t was 237418.4±66054.8 μg/L·h.The AUC ratio was the 274th to first day. For Ben the AUC ratio were 1.0、1.3、 1.0 and 1.3, respectively. For benazeprilat the AUC ratio were 0.5、1.0、0.7 and 0.9, respectively. For LER the AUC ratio were 1.1、 3.7、3.9 and 10.5, respectively.4. In the L group, M group, H group, LER group and BEN group, the rats had none abnormal reaction except the main toxic organs, such as liver and kidney.BEN quickly transformed to benazeprilat in vivo of rats. The plasma concentration of BEN is lower than LLOQ. On the first day after administration, the main toxickinetics parameters of benazeprilat in L group, M group, H group were as follow:AUC0-1 were 10189.6±6609,30954.7±14906.1 and 97434.9±18599.8 μg/L·h. The main toxickinetics parameters of LER in L group, M group, H group were as follow:AUC0-t were 1653.7±540.7,4391.4±1506.7 and 12553.9±3143.6 μg/L·h. The main toxickinetics parameters of LER in LER group were as follow:AUC0-t was 17302.5±4095.5 μg/L·h. The main toxickinetics parameters of benazeprilat in BEN group were as follow:AUC0-t was 42329.7±15679.7 μg/L·h.On the 183 rd day after administration, the main toxickinetics parameters of benazeprilat in L group, M group, H group were as follow:AUC0-t were 17849.5±6241.5, 41423.6±13415.3 and 101125.6±23839.8 μg/L·h. The main toxickinetics parameters of LER were as follow:AUC0-t were 1509.2±786.2,6444.4±1068.1 and 19625.2±6899.2 μg/L·h. The main toxickinetics parameters of LER in LER group were as follow:AUC0-t was 18579.1±3855.2 μg/L·h. The main toxickinetics parameters of benazeprilat in BEN group were as follow:AUC0-t was 114166.6±22773.8 μg/L·h.The AUC ratio was the 274th to first day. For Ben the AUC ratio were 1.83、1.3、1.0 and 2.7, respectively. For LER the AUC ratio were 0.9、1.5、1.6 and 1.1, respectively.Conclusions 1. The established method was suitable for Benazepril Hydrochloride and Lercanidipine Hydrochloride Tablets toxicokinetics studies.2. The safe dose in Beagle dogs was (6+18) mg/kg/d and the main toxic organs were kidney and liver. When the doses of administration were more than 6 mg/kg/d in Beagle dogs, LER were accumulated. Compared with single administration, on the first day after adminstration, the concentration in plasma and exposure of BEN, benazeprilat and LER were increased in compound groups. There were no significant differences on the 274th day. The safe dose in Beagle dogs was (4+12) mg/kg/d and the main toxic target organs were kidney and liver. None administration doses in rats may cause accumulation. Compared with single administration, concentration in plasma and exposure of benazeprilat in compound groups were increased on the first day after administration. There were no significant differences on the 183rd day. |
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