Effects of preparing and ligand-binding methods of small unilamellar liposomes on their blood elimination and tissue distribution in rats

The effects of two methods of preparing small unilamellar vesicles (SUV) (detergent removal or sonication) on their in vivo elimination and tissue distribution was investigated in rats. The SUV prepared by either method had the same size distribution and lipid composition (egg yolk phosphatidylcholine/cholesterol/dipalmitoyl phosphatidylethanolamine or palmitic acid = 20/10/0.3, molar ratio). Three types of SUV made by either method were prepared. These contained one of three different surface ligand-binding functional groups (N-hydroxysuccinimide ester of palmitic acid, NHSP; glutaraldehyde-phosphatidylethanolamine, GA-PE; N-[4-(p-maleimidophenyl)butyryl]phosphatidylethanolamine, MPB-PE). SUV prepared by detergent removal were eliminated slowly from the circulation, and exhibited a low liver uptake and little leakage of [3H]inulin. There was no significant difference in elimination of the NHSP-SUV, GA-SUV or MPB-SUV prepared by detergent removal and their tissue distribution was similar. In contrast, the sonicated SUV were eliminated from the circulation much more rapidly mainly by liver uptake. The leakage of [3H]inulin from sonicated SUV into urine was relatively large. When sonicated control-SUV were prepared in the presence of the antioxidant, alpha-tocopherol (alpha-T-SUV), which reduces lipid peroxidation during sonication, the alpha-T-SUV were eliminated slowly with only a low liver uptake. Our results indicate that the rapid elimination and greater liver uptake of sonicated SUV is partly due to lipid peroxidation during preparation. These findings have relevance to the use of liposomes as a drug delivery system.     

Metabolic fate of [14C]triglyceride-entrapped lactosylceramide-bearing liposomes after intravenous injection into mouse.

We investigated the distribution and fate of liposomes after their intravenous injection into a mouse. Liposomes were composed of dimyristoylphosphatidylcholine, cholesterol and dicetylphosphate (7:2:1, molar ratio) with or without lactosylceramide. They were characterized as small unilamellar vesicles, approximately 100 nm in diameter, using gel-exclusion chromatography on a Sephacryl S-1000, freeze-fracture electron microscope and dynamic light scattering method. Liposomes were very stable in serum as seen by the results of leakage of the entrapped marker and electrophoresis experiments. We demonstrated that liposomes were internalized by way of an endocytotic process via coated vesicles detected in the electron microscope. The increase in liver uptake of lactosylceramide-bearing liposomes was mostly accounted for by enhanced uptake in the parenchymal cells, while uptake by non-parenchymal cells was only slightly increased. This observation supported the notion that a galactose-specific receptor was involved in liver uptake of lactosylceramide liposomes. The lactosylceramide-bearing liposomes were preferentially recovered in the liver and were found to first be predominantly localized in the mitochondria-lysosomal fraction. They were then decomposed by lysosomal enzymes, and the hydrolyzed components were reincorporated into membrane phospholipids in the microsomal fraction. At the same time, a rapid and reversible exchange of phosphatidylcholine between microsomes and mitochondria was demonstrated.     

Pharmacological properties of galenical preparation. XV. Pharmacokinetics study of evocarpine and its metabolite in rats.

It is known that when methanol extract of Evodia fruit is orally administered, 5-(1,4-dihydro-1-methyl-4-oxo-2-quinolin-2-yl) pentanoic acid (EVCA) is excreated as a matabolite in rat urine. In this study, we separated Evodia fruit extract into major alkaloids administered each alkaloid individually to male Wistar rats. Consequently, it was demonstrated that the original substance of the metabolite are evocarpine and its analogues, dihydroevocarpine and 1-methyl-2-undecenyl-4(1H)-quinolone. Investigation of a blood sample after oral administration of evocarpine by high performance liquid chromatography confirmed that the substance was absorbed without alteration. Pharmacokinetics of evocarpine after intravenous injection was expressed in a one-compartment model, showing a linear elimination of plasma evocarpine up to a dosage of 75 mg/kg. Total plasma clearance (CL), volume of distribution (Vd), and half-life (T1/2) of evocarpine were 60 ml/min.kg, 3.21/kg and 0.6 h-1, respectively. Metabolic ratio of evocarpine into EVCA after intravenous injection was 15.4%, and absorption ratio of the unaltered compound calculated from the levels of AUC after oral administration and intravenous injection was 4.7%. In this paper, it is shown that evocarpine is absorbed amount 100% when it is administered orally.     

Cyclodextrin-mediated entrapment of curcuminoid 4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid] or CLEFMA in liposomes for treatment of xenograft lung tumor in rats

We recently reported a novel curcuminoid 4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid] or CLEFMA as a potent anti-proliferative agent, and showed that it induces autophagic cell death in lung cancer cells. We are now reporting a drug-in-CD-in-liposome approach to formulate CLEFMA liposomes that could be labeled with Tc-99m radionuclide for non-invasive imaging of their biodistribution. CLEFMA encapsulation was enabled by hydroxypropyl-β-cyclodextrin. In vitro studies showed that CLEFMA possessed more potent anti-proliferative activity in lung adenocarcinoma H441 cells than naturally occurring curcumin. At the same time, it had no effect on the proliferative capacity of normal lung fibroblasts. CLEFMA liposomes retained the antiproliferative potency of free CLEFMA, while maintaining its non-toxic nature in normal lung fibroblasts. In nude rats bearing xenograft H441 tumors, the tumor volume significantly reduced after i.v. treatment with CLEFMA liposomes (p<0.05); the tumor inhibition was determined to be 94%. The anti-tumor activity of CLEFMA liposomes was confirmed by the observation that F-18-fluorodeoxyglucose uptake in tumors of treated rats was reduced as compared to those of control rats. Tc-99m-labeled CLEFMA liposomes accumulated in liver (33.7%); spleen showed the largest accumulation on per gram tissue basis (6.2%/g). Upon histopathological examination of liver, lung and kidney, we found no apparent toxicity from multiple CLEFMA liposome administrations. The results demonstrate the utility of liposomes to serve as a carrier for CLEFMA. This study is the first to demonstrate the efficacy of novel curcuminoid CLEFMA in a preclinical model.     

甘草次酸修饰海藻酸钠纳米给药系统的肝靶向性评估

通过EDC/NHS偶联反应将疏水性肝靶向小分子甘草次酸(GA)连接到天然多糖海藻酸钠(ALG)上,制备了具有双亲性肝靶向药物载体材料(GA-ALG).采用乳化法对广谱抗癌药物阿霉素(DOX)进行包载,得到肝靶向载药纳米粒子( DOX/GA-ALG NPs).利用单光子发射型计算机断层成像技术(SPECT)和药物体内分布...     

Determination of glycyrrhizin in rabbit plasma by high-performance liquid chromatography with photodiode-array ultraviolet detection and its pharmacokinetics application.

A simple and sensitive high-performance liquid chromatographic method for the determination of glycyrrhizin in rabbit plasma has been developed. Up to 0.1 ml of plasma containing glycyrrhizin was deproteinized by acetonitrile, which contained an internal standard (indomethacin). The supernatant was injected onto a LiChrospher RP-18 column using a methanol-water-ammonia solution (80:20:0.1, v/v, pH 3.0-3.2, adjusted with perchloric acid) as the mobile phase and ultraviolet detection at 254 nm, followed by ultraviolet spectrum identification (between 200 and 380 nm) with a photodiode-array detector. The method is rapid, easily reproduced, selective and sensitive. It was applied to pharmacokinetic studies of glycyrrhizin in rabbit, after a 2 mg/kg intravenous administration. A biphasic phenomenon with a rapid distribution followed by a slower elimination phase was observed from the plasma concentration-time curve. Compartmental analysis yielded a two-compartment model.     

Liposomes prepared from synthetic amphiphiles. I. Their technetium labeling and stability

In order to apply liposomes prepared from synthetic amphiphiles containing amino acid residues to radiopharmaceuticals, their labeling with 99Tc or 99mTc and stability in saline or in serum were investigated. These liposomes were highly labeled by embedding stearylamine-diethylenetriamine pentaacetic acid as a ligand of technetium. The labeling was more efficient at pH 7.0 than at pH 4.0 or 8.5. Among these technetium-labeled liposomes tested, liposomes containing the alanine residue were stable in saline or in 50% serum at 37 degrees C for at least 24 h, in contrast to liposomes (phosphatidylcholine: cholesterol = 1:1 molar ratio) whose stability had been enhanced by adding cholesterol.     

Uptake of transition metal ions using liposomes containing dicetylphosphate as a ligand.

The uptake of Cu2+ was investigated using various types of liposomes composed of phosphatidylcholine (PC), cholesterol (Chol) and dicethylphosphate (DCP). DCP played a role as a ligand for Cu2+. Multilamellar vesicles (MLVs) were more effective for the uptake of Cu2+ compared to unilamellar vesicles prepared by the extrusion technique. The uptake efficiency of MLVs for Cu2+ was dependent on the molar ratio of DCP in MLVs. The uptake percent of Cu2+ was 92% using MLVs having a PC:DCP:Chol molar ratio of 4:3:3; 95% of the total vesicle Cu2+ was bound to DCP of the outer membrane surface of the MLVs, and the remaining 5% of the total Cu2+ was distributed into the interior side of the MLVs. MLVs having a PC:DCP:Chol molar ratio of 4:3:3 were also effective as separation media for Mn2+, Co2+, Ni2+ and Zn2+. The uptake efficiency of the MLVs for the transition-metal ions increased in the order Co2+ < Zn2+ < Ni2+ < Mn2+ < Cu2+.     

Evaluation of the immunity activity of glycyrrhizin in AR mice

In this study, we evaluated effect of glycyrrhizin on immunity function in allergic rhinitis (AR) mice. The AR mice model were induced by dripping ovalbumin in physiological saline (2 mg mL?1, 10 μL) into the bilateral nasal cavities using a micropipette. After the AR model was induced, mice were randomly divided into six groups: the normal control, model, lycopene 20 mg kg?1 (as positive control drug) group, and glycyrrhizin 10, 20, 30 mg kg?1 groups. After the sensitization day 14, lycopene (20 mg/kg BW) and glycyrrhizin (10, 20 and 30 mg/kg BW) were given orally for 20 days once a day. Mice in the normal control and model groups were given saline orally once a day for 20 days. Results showed that glycyrrhizin treatment could dose-dependently significantly reduce blood immunoglobulin E (IgE), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), nitrous oxide (NO), tumor necrosis factor-alpha (TNF-α) levels and nitrous oxide synthase (NOS) activity and enhance blood immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM), interleukin-2 (IL-2) and interleukin-12 (IL-12) levels in AR mice. Furthermore, glycyrrhizin treatment could dose-dependently significantly enhance acetylcholinesterase (AchE) activity and reduce substance P (SP) level in peripheral blood and nasal mucosa of AR mice. We conclude that glycyrrhizin can improve immunity function in AR mice, suggesting a potential drug for the prevention and therapy of AR.     

Preparation and pharmaceutical evaluation of liposomes entrapping salicylic acid/gamma-cyclodextrin conjugate

To evaluate the potential use of a drug/cyclodextrin (CyD) conjugate for efficient entrapment in liposomes and prolonged residence of a drug in tissues, we synthesized a salicylic acid (SA) conjugate bound covalently with gamma-cyclodextrin (SA/gamma-CyD conjugate), a model drug/CyD conjugate, and then liposomes entrapping the conjugate (conjugate-in-liposome) were prepared by a freezing-thawing method. The chemical and physicochemical properties of the SA/gamma-CyD conjugate in solution and solid state were investigated and then the physicochemical properties of conjugate-in-liposome, in vitro cellular uptake/release and in vivo disposition of SA/gamma-CyD conjugate after intravenous administration of aqueous suspension containing conjugate-in-liposome in rats, were evaluated, comparing with those of the liposome-entrapped SA alone (SA-in-liposome) or the liposome-entrapped noncovalent SA/gamma-CyD complex (complex-in-liposome). As a result, it was found that the conjugate was amorphous powder and the release of SA from the conjugate in phosphate-buffered saline (PBS) was tolerated to chemical and enzymatic degradation. Meanwhile, the particle sizes and stability of these liposomes were almost identical, and the entrapment ratio of SA/gamma-CyD conjugate in liposomes was higher than those of SA alone and SA/gamma-CyD complex. The cellular uptake of these liposomes was almost equivalent, but the release of SA/gamma-CyD conjugate from RAW264.7 cells was markedly slower, compared with that of SA from cells following cellular uptake of the SA-in-liposome and complex-in-liposome. The disposition of SA or SA/gamma-CyD conjugate following intravenous administration of aqueous suspensions containing each liposome system in rats was comparable, but the residence time of the conjugate in tissues significantly prolonged, compared with that of the SA-in-liposome and complex-in-liposome systems. These results suggest the potential use of SA/gamma-CyD conjugate for efficient entrapment in liposomes as well as of liposomes containing SA/gamma-CyD conjugates for prolonged residence of drugs in tissues.     

Mannan-coated liposome delivery of gadolinium-diethylenetriaminepentaacetic acid, a contrast agent for use in magnetic resonance imaging.

Gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA), a paramagnetic contrast agent for use in magnetic resonance imaging (MRI) was bound to stearylamine and incorporated into the liposomal membranes (Gd-DTPA liposomes). In addition, the Gd-DTPA liposomes were coated with mannan (cholesterol-aminoethylcarbamylmethyl mannan), a polysaccharide, to obtain the mannan-coated liposomes. An in vitro MRI study showed that the Gd-DTPA liposomes produced a greater intensity of contrast than did the Gd-DTPA solution with a reduced T1 relaxation time. Intravenous injection of the Gd-DTPA liposomes containing 153Gd or liposomes containing 153Gd or 14C-DTPA to mice showed an accumulation of Gd-DTPA primarily in the liver and lung. When the mannan-coated liposomes were administered, an increased uptake of Gd-DTPA by these tissues was demonstrated. The mannan-coated liposomes may enhance contrast of the liver in MRI at a lower dose of Gd-DTPA.     

Liposomes prepared from synthetic amphiphiles. II. Their interaction with Ehrlich ascites tumor cells and tissue distribution in Ehrlich solid tumor-bearing mice

The interaction of 99mTc-labeled liposomes prepared from synthetic amphiphiles containing amino acid residues with Ehrlich ascites tumor cells in vitro and their tissue distributions in Ehrlich solid tumor-bearing mice were investigated. The amphiphiles used were N,N-didodecyl-N alpha-[6-(trimethylammonio)hexanoyl]-L-alaninamide bromide N+C5Ala2C12), N,N-didodecyl-N alpha-(6-[dimethyl(2-carboxyethyl)ammonio]hexanoyl)-L- alaninamide bromide (CAC2N+C5Ala2C12) and S-[1-carboxy-2- ([2,3-bis(hexadecyloxy)propoxy]carbonyl)ethyl]homocysteine (HcyM-G2C1 6). Most of the radioactivity of N+C5Ala2C12 and CAC2N+C5Ala2C12 liposomes was firmly bound to Ehrlich ascites tumor cells in vitro. On the other hand, the accumulation of three 99mTc-labeled liposomes in the tumor of Ehrlich solid tumor-bearing mice was low (about 1% dose per gram of tissue), and most of the liposomes were taken up highly in the liver and spleen of the tumor-bearing mice. However, the radioactivity of the liposomes in the tumor, especially that of N+C5Ala2C12 and CAC2N+C5Ala2C12 liposomes, decreased more slowly with time than in the liver in up to 24 h after injection, suggesting that these liposomes were hard to separate from the tumor cells.     

Determination of the antiplatelet agent. KB-3022, and its metabolite by high-performance liquid chromatography

A rapid, accurate and reproducible high-performance liquid chromatographic method for the simultaneous determination of a new antiplatelet agent, ethyl 2-[4,5-bis(4-methoxyphenyl)thiazol-2-yl]pyrrol-1-ylacetate (KB-3022), and its main metabolite, 2-[4,5-bis(4-methoxyphenyl)thiazol-2-yl]pyrrol-1-ylacetic acid (desethyl KB-3022), in biological fluids has been developed. KB-3022 and desethyl KB-3022 in plasma or urine were extracted with a mixture of n-hexane and dichloromethane (1:1), separated on a reversed-phase C18 column with a mixture of 0.01 M (NH4)2HPO4 (pH 3.0), acetonitrile and isopropyl alcohol as a mobile phase, and quantitated by ultraviolet absorbance measurement at 340 nm. 4-[2-(4,5-bis(4-methoxyphenyl)thiazol-2-yl)pyrrol-1-yl]butyric acid was used as an internal standard. The detection limit of KB-3022 in plasma was 3 ng/ml, and that of KB-3022 in urine and desethyl KB-3022 in plasma or urine was 1 ng/ml. The coefficients of variation for the determination of KB-3022 or desethyl KB-3022 in plasma or urine were below 5.6%. This method was applied to the determination of the plasma concentration of KB-3022 and desethyl KB-3022 after intravenous administration to rats.     

Synthesis and hepatic transport of strongly fluorescent cholephilic dipyrrinones

A new class of highly fluorescent (phi(F) 0.3-0.8) low molecular weight water-soluble cholephilic compounds has been synthesized in two steps from dipyrrinones. The dipyrrinone nitrogens are first bridged by reaction with 1,1'-carbonyldiimidazole to form an N,N'-carbonyldipyrrinone (3H,5H-dipyrrolo[1,2-c:2',1'-f]pyrimidine-3,5-dione) nucleus, and a sulfonic acid group is then introduced at C(8) by reaction with concd H(2)SO(4). The resulting sulfonated N,N'-carbonyl-bridged dipyrrinones ("sulfoglows") are isolated as their sodium salts. When the alkyl substituents of the lactam ring are lengthened from ethyl to decyl, sulfoglows become increasingly lipophilic while maintaining water solubility. Low molecular weight sulfoglows were rapidly excreted intact in both bile and urine after intravenous infusion into rats, but higher molecular weight sulfoglows were excreted more selectively in bile. Hepatobiliary excretion of sulfoglows was partially, but not completely, blocked in mutant rats deficient in the multidrug-resistance associated transport protein Mrp2 (ABCC2). These observations point to the feasibility of developing simple sulfoglows with clinical diagnostic potential that are normally excreted in bile but appear in urine when hepatic elimination is impaired by cholestatic liver disease.     

Glucuronate-modified liposomes with prolonged circulation time

For the purpose of obtaining liposomes with long circulation time in blood, we synthesized 1-O-palmityl-D-glucuronic acid (PGlcUA) and incorporated it into the liposomal membranes. The clearance of the PGlcUA-liposomes composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol, and PGlcUA (40:40:20 as a molar ratio) from blood and their tissue distribution were compared with those of dipalmitoylphosphatidylglycerol (DPPG)-liposomes (DPPC/cholesterol/DPPG = 40:40:20). When [3H]inulin-loaded PGlcUA-liposomes and DPPG-liposomes were intravenously injected into rats, the half-life of the PGlcUA-liposomes in the blood appeared to be 1.7-fold longer than that of DPPG-liposomes. Radioactivities present in plasma and various tissues were measured 22 h after administration of these liposomes, and radioactivity remaining in the plasma was 2.5-fold greater when PGlcUA-liposomes were injected. The distribution pattern of [3H]inulin in PGlcUA-liposomes was similar to that in DPPG-liposomes. The radioactivity recovered in urine was 25% lower in rats treated with PGlcUA-liposomes than in those treated with DPPG-liposomes. Since both PGlcUA- and DPPG-liposomes exhibited similar size distribution and zeta-potential, glucuronic acid, rather than negative charge, on the liposomal surface appears to endow liposomes with a longer circulation time in the bloodstream.     

Pharmacokinetics of [6]-gingerol after intravenous administration in rats with acute renal or hepatic failure.

The pharmacokinetics of [6]-gingerol were investigated in rats with acute renal failure induced by bilateral nephrectomy, or those with acute hepatic failure induced by a single oral administration of carbon tetrachloride (CCl4), to clarify the contribution of the kidney and liver to the elimination process of [6]-gingerol. After bolus intravenous administration, a plasma concentration-time curve of [6]-gingerol was illustrated by a two-compartment open model. There was no significant difference in either the plasma concentration-time curve or any pharmacokinetic parameters between the control and nephrectomized rats. It is suggested, therefore, that renal excretion does not contribute at all to the disappearance of [6]-gingerol from plasma in rats. In contrast, hepatic intoxication with CCl4 elevated the plasma concentration of [6]-gingerol at the terminal phase. Its elimination half-life increased significantly, from 8.5 to 11.0 min, in CCl4-intoxicated rats. The extent of [6]-gingerol bound to serum protein was more than 90% and was affected very slightly by the CCl4-intoxication. These aspects indicate that [6]-gingerol is eliminated partly by the liver.     

Preparation of giant unilamellar CdTe quantum dot vesicles and their metabolic pathway in vivo

Glutathione (GSH) capped CdTe quantum dots (QDs) with photoluminescence quantum yields of 61% and the maximum emitting at 601.2 nm were prepared in water phase. Giant unilamellar CdTe quantum dot vesicles (GUVs-CdTe), with diameters larger than 1.5 μm, were obtained using lower-pressure evaporation techniques with soybean lecithin. Compared with other QD liposomes, the entrapment efficiency of GUVs-CdTe for QDs has been significantly improved to 86.3%. After GUVs-CdTe were injected into mice through the tail vein, the fluorescence microscopy of tissue sections showed that GUVs-CdTe could not pass through the blood-brain barrier and air-blood barrier, which were removed mostly by the reticuloendothelial system and were widely distributed in the spleen and the liver. This behavior is the same as the character of the metabolic pathway of giant unilamellar vesicles by intravenous injections in mice.     

Separation and analysis of glycyrrhizin, 18beta-glycyrrhetic acid and 18alpha-glycyrrhetic acid in liquorice roots by means of capillary zone electrophoresis

Glycyrrhizin is the main active compound of Glycyrrhiza glabra root extracts; according to recent studies, glycyrrhizin and its aglycon, glycyrrhetic acid, have interesting therapeutic properties. A new capillary electrophoretic method has been developed for the separation and quantification of glycyrrhizin, beta-glycyrrhetic acid and its isomer a-glycyrrhetic acid. Separation of the analytes was achieved in less than 3 min on a fused silica capillary, by injecting the samples at the short end of the capillary (effective length: 8.5 cm). The background electrolyte was composed of pH 10.0 carbonate buffer, methanol and ethylene glycol (80/10/10) and contained 0.4% beta-cyclodextrin; indomethacin was used as the internal standard. Diode array detection was used, with quantitative assays carried out at 254 nm. Linearity was found over the 5-200 and 2.5-100 microg mL(-1) concentration ranges for glycyrrhizin and glycyrrhetic acid, respectively. This method has been applied to the determination of the analytes in different matrices (liquorice roots and commercial confectionery products), and to the purity control of beta-glycyrrhetic acid obtained from the hydrolysis of glycyrrhizin. When analysing beta-glycyrrhetic acid and its epimer in roots, the samples were purified by means of a suitable solid-phase extraction (SPE) procedure with Oasis HLB cartridges, which granted good selectivity, eliminating matrix interference.     

Targeting of mitomycin C to the liver by the use of asialofetuin as a carrier.

Desialylated fetuin (asialofetuin) was adopted as a carrier for introducing drugs in parenchymal liver cells. Mitomycin C, as a model guest-compound, was covalently attached to asialofetuin through a spacer of the succinyl group. The asialofetuin-mitomycin C conjugate contained 4.4 w/w% of mitomycin C and liberated it gradually at physiological conditions (t1/2 = 37 h). The survival time of the conjugate in rat blood circulation was significantly smaller than that of the non-desialylated fetuin conjugate; the elimination half-life was 7.3 min after intravenous injection. At 30 min after injection of the conjugate in rats, 40% of the dose was present in the liver. Parenchymal cells in the liver selectively took up the conjugate, which was highly distributed to the lysosomal fraction in the cell. The greater uptake of the conjugate by hepatocytes reflected the increased excretion in the bile; totally 10.4% of the dose was recovered.     

高效毛细管电泳法测定银翘解毒片中的氯原酸、甘草酸和甘草次酸

建立了同时测定银翘解每片中氯原酸、甘草酸和甘草次酸的高效毛细管电泳法,电解缓冲液为20mmol／L磷酸二氢钠和5mmol／L硼砂的混合溶液（pH7.0）。方法简便快速,具有良好的精密度、回收率和线性关系,并测定了很翘解毒片中3组分的含量。