Determination of Phytomarkers in Pharmaceutical Preparations of Hemidesmus indicus Roots by Micellar Electrokinetic Chromatography and High-Performance Liquid Chromatography–Mass Spectrometry

Micellar electrokinetic chromatography was applied to the determination of the major phytomarkers, namely 2-hydroxy-4-methoxybenzaldehyde, 2-hydroxy-4-methoxybenzoic acid, and 3-hydroxy-4-methoxybenzaldehyde, of Hemidesmus indicus root, an Indian medicinal plant. H. indicus bioactive preparations were analyzed by reverse flow micellar electrokinetic chromatography (MEKC) using sodium taurodeoxycholate as the surfactant. A pH 2.5 phosphate buffer (50 mM) was supplemented with 65 mM of sodium taurodeoxycholate to produce the MEKC pseudostationary phase; because of the suppression of the electroosmotic flow, the migration of the partitioned analytes was toward the capillary anodic end. The use of a short fused-silica capillary (8.5 cm effective length; 50 μm i.d.) allowed the separation of phytomarkers, including vanillin and salicylaldehyde (reported as additional metabolites of H. indicus roots), in less than 8 min. The method showed good validation parameters and was applied to the analysis of methanol extracts and a root decoction of H. indicus, a promising botanical drug. The obtained results were compared to those from an independent high-performance liquid chromatography–mass spectrometry method. The 2-Hydroxy-4-methoxybenzaldehyde, 2-hydroxy-4-methoxybenzoic acid, and 3-hydroxy 4-methoxybenzaldehyde were found in all samples confirming their roles as phytomarkers. The absence of vanillin and salicylaldehyde suggested that these latter compounds should not be regarded as characteristic components of the bioactive preparations from the plant roots.     

Stereospecific analysis of flurbiprofen and its major metabolites in plasma and urine by chiral-phase liquid chromatography

Summary Direct chiral-phase HPLC methods have been developed for the determination of flurbiprofen and its major metabolites, namely 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen, in biological fluids using a derivatized amylose chiral stationary phase (CSP; Chiral-pak AD). Quantification of all three analytes, both free and conjugated, in urine was carried out following liquid-liquid extraction using tandem ultraviolet (UV) and fluorescence detection. Determination of flurbiprofen and the 4′-hydroxy-metabolite in plasma utilized the same CSP but required modification in the mobile phase composition and sole use of fluorescence detection. The urine assay was linear (r>0.998) between 0.05–10 μg mL⁻¹, 0.1–20 μg mL⁻¹ and 0.01–2 μg mL⁻¹ for the enantiomers of flurbiprofen, 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen respectively. The plasma assay was linear (r>0.997) between 0.1–6 μg mL⁻¹ and 0.01–0.6 μg mL⁻¹ for the enantiomers of flurbiprofen and 4′-hydroxyflurbiprofen respectively. Both assays, typically yielded within- and between-day imprecision and accuracy values less than 10% for the enantiomers of the different analytes. Initial volunteer studies suggest that the disposition of flurbiprofen displays modest enantioselectivity in humans.     

Automated Solid-Phase Extraction and Liquid Chromatography Tandem Mass Spectrometry Determination of N-methyl-2-pyrrolidone and its Metabolites in Urine and Plasma

A method for the simultaneous determination of N-methyl-2-pyrrolidone (NMP) and its metabolites 5-hydroxyl-N-pyrrolidone (5HNMP), N-methylsuccinimide (MSI) and 2-hydroxy-N-methylsuccinimide (2HMSI) in plasma and urine has been developed. Samples were purified by SPE using an ASPEC XL4. Analysis was performed using LC–MS equipped with an APCI interface. The analysis provided linear responses in the range of 0.125–12 μg mL⁻¹ for all of the analytes and up to 150 μg mL⁻¹ for 5HNMP and 2HMSI. The within day precision was in the range of 0.9–19.1% for plasma samples and 1.9–10.4% for urine samples whereas the between day precisions were 4.5–11.9% and 1.2–17.5%, respectively. The method was deemed to be suitable for monitoring the levels of NMP and its metabolites in the plasma and urine of occupationally exposed persons.     

Study of the metabolism on tobacco-specific N-nitrosamines in the rabbit by solid-phase extraction and liquid chromatography–tandem mass spectrometry

Metabolism of four tobacco-specific N-nitrosamines (TSNAs), N′-nitrosonornicotine (NNN), N′-nitrosoanatabine (NAT), N′-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) has been studied by solid-phase extraction (SPE) and liquid chromatography–tandem mass spectrometry (LC–MS–MS). 4-(Methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) was used as internal standard. SPE and LC–MS–MS was found to be a rapid, simple, sensitive, and selective method for analysis of TSNAs in rabbit serum. The relative standard deviation (R.S.D., n = 6) for analysis of 5 ng mL⁻¹ and 0.5 ng mL⁻¹ standards and of serum sample spiked with 5 ng mL⁻¹ standards of five TSNAs was 2.1–11% and recovery of 5 ng mL⁻¹ standards from serum was 100.2–112.9%. A good linear relationship was obtained between peak area ratio and concentration in the range of 0.2–100 ng mL⁻¹ for NNAL and 0.5–100 ng mL⁻¹ for other four TSNAs, with correlation coefficients (R ²) >0.99 (both linear and log–log regression). Detection limits for standards in solvent were between 0.04 and 0.10 ng mL⁻¹. Doses of TSNAs administered to rabbits via the auricular vein were 4.67 μg kg⁻¹ and 11.67 μg kg⁻¹, in accordance with the different levels in cigarettes. Metabolic curves were obtained for the four TSNAs and for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a metabolite of NNK; on the basis of these curves we modeled metabolic kinetic equations for these TSNAs by nonlinear curve fitting.     

Simultaneous separation and determination of Cu(II), Fe(III), and Pb(II) by reversed-phase ion-pair chromatography withN,N,N′, N′-ethylene-diaminetetrakis(methylenephosphonic Acid)

Summary A reversed-phase ion-pair chromatographic (RPIPC) method withN,N,N′, N′-ethylenediaminetetrakis(methylenephosphonic acid) (EDTMP) as coordinating agent has been developed for simultaneous separation and detection of Cu(II), Fe(III), and Pb(II) ions. Response is linearly dependent on amount of sample over the range 9.52–50.8 μg mL⁻¹ for Cu(II), 8.31–41.8 μg mL⁻¹ for Fe(III), and 37.3–51.8 μg mL⁻¹ for Pb(II). The method has been applied successfully to an artificial mixed-ore sample.     

Isocratic Reversed-Phase HPLC for Simultaneous Separation and Determination of Seven Antiepileptic Drugs and Two of their Active Metabolites in Human Plasma

A simple reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of the antiepileptic drugs (AEDs) zonisamide (ZNS), primidone (PRI), lamotrigine (LTG), phenobarbital (PB), phenytoin (PHT), oxcarbazepine (OXC), and carbamazepine (CBZ) and two of their active metabolites, monohydroxycarbamazepine (MHD) and carbamazepine 10,11-epoxide (CBZE) in human plasma. Plasma (100 μL) was pretreated by deproteinization with 300 μL methanol containing 20 μg mL⁻¹ propranolol hydrochloride as internal standard. HPLC was performed on a C₈ column (4.6 mm × 250 mm; particle size 5 μm) with methanol–acetonitrile–0.1% trifluoroacetic acid, 235:120:645 (v/v), as mobile phase at a flow rate of 1.5 mL min⁻¹. ZNS, OXC, and CBZ were monitored by UV detection at 235 nm, and PRI, LTG, MHD, PB, PHT, and CBZE by UV detection at 215 nm. Relationships between response and concentration were linear over the concentration ranges 1–80 μg mL⁻¹ for ZNS, 5–50 μg mL⁻¹ for PRI, 1–25 μg mL⁻¹ for LTG, 1–50 μg mL⁻¹ for MHD, 5–100 μg mL⁻¹ for PB, 1–10 μg mL⁻¹ for CBZE, 0.5–25 μg mL⁻¹ for OXC, 1–50 μg mL⁻¹ for PHT, and 1–25 μg mL⁻¹ for CBZ. Intra-day and inter-day reproducibility were adequate (coefficients of variation were ≤11.6%) and absolute recovery ranged from 95.2 ± 6.13 to 107.7 ± 7.76% for all the analytes; for the IS recovery was 98.69 ± 1.12%. The method was proved to be accurate, reproducible, convenient, and suitable for therapeutic monitoring of the nine analytes.     

Simultaneous First Derivative Spectrophotometric Determination of Palladium and Nickel Using 2-(2-Thiazolylazo)-5-Dimethylaminobenzoic Acid as an Analytical Reagent

 A simple, rapid, selective, sensitive and economical method has been developed for the simultaneous determination of trace amounts of palladium and nickel in aqueous methanolic medium using 2-(2-thiazolylazo)-5-dimethylam inobenzoic acid as an analytical reagent by first derivative spectrophotometr y. Palladium is determined by measuring base to peak distance at λ=695.0 nm while nickel is estimated by zero crossing method in the mixture. The linearity is maintained between 0.12–1.75 μg mL⁻¹ for palladium and 0.07–1.60 μg mL⁻¹ for nickel in the pH range 2.8–7.2 and 3.4–8.8 respectively. Seven replicate determinations of 1.0 μ g mL⁻¹ of palladium and 0.8 μg mL⁻¹ of nickel in a mixture give a mean signal height of 0.391 for Pd and 0.541 for Ni with relative standard deviations of 0.9% and 1.2%, respectively. The sensitivity of the proposed method is 0.391 (dA/dλ)/(μg mL⁻¹) for palladium and 0.685 (dA/dλ)/(μg mL⁻¹) for nickel. Various parameters have been optimised for the simultaneous determination of palladium and nickel in various complex samples. Received March 30, 1999. Revision November 25, 1999.     

Simultaneous HPLC Analysis of Olmesartan and Hydrochlorothiazide in Combined Tablets and in vitro Dissolution Studies

A simple, rapid and reproducible HPLC method was developed and validated for the simultaneous determination of olmesartan (OLM) medoxomil and hydrochlorothiazide (HCT) in combined tablets. Chromatography was carried out on a 4.6 mm I.D × 200 mm, 5 μm cyano column with methanol–10 mM phosphoric acid containing 0.1% triethylamine (pH 2.5, 50:50 v/v) at a flow rate of 1.0 mL min⁻¹ and UV detector was set at 260 nm. Valsartan (VAL) was used as internal standard (IS). A linear response was observed in the range of 0.2–6 μg mL⁻¹ (r ² = 0.9998) for OLM and 0.1–4 μg mL⁻¹ (r ² = 0.9999) for HCT, respectively. The method showed good recoveries (99.56% for OLM and 99.48% for HCT) and the relative standard deviation (RSD) values for intra- and inter-day precision were 0.70–1.59 and 0.80–2.00% for OLM and 1.20–1.37 and 1.63–1.93% for HCT, respectively. The developed method was applied successfully for quality control assay of OLM and HCT in combined tablets and in vitro dissolution studies.     

A Simple and Validated LC Method for the Simultaneous Determination of Three Compounds in Mikania laevigata Extracts

The Mikania genus is widely known as guaco and is used to treat fever, rheumatism, influenza and respiratory diseases. This article deals with the simultaneous quantification of three commercially available phenolic markers (o-coumaric acid, coumarin and syringaldehyde) in M. laevigata extracts, through LC-PDA. The validation data show that the method is specific, accurate, precise and robust, and also indicative of the stability of guaco extract. The method was linear, over a range of 1.25–20.0 μg mL⁻¹ for o-coumaric acid, 2.5–40.0 μg mL⁻¹ for coumarin, and 0.25–4.0 μg mL⁻¹ for syringaldehyde. The range of recovery was 94.3–96.4% for all the components, at a level of 100%.     

Determination and Pharmacokinetic Study of 10-O-Dimethylaminoethylginkgolide B in Rat Plasma by LC–MS

To evaluate the pharmacokinetics of a novel analogue of ginkgolide B, 10-O-dimethylaminoethylginkgolide B (XQ-1) in rat plasma in pre-clinical studies, a sensitive and specific liquid chromatographic method with electrospray ionization mass spectrometry detection (LC–ESI–MS) was developed and validated. After a simple extraction with ethyl acetate, XQ-1 was analyzed on a Shim-pack C18 column with a mobile phase of a mixture of 1 μmol L⁻¹ ammonium acetate containing 0.02% formic acid and methanol (55:45, v/v) at a flowrate of 0.3 mL min⁻¹. Detection was performed in selected ion monitoring (SIM) mode using target ions at [M + H]⁺ m/z 496.05 for XQ-1 and m/z 432.10 for the internal standard (lafutidine). Linearity was established for the concentration range from 2 to 1,000 ng mL⁻¹ . The extraction recoveries ranged from 86.0 to 89.9% in plasma at concentrations of 5, 50, and 500 ng mL⁻¹. The lower limit of quantification was 2 ng mL⁻¹ with 100 μL plasma. The validated method was successfully applied to a pharmacokinetic study after intragastic administration of XQ-1 mesylate in rats at a dose of 20 mg kg⁻¹.     

LC Determination of 4-Bromoaniline in Green Algae Chlamydomonas reinhardtii After Continuous-Flow Microextraction

In this study, the determination of 4-Bromoaniline (4-BA) in green algae Chlamydomonas reinhardtii (C. reinhardtii) was investigated by applying continuous-flow microextraction (CFME) combined with high-performance liquid chromatography (HPLC). Continuous-flow microextraction was conducted in a homemade glass chamber, i.e. the sample solution flowed through a constant volume drop of solvent in the chamber at a constant flow rate. The effects of different factors on extraction efficiencies were also investigated and these factors included the kind of extraction solvent, solvent drop volume, sample flow rate, extraction time and addition amount of salt. Under the optimum extraction conditions (extraction solvent, carbon tetrachloride; solvent drop volume, 3.5 μL; sample flow rate, 1.0 mL min⁻¹; extraction time, 10 min; no addition of salt), the calibration plot was set up by plotting peak area against a series of 4-Bromoaniline concentrations (0.01–10 μg mL⁻¹) in aqueous solution. The correlation coefficient (r) was 0.9990. The limit of detection (LOD) was 0.6 ng mL⁻¹. The precision of this method was obtained by successive five time analyses of 100-ng mL⁻¹ standard solution of 4-Bromoaniline, and the relative standard deviation (RSD) was 3.5%. The concentration factor was calculated by the ratio of peak area of the analyte obtained after and before extraction and found to be 10.6. 4-Bromoaniline residues in Chlamydomonas. reinhardtii cells and tap water samples were satisfactorily analyzed according to the method described above.     

Simultaneous RP-LC Determination of Losartan Potassium, Ramipril, and Hydrochlorothiazide in Pharmaceutical Preparations

A simple, rapid, and precise reversed-phase high-performance liquid chromatographic method has been developed for simultaneous determination of losartan potassium, ramipril, and hydrochlorothiazide. The three drugs were separated on a 150 mm × 4.6 mm i.d., 5 μm particle, Cosmosil C₁₈ column. The mobile phase was 0.025 m sodium perchlorate–acetonitrile, 62:38 (v/v), containing 0.1% heptanesulphonic acid, pH adjusted to 2.85 with orthophosphoric acid, at a flow rate of 1.0 mL min⁻¹. UV detection was performed at 215 nm. The method was validated for linearity, accuracy, precision, and limit of quantitation. Linearity, accuracy, and precision were acceptable in the ranges 35–65 μg mL⁻¹ for losartan, 1.75–3.25 μg mL⁻¹ for ramipril, and 8.75–16.25 μg mL⁻¹ for hydrochlorothiazide.     

Resolution of an intense sweetener mixture by use of a flow injection sensor with on-line solid-phase extraction

An integrated solid-phase spectrophotometry–FIA method is proposed for simultaneous determination of the mixture of saccharin (1,2-benzisothiazol-3(2H)-one-1,1-dioxide; E-954) (SA) and aspartame (N-l-α-aspartyl-l-phenylalanine-1-methyl ester; E-951) (AS). The procedure is based on on-line preconcentration of AS on a C₁₈ silica gel minicolumn and separation from SA, followed by measurement, at λ=210 nm, of the absorbance of SA which is transiently retained on the adsorbent Sephadex G-25 placed in the flow-through cell of a monochannel FIA setup using pH 3.0 orthophosphoric acid–dihydrogen phosphate buffer, 3.75×10^–3 mol L⁻¹, as carrier. Subsequent desorption of AS with methanol enables its determination at λ=205 nm. With a sampling frequency of 10 h⁻¹, the applicable concentration range, the detection limit, and the relative standard deviation were from 1.0 to 200.0 μg mL⁻¹, 0.30 μg mL⁻¹, and 1.0% (80 μg mL⁻¹, n=10), respectively, for SA and from 10.0 to 200.0 μg mL⁻¹, 1.4 μg mL⁻¹, and 1.6% (100 μg mL⁻¹, n=10) for AS. The method was used to determine the amounts of aspartame and saccharin in sweets and drinks. Recovery was always between 99 and 101%. The method enabled satisfactory determination of blends of SA and AS in low-calorie and dietary products and the results were compared with those from an HPLC reference method.     

Determination and Pharmacokinetic Study of Calycosin-7-O-β-d-glucoside in Rat Plasma After Intravenous Administration of Aidi Lyophilizer

Aidi injection is a clinical medicine used in China for the treatment of cancer. Calycosin-7-O-β-d-glucoside is the main effective components of the formulas. In this study, a high performance liquid chromatographic (LC) method was developed to quantify calycosin-7-O-β-d-glucoside in rat plasma using a liquid–liquid extraction and ultraviolet (UV) absorbance detection. LC analysis was performed on a Diamonsil C₁₈ column (200 × 4.6 mm i.d., 5 μm particle size) with isocratic mobile phase consisting of acetonitrile–0.05% phosphoric acid (19.5:80.5, v/v) of a flow rate of 1.0 mL min⁻¹. The linear range was 0.11–17.6 μg mL⁻¹ and the low quantification limit was 0.11 μg mL⁻¹ (S/N = 10). The intra- and inter-day relative standard deviations (RSD) in the measurement of quality control (QC) samples 0.11, 0.22, 1.32 and 8.80 μg mL⁻¹ ranged from 4.1 to 6.3 and 4.3 to 6.2%, respectively. The accuracy was from −6.7 to 4.3% in terms of relative error (RE). Calycosin-7-O-β-d-glucoside was stable in storage at −20 °C for 2 weeks and stable after three freeze–thaw cycles in rat plasma. This method was validated for specificity, accuracy, precision and was successfully applied to pharmacokinetic study of calycosin-7-O-β-d-glucoside in rat plasma after intravenous administration of Aidi lyophilizer.     

LC Determination of Four Isoflavone Aglycones in Red Clover (Trifolium pratense L.)

Red clover (Trifolium pratense L.) is an important forage plant that contains the isoflavones daidzein, genistein, formononetin, and biochanin A. These compounds have been studied lately due to their human health benefits. The aim of this study was to develop and validate an HPLC method with simplified sample preparation to quantify daidzein, genistein, formononetin and biochanin A simultaneously in red clover leaves. The validation showed that the method is specific, accurate, precise and robust, not to mention that the sample preparation is easier and faster than those described earlier. The response was linear over a range of 0.01–0.2 μg mL⁻¹ for daidzein, 0.05–0.5 μg mL⁻¹ for genistein, 4–40 μg mL⁻¹ for formononetin and 2–20 μg mL⁻¹ for biochanin A. The range of recoveries was 85.6–101.0%. The RSD for intra- and inter-day precision were <2.54 and <7.22%, respectively. Five populations of red clover, from the National Plant Germplasm System-USDA were analyzed and the content of daidzein, genistein, formononetin and biochanin A ranged from 7.87–91.31, 51.60–131.30, 6568.33–23461.82, to 2499.55–10337.33 μg g⁻¹ of dried material, respectively.     

Simultaneous Determination of Triptolide and Tripdiolide in Extract of Tripterygium wilfordii Hook. f. by LC–APCI-MS

A novel method using liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry in the negative selected ion monitoring mode has been developed and validated for rapid simultaneous determination of triptolide and tripdiolide in the extract of Tripterygium wilfordii Hook. f. The molecular ions m/z [M–H]⁻ 359 and 375 were selected for the quantification in selected ion monitoring mode for triptolide and tripdiolide. Standard calibration curve was linear over the concentration range of 0.12–24 and 0.15–30 μg mL⁻¹ for triptolide and tripdiolide. The relative standard deviations of intra- and inter-day were in the range of 4.7–9.9 and 8.9–12.6%. The average recoveries were between 96.4 and 104.6%. The limits of quantitation were 2.0 × 10⁻³ and 2.5 × 10⁻³ μg mL⁻¹ for triptolide and tripdiolide.     

Simple and rapid determination of piperacillin and ceftazidime in human plasma samples by HPLC

Summary A simple and rapid liquid chromatographic method has been developed for the determination of therapeutic levels of piperacillin (I) and ceftazidime (II) in human plasma. Plasma and p-propionamidophenol (internal standard) were precipitated with methanol (I) or 20% trichloroacetic acid (II). The supernatant was analysed on a 5 μm Spherisorb ODS C₁₈ column with acetonitrile-0.05 M phosphate buffer pH 3.8 as mobile phase and ultraviolet detection at 254 nm. The calibration graph was linear from 10 to 250 μg mL⁻¹, for (I), and from 5 to 200 μg mL⁻¹ for (II). Intra and inter-day CV did no exceed 2.29% for (I), and were 10.76–11.13%–2.00–5.62 for (II) at concentrations of 10 μg mL⁻¹ and 250 μg mL⁻¹.     

Determination of 5-n-Butyl-4-{4-[2-(1H-tetrazole-5-yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6-dichloridephenyl)-3H-1,2,4-triazol-3-one, a New Angiotensin Type 1 Receptor Antagonist in Rat Plasma by LC-ESI-MS: Application to Pharmacokinetic Studies

A simple and sensitive reversed-phase LC-ESI-MS method to identify and quantitate 5-n-butyl-4-{4-[2-(1H-tetrazole-5-yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6-dichloridephenyl)-3H-1,2,4-triazol-3-one (1b), a new Angiotensin II type 1 receptor antagonist in rat plasma has been developed and validated. Sample preparation used a simple liquid–liquid extraction with ethyl acetate. Separation was achieved by gradient elution on a C₁₈ column. The mobile phase consisted of acetonitrile and water (0.05% triethylamine and 0.05% acetic acid) at a flow rate of 0.2 mL min⁻¹. The detection utilized selected ion monitoring (SIM) in the negative mode at m/z 507.1 and m/z 407.2 for the deprotonated molecular ions of 1b and the internal standard irbesartan, respectively. The lower limit of quantification was reproducible at 5 ng mL⁻¹ with 100 μL of plasma and the good linear was observed in the 5–500 ng mL⁻¹ range. This concentration range corresponded well with the plasma concentrations of 1b in pharmacokinetic studies. Recoveries of 1b in rat plasma were 76.1, 74.6 and 79.0% at 5, 50 and 500 ng mL⁻¹. The RSD of intra-assay and inter-assay variations were all less than 5%. This validated LC-ESI-MS assay is an economic, quick, precise and reliable method for the analysis of 1b in pharmacokinetic studies.     

Simultaneous Determination of Baicalin, Baicalein, Wogonin, Oxysophocarpine, Oxymatrine and Matrine in the Chinese Herbal Preparation of Sanwu-Huangqin-Tang by Ion-Paired HPLC

A sensitive and reliable ion-paired high-performance liquid chromatographic method has been established for the simultaneous quantification of six major active ingredients, namely baicalin, baicalein, wogonin, oxysophocarpine, oxymatrine and matrine in the Chinese herbal preparation, Sanwu-Huangqin-Tang. HPLC analyses were performed on a Phenomenex luna C₁₈ column with mobile phase of methanol–acetonitrile–aqueous phosphoric acid at a flow rate of 0.9 mL min⁻¹. The complete separation was achieved within 35 min for the six target constituents. A good linear regression relationship between peak-areas and concentrations was obtained over the range of 12.10–242.0 μg*mL⁻¹ for baicalin, 5.05–101.0 μg*mL⁻¹ for baicalein, 0.95–19.0 μg*mL⁻¹ for wogonin, 2.75–55.0 μg*mL⁻¹ for oxysophocarpin, 2.75–55.0 μg*mL⁻¹ for oxymatrine and 4.90–98.0 μg*mL⁻¹ for matrine, respectively. The repeatability was evaluated by intra- and inter-day assays with relative standard deviation (RSD) being less than 5.1%. The recoveries, measured at three concentration levels, varied from 93.8 to 102.1%. The assay was successfully applied for determination of six bioactive compounds in Sanwu-Huangqin-Tang. The interaction of chemical constituents was observed when the herbs were used in compatibility. The results indicated that the developed assay method was rapid, accurate and could be readily utilized as a quality control method for Sanwu-Huangqin-Tang.     

Reversed Capillary Electrochromatography: Investigation of Peak Symmetry for Eluting Curves of Solutes

Summary A clean method without use of organic solvents has been developed for isolation and high-performance liquid chromatographic (HPLC) determination of oxytetracycline (OTC) and sulphadimidine (SDD) in cow's milk. Isolation is rapid and simple—homogenization with an inorganic acid solution by means of a handy ultrasonic homogenizer, which is easy-to-use and portable, followed by centrifugation. Reversed-phase HPLC was performed on a C₄ column, with 1.25 mmol L⁻¹ succinic acid solution as mobile phase, and identification was by means of a photodiode-array detector. Separation of the analytes was achieved in less than 8 min. Significant linearity was established over the concentration range of 0.1–1.0 μg mL⁻¹ for both target compounds (r>0.99,P<0.01). Average recoveries of OTC and SDD (each spiked at 0.1–1.0 μg mL⁻¹) were ≥88.8, and inter- and intra-assay variability was ≤2.8%. The total time required for analysis of one sample was <20 min. The limits of quantitation of the method (μg mL⁻¹ in milk) were 0.044 for OTC and 0.023 for SDD. No organic solvent was used at any stage of the analysis.