Isolation and Simultaneous Determination of Coumarin Compounds in Radix Angelica dahurica

For the first time simple, rapid, and systematic methods have been established for preparative isolation and purification of coumarin compounds in an important traditional Chinese Medicine, Radix Angelica dahurica, and for simultaneous determination of several of the compounds in the medicine. Bergapten, imperatorin, and cnidilin, three of the biologically active coumarin compounds, were isolated from the chloroform-soluble fraction of the ethanol extract of Radix Angelica dahurica. After further purification by open column ODS chromatography the purified components were simultaneously determined, with two other coumarins (osthole and isoimperatorin), by reversed phase high-performance liquid chromatography (RP-HPLC) on a C₁₈ column, with methanol–water, 66:34 (v/v), as mobile phase at a flow rate of 0.8 mL min⁻¹. The compounds were detected by UV absorption at 310 nm. Calibration plots for all the coumarins had correlation coefficients close to unity. Limits of detection (S/N = 3) were <92 ng mL⁻¹ and limits of quantification (S/N = 10) were <259 ng mL⁻¹. Mean recovery of the coumarins was in the range 96.7–101.9% and the intra-day and inter-day precision, as relative standard deviation, was <2.3 and <2.9%, respectively. This simple, sensitive, and reproducible method can be used for quality control of Radix Angelica dahurica.     

Quantitative HPLC Determination and Stability Studies of Pyridostemin in Extracts and Water Dispersible Granule Formulations of Stemona curtisii

A reversed-phase high-performance liquid chromatographic method has been developed and validated for the determination of pyridostemin, the major pesticidal alkaloid found in Stemona curtisii. This methodology was applied to the investigation of plant extracts and water dispersible granule formulations. Stability indicating procedures have also been carried out. The chromatographic separation was on a C₁₈ column with a mixture of acetonitrile–water–triethylamine (30:70:0.12, v/v/v), using UV detection at 300 nm. Validation procedures showed that the method was specific, accurate and precise. The response was linear over a range of 5–25 μg mL⁻¹ with recoveries in the range of 98.28–102.85%. The RSD for intra- and inter-day precision were <0.72 and <1.29%, respectively. Extraction of plant material with dichloromethane gave a significantly higher pyridostemin content in the crude extracts when compared with extractions in methanol. Partial purification of the crude extracts by silica gel column chromatography was used to concentrate the mixture about fourfold. Degradation behavior of pyridostemin in the partially purified extracts followed first-order kinetics. The main pathways for its decomposition were base hydrolysis and oxidation.     

Analysis of Phytochelatins and Other Thiol-Containing Compounds by RP-HPLC with Monobromobimane Precolumn Derivatization

In this article, a method for quantitative determination of phytochelatins (PC _n being the classic example) and other thiol-containing compounds in mixed standard solution and plant tissues is presented. Thiols were converted to fluorescent derivatives by precolumn derivatization with monobromobimane. The results showed that PC _n and other thiol-containing compounds in standard mixed solutions were rapidly separated within 15 min by using a ACN 0.1% trifluoroacetic acid binary gradient elution. Glutathione was representatively selected to test the precision of this method. The calibration curve was linear in the range of 1.25–160 ng μl⁻¹ (regression coefficient r ²=0.9999). It was confirmed that this method was rapid, simple, highly sensitive, stable, and had the property of simultaneous determination of PC _n and other thiol-containing compounds. This method was applied to determine PC _n and other thiol-containing compounds in a Cd hyperaccumulator Sedum alfredii in response to Cd. It was found that no PC _n was detected in any tissue at any Cd treatment, suggesting that Cd hyperaccumulation and detoxification in this plant is not based on PC synthesis. Translated from Journal of Nanjing University (Natural Science Edition), 2005, 41(3) (in Chinese)     

Simultaneous Determination of Seven Active Compounds in Radix Salviae Miltiorrhizae by Temperature-Controlled Ultrasound-Assisted Extraction and HPLC

A simple and effective high-performance liquid chromatographic (HPLC) method has been developed for simultaneous quantification of three phenolic acids (3,4-dihydroxyphenyllactic acid (Chinese name danshensu), protocatechuic aldehyde, and salvianolic acid B) and four diterpenes (dihydrotanshinone I, cryptotanshinone, tanshinone I, and tanshinone II_A) in radix salviae miltiorrhizae. Chromatography was performed on a 250 mm × 4.6 mm i.d., 5-μm particle size, C₁₈ column. The mobile phase was a linear gradient prepared from 0.1% (v/v) aqueous formic acid and acetonitrile at a flow-rate of 1.0 mL min⁻¹. All the target components were well separated with high resolution and without interference. Good linearity (R ² > 0.999) was observed over the concentration ranges investigated, and intra-day and inter-day precision were high. Temperature-controlled ultrasound-assisted extraction was used to prevent hydrolysis of thermally unstable components during the sample-extraction procedure, and the extraction conditions were carefully optimized. Recovery of the seven components was from 98.45 to 100.63% and relative standard deviations were always <1.5%. The validated method was successfully used for simultaneous quantification of the three phenolic acids and the four diterpenes in radix salviae miltiorrhizae of different geographic origins.     

Quantitative TLC Analysis of Sterol (24β-Ethylcholesta-5,22E,25-triene-3β-ol) in Agnimantha (Clerodendrum phlomidis Linn)

A quantitative method using silica gel 60F₂₅₄ high performance thin layer chromatography plates, automated bandwise sample application, and automated visible mode densitometric method has been developed for the determination of 24β-ethylcholesta-5,22E,25-triene-3β-ol (ECTO) in the aerial part of Clerodendrum phlomidis. ECTO was used as a chemical marker for the standardization of C. phlomidis plant extracts. The separation was performed on silica gel 60F₂₅₄ TLC plates using chloroform-methanol (98.5: 1.5, v/v) as mobile phase. The quantitation of ECTO was carried out using the densitometric reflection/absorption mode at 650 nm after post chromatographic derivatization with anisaldehyde reagent. A precise and accurate quantification can be performed in the linear working concentration range of 150–400 ng band⁻¹ with good correlation (r ² = 0.996). The method was validated for peak purities, precision, robustness, limit of detection (LOD) and quantitation (LOQ), etc. as per ICH guidelines.     

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.     

Determination of the Active Metabolite of Prulifloxacin in Human Plasma by HPLC with Fluorescence Detection

A cheap, simple and rapid sample preparation method has been developed for quantification of ulifloxacin, the active metabolite of prulifloxacin in human plasma, by HPLC with fluorescence detection using lemefloxacin as the internal standard. One-step protein precipitation with 10% perchloric acid (2:1, v/v) on a 200 μL sample was used. The separation was performed at 30 °C on a C18 column using an eluent of acetonitrile-0.5% triethylamine buffer. The compounds were monitored at λ _ex of 280 nm, λ _em of 425 nm. The calibration curve for ulifloxacin in human plasma was linear over the range 0.01–1.00 μg mL⁻¹. The lower limit of quantification is 0.01 μg mL⁻¹. The intra- and inter-day precision ranged from 3.0 to 6.7%, respectively. The method had been used for clinical pharmacokinetic studies of prulifloxacin formulation product after oral administration to healthy volunteers. Jun Wen and Zhenyu Zhu have equal contribution to this work.     

A Validated HPLC Method for Simultaneous Determination of 2-Hydroxy-4-methoxybenzaldehyde and 2-Hydroxy-4-methoxybenzoic Acid in Root Organs of Hemidesmus indicus

A reverse phase HPLC method was developed and validated for the simultaneous determination of 2-hydroxy-4-methoxybenzaldehyde and 2-hydroxy-4-methoxybenzoic acid in the root extracts of Hemidesmus indicus. A comprehensive validation of the method including sensitivity, linearity, reproducibility, accuracy, limit of detection (LOD) and limit of quantification (LOQ) was conducted using the optimized chromatographic conditions. The method was found to be linear (r > 0.998) in the range of 5–350 μg mL⁻¹ for 2-hydroxy-4-methoxybenzaldehyde and for 2-hydroxy-4-methoxybenzoic acid (r > 0.999) in the range 10–300 μg mL⁻¹. The method was found to be precise with inter-day precision values (% RSD) in the ranges of 0.61–1.76% for 2-hydroxy-4-methoxybenzaldehyde and 1.3–2.8% for 2-hydroxy-4-ethoxybenzoic acid while intra-day precisions (% RSD) of two analytes were in the range of 0.41–1.07 and 0.95–2.5%. The limits of detection (LODs) for 2-hydroxy-4-methoxybenzaldehyde and 2-hydroxy-4-methoxybenzoic acid were 0.84 and 2.34 μg mL⁻¹. The described method was fast, sensitive and reproducible, and thus well suited for routine analysis of these two compounds from root extracts of H. indicus and other plants.     

1,3,5,7-Tetramethyl-8-(<Emphasis Type="Italic">N</Emphasis>-hydroxysuccinimidyl butyric ester)difluoroboradiaza-<Emphasis Type="Italic">s</Emphasis>-indacene as a new fluorescent labeling reagent for HPLC determination of amino acid neurotransmitters in the cerebral cortex of mice

1,3,5,7-Tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)difluoroboradiaza-s-indacene (TMBB-Su), a new BODIPY-based fluorescent probe, was designed and synthesized for the labeling of amino compounds. It was used as a pre-column derivatizing reagent for determination of amino acid neurotransmitters by high-performance liquid chromatography (HPLC). The fluorescence quantum yield in acetonitrile increased from 0.84 to 0.95 when it reacted with amino acid neurotransmitters. Derivatization of TMBB-Su with seven amino acid neurotransmitters was completed within 30 min at 25 °C in 24.0 mmol L⁻¹ pH 7.8 boric acid buffer. The separation was performed on a C₁₈ column with methanol–water–buffer 55:35:10 (v/v) as mobile phase (buffer: 0.10 mol L⁻¹ H₃Cit–0.10 mol L⁻¹ NaOH). Interference from the other concomitant amino acids was eliminated successfully by means of pH gradient elution. With fluorescence detection at 494 and 504 nm for excitation and emission, respectively, the limits of detection (signal-to-noise ratio = 3) were from 2.1 to 12.0 nmol L⁻¹. The proposed method has been used to determine amino acid neurotransmitters in the cerebral cortex of mice with cerebral ischemia at the convalescence stage with satisfactory recoveries varying from 94.9 to 105.2%.     

Development and Validation of a Stability-Indicating LC Method for Determination of Ebastine in Tablet and Syrup

A simple stability-indicating reversed-phase liquid chromatographic method with diode-array detection was developed and validated for the quantitative determination of ebastine in tablets and syrup. The LC method was carried out on a C₁₈ column with acetonitrile:phosphoric acid 0.1% pH 3.0 (55:45, v/v) as mobile phase, at a flow rate of 1.2 mL min⁻¹. Ultraviolet detection of ebastine was at 254 nm. A linear response (r = 0.9999) was observed in the range of 10–80 μg mL⁻¹. The RSD values for intra- and inter-day precision studies showed good results (RSD < 2%) and accuracy was greater than 98%. Validation parameters such as specificity and robustness were also determined. The method was found to be stability-indicating and can be applied to quantitative determination of ebastine in tablets and syrup.     

Determination of Apigenin by LC with Electrochemical Detection

A rapid, sensitive, and specific method for quantification of olmesartan, the prodrug of olmesartan medoxomil, in human plasma, using zidovudine as internal standard, is described. Sample preparation involved a simple solid-phase extraction procedure. The extract was analyzed by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry (LC–MS–MS). Chromatography was performed isocratically on a 5 μm C₁₈ analytical column (50 mm × 4.6 mm i.d.) with water–acetonitrile–formic acid 20:80:0.1 (v/v) as mobile phase. The response to olmesartan was a linear function of concentration over the range 4.82–1,928 ng mL⁻¹. The lower limit of quantification in plasma was 4.82 ng mL⁻¹. The method was successfully applied in a bioequivalence study of an olmesartan formulation after administration as a single oral dose.     

Determination of (E)-3,5,4′-Trimethoxystilbene in Rat Plasma by LC with ESI-MS

(E)-3,5,4′-trimethoxystilbene (BTM-0512) is a resveratrol analog with a variety of pharmacological action, including anti-cancer properties, anti-allergic activity, estrogenic activity, antiangiogenic activity, and vascular-targeting activity against microtubule-destabilization. There is, however, no validated analytical method for quantification of (E)-3,5,4′-trimethoxystilbene in biological matrices, so pharmacokinetic data and suitable methods for determination of the compound in plasma are currently lacking. A rapid and sensitive liquid chromatographic–mass spectrometric method for determination of (E)-3,5,4′-trimethoxystilbene in rat plasma, using carbamazepine as internal standard, has been developed and validated. Plasma samples were treated with acetonitrile to precipitate proteins. Samples were then analyzed by HPLC on a 250mm × 4.6 mm i.d., 5-μm particle, C₁₈ column with methanol–water, 80:20 (v/v), containing 10 mm ammonium acetate and 0.2% formic acid (pH 3.0), as mobile phase, delivered at 0.85 mL min⁻¹. A single-quadrupole mass spectrometer with an electrospray interface operated in selected-ion monitoring mode was used to detect [M + H]⁺ ions at m/z 271.3 for (E)-3,5,4′-trimethoxystilbene and m/z 237.5 for the internal standard. (E)-3,5,4′-trimethoxystilbene and the internal standard eluted as sharp, symmetrical peaks with retention times of 8.9 and 4 min, respectively. Calibration plots for (E)-3,5,4′-trimethoxystilbene in rat plasma at concentrations ranging from 0.01 to 5.0 μg mL⁻¹ were highly linear. Intra-day and inter-day precision, as RSD, was <12.9%, and accuracy was in the range 94.8–104.7%. The limit of detection in plasma was 0.005 μg mL⁻¹. The method was successfully used to determine the concentration of (E)-3,5,4′-trimethoxystilbene after oral administration of 86 mg kg⁻¹ of the drug to Sprague–Dawley rats and can be used to investigate the pharmacokinetics of the compound.     

LC–ESI–MS for Rapid and Sensitive Determination of Aripiprazole in Human Plasma

A rapid, sensitive, and accurate high-performance liquid-chromatographic–mass spectrometric (HPLC–MS) method, with estazolam as internal standard, has been developed and validated for determination of aripiprazole in human plasma. After liquid–liquid extraction the compound was analyzed by HPLC on a C₁₈ column, with acetonitrile—30 mm ammonium acetate containing 0.1% formic acid, 58:42 (v/v), as mobile phase, coupled with electrospray ionization mass spectrometry (ESI-MS). The protonated analyte was quantified by selected-ion recording (SIR) with a quadrupole mass spectrometer in positive-ion mode. Calibration plots were linear over the concentration range 19.9–1119.6 ng mL⁻¹. Intra-day and inter-day precision (CV%) and accuracy (RE%) for quality-control samples (37.3, 124.4, and 622.0 ng mL⁻¹) ranged between 2.5 and 9.0% and between 1.3 and 3.5%, respectively. Extraction recovery of aripiprazole from plasma was in the range 75.8–84.1%. The method enables rapid, sensitive, precise, and accurate measurement of the concentration of aripiprazole in human plasma.     

Quantitative Determination of Acyclovir in Aqueous Humor by LC-MS

A specific, sensitive and precise liquid chromatographic assay method was established using LC-MS for the determination of acyclovir (ACV) in aqueous humor (AH), which was directly injected onto an Inertsil ODS-3 C₁₈ column without any pretreatment. The Agilent 1100 series LC–MS system was operated under the electrospray ionization mode (ESI). The analyte was separated from endogenous substances with a mobile phase of methanol: water: acetic acid (5:95:0.1, v/v) at a flow-rate of 0.3mL min⁻¹. A linear response was observed over the concentration range from 5 to 50ng mL⁻¹ (r=0.9993). Intra- and inter-day coefficients of variation were in the ranges 5.2–9.0% and 5.8–8.2%, respectively. The recovery was > 91.0%, and the limit of detection was approximate 1ng mL⁻¹. The pharmacokinetics of topically applied eye-drop and thermosetting gel were compared in rabbits utilizing the present method, the results demonstrated that LC-MS was a powerful tool for the detection of ACV in AH.     

Determination and Validation of an LC Method for Fluvoxamine in Tablets

A new, simple, rapid and specific reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for the determination of fluvoxamine in pharmaceutical dosage forms. The HPLC separation was achieved on a C₁₈ μ-Bondapack column (250 mm × 4.6 mm) using a mobile phase of acetonitrile–water (80:20, v/v) at a flow rate of 1 mL min⁻¹. Proposed method is based on the derivatization of fluvoxamine with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS) in borate buffer of pH 8.5 to yield a orange product. The HPLC method is based on measurement of the derivatized product using UV-visible absorbance detection at 450 nm. The method was validated for specificity, linearity, precision, accuracy, robustness. The degree of linearity of the calibration curves, the percent recoveries of fluvoxamine, the limit of detection and quantification, for the HPLC method were determined. The assay was linear over the concentration range of 45–145 ng mL⁻¹ (r = 0.9999). Limit of detection and quantification for fluvoxamine were 15 and 50 ng mL⁻¹, respectively. The results of the developed procedure (proposed method) for fluvoxamine content in tablets were compared with those by the official method. The method was found to be simple, specific, precise, accurate, reproducible and robust.     

Quantitative Determination of Four Water-Soluble Compounds in Herbal Drugs from Lamiaceae Using Different Chromatographic Techniques

HPTLC-densitometric and HPLC–UV techniques were used for qualitative and quantitative determination of luteolin-7-O-glucuronide, lithospermic acid, rosmarinic acid and caffeic acid in several herbal drugs from the Lamiaceae family: Thymi herba, Serpylli herba, Majoranae herba and Menthae piperitae folium. Unmodified silica gel (HPTLC Si60) and silica gel chemically modified with aminopropyl groups (HPTLC NH₂) were used during the investigation process. Among HPTLC methods the best resolution and selectivity was achieved with mobile phases: diisopropyl ether–acetone–formic acid–water (50:30:10:10, v/v/v/v) and acetone–formic acid (85:15, v/v), respectively. Plates were densitometrically evaluated. Contents of analyzed compounds in the studied aqueous extracts prepared from herbal drugs were established using both techniques. The results from the HPTLC-densitometric analysis have been compared with those from HPLC–UV on a C18 column with acetonitrile–water–formic acid as a mobile phase. The chromatographic methods were validated for linearity, LOD, LOQ, repeatability, intermediate precision and recovery. An analysis of variance showed that the HPTLC-densitometric and HPLC–UV methods are equivalent and sufficiently precise for the estimation of polyphenolic compounds mentioned above, in investigated herbal drugs. All of the suggested methods (HPTLC NH₂, HPTLC Si60 and HPLC RP18) give results with good agreement.     

Development and Validation of an HPLC Method for Determination of Ceftiofur Sodium

An isocratic high-performance liquid chromatographic method has been developed for assay of ceftiofur sodium in drug substance and in sterile powder for injection. Chromatography was performed on a 250 mm × 4.6 mm, 5 μm particle, C₁₈ column with a 78:22 (v/v) mixture of 0.02 m disodium hydrogen phosphate buffer (pH adjusted to 6.0 with 85% orthophosphoric acid) and acetonitrile as mobile phase, at a flow rate of 1.0 mL min⁻¹. The separation was monitored by UV detection at 292 nm. Validation of the method for linearity and range, intra- and inter-day precision, accuracy, specificity, recovery, robustness, and limits of quantification and detection yielded good results. The calibration plot was linear from 20.0–120.0 μg mL⁻¹ and the correlation coefficient was 0.9999. It was shown that ceftiofur was degraded under acidic, alkaline, oxidative, and photolytic conditions. The method was found to be stability-indicating and could be used for routine analysis of ceftiofur sodium for injection.     

Quantitative Determination of Three Protoberberine Alkaloids in Jin-Guo-Lan by HPLC-DAD

An accurate and simple HPLC method for the simultaneous determination of three protoberberine alkaloids (columbamine, jatrorrhizine, and palmatine) contained in Chinese medicine Jin-Guo-Lan (Tinospora sagittata Oliv. and Tinospora capillipes Gagnep) is presented in this study. The herb samples from six main origins and three herb markets were investigated. The separation was performed on a YMC-C₁₈ ODS column at 30°C with a gradient elution program. Acetonitrile and phosphate buffer (0.02 mol L⁻¹ sodium dihydrogen phosphate and 0.01 mol L⁻¹ triethylamine, pH 3) were used as mobile phases and the flow rate was set at 1 mL min⁻¹. The recovery of the method was in the range of 99.43–100.96%, and all the alkaloids showed good linearity (r ² > 0.9997) in the relatively wide concentration ranges. The developed method was applied to the determination of these alkaloids in the collected herb samples, and the results showed that the contents of these components in Jin-Guo-Lan varied greatly from habitat to habitat. It was demonstrated that the proposed method was helpful for the quality evaluation of Chinese medicine Jin-Guo-Lan.     

Simultaneous chemical fingerprint and quantitative analysis of Ginkgo biloba extract by HPLC–DAD

A reverse-phase liquid chromatography method with diode array detection was developed to evaluate the quality of Ginkgo biloba extract through establishing chromatographic fingerprint and simultaneous determination of eight flavonoid compounds, namely rutin, myricetin, quercitrin, quercetin, luteolin, kaempferol, apigenin, and isorhamnetin. The chromatographic separation was performed on an Agilent SB-C18 column (250 × 4.6 mm, 5.0 μm) with a gradient elution program using a mixture of methanol and 0.1% formic acid (v/v) as mobile phase within 55 min at 360-nm wavelength. The correlation coefficients of similarity for different batches of G. biloba extract from the same manufacturer and G. biloba extract from different manufacturers were determined from the LC fingerprints, and they shared a close similarity. The eight flavonoid compounds showed good regression (R ² > 0.9995) within test ranges, and the recovery of the method was in the range of 94.1–101.4%. In addition, the content of those eight flavonoid compounds in G. biloba extract prepared by different manufacturers of China was determined to establish the effectiveness of the method. The results indicated that the developed method by having a combination of chromatographic fingerprint and quantification analysis could be readily utilized as a quality control method for G. biloba extract and its related traditional Chinese medicinal preparations.     

Qualitative and Quantitative LC Profile of Embelin and Rapanone in Selected Lysimachia Species

Qualitative and quantitative determination of the pharmacologically active benzoquinones, embelin and rapanone, in different organs of eight Lysimachia species has been conducted by reversed-phase high-performance liquid chromatography. An analytical Hypersil BDS C-18 column and a mobile phase of water containing 0.1% v/v H₃PO₄ and acetonitrile (10:90) at a flow rate of 1.0 mL min⁻¹ were used. UV detection was at 286 nm. The recovery of the method was 81.5% for embelin and 80.5% for rapanone. Good linearity (r > 0.999) was obtained for both compounds. The leaves of L. ephemerum had the highest amount of rapanone (1.69%) while the roots of L. punctata had the highest amount of embelin (1.28%).