Simultaneous determination of three Aconitum alkaloids in six herbal medicines by high-performance liquid chromatography

To simultaneously determine three components of aconitine, mesaconitine, and hypaconitine in six species of Aconitum genus, an extraction condition for the total alkaloids was specifically optimized and a simple analytical method of reversed-phased highperformance liquid chromatography (HPLC) was developed. The extraction rate of total alkaloids in A. szechenyianum Gay was 98.3% for repeated extracting three times with an acidic alcohol solution (alcohol: pH 3.0 HAc = 85:15, v/v). The chromatography was carried out on a Phenomenex Luna C(18) column by gradient elution with a mobile phase of 0.03 mol/mL ammonium bicarbonate (pH = 9.50) -acetonitrile at a flow rate of 1.0 mL/min. The method for all three alkaloids had good linear relationships (r > 0.999) in the concentration range of 1.0-200.0 μg/mL. The average recoveries were 96.6-103.1%, and the LOQ and LOD were in the range of 25-37 ng/mL and 9-12 ng/mL, respectively. The quantitative results indicated that contents of the three alkaloids varied significantly among crude aconite roots, so quality control of traditional Chinese medicines containing aconite roots should be taken into account.     

Simultaneous determination of three naturally occurring estrogens in environmental waters by high-performance liquid chromatography

A simple, sensitive and accurate reversed-phase high-performance liquid chromatographic (HPLC) method for simultaneous determination of three naturally occurring estrogenic steroids including estrone (E1), 17β-estradiol (E2) and estriol (E3) in environmental water samples was developed. Analytes were extracted with ethyl acetate solvents and preconcentrated prior to HPLC analysis. Separations were accomplished in <20 min using a reversed-phase C(18) column (4.6×250 mm id, 5 μm) with a gradient elution of mobile phase containing 3.0 mM ammonium acetate/acetonitrile mixtures (flow rate, 1.0 mL/min). UV light absorption responses at 205 nm were linear over a wide concentration range from 100,000 μg/L to the detection limits of 0.96 μg/L E1, 0.64 μg/L E2 and 0.78 μg/L E3. Quantitation was carried out by the peak area method. The relative standard deviation for the analysis of three estrogens was <3.0%. This method was applied for the simultaneous determination of estrogens in environmental water samples collected in Zhejiang, China. The higher concentrations of both E2 and E3 were found in Tang River and West Lake waters, and E1 was detected in lake water only. All three estrogens were below the detection limits in rain waters.     

Simultaneous determination of fluzinamide and three of its active metabolites in plasma by high-performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic method has been developed for a new anticonvulsant, fluzinamide, and three of its active metabolites. This method requires only 0.5 ml of plasma, and it involves a single extraction with a mixture of hexane--dichloromethane--butanol (55:40:5). The plasma extract is chromatographed on a 10-micron, C18 reversed-phase column and quantitated by ultraviolet absorbance at 220 nm. The concentration--response curves for all four compounds are linear from 0.05 micrograms/ml to at least 10 micrograms/ml. The extraction efficiency of this method is greater than 90%. The accuracy and precision of the method were tested by analyzing spiked unknown samples that had been randomly distributed across the concentration range. The mean concentrations found were within +/- 9% of the various amounts added with a standard deviation of +/- 3.5%. This method has been successfully applied to the analysis of samples obtained from fluzinamide-dosed dogs, healthy unmedicated volunteers, and patients who were at steady state with phenytoin, carbamazepine, and fluzinamide.     

Simultaneous determination of aceclofenac and three of its metabolites in human plasma by high-performance liquid chromatography

Aceclofenac [[2-(2',6'-dichlorophenyl)amino]phenylacetoxyacetic acid] is a phenylacetic acid derivative with potent analgesic and anti-inflammatory properties and an improved gastro-intestinal tolerance. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of aceclofenac and three of its metabolites (4'-hydroxy-aceclofenac, diclofenac, 4'-hydroxy-diclofenac) in human plasma. The analytes were separated using an acetonitrile-phosphate buffer gradient at a flow rate of 1 mL/min, and UV detection at 282 nm. The retention times for aceclofenac, diclofenac, 4'-hydroxy-aceclofenac, 4'-hydroxy-diclofenac and ketoprofen (internal standard) were 69.1, 60.9, 46.9, 28.4 and 21.2 min, respectively. The validated quantitation range of the method was 10-10000 ng/mL for aceclofenac, 4'-hydroxy-aceclofenac and diclofenac, and 25-10000 ng/mL for 4'-hydroxy-diclofenac. The developed procedure was applied to assess the pharmacokinetics of aceclofenac and its metabolites following administration of a single 100 mg oral dose of aceclofenac to three healthy male volunteers.     

Simultaneous determination of retinol and tocopherols by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method to determine retinol and all four tocopherols (alpha-, beta-, gamma- and delta-) simultaneously was established using a reversed-phase column (YMC-PACK A-302 S-5 120A ODS). The HPLC conditions were mobile phase 65% isopropanol, sample solvent 99.5% methanol and temperature 30 degrees C. Retinol and tocopherols were measured in rat liver.     

Simultaneous determination of four antiepileptic drugs in serum by high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed for the simultaneous determination of oxcarbazepine, 10-hydroxycarbamazepine, epoxycarbamazepine, carbamazepine, phenobarbital and phenytoin. After protein precipitation by acetonitrile, the supernatant was analysed on a C18 reversed-phase HPLC column. Antiepileptic drugs and oxazepam (internal standard) were detected by ultraviolet absorbance at 240 nm. Linearity was established for the whole concentration range for each compound. Quantitation limits of oxcarbazepine, 10-hydroxycarbamazepine, epoxycarbamazepine, carbamazepine, phenobarbital and phenytoin were 0.58, 3.5, 2.35, 0.66, 1.02 and 3.13 microg/mL, respectively, and mean recoveries added to serum were 105.15, 84.76, 94, 45, 96.52, 98.62 and 95.08%, respectively. This method has been used for the simultaneous determination of steady-state serum concentration of antiepileptic drugs in patients treated by one or more anticonvulsive treatment.     

Simultaneous determination of naphthoquinone derivatives in Boraginaceous herbs by high-performance liquid chromatography

A high-performance liquid chromatographic method using diode-array detection (HPLC-DAD) has been developed for the simultaneous quantification of eight naphthoquinone derivatives namely shikonin, acetylshikonin, deoxyshikonin, β-acetoxyisovalerylshikonin, isobutylshikonin, β,β-dimethylacrylshikonin, 2-methyl-n-butyrylshikonin and isovalerylshikonin in nine species of the Boraginaceae family. These species, coming from different areas of China, are all used as interchangeable sourcing plants for the Chinese Materia Medica known as “Zicao”, and are Arnebia euchroma (Royle) Johnston., A. guttata Bunge, Lithospermum erythrorhizon Sieb. et Zucc., Onosma paniculatum Bur. et Franch., O. exsertum Hemsl., O. confertum W.W. Smith, O. hookerii Clarke var. longiflorum Duthie, O. hookerii Clarke and O. waltonii Duthic. Quantification of the eight naphthoquinones in all the Zicao samples are reported and compared with each other. Furthermore, two positional isomers, 2-methyl-n-butyrylshikonin and isovalerylshikonin, were successfully separated and quantified for the first time in the present study. The results showed that, besides the three officially used species (namely, A. euchroma, A. guttata and L. erythrorhizon) that were listed in Chinese pharmacopoeia as interchangeable sourcing plants for Zicao, other six species of Onosma used by native peoples in Tibet and Yunnan Province also contain various types and considerable amounts of naphthoquinones and that O. waltonii contains the most. Therefore, these species of Onosma could be developed as new sources of naphthoquinones. The entire analytical procedure is reproducible and suitable for the quantification of naphthoquinones in all related Boraginaceous plants for quality assessment purposes.     

Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C18 and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5-21.5 nmol/ml for NEF and 0.4-9.5 nmol/ml for metabolites in serum and 4-86 nmol/ml for NEF and 8-190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

Simultaneous determination of four bufadienolides in human liver by high-performance liquid chromatography

A high-performance liquid chromatographic method for the simultaneous quantitation of four bufadienolides-cinobufotalin, bufalin, cinobufagin and resibufogenin-in human liver was investigated. The procedure involved solid phase extraction of human liver with an Oasis HLB cartridge coupled with reversed-phase HPLC and photodiode array detection. Recoveries obtained from spiked liver for the bufadienolides were better than 70%. The linearity was studied up to 1.2 mg/kg and the detection limits of the method were 0.4 ng for cinobufotalin and bufalin and 0.5 ng for cinobufagin and resibufogenin. The developed method was successfully applied to a lethal poisoning case.     

Simultaneous determination of residual synthetic antibacterials in fish by high-performance liquid chromatography

A simple and rapid high-performance liquid chromatographic (HPLC) method for the simultaneous determination of sulphamonomethoxine (SMMX), sulphadimethoxine (SDMX), sulphisozole (SIZ), nalidixic acid (NA), oxolinic acid (OXA), piromidic acid (PMA), furazolidone (FZ) and sodium nifurstyrenate (NFSA) in cultured fish was developed. The drugs were extracted with 0.2% metaphosphoric acid-methanol (6:4), followed by a Bond Elut C18 clean-up procedure. The HPLC separation was carried out on an Inertsil ODS column (150 x 4.6 mm I.D.) using 5 mM aqueous oxalic acid-acetonitrile (55:45) as the mobile phase with detection at 265 nm (0.04 a.u.f.s.). The calibration graphs were rectilinear from 1 to 20 ng for OXA, from 2 to 50 ng for SMMX, SDMX, SIZ, NA, PMA and FZ and from 5 to 100 ng for NFSA. The recoveries of each drug added to fish were 65.0-89.5%. The detection limits were 0.02 micrograms/g for OXA, 0.05 micrograms/g for SMMX, SDMX, SIZ, NA, PMA and FZ and 0.1 micrograms/g for NFSA.     

Simultaneous determination of itraconazole and hydroxyitraconazole in human plasma by high-performance liquid chromatography

The development and validation of a high-performance liquid chromatography (HPLC) method for the simultaneous determination of itraconazole and its metabolite, hydroxyitraconazole, in human plasma is described. The method involved liquid-phase extraction of itraconazole and hydroxyitraconazole using a hexane-dichloromethane (70:30) mixture, after addition of loratidine as an internal standard (IS). Separation was achieved with a reversed-phase C18 column (250 mm x 4.6 mm) employing fluorescence detection (excitation: 264 nm, emission: 380 nm). The mobile phase consisted of [0.01% triethylamine solution adjusted to pH 2.8 with orthophosphoric acid-acetonitrile (46:54)]-isopropanol (90:10, v/v) at a flow rate of 1.0 ml/min. For both the drug and metabolite, the standard curve was linear from 5.0 to 500 ng/ml with goodness of fit (r2) greater than 0.98 observed with four precision and accuracy batches during validation. An observed recovery was more than 70% for drug, metabolite and internal standard. The applicability of this method to pharmacokinetic studies was established after successful application during 35 subjects bioavailibity study. The method was found to be precise, accurate and specific during the study.     

Simultaneous determination of trimipramine and its major metabolites by high-performance liquid chromatography

Trimipramine is a tricyclic antidepressant drug often assayed by gas chromatographic or gas chromatographic-mass spectrometry techniques. A high-performance liquid chromatographic method with electrochemical detection is described for the assay of trimipramine and its major metabolites, monodesmethyltrimipramine and 2-hydroxytrimipramine, in plasma. The method is sensitive, accurate and robust and thus suitable for routinely assaying samples following single doses of trimipramine to man. The assay was applied to plasma samples obtained following a single 50-mg dose of trimipramine to healthy volunteers.     

Simultaneous determination of triterpene saponins in ginseng drugs by high-performance liquid chromatography

A HPLC method for the simultaneous determination of 11 triterpene saponins with four-type aglycones (protopanaxadiol, protopanaxatriol, ocotillol and oleanolic acid types) in Ginseng drugs was developed and validated. Using a gradient of acetonitrile and 10 mM K-phosphate buffer (pH 5.80) as the mobile phase and UV detection at 196 nm, more than 18 ginsenosides with different aglycones were separated satisfactorily within 60 min. The detection limits (signal/noise> or =3) were 0.1 microg for ginsenosides Rb1, Rc, Rd, Re and Rg1, chikusetsusaponin III, and notoginsenoside R2, 0.2 microg for gisenoside Ro and chikusetsusaponin IVa, 0.3 microg for chikusetsusaponin IV, and 3 microg for majonoside R2. The calibration curve of each saponin had a correlation coefficient close to 1. Intra- and interday precisions were less than 2.1% (n=5) and 3.3% (n=15), respectively. The recovery rates of extraction were in the range of 96.4-102.7% for all ginsenosides. By adopting this method, the determinations of 11 ginsenosides in three Ginseng drugs derived from Panax ginseng, Panax vietnamensis var. fuscidiscus and Panax japonicus (Japan) were achieved.     

Simultaneous determination of rifampicin and sulbactam in mouse plasma by high-performance liquid chromatography

A simple and rapid high-performance liquid chromatographic (HPLC) method with ultraviolet detection has been developed and validated for the simultaneous determination of rifampicin and sulbactam in mouse plasma. Plasma samples were deproteinized with acetonitrile and separated by HPLC on a RP-18 (125 x 4 mm, 5 microm) column and gradient elution with potassium dihydrogen phosphate solution (pH 4.5; 50 mm) and acetonitrile at a flow-rate of 1.0 mL/min. Rifampicin and sulbactam were monitored at 230 nm and confirmed by means of their UV spectra using a diode-array detector. The method was linear at plasma levels from 1 to 100 microg/mL for rifampicin and from 5 to 200 microg/mL for sulbactam. The limits of quantification were 0.6 microg/mL for rifampicin and 4.2 microg/mL for sulbactam. The intra- and inter-day precisions of the method (RSD) were lower than 5% for both compounds. Average recoveries of rifampicin and sulbactam from mice plasma were 98.2 and 89.3%, respectively. The developed method was successfully applied to the determination of the pharmacokinetic profile of both compounds in mice.