Optimisation of infrared-assisted extraction of rutin from crude Flos Sophorae Immaturus using response surface methodology and HPLC analysis.

INTRODUCTION: Rutin, one of main constituents in Flos Sophorae Immaturus, has been proven to possess several pharmacological properties such as anti-oxidant, anti-platelet, anti-inflammatory effects and so on. However, optimisation of the extraction of rutin from Flos Sophorae Immaturus has rarely been reported. Thus, it is important to develop an effective method to obtain maximum yields of rutin from Flos Sophorae Immaturus. OBJECTIVE: To develop an infrared-assisted extraction method for maximum rutin yield from crude Flos Sophorae Immaturus using response surface methodology and HPLC analysis. METHODOLOGY: Through single factor experiments, ranges of the main variables (including methanol concentration, liquid:solid ratio, extraction time and infrared power) affecting the extraction yield of rutin were confirmed. A Box-Behnken design consisting of 24 experimental runs and five replicates at zero point was then applied and a regression model was obtained to predict the optimal extraction yield. RESULTS: The ANOVA analysis indicated that the regression equation fits very well with the actual situation. The optimal conditions were as follows: infrared power 204.90 W, liquid:solid ratio 30.00 mL/g, methanol concentration 70.00% and extraction time 4.80 min. Under optimal conditions the predicted maximum yield (125.70 mg rutin/0.5 g raw material) was consistent with the experimental value (126.32 ± 0.67 mg rutin/0.5 g raw material) (n = 3). CONCLUSION: The application of response surface methodology was reliable and feasible in the optimisation of infrared-assisted extraction of rutin from crude Flos Sophorae Immaturus.

Improved liquid chromatography fingerprint of fat-soluble Radix isatidis extract using multi-wavelength combination technique.

We first investigated liquid chromatography (LC) fingerprint method using multi-wavelength combination technique, and successfully used this method for the analysis of a fat-soluble extract from Radix isatidis. LC fingerprints of fat-soluble R. isatidis extracts from 11 origins were established using the Origin software and Similarity Evaluation System for Chromatographic Fingerprints of traditional Chinese medicine (TCM). The typical LC fingerprints of fat-soluble extracts from R. isatidis were first established, and the reference chromatogram was also generated with 24 common peaks showing large peak areas and good separation from adjacent peaks. Seven common characteristic peaks were identified for the first time: anthranilic acid, syringic acid, benzoic acid, salicylic acid, tryptanthrin, indigo and indirubin. The total peak areas of 24 common peaks were more than 80% of the total peak areas. Hierarchical clustering analysis (HCA) of 11 R. isatidis samples was performed, and the results show that the differences between 11 origin R. isatidis were large. Principal component analysis (PCA) on 24 common peaks was obtained to find the possible chemical markers for the discrimination of different samples. The loading plot indicated that peaks 8, 11, 13 and 14 may have more influence on the discrimination of the samples. All these were useful for evaluating and controlling the quality of R. isatidis. Our work provides a general model of chromatogram combination at multi-wavelength detection to study the complex or the undeveloped materials, which can be used to scientifically ensure the quality of such samples and deeply do qualitative, quantitative and multicomponent pharmacodynamic research combined with modern advanced chromatographic technique.

High-performance liquid chromatographic determination of memantine hydrochloride in rat plasma using sensitive fluorometric derivatization.

In this study, we investigated a simple, sensitive and reliable liquid chromatography-fluorescence detection method for the determination of memantine hydrochloride in rat plasma which was based on derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl). For the first time, FMOC-Cl was introduced into derivatization of memantine hydrochloride in rat plasma. The amino groups of memantine hydrochloride and amantadine hydrochloride (internal standard) were trapped with FMOC-Cl to form memantine hydrochloride-FMOC-Cl and amantadine hydrochloride-FMOC-Cl compositions, which can be very compatible for LC-FLD. Precipitation of plasma proteins by acetonitrile was followed by vortex mixing and centrifugation. Chromatographic separation was performed on a C(18) column (DIAMONSIL 150 × 4.6 mm, id 5 µm) with a mobile phase consisting of acetonitrile and water at a flow rate of 1.0 mL/min. The retention times of memantine hydrochloride-FMOC-Cl and amantadine hydrochloride-FMOC-Cl compositions were 23.69 and 40.27 min, respectively. Optimal conditions for the derivatization of memantine hydrochloride were also described. The limit of quantification (LOQ) was 25 ng/mL for memantine hydrochloride in plasma, the linear range was 0.025-5.0 µg/mL in plasma with a correlation coefficient (r) of 0.9999. The relative standard deviations (RSDs) of intra-day and inter-day assays were 4.46-12.19 and 5.23-11.50%, respectively. The validated method was successfully applied to the determination of memantine hydrochloride in rat plasma samples.

High performance liquid chromatography for the determination of glucosamine sulfate in human plasma after derivatization with 9-fluorenylmethyl chloroformate.

In this study, we developed a simple, rapid, sensitive, and reliable method for the determination of glucosamine sulfate in human plasma, which was based on derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) followed by reverse-phase HPLC-FLD. For the first time, FMOC-Cl was introduced into derivatization of glucosamine sulfate in human plasma. The amino groups of glucosamine sulfate and vertilmicin sulfate (the internal standard) were trapped with FMOC-Cl to form glucosamine-FMOC-Cl and vertilmicin-FMOC-Cl adducts, which can be very suitable for HPLC-FLD. Precipitation of plasma proteins by acetonitrile was followed by vortex mixing and centrifugation. Chromatographic separation was performed on a C18 column (DIAMONSIL 150 x 4 mm id, 5 microm) with a mobile phase gradient consisting of acetonitrile and water at a flow-rate of 1 mL/min. The retention times of glucosamine-FMOC-Cl and vertilmicin-FMOC-Cl adducts were 8.9 and 21.2 min, respectively. This method was shown to be selective and sensitive for glucosamine sulfate. The limit of detection was 15 ng/mL for glucosamine sulfate in plasma and the linear range was 0.1-10 mg/mL in plasma with a correlation coefficient (r) of 0.9999. The relative standard deviations (RSDs) of intra-day and inter-day assays were 5.2-8.1% and 6.1- 8.5%, respectively. Extraction recoveries of glucosamine sulfate in plasma were greater than 90%. The validated method was successfully applied to the determination of glucosamine sulfate in human plasma samples.

Liquid chromatography with electrospray ionization mass spectrometry method for the assay of glucosamine sulfate in human plasma: validation and application to a pharmacokinetic study.

A liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method was developed and validated for the assay of glucosamine sulfate in human plasma. Plasma proteins were precipitated by acetonitrile, followed by vortex mixing and centrifugation. The supernatant was transferred and derivatized with phenyl iso-thiocyanate in acetonitrile at 60 degrees C for 40 min. Chromatographic separation was performed on a C(18) column (Inertsil ODS-3 150 x 2.1 mm i.d., 5 microm, JP) with a mobile phase gradient consisting of 0.2% acetic acid (aqueous) and methanol at a flow-rate of 0.3 mL/min. MS detection using electrospray ionization (ESI) as an interface was used in single ion monitoring mode to determine positive ions at m/z 297. This method was shown to be selective and sensitive for glucosamine sulfate. The limit of detection was 35 ng/mL for glucosamine sulfate in plasma and the linear range was 0.1-20 microg/mL in plasma with a correlation coefficient (r) of 0.9991. The relative standard deviations (RSDs) of intra-day and inter-day assays were 8.7-11.4 and 9.8-12.6%, respectively. Extraction recoveries of glucosamine sulfate in plasma were greater than 73%. This method proved to be simple, reproducible and feasible for pharmacokinetic studies of glucosamine sulfate in healthy volunteers after a single oral administration (1500 mg). The pharmacokinetic parameters and relative bioavailabilities were investigated for both domestic glucosamine sulfate tablet and capsule preparations compared with an imported capsule product.

Stereoselectivity in metabolic 3-reduction of tibolone in healthy Chinese female volunteers.

AIM: To investigate the stereoselectivity in human metabolic 3-reduction of tibolone. METHODS: Twenty healthy Chinese female volunteers were given a single oral dose of tibolone (2.5 mg), and serial blood samples were collected after treatment. The plasma concentrations of the two pharmacologically active 3-hydroxyl metabolites of tibolone, 3alpha-hydroxyl-7-methyl- norethynodrel (3alpha-HMN) and 3beta-hydroxyl-7-methyl- norethynodrel (3beta-HMN) in plasma were determined by using a validated liquid chromatography-mass spectrometry (LC-MS) method. RESULTS: The apparent elimination half-life (T(1/2) of 3alpha-HMN was 1.43+/-0.52 h, and that of 3beta-HMN was 1.53+/-0.60 h. Maximum plasma concentrations (C(max)) were found to be 8.75+/-4.36 microg/L for 3alpha-HMN and 3.59+/-1.81 microg/L for 3beta-HMN. Areas under the plasma concentration versus time curve (AUC(0-t)) were 26.30+/-12.14 microg.h(-1).L(-1) for 3alpha-HMN and 9.89+/-4.93 microg.h(-1).L(-1) for 3beta-HMN. CONCLUSION: Stereo-selective differences exist in the pharmacokinetics of tibolone metabolism in humans.

High-performance liquid chromatographic determination of vertilmicin in rat plasma using sensitive fluorometric derivatization.

A sensitive and reliable high-performance liquid chromatographic method was developed for the determination of vertilmicin in rat plasma. Derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) followed by C(18) reversed-phase chromatography allowed the fluorimetric detection of vertilmicin. Optimal conditions for the derivatization of vertilmicin are described. The limit of quantification was 0.02 mg/L. The pharmacokinetics of vertilmicin was studied in 24 rats following intramuscular injection (i.m.) of different doses (4, 8, 16, 32 mg/kg of body weight). The pharmacokinetic parameter values were estimated by use of 3P97 program. In this study, we assessed the dose proportionality of vertilmicin after single intramuscular injection doses and obtained new information on the pharmacokinetics of the compound.

p-Toluenesulfonyl isocyanate as a novel derivatization reagent to enhance the electrospray ionization and its application in the determination of two stereo isomers of 3-hydroxyl-7-methyl-norethynodrel in plasma.

In this paper, p-toluenesulfonyl isocyanate has been reported as a novel derivatization reagent with strong nuclephilic reactivity for the hydroxyl compounds. The derivatization for the two pharmacologically active 3-hydroxyl metabolites, 3alpha-hydroxyl-7-methyl-norethynodrel and 3beta-hydroxyl-7-methyl-norethynodrel by p-toluenesulfonyl isocyanate can be accomplished in 2 min under room temperature. The offline derivatization procedure introduced an easily ionizable sulfonylcarbamic ester moiety to the metabolites. This greatly improved the analyte's sensitivity in negative electrospray ionization and enabled us to achieve the desired lower limit of quantitation at 100 pg/ml in plasma. Therefore, a sensitive high performance liquid chromatography-mass spectrometry (HPLC-MS) method for the analysis of the two stereo isomers was developed. The method had been validated to be accurate, precise, and sensitive, and can be used for the metabolism pharmacokinetic study of tibolone in human subjects.

Simultaneous determination of ropivacaine and antipyrine by high performance liquid chromatography and its application to the in vitro transplacental study.

A simple and reliable RP-HPLC method has been developed for the simultaneous determination of ropivacaine and antipyrine in perfusate samples. Samples were analyzed on an ODS column with UV detection at 210 nm after liquid-liquid extraction. The mobile phase consisted of potassium dihydrogenphosphate (25 mM, adjusted to pH 5.0 with phosphoric acid)-acetonitrile (79:21, v/v). The method has been validated to be precise, accurate and linear. It has been applied to the investigation of placental transfer of ropivacaine via a dually perfused cotyledon model of human placenta in vitro.

Pharmacokinetic difference between S-(+)- and R-(-)-etodolac in rats.

AIM: To study whether etodolac enantiomers have pharmacokinetic difference after oral administration. METHODS: Fourteen rats, divided into two groups randomly, were orally given S-(+)- or R-(-)-etodolac at a single dose of 20 mg/kg, respectively. Blood samples were collected before and at 5, 10, 20, 30 min and 1, 3, 6, 12, 24, 48, 72 h after treatment. The plasma samples were analyzed with a high-performance liquid chromatographic method. RESULTS: The calibration curves were linear in the range of 0.5-50.0 mg/L (r=0.9999) to S-(+)-etodolac and 2.0-200.0 mg/L (r=0.9999) to R-(-)-etodolac, respectively. The main pharmacokinetic parameters of S-(+)- and R-(-)-etodolac were as follows: t1/2(lambdaz) 18+/-4 h vs 19.4+/-2.2 h, tmax 3.3+/-2.6 h vs 4+/-4 h; Cmax 29+/-6 mg/L vs 97+/-14 mg/L, AUC0-t 706+/-100 h.mg.L(-1) vs 2940+/-400 h mg.L(-1), CL(s) 0.030+/-0.006 L.kg(-1).h(-1) vs 0.0065+/-0.0010 L.kg(-1).h(-1) and V/F 0.25+/-0.22 L.kg(-1) vs 0.03+/-0.05 L.kg(-1). There was no significant difference in t1/2(lambdaz), and tmax between S-(+)- and R-(-)-etodolac (P>>0.05). The Cmax, and AUC0-t of R-(-)-etodolac were markedly higher (P<0.05), while the CL(s) and V/F were markedly lower than those of S-etolodac (P<0.05). CONCLUSION: There is pharmacokinetic difference between S-(+)- and R-(-)- etodolac enantiomers in rats after oral administration.

High-performance liquid chromatographic method for determination of leucovorin in plasma: validation and application to a pharmacokinetic study in healthy volunteers.

A sensitive and reliable high-performance liquid chromatographic (HPLC) method, using a solid-phase extraction (SPE), was developed and validated for determination of leucovorin (LV) in human plasma. Plasma sample was extracted by using a Sep-Pak cartridge which could be renewable. The sample was analyzed by HPLC with UV detection at 286 nm. The method was shown to perform selectively and sensitively for LV. The main metabolite of LV, 5-methyltetrahydrofolic acid, and endogenous substances in plasma did not show any interference in the analysis. The limit of detection was 10 ng/mL for LV in plasma and the linear range was 50-1500 ng/mL in plasma. The relative standard deviation (RSD) of intra-day and inter-day assays was 2.8-6.1% and 2.4-5.3%, respectively. The extraction recoveries of LV in plasma were over 90%. The method was proved to be applicable to the pharmacokinetic study of LV in healthy volunteers after a single oral administration (75 mg). The pharmacokinetic parameters and relative bioavailability were investigated for domestic LV tablet and capsule vs an imported tablet.