Development and Validation of an LC Method for Simultaneous Determination of Ascorbic Acid and Three Phenolic Acids in Sustained Release Tablets at Single Wavelength

A simple, rapid and sensitive column liquid chromatographic method was developed and validated to measure simultaneously the amount of ascorbic acid and phenolic acids at single wavelength (240 nm) in order to assess drug release profiles and drug-excipients compatibility studies for a new sustained release tablet formulation and its subsequent stability studies. A combined isocratic and linear gradient reversed-phase LC method was carried out at 240 nm. Quantification was achieved with reference to the external standards. The linearity for concentrations between 0.042 and 0.150 mg mL^?1 for ascorbic acid, 0.084–0.250 mg mL^?1 for chlorogenic acid, 0.053–0.360 mg mL^?1 for caffeic acid, and 0.016–0.250 mg mL^?1 for ferulic acid (r > 0.99 for all analytes) were established. The recovery of the active ingredients from the samples was at the range of 92.3–102.9%. Intra- and inter-day precisions were less than 2.5%. The limits of detection and quantification were 8 and 24 μg mL^?1 for ascorbic acid, 18 and 54 μg mL^?1 for chlorogenic acid, 37 and 112 μg mL^?1 for caffeic acid, and 11 and 34 μg mL^?1 for ferulic acid. The determination of the four active ingredients was not interfered by the excipients of the products. Samples were stable in the release mediums (37 °C) at least for 12 h.     

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.     

Determination of reference interval values for inorganic elements in whole blood samples of humans and laboratory animals by X-ray fluorescence spectrometry

Inorganic elements are responsible for essential bodily functions, such as osmotic regulation, cardiac frequency and contractibility, blood clotting and neuromuscular excitability. The determination of inorganic elements in corporeal fluids such as blood, serum, plasma and urine is used as a monitor for a part or the whole organism; their values, then, are compared with reference interval values. In this study, the energy dispersive X-ray fluorescence spectrometry (EDXRF), applying the Fundamental Parameters method, for the determination of inorganic elements in whole blood samples from humans and laboratory animals, was used. Peripheral blood samples were collected and, before coagulation, 100 μL of sample were deposited onto Whatman No. 41 filter paper and dried, using infrared spotlight. The reference interval values for healthy Brazilian population of Na were found to be 1,788–1,826 μg g^?1, of Mg 63–75 μg g^?1, of P 602–676 μg g^?1, of S 1,519–1,718 μg g^?1, of Cl 2,743–2,867 μg g^?1, of K 1,508–1,630 μg g^?1, of Ca 214–228 μg g^?1, of Fe 170–184 μg g^?1, of Cu 4–6 μg g^?1 and of Zn 1–3 μg g^?1. The reference interval values for golden hamster (Mesocricetus auratus) of Na were found to be 1,714–1,819 μg g^?1, Mg 51–79 μg g^?1, P 970–1,080 μg g^?1, S 1,231–1,739 μg g^?1, Cl 2,775–2,865 μg g^?1, of K 1,968–2,248 μg g^?1, of Ca 209–257 μg g^?1, of Fe 145–267 μg g^?1, of Cu 4–6 μg g^?1 and of Zn 3–5 μg g^?1. A comparative study between EDXRF and instrumental neutron activation analysis data was carried out and the results for both techniques are statistically equal (α = 0.05). The results contribute for the establishment of reference interval values for Na, Mg, P, S, Cl, K, Ca, Cu and Zn in the healthy Brazilian population and the referred laboratory animal species.     

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.     

In Situ Biphasic Extractive Fermentation for Hexanoic Acid Production from Sucrose by Megasphaera elsdenii NCIMB 702410

Hexanoic acid production by a bacterium using sucrose as an economic carbon source was studied under conditions in which hexanoic acid was continuously extracted by liquid–liquid extraction. Megasphaera elsdenii NCIMB 702410, selected from five M. elsdenii strains, produced 4.69 g l^?1 hexanoic acid in a basal medium containing sucrose. Production increased to 8.19 g l^?1 when the medium was supplemented by 5 g l^?1 sodium butyrate. A biphasic liquid–liquid extraction system with 10 % (v/v) alamine 336 in oleyl alcohol as a solvent was evaluated in a continuous stirred-tank reactor held at pH 6. Over 90 % (w/w) of the hexanoic acid in a 0.5 M aqueous solution was transferred to the extraction solvent within 10 h. Cell growth was not significantly inhibited by direct contact of the fermentation broth with the extraction solvent. The system produced 28.42 g l^?1 of hexanoic acid from 54.85 g l^?1 of sucrose during 144 h of culture, and 26.52 and 1.90 g l^?1 of hexanoic acid was accumulated in the extraction solvent and the aqueous fermentation broth, respectively. The productivity and yield of hexanoic acid were 0.20 g l^?1 h^?1 and 0.50 g g^?1 sucrose, respectively.     

Antioxidant potential of indigenous cyanobacterial strains in relation with their phenolic and flavonoid contents

Antioxidant activities of eight indigenous cyanobacterial strains belonging to the genera Oscillatoria, Chroococcidiopsis, Leptolyngbya, Calothrix, Nostoc and Phormidium were studied in relation with their phenolic and flavonoid contents, ranging 3.9–12.6 mg GAE g^?1 and 1.7–3.44 mg RE g^?1. The highest activities were shown by Leptolyngbya sp. SI-SM (EC₅₀ = 63.45 and 67.49 μg mL^?1) and Calothrix sp. SI-SV (EC₅₀ = 65.79 and 69.38 μg mL^?1) calculated with ABTS and DPPH assays. Significant negative correlations were seen between total phenolic and flavonoid contents and the antioxidant activities in terms of EC₅₀ values. Furthermore, HPLC detected 15 phenolic compounds with total concentrations ranging from 277.3 to 829.7 μg g^?1. The prevalent compounds in most of the strains were rutin, tannic acid, orcinol, phloroglucinol and protocatechuic acid. Cyanobacterial strains showed high potential as a good source of phenolic compounds with potent antioxidative potential which could be beneficial for food, cosmetic and pharmaceutical industries.     

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.     

A Validated LC Method for the Quantitation of Cefotaxime in pH-Sensitive Nanoparticles

A sensitive and rapid routine LC method was validated for measuring cefotaxime incorporated in three different pH-sensitive nanoparticles. The drug was chromatographed on a C18 reversed-phase column; the mobile phase used was 0.05 M aqueous ammonium acetate, acetonitrile and tetrahydrofuran (87:11:2, v/v) adjusted to pH 5.5 with acetic acid. The flow rate was 1 mL min^?1 and cefotaxime was quantified at 254 nm, with a sensitivity range of 0.005 AUFS. The validated method was specific, linear (R ² ≥ 0.999), precise and accurate in a concentration range of 0.2–50.0 μg mL^?1. The method was rapid, selective and suitable for evaluation of cefotaxime in pH-sensitive Eudragit nanoparticles.     

LC Method for Quantification of ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic Acid in Rabbit Plasma: Validation and Application to a Pharmacokinetic Study

ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic acid (5F), a diterpenoid isolated from the Chinese herb Pteris semipinnata L, has been suggested to show antitumor properties. A simple and sensitive LC method was developed for the determination of 5F in rabbit plasma. The method involved liquid–liquid extraction using ethyl acetate under acidic conditions using naproxen as an internal standard. Separations were performed on a reversed-phase column with a mixture of 1% (v/v) glacial acetic acid and methanol (45:55, v/v) as mobile phase and UV detection was utilized at 242 nm. The calibration plot was linear in the range 0.20–10.0 μg mL^?1 (correlation coefficients r ²  > 0.998). The detection limit was 0.20 μg mL^?1, mean extraction recovery was above 82%, intra-day precision of the method was less than 6.4%, and inter-day precision was better than 8.7%, respectively. The validated assay was found to be suitable for the pharmacokinetic study of 5F in rabbits.     

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%.     

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%.     

RP-LC Quantification and Pharmacokinetic Study of Iriflophenone 2-O-α-Rhamnopyranoside in Rat Plasma

Iriflophenone 2-O-α-rhamnopyranoside (IP2R) is one of the main bioactive constituents of the leaves of Aquilaria sinensis (Lour.) Gilg, used in traditional Chinese medicines. A simple, rapid, and sensitive reversed-phase high-performance liquid chromatographic method has been developed for analysis of IP2R in rat plasma after intravenous administration. The analyte was extracted from plasma samples with methanol as deproteinization agent. Analysis was performed on an 250 mm × 4.6 mm i.d., 5-μm particle, C₁₈ column with a 8 mm × 4.6 mm i.d., 5-μm particle, RP-18 guard column; the mobile phase was acetonitrile–H₂O–acetic acid 22:78:0.01 (v/v) at a flow-rate of 1.0 mL min^?1. UV detection was at 289 nm. The calibration plot was linear in the range 0.01–33.33 μg mL^?1 (r = 0.9997, n = 5) in rat plasma. The lower limits of detection and quantification were 0.004 and 0.01 μg mL^?1. Intra-day and inter-day precision were 1.18–3.96 and 1.29–2.81%, respectively. Average extraction recovery from plasma was more than 95%. This assay method was successfully used to study the pharmacokinetics of IP2R in rats after a single dose of 25 mg kg^?1 by intravenous administration; the plasma concentration–time curve of IP2R conformed to a two-compartment open model.     

Fatty acids profiling of avocado seed and pulp using gas chromatography–mass spectrometry combined with multivariate chemometric techniques

In the present study, the profiling of 17 fatty acids (FAs) in avocado seed and pulp was investigated. The fatty acids were extracted with vortex-assisted extraction, methyl esterified and finally preconcentrated by dispersive liquid–liquid microextraction. The preconcentrated fatty acid methyl esters (FAMEs) were analyzed using gas chromatography–mass spectrometry (GC–MS) to obtain qualitative and quantitative information. The GC–MS data were analyzed using multivariate curve resolution-alternating least squares (MCR-ALS) method to overcome general chromatographic problems such as overlapped peaks, background interference and peak shifts. The calibration data were prepared using pure analytical information obtained by MCR-ALS. The linear dynamic ranges and regression coefficients (R ²) for FAMEs were in the range of 0.19–65 mg L^?1 and 0.990–0.999, respectively. The relative standard deviation (RSD%) for determination of FAs in avocado seed and pulp was 0.17–8.84 and 0.64–17.93, respectively. The main FAs in the avocado pulp were: oleic acid (74.25 g Kg^?1), linoleic acid (26.87 g Kg^?1), palmitic acid (26.02 g Kg^?1), palmitoleic acid (1.22 g Kg^?1) and stearic acid (0.05 g Kg^?1). And, the main FAs in the avocado seed were: linoleic acid (1.09 g Kg^?1), palmitic acid (0.47 g Kg^?1), oleic acid (0.33 g Kg^?1), linolenic acid (0.12 g Kg^?1), and palmitoleic acid (0.04 g Kg^?1).     

Simultaneous Determination of Paracetamol and Caffeine in Human Plasma by LC–ESI–MS

A rapid, selective and convenient liquid chromatography–mass spectrometric method for the simultaneous determination of paracetamol and caffeine in human plasma was developed and validated. Analytes and theophylline [internal standard (I.S.)] were extracted from plasma samples with diethyl ether-dichloromethane (3:2, v/v) and separated on a C₁₈ column (150 × 4.6 mm ID, 5 μm particle size, 100 Å pore size). The mobile phase consisted of 0.2% formic acid–methanol (60:40, v/v). The assay was linear in the concentration range between 0.05 and 25 μg mL^?1 for paracetamol and 10–5,000 ng mL^?1 for caffeine, with the lower limit of quantification of 0.05 μg mL^?1 and 10 ng mL^?1, respectively. The intra- and inter-day precision for both drugs was less than 8.1%, and the accuracy was within ±6.5%. The single chromatographic analysis of plasma samples was achieved within 4.5 min. This validated method was successfully applied to study the pharmacokinetics of paracetamol and caffeine in human plasma.     

LC Simultaneous Determination of Thioctic Acid, Benfotiamine and Cyanocobalamin in Thiotacid Compound Capsules

A simple, selective, sensitive, precise, simultaneous liquid chromatographic analysis of capsules containing thioctic acid, benfotiamine and cyanocobalamin was described. Good chromatographic separation was achieved using a Zorbax C18 (4.6 cm × 250 mm, 5 μm) and a mobile phase consisting of acetonitrile–phosphate buffer pH 3.5 (15:85, v/v) at a flow rate of 0.9 mL min^?1. The ultraviolet detector was set a wavelength of 280 nm. Thioctic acid, benfotiamine and cyanocobalamin were eluted at 2.869, 3.752 and 13.689 min, respectively. The linear ranges for thioctic acid, benfotiamine and cyanocobalamin were 30–180, 4–24 and 0.025–0.150 μg mL^?1, respectively. The recoveries of thioctic acid, benfotiamine and cyanocobalamine in pharmaceutical preparation were all greater than 98% and their relative standard deviations were less than 2.0%. The limits of detection were 2.57, 0.19 and 0.003 μg mL^?1 for thioctic acid, benfotiamine and cyanocobalamin, respectively.     

LC Determination and Pharmacokinetic Study of Gemfibrozil in Human Plasma

A simple and sensitive LC method for the quantitative determination of gemfibrozil in human plasma samples is described. Mometasone furoate was used as the internal standard. Plasma samples were pretreated by protein precipitation using methanol. Separation was performed at 40 °C on a YMC^® ODS-A reverse phase column (5 μm particle size, 150 mm × 4.6 mm i.d.) using 0.2% (v/v) triethylamine in water (adjusting to pH 4.0 with phosphoric acid) and acetonitrile (45:55, v/v) as mobile phase which was delivered at 1.5 mL min^?1. Ultraviolet detection was performed at 230 nm. The linear concentration range for gemfibrozil was 0.25–50 μg mL^?1. The detection limit of this method was 0.1 μg mL^?1. Intra- and inter-assay RSD ranged from 0.63 to 2.04% and 1.37 to 4.27%, respectively. The method was sensitive, simple and repeatable enough to be used in pharmacokinetic studies.     

Stereoselective Determination of Ornidazole Enantiomers in Human Plasma and Urine Samples by Chiral LC: Application to Pharmacokinetic Study

A stereoselective liquid chromatographic method to determine the enantiomers of ornidazole in human plasma and urine has been developed and validated. After addition of the internal standard (naproxen), samples were acidified and extracted with diethyl ether. The separation was performed on a Chiralcel OB-H column, using hexane-ethanol- glacial acetic acid (94:6:0.08, v/v) as the mobile phase. The method was validated for specificity, linearity, sensitivity, precision, accuracy and stability. For each enantiomer of ornidazole, linear calibration curves were obtained over the concentration range of 0.16–20 μg mL^?1 in plasma and 0.32–20 μg mL^?1 in urine. For both enantiomers of ornidazole in plasma and urine, the coefficient of variation for precision were consistently less than 12% and accuracy were within ±14% in terms of relative error. Application of the method to a preliminary pharmacokinetic study showed that this validated method was qualified for the direct determination of ornidazole enantiomers in human plasma and urine.     

LC-DAD and LC–MS–MS Analysis of Piperidine Alkaloids of Lobelia inflata L. (In Vitro and In Vivo)

An LC-DAD method was developed for determination of lobeline from in vitro and in vivo cultures of Lobelia inflata. Samples were extracted with 0.1 N HCl–acetonitrile (1:1, v/v), and purified by solid-phase extraction. Optimized conditions resulted in high recovery. LC separations were performed on an Eurosphere C8 reversed-phase column using 30:70 (v/v) acetonitrile–0.1% trifluoroacetic acid as a mobile phase. Quantitative determination of lobeline was performed by external standard method at 250 nm, in the range of 2.4–80 μg mL⁻¹. Validation studies proved that the repeatability of the method was good and the recovery was satisfactory. In vitro organized cultures contained considerable amount of lobeline (herb: 175 μg g⁻¹, root: 100 μg g⁻¹). When these cultures were transplanted into the open field, the lobeline content increased significantly (herb: 323 μg g⁻¹, root: 833 μg g⁻¹). Plants obtained from seed propagation contained 382 μg g⁻¹ lobeline in the herb. For direct characterization of di-substituted piperidine alkaloids in extracts of L. inflata, tandem mass spectrometric method was developed using electrospray ionization. Analysis was performed in the positive ion mode on a triple quadropole LC–MS system. LC separations were achieved on Eurosphere C8 column with a modified mobile phase (acetonitrile–30 mM ammonium formate, pH 2.80) to ensure proper molecular ionization. The identification and structural elucidation of the alkaloids were performed by comparing their changes in molecular mass (ΔM), full-scan MS–MS spectra with those of lobeline, norlobelanine and lobelanidine. These alkaloids and ten other derivatives were identified in the plant extracts. Three piperidine alkaloids were reported in L. inflata for the first time.

     

Extraction and Hydrolysis Parameters for Determination of Quercetin in Hypericum perforatum

The optimum conditions for extraction of rutin and quercetin from Hypericum perforatum were investigated. The best efficiency of extraction was achieved with aqueous methanol 40–80% (v/v). For quercetin analysis as aglycone the effect of acid concentration and hydrolysis time on the extraction recovery were also studied. Hydrolysis for 5 min in the presence of 2.8 mol L^?1 HCl as well as for 10 min with 1.1 mol L^?1 HCl efficiently released quercetin from rutin. The content of quercetin?3-O-glycosides (rutin, hyperoside and quercetrin) and quercetin aglycone as well as chlorogenic and caffeic acids in H. perforatum leaves and flowers were determined by HPLC with photodiode-array detection and confirmed by electrospray mass spectrometry.     

Rapid Determination of Polyphenols in Tobacco by MLC

A rapid, simple and reliable analytical procedure has been developed for the determination of polyphenols, including chlorogenic acid, rutin and scopoletin, in tobacco samples. After extraction, the polyphenols were analyzed by micellar liquid chromatography coupled with UV detection at 340 nm without sample preparation step. The compositions of the micellar mobile phase for the separation of these polyphenols were selected adequately by a chemometric method. The optimal resolution and satisfactory peak shapes were achieved with 0.022 mol L^?1 sodium dodecyl sulfate and 0.45% (volume fraction) 1-propanol solution at pH 5.0 using Eclipse XDB-C18 (250 mm × 3.0 mm, 5 μm) column. The limits of detection for chlorogenic acid, rutin and scopoletin were 0.15, 0.29 and 0.22 μg mL^?1, respectively. This validated method was successfully applied to qualitatively and quantitatively analyze the polyphenols in tobacco in less than 10 min.