Simultaneous Determination of Five Phenolic Compounds in Dried Flowers by LC Using DAD Combined Electrochemical Detection

A simple, sensitive and accurate method for the simultaneous separation and determination of apigenin and four phenolic acids including chlorogenic acid, caffeic acid, p-coumaric acid and ferulic acid in four dried flowers by high performance liquid chromatography with electrochemical detection (ECD) and diode array detection (DAD) has been established. The detection limits of caffeic acid, p-coumaric acid and ferulic acid obtained with ECD were 3, 1 and 4 ng mL^?1, and LOD of apigenin and chlorogenic acid obtained with DAD were 1 × 10^?2 and 6 × 10^?2 μg mL^?1. The detection and quantification limits of three phenolic compounds obtained with ECD were two to ninefold greater than those obtained with DAD. As electrochemically inactive compounds, apigenin and chlorogenic acid were detected by DAD. All calibration curves showed good linearity (r ≥ 0.9992) within the test ranges. The recoveries ranged from 95.3 to 101.4% (RSD ≤ 2.9%). This approach could provide scientific evidence for comprehensive evaluation about the effect of the medicine and ensure nutrient status of dried flowers.     

Determination of Five Polyphenols by HPLC/DAD and Discrimination of Apple Varieties

The objective of this study was to set up a method to detect five compounds in fresh smashed apples by HPLC/DAD simultaneously. Different methods have been tested to control browning and ascorbic acid with ultrasonication was adopted. Methanol–water–acetic acid (30:69:1, v/v) containing 2.0 g of ascorbic acid L^?1 was chosen as the extract solvent. The method effectively simplified the sample treatment compared with the traditional ways. And primarily, the results were used to identify between different varieties. The chromatographic separation was performed on an Atlantis C18 (250 mm × 4.5 mm, particle size 5 μm) with a gradient elution program using a mixture of acetonitrile and 2% aqueous acetic acid (v/v) as mobile phase within 20 min at 270 nm wavelength. The variation of the content of five compounds was gallic acid (ND ~1.81 μg g^?1), protocatechuic acid (ND ~1.79 μg g^?1), chlorogenic acid (13.81–189.4 μg g^?1), caffeic acid (6.82–45.02 μg g^?1) and rutin (0.96–18.55 μg g^?1). The results could successfully be used to discriminate between different apple varieties (Gala, Fuji, Delicious, 8th Apple US, Golden Apple, Green Apple and Red Rose); chlorogenic acid and rutin being the polyphenols that contribute most to the differentiation.     

Simultaneous LC Determination of Chlorogenic Acid and Hydrochlorthiazide in Zhenju Jiangya Tablets

A method has been developed to determine chlorogenic acid and hydrochlorothiazide simultaneously in Zhenju Jiangya tablets by HPLC using an isocratic mode and UV detection. A solution of acetonitrile-0.4% phosphoric acid (13:87, v/v) was used as mobile phase. Results showed that the two compounds were well separated. Chlorogenic acid and hydrochlorothiazide had good linearities in the range of 0.64～6.4 μg mL^?1 and 0.08～0.16 mg mL^?1, respectively with average recoveries of 96.5～99.3 and 97.7～101.0%.     

Identification and Quantification of Heterologous Compounds Parthenin and Organic Acids in Parthenium Hysterophorus L. Using HPLC-PDA-MS-MS

Parthenium hysterophorus L., is an obnoxious weed known for its environmental health hazards and medicinal uses. These characteristics are due to presence of sesquiterpene lactones and organic acids; therefore a rapid and sensitive analytical procedure using HPLC-PDA-MS-MS was developed and optimized for separation, identification, and quantification of parthenin and six organic acids. Separation and characterization of compounds was achieved on a RP-C18 column with 1% acetic acid in water (A) and acetonitrile (B) as a mobile phase at a flow rate of 0.6 mL min^?1 and by matching their UV and mass spectra with reference compounds. Six organic acids (ferulic acid, 0.1 mg g^?1 to coumaric acid, 13.6 mg g^?1) and parthenin (27.4 mg g^?1) were characterized within 26 minutes of chromatographic separation in plant extract. The calibration curves are linear with correlation coefficients from 0.985 to 0.998, limit of detection and quantification ranged between 1.0 µg mL^?1 (anisic acid) to 2.2 µg mL^?1 (parthenin) and 2.5 µg mL^?1 (coumaric acid) to 5.2 µ g mL^?1 (parthenin) and recovery ranged between 90.9% to 97.3%. To the best of our knowledge this is the first report for the simultaneous separation of parthenin and organic acids. The method is applicable for screening of commercial crops, medicinal plants, and their products which might be mixed with P. hysterophorus during harvesting period.     

Simultaneous Determination of Ibuprofen and Diphenhydramine Citrate in Tablets by Validated LC

A stability-indicating LC method was developed for the simultaneous determination of ibuprofen and diphenhydramine citrate in pharmaceutical dosage forms. The chromatographic separation was achieved on an Inertsil ODS 3V, 150 × 4.6 mm, 5 μm, column. The mobile phase contained a mixture of 50 mM potassium dihydrogen phosphate buffer:acetonitrile:triethylamine:glacial acetic acid (55:45:0.2:0.2, v/v/v/v). This method allowed the determination of 2.85–9.14 mg mL^?1 of ibuprofen and 0.54–1.73 mg mL^?1 of diphenhydramine citrate, in a diluent consisting of pH 7.2, 50 mM potassium dihydrogen phosphate buffer:acetonitrile (40:60, v/v). The flow rate was 1.2 mL min^?1 and the detection wavelength was 260 nm. The limit of detection for ibuprofen and diphenhydramine citrate was 1.72 and 0.54 μg mL^?1 and the limit of quantification was 5.73 and 1.64 μg mL^?1, respectively. This method was validated for accuracy, precision and linearity. The method was also found to be stability indicating.     

Optimization of the Extraction Conditions and Simultaneous Quantification of Six Flavonoid Glycosides in Flos Chrysanthemi by RP-LC

A sensitive, accurate and reliable reversed-phase liquid chromatographic method coupled with DAD (278 nm) was established for simultaneous quantification of six compounds in 20 cultivars of Flos Chrysanthemi. The method was carried out by using a Kromasil 100-5 C₁₈ column with methanol–acetonitrile—1.414 × 10^?2 mol L^?1 aqueous phosphoric acid as a gradient mobile phase. The contents of the six flavonoid glycosides in Flos Chrysanthemi could be determined within 120 min. The linear calibration ranges for these were 0.42–126.00, 11.44–220.00, 0.53–530.00, 4.80–195.00, 11.00–220.00, and 0.12–200.00 μg mL^?1. Their recoveries were 95.33–105.33% with RSDs from 0.10 to 2.00%. Their lower limits of quantification were 0.420, 1.144, 0.250, 0.480, 0.242, and 0.120 μg mL^–1. The method can be used for analysis of the six flavonoid glycosides in Flos Chrysanthemi.     

Establishment of an LC-Based Assay to Determine Caffeic Acid Tetramer in Rat Plasma and Its Application to a Pharmacokinetic Study

A facile, sensitive, and accurate liquid chromatographic method with ultraviolet detection was developed for the determination of caffeic acid tetramer in rat plasma. Chromatographic separation was performed on an YMC C18 10 μm column (250 × 4.6 mm) using acetonitrile and phosphate buffer (19:81, v/v) as mobile phase at a flow rate of 1 mL min^?1. The UV detection wavelength was set at 252 nm. The method showed good linearity in the range of 1–150 μg mL^?1 with a satisfactory correlation coefficient (r) of 0.997. The limit of detection was 20 ng mL^?1 while inter- and intra-day precisions were less than 5.39 and 5.48%, respectively. Furthermore, the accuracy ranged from 98.27 to 103.76% with high extraction recoveries of caffeic acid tetramer from plasma greater than 88.0%. This practical methodology opens a facile and effective pathway for a pharmacokinetic study.     

UPLC-PDA Analysis for Simultaneous Quantification of Four Active Compounds in Crude and Processed Rhizome of Polygonum multiflorum Thunb

A novel, rapid and specific ultra performance liquid chromatography-photo diode array detection method was developed for the simultaneous determination of 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (TSG), emodin-8-O-β-d-glucoside (EMG), emodin (EM) and physcion (PS). The chromatographic separation was performed on an Acquity BEH C₁₈ column (100 × 2.1 mm i.d., 1.7 μm). The mobile phase was a mixture of 0.3% acetic acid–water and 0.3% acetic acid–acetonitrile employing gradient elution at the flow rate of 0.4 mL min^?1. The four compounds behaved linearly in the concentration range between 60.80–3040.00 μg mL^?1 (TSG), 0.50–25.00 μg mL^?1 (EMG), 2.16–108.00 μg mL^?1 (EM) and 1.56–78.00 μg mL^?1 (PS), respectively with correlation coefficients >0.999. The precision of the method were below 5% RSD. Recoveries of the four compounds ranged from 95.71 to 102.97%, with RSD values less than 2%.     

Development and Validation of a LC Method for the Analysis of Phenolic Acids in Turkish Salvia Species

An accurate, simple, reproducible, and sensitive method for determination of rosmarinic, caffeic, chlorogenic, and gallic acids in 12 Salvia species growing naturally in Anatolia, has been developed and validated. The phenolic acids were separated using a μBondapack C₁₈ column by gradient elution with a flow rate of 1.0 mL min⁻¹, which was adjusted to deliver firstly o-phosphoric acid 0.085% in water, 0.085% in methanol, and 0.085% in 2-propanol (80:10:10, v/v/v), then decreased gradually (60:20:20, v/v/v) during 20 min with a flow rate of 1.0 mL min⁻¹. The samples were monitored at 220 nm for gallic acid and 330 nm for rosmarinic, caffeic, and chlorogenic acids using photo-diode array detection. The linear range of detection for gallic, chlorogenic, caffeic, and rosmarinic acids were between 0.051–101.4, 0.207–103.6, 0.100–100, and 0.201–100.5 μg mL⁻¹, respectively. The linearity, range, peak purity, selectivity, system performance parameters, precision, accuracy, and robustness had also acceptable values. The developed method was applied to the flower, leaf, stem, and root parts of the Salvia species.

     

Gas Chromatographic Determination of Guanidino Compounds Using Hexafluoroacetylacetone and Ethyl Chloroformate as Derivatizing Reagents

Guanidino compounds guanidine, methylguanidine, guanidinoacetic acid, guanidinobutyric acid, guanidinopropionic acid, and guanidinosuccinic acid after derivatization with hexafluoroacetylacetone and ethyl chloroformate at pH 9 in aqueous phase, eluted, and separated from gas chromatographic column HP-5 (30 m × 0.32 mm id) with film thickness of 0.25 μm at an initial column temperature 90 °C for 2 min, followed by heating rate of 10 °C min^?1 up to 220 °C with nitrogen flow rate of 1 mL min^?1. The detection was by flame ionization detector. The linear calibration ranges of each of guanidino compounds were obtained within 1–10 μg mL^?1, and the limit of detection was within 0.014–0.19 μg mL^?1. The derivatization and gas chromatography elution and separation were repeatable in terms of retention time and peak height/peak area with relative standard deviation (RSD) (n = 4) within 1.7–2.9 % and 1.4–2.8 %, respectively. The method was applied for the determination of guanidino compounds from deproteinized serum of uremic patients and healthy volunteers, and was found in the range below the limit of quantitation (BLOQ) to 1.25 μg mL^?1 with RSD within 1.4–3.6 %, and BLOQ to 0.4 μg mL^?1 with RSD 1.3–3.4 %, respectively. A number of pharmaceutical additives did not effect the determination with RSD within ±3.1 %.     

HPLC and UPLC Methods for the Simultaneous Determination of Enalapril and Hydrochlorothiazide in Pharmaceutical Dosage Forms

High efficiency and less elution are the basic requirements of high-speed chromatographic separation. In this study, a new gradient reverse phase chromatographic methods were developed using HPLC and UPLC systems for simultaneous determination of enalapril maleate (ENL) and hydrochlorothiazide (HCZ) in pharmaceutical dosage forms. The chromatographic separations of ENL and HCZ were achieved on a Waters μ-Bondapak C 18, (300 × 3.9 mm, 10 μm) and Waters Acquity BEH C18 (100 × 2.1 mm, 1.7 μm) columns for HPLC within 5.30 min and UPLC within a short retention time of 1.95 min, respectively. A linear response was observed over the concentration range 0.270–399 μg mL^?1 of ENL, 0.260–399 μg mL^?1 of HCZ for HPLC system and 0.270–399 μg mL^?1 of ENL and 0.065–249 μg mL^?1 of HCZ for UPLC system. Also, limit of detection for ENL was 1.848 ng mL^?1 and 31.477 ng mL^?1 for HCZ, 2.804 ng mL^?1 for ENL and 2.943 ng mL^?1 for HCZ using HPLC and UPLC, respectively. The proposed methods were validated according to ICH guideline with respect to precision, accuracy, and linearity. Forced degradation studies were also performed for both compounds in bulk drug samples to demonstrate the specificity and stability indicating power of the HPLC method. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency, and resolution.     

RP-LC Determination and Pharmacokinetic Study of Ferulic Acid and Isoferulic Acid in Rat Plasma After Taking Traditional Chinese Medicinal-Preparation: Guanxinning Lyophilizer

A sensitive, simple, and accurate method for determination and pharmacokinetic study of ferulic acid and isoferulic acid in rat plasma was developed using a reversed-phase column liquid chromatographic (RP-LC) method with UV detection. Sample preparations were carried out by protein precipitation with the addition of methanol, followed by evaporation to dryness. The resultant residue was then reconstituted in mobile phase and injected into a Kromasil C₁₈ column (250 × 4.6 mm i.d. with 5 μm particle size). The mobile phase was methanol-1% formic acid (33:67, v/v). The calibration plots were linear over the range 5.780–5780 ng·mL^?1 for ferulic acid and 1.740–348.0 ng·mL^?1 for isoferulic acid. Mean recoveries were 85.1% and 91.1%, respectively. The relative standard deviations (RSDs) of within-day and between-day precision were not above 15% for both of the analytes. The limits of quantification were 5.780 ng·mL^?1 for ferulic acid and 1.740 ng·mL^?1 for isoferulic acid. This RP-LC method was used successfully in pharmacokinetic studies of ferulic acid and isoferulic acid in rat plasma after intravenous injection of Guanxinning Lyophilizer.     

Simultaneous Determination of Acetaminophen, Caffeine, and Chlorphenamine Maleate in Paracetamol and Chlorphenamine Maleate Granules

A simple and rapid HPLC method using phenacetin (PHN) as internal standard has been developed for simultaneous determination of acetaminophen, caffeine, and chlorphenamine maleate in the product compound paracetamol and chlorphenamine maleate granules. Separation and quantitation were achieved on a 250 mm × 4.6 mm, 5 μm particle, C₁₈ column. The mobile phase was methanol 0.05 mol L^?1 aqueous KH₂PO₄ solution, 45:55 (v/v), containing 0.1% triethylamine and adjusted to pH 3.6 by addition of phosphoric acid; the flow rate was 1.0 mL min^?1. Detection of all compounds was by UV absorbance at 260 nm and elution of the analytes was achieved in less than 12 min. The linearity, accuracy, and precision of the method were acceptable to good over the concentration ranges 6.4–153.6 μg mL^?1 for acetaminophen, 5.0–120.0 μg mL^?1 for caffeine, and 9.6–230.4 μg mL^?1 for chlorphenamine maleate.     

Determination of Trace Amounts of Chlorogenic Acid and Three of Its Metabolites Using Time-Resolved LPME and LC-UV Detection in Biological Specimens

Chlorogenic acid (CGA) is an effective antitumor, anti-inflammatory and antimicrobial agent. Since the absorption and metabolism of CGA remains controversial, time-resolved binary-solvent synergy liquid-phase microextraction (TRBSS-LPME) using hollow fiber was developed for the extraction of CGA and its metabolites: caffeic acid, p-hydroxycinnamic acid and ferulic acid, from biological specimens. In this technique, the target drugs were extracted into a binary-solvent immobilized in the wall pores of hollow fiber. The extraction occurred due to a pH gradient between the two sides of the fiber. After extraction, an aliquot was analyzed by LC. Under the optimal conditions, the CGA, caffeic acid, p-hydroxycinnamic acid and ferulic acid had good correlation of determination values (R > 0.97) and the detection limits (LODs) were 1.0, 1.0, 2.0, and 5.0 ng mL^?1 in plasma; and 1.0, 50, 10, and 50 ng mL^?1 in urine. The mean recoveries in plasma were 90.8–119.8% for CGA and its metabolites: caffeic acid, p-hydroxycinnamic acid and ferulic acid evaluated and the mean recoveries of caffeic acid and p-hydroxycinnamic acid in urine were 81.6–111.6%. Finally, TRBSS-LPME was successfully used for the determination of target drugs in biological specimens. It not only extended the linear range of CGA determination in biological samples and improved the sensitivity, but also eliminated interferences from complex constituents in the biological specimens and reduced the LOD.     

Electrochemical investigation of the interaction between lysozyme-shelled microbubbles and vitamin C

We report loading of vitamin C (ascorbic acid) on to lysozyme-shelled microbubbles. The interaction between lysozyme-shelled microbubbles and vitamin C was studied by use of cyclic and differential pulse voltammetry, zeta potential measurements, and scanning electron microscopy. The effect of microbubbles on electrochemical measurement of ascorbic acid was evaluated. The linear range for ascorbic acid obtained for differential pulse measurement in the presence of 1 mg mL^?1 microbubbles was 1–50 μmol L^?1 (y?=?0.067x?+?0.130, r ²?=?0.995), with a detection limit of 0.5 μmol L^?1. The experimental conditions, i.e., pH and ionic strength, were optimized to improve the interaction between ascorbic acid and lysozyme-shelled microbubbles. The results were satisfactory when the interaction was performed for 1 h in aqueous solution at pH 6. The amount of vitamin C loaded on the microbubbles (90 % of the analyte added, RSD _inter-expt. = 3 %, n?=?6) and the stability of microbubbles–ascorbic acid complex (until 72 h at 25 °C) were also evaluated by use of differential pulse voltammetry and zeta potential measurements.

Simultaneous Determination of Five Major Compounds in Polygonum cuspidatum by HPLC

An RP-HPLC method was developed for the first time to simultaneously determine five major compounds in Polygonum cuspidatum, namely resveratrol, polydatin, anthraglycoside B, emodin and physcion with UV detection at 306 nm. The column was an Agilent Zorbax SB-C₁₈ (250 × 4.6 mm i.d., 5 μm). The separation was carried out with a gradient program. The mobile phase was acetonitrile–water (containing 0.1% formic acid) at a flow rate of 1.0 mL min^?1. The standard curve was rectilinear in the range of 2.04–62.96 μg mL^?1 (r = 0.9998) for resveratrol, 20.13–239.7 μg mL^?1 (r = 0.9998) for polydatin, 7.19–71.92 μg mL^?1 (r = 1.0000) for anthraglycoside B, 2.68–83.68 μg mL^?1 (r = 0.9998) for emodin and 0.60–14.37 μg mL^?1 (r = 0.9997) for physcion. The recoveries of the markers were 96.0, 106.5, 97.8, 97.9 and 98.1%, respectively. The relative standard deviation of intra-day and inter-day were less than 5.0 and 2.3%. This method was simple, accurate and reproducible. The developed method was successfully applied to analyze five compounds in P. cuspidatum of 20 commercial brands.     

Development and Validation of a Stability-Indicating LC Method for Simultaneous Analysis of Aceclofenac and Paracetamol in Conventional Tablets and in Microsphere Formulations

A stability-indicating reversed-phase LC method for analysis of aceclofenac and paracetamol in tablets and in microsphere formulations has been developed and validated. The mobile phase was 80:20 (v/v) methanol–phosphate buffer (10 mM at pH 2.5 ± 0.02). UV detection was at 276 nm. The method was linear over the concentration ranges 16–24 and 80–120 μg mL^?1 for aceclofenac and paracetamol, respectively, with recovery in the range 100.9–102.22%. The limits of detection and quantitation for ACF were 0.0369 and 0.1120 μg mL^?1, respectively; those for PCM were 0.0631 and 0.1911 μg mL^?1, respectively.     

Ion-Pair LC Method for Simultaneous Determination of Aliskiren Hemifumarate,Amlodipine Besylate and Hydrochlorothiazide in Pharmaceuticals

A rapid and precise LC method was developed for the simultaneous determination of aliskiren hemifumarate (ALS), amlodipine besylate (AML) and hydrochlorothiazide (HCZ) using acetonitrile:25 mM octane sulfonic acid sodium salt monohydrate in water (60:40 v/v) as the mobile phase. The flow rate was maintained at 1.2 mL min^?1 on a stationary phase composed of Supelco, Discovery^® HS (C18) column (25 cm × 4.6 mm, 5 μm). Isocratic elution was applied throughout the analysis. Detection was carried out at λ _max (232 nm) at ambient temperature. The method was validated according to ICH guidelines. Linearity, accuracy and precision were satisfactory over the concentration ranges of 32–320, 2–44 and 4–64 μg mL^?1 for ALS, AML and HCZ, respectively. LOD and LOQ were estimated and found to be 0.855 and 2.951 μg mL^?1, respectively, for ALS, 0.061 and 0.202 μg mL^?1, respectively, for AML as well as 0.052 and 0.174 μg mL^?1, respectively, for HCZ. The method was successfully applied for the determination of the three drugs in their co-formulated tablets. The results were compared statistically with reference methods and no significant difference was found. The developed method is specific and accurate for the quality control and routine analysis of the cited drugs in pharmaceutical preparations.     

Determination of Nateglinide in Human Plasma by LC-ESI-MS and Its Application to Bioequivalence Study

To evaluate the bioequivalence of nateglinide, a rapid and specific liquid chromatographic-electrospray ionization mass spectrometric method was developed and validated to determine nateglinide for human plasma samples. The analyte was detected using electrospray positive ionization mass spectrometry in the selected ion monitoring mode. Tinidazole was used as the internal standard. A good linear relationship obtained in the concentration ranged from 0.05 to 16 μg mL^?1 (r ² = 0.9993). Lower limit of quantification was 0.05 μg mL^?1 using 100 μL of plasma sample. Intra- and inter-day relative standard deviations were 2.1–7.5 and 4.7–8.9%, respectively. Among the pharmacokinetic data obtained, T _max was 2.09 ± 1.06 h for reference formulation and 2.40 ± 0.97 h for test formulation. C _max was 4.17 ± 1.31 μg mL^?1 for reference formulation and 4.37 ± 1.53 μg mL^?1 for test formulation. The half-life (t _½) was 1.93 ± 0.44 h for reference formulation and 1.92 ± 0.29 h for test formulation. AUC_0–10h was 13.67 ± 4.36 μg h mL^?1 for reference formulation and 13.21 ± 4.09 μg h mL^?1 for test formulation. This method was successfully applied to the pharmacokinetic study in human plasma samples.     

Capillary GC Analysis of Glyoxal and Methylglyoxal in the Serum and Urine of Diabetic Patients After Use of 2,3-Diamino-2,3-dimethylbutane as Derivatizing Reagent

The dicarbonyl compounds glyoxal, methylglyoxal, and dimethylglyoxal have been separated by capillary GC on a 30 m × 0.32 mm i.d. HP-5 column after precolumn derivatization with 2,3-diamino-2,3-dimethylbutane at pH 4. Chromatographic separation was complete in 6 min. Nitrogen was used as carrier gas at a flow rate of 2 mL min^?1. Split injection was performed with a split ratio of 10:1 (v/v). The derivatives were monitored by flame-ionization detection, and linear calibration plots were obtained in the ranges 0.06–0.69, 0.05–1.01, and 0.07–1.33 μg mL^?1 for glyoxal, methylglyoxal, and dimethylglyoxal, respectively; the respective detection limits were 20, 10, and 10 ng mL^?1. Glyoxal and methylglyoxal were analyzed in serum and urine from diabetics and from healthy volunteers. Amounts of glyoxal and methylglyoxal in serum from diabetic patients were 0.19–0.33 and 0.20–0.29 μg mL^?1, respectively, with respective relative standard deviations (RSD) of 0.8–1.0 and 0.8–1.1%. Amounts of glyoxal and methylglyoxal in serum from healthy volunteers were 0.05–0.08 and 0.04–0.10 μg mL^?1, respectively, with respective RSD of 0.9–1.2 and 1.0–1.2%. Levels of glyoxal and methylglyoxal in urine from diabetic patients were 0.18–0.40 and 0.25–0.36 μg mL^?1, respectively.