Rapid HPLC-DAD Research Articles

Simultaneous determination of 11 water-soluble synthetic colorants in foods consumed in Chile by high-performance liquid chromatography with diode Array detection

In Chile, limited information is available on colorants in commonly consumed foods among vulnerable age groups. We developed and validated a rapid HPLC-DAD method to simultaneously evaluate 11 synthetic colorants in candies, beverages, ice cream, and cereals. The method exhibited excellent analytical performance for all 11 colorants with LOD (0.44 - 1.55 mgL-1), LOQ v(1.32 - 4.70 mgL-1), precision (4.0 and 7.3% RSD), and recovery (80 - 105%) in fortified matrices (10-50-100 mgL-1). The highest detection frequencies were as follows: cereals > candies > beverages > ice cream. Sunset Yellow was the most prevalent colorant in all food matrices, followed by Allura Red and Azorubine. Positive samples contained between 1 and 5 synthetic colorants. With the exception of cereals, the colorant concentrations in the remaining matrices exceeded the Codex Alimentarius regulations and the values reported in other studies worldwide, indicating the Chilean population is at risk.

A rapid HPLC–DAD method for quantification of amikacin in pharmaceuticals and biological samples using pre-column derivatization with Hantzsch reagent

Amikacin (AMK) is an important member of aminoglycoside class, and its determination has therapeutic importance due to its matchless potency against gram –ve pathogens. Due to narrow therapeutic window, its monitoring in clinical samples is inevitable. Direct determination of AMK using HPLC with UV–visible detection is not possible because of its limited absorbance. Herein, Hantzsch reagent (mixture of acetylacetone, formaldehyde and acetate buffer) was used as pre-column derivatization for AMK. UV–visible detection was performed at 340 nm. Separation and identification of derivatized drug (amikacin) were carried out using C-18 column Kromasil 100 (15 cm × 0.46 mm, 5 μm) with isocratic mobile phase elution of pH 5 (acetate buffer 0.01 M):acetonitrile (30:70 v/v) with flow rate of 1 ml/min. The procedure was able to resolve AMK from endogenous compounds and from cephalosporin drug (most prescribed combination) with run time of 10 min. Under optimized conditions; calibration curve was linear in the range 0.10–25.0 µg/mL with LOD and LOQ values of 0.024 and 0.071 µg/mL. Method was also validated for reproducibility, ruggedness and accuracy. The procedure was found sensitive, robust and precise for the comprehensive analysis (qualitative and quantitative) that was applied for determination of AMK in pharmaceuticals, urine and blood samples.

A sensitive and rapid HPLC-DAD method for the determination of 3-hydroxy-1,2-dimethyl-4-pyridone and its distribution in rats

Purpose: To establish a sensitive and rapid method for the determination of the tissue distribution of 3-hydroxy-1,2-dimethyl-4-pyridone (L1) in vivo, and its plasma protein binding capacity.Methods: This study optimized a reverse-phase HPLC method for specific and sensitive determination of L1 as well as its plasma and tissue distributions. The optimized method was used to determine the plasma protein-binding capacity of L1 in Wistar rats.Results: A rapid, sensitive and simple HPLC-DAD method was established for studying the plasma and tissue distribution of L1. Following TI administration, its liver concentrations peaked at 60 min and 360min, followed 360 min later with peak level in the kidney (second highest). The L1 concentration was significantly lower after 360 min than after 60 min, and values of its mean binding to plasma proteins was 5.2 % at different L1 concentrations.Conclusion: These results indicate that L1 is a drug with rapid-absorption and rapid-elimination thath is distributed widely in vivo in rats. Moreover, the drug has a weak plasma protein-binding capacity.
 Keywords: 3-Hydroxy-1,2-dimethyl-4-pyridone, Distribution, Alzheimer’s disease, Therapy

Determination of Sorbic Acid in Cheese Samples by Rapid HPLC-DAD Method.

The sorbic acid and its salts have been widely used in the food industries for many years as important food preservatives in order to inhibit the growth of various bacteria, yeasts, and fungi in acidic media. The health effects have led to limitation on the concentrations that can be used in food. Because of that, the analytical determination of these preservatives is important for consumer interest and protection. The purpose of this study is to determine the concentration of sorbic acid in cheese samples by using HPLC. For HPLC analysis, an X-Terra RP-18 (150 × 4.60 mm i.d. × 5 μm) column was selected as the stationary phase at 25°C. Analysis time is about 3 minutes. The developed method was applied to 10 different cheese samples collected from the Turkish market. The levels of sorbic acid in the analyzed samples were between 21.3 mg/kg and 511.3 mg/kg.

Quantification of Ioversol in Injection Dosage form Using HPLC: Stability Indicting Method Development and Validation

Background: Ioversol (IVL) is a radiographic contrast agent employed in the diagnostic radiography. In this investigation, our aim was to develop and validate a simple and rapid HPLC-DAD method for determination of IVL in bulk and injection dosage form. Methods: IVL separation and analysis was performed on Zodiac phenyl C18 column (250 mm × 4.5 mm; 5 µ particle size) using water-methanol (90:10 by volume) as mobile phase system and with detection at 254 nm. Results: The retention value of IVL was 4.11 min. The method linearity range was found 254.5-763.5 µg/ml with LOQ and LOD values of 2.376 µg/ml and 0.729 µg/ml, respectively. The accuracy (̴ 100%) and precision (< 2.0%) were within the acceptance criteria. Stability indicating ability of the method was proved by stress degradation studies. Adoptability of this method was assessed with application to marketed injection dosage form with good accuracy (recovery 100.49%) and precision (RSD 0.715%). Conclusion: By adopting this method one can analyze IVL in injection dosage form in less than 10 min and hence this method is time saving and enables the estimation of large number of samples.

Chromatographic fingerprint analysis and quantitative evaluate the rhizomes of Alpinia officinarum Hance (lesser galangal)

The rhizome of Alpinia officinarum Hance is a well-known traditional Chinese medicine (TCM) and has been widely used for the remedy of gastrointestinal diseases. In the present study, a simple and rapid HPLC-DAD was developed for the quality control of the rhizomes of A. officinarum. Its chemical fingerprint was established using raw material of 15 different origins in China. Similarity analysis (SA) and hierarchical clusting analysis (HCA) were applied to select the qualitative markers. Principal components analysis (PCA) was conducted to select the quantitative markers of the rhizomes of A. officinarum samples from different origins. The constituents were confirmed by (+) electrospray ionization LC-MS. 12 constituents were selected as common peaks and 10 of them were confirmed by (+) electrospray ionization LC-MS. Six bioactive constituents including DPHA, galangin flavanone, galangin, galangin 3-methylether, DPHB and DPHC were simultaneous determination by using the HPLC-DAD analysis. The developed method was able to determine the bioactive components with excellent resolution, precision and recovery. The results indicated that chromatographic fingerprint combination with multi-components determination method is suitable for quality assessment of the rhizomes of A. officinarum.

β-Cyclodextrin-assisted extraction and green chromatographic analysis of Hibiscus sabdariffa L. anthocyanins and the effects of gallic/ferulic/caffeic acids on their stability in beverages

This study aimed to develop a green approach for extraction and determination of anthocyanins from Hibiscus sabdariffa calyx using a hydroxypropyl-β-cyclodextrin (HPβ-CD)-assisted extraction technique coupled with HPLC-DAD analysis and to investigate the effects of gallic/ferulic/caffeic acids on the stability of H. sabdariffa anthocyanins in beverages. HPβ-CD-assisted extraction of total anthocyanins from H. sabdariffa calyx was optimized using a Box–Behnken design-response surface methodology, and extraction at 54 °C for 53 min using HPβ-CD aqueous solution (3.7 g/100 mL) gave the maximum extraction yield of total anthocyanins (4.82 ± 0.13 mg/g). A work-safe and rapid HPLC-DAD method was developed for analysing H. sabdariffa anthocyanins using ethanol and a tartaric acid aqueous solution as mobile phases, and the main anthocyanin peaks were determined as delphinidin-3-O-sambubioside and cyanidin-3-O-sambubioside using a two-dimensional HPLC-DAD/ESI-MS method. Method validation showed that the developed method was accurate, stable and reliable for the analysis of H. sabdariffa anthocyanins. Additionally, the degradation of delphinidin-3-O-sambubioside, cyanidin-3-O-sambubioside and total anthocyanins from H. sabdariffa calyx ideally followed a first-order model (R2 > 0.98) in beverages alone or with additional phenolic acids. Gallic acid improved the stability of H. sabdariffa anthocyanins better than ferulic and caffeic acids, and adding/increasing ferulic and caffeic acids accelerated anthocyanin degradation.

A simple and rapid HPLC-DAD method for simultaneously monitoring the accumulation of alkaloids and precursors in different parts and different developmental stages of Catharanthus roseus plants.

A rapid and simple reversed phase liquid chromatographic system has been developed for simultaneous analysis of terpenoid indole alkaloids (TIAs) and their precursors. This method allowed separation of 11 compounds consisting of eight TIAs (ajmalicine, serpentine, catharanthine, vindoline, vindolinine, vincristine, vinblastine, and anhydrovinblastine) and three related precursors i.e., tryptophan, tryptamine and loganin. The system has been applied for screening the TIAs and precursors in Catharanthus roseus plant extracts. In this study, different organs i.e., flowers, leaves, stems, and roots of C. roseus were investigated. The results indicate that TIAs and precursor accumulation varies qualitatively and quantitatively in different organs of C. roseus. The precursors showed much lower levels than TIAs in all organs. Leaves and flowers accumulate higher level of vindoline, catharanthine and anhydrovinblastine while roots have higher level of ajmalicine, vindolinine and serpentine. Moreover, the alkaloid profiles of leaves harvested at different ages and different growth stages were studied. The results show that the levels of monoindole alkaloids decreased while bisindole alkaloids increased with leaf aging and upon plant growth. The HPLC method has been successfully applied to detect TIAs and precursors in different types of C. roseus samples to facilitate further study of the TIA pathway and its regulation in C. roseus plants.

Study of the Biotransformation of Tongmai Formula by Human Intestinal Flora and Its Intestinal Permeability across the Caco-2 Cell Monolayer.

Tongmai formula (TMF) is a well-known Chinese medicinal preparation that contains isoflavones as its major bioactive constituents. As traditional Chinese medicines (TCMs) are usually used by oral administration, their fate inside the intestinal lumen, including their biotransformation by human intestinal flora (HIF) and intestinal absorption deserves study. In this work TMF extract was incubated with human intestinal bacteria under anaerobic conditions and the changes in the twelve main constituents of TMF were then investigated. Their intestinal permeabilities, i.e., the transport capability across the intestinal brush border were investigated with a human colon carcinoma cell line (Caco-2) cell monolayer model to predict the absorption mechanism. Meanwhile, rapid HPLC-DAD methods were established for the assay. According to the biotransformation curves of the twelve constituents and the permeability coefficients, the intestinal absorption capacity of the typical compounds was elevated from the levels of 10−7 cm/s to 10−5 cm/s from those of the original compounds in TMF. Among them the main isoflavone glycosides puerarin (4), mirificin (6) and daidzin (7) were transformed into the same aglycone, daidzein (10). Therefore it was predicted that the aglycone compounds might be the real active ingredients in TMF. The models used can represent a novel path for the TCM studies.

Simultaneous estimation of three triterpenoids from Carissa carandas using validated high performance liquid chromatography

Background: Carissa carandas L. (Apocynaceae), an important fruit of tropical and subtropical arid region, has a great potential for the development of a new phytotherapeutic medicine. Due to an increase in commercial products of this fruit in the global market, an extensive phytochemical comparison seems imperative to understand its internal quality. Aims and Objective: To develop and validate an HPLC-DAD method for simultaneous estimation of three triterpenoids from C. carandas. Materials and Methods: A simple, sensitive and rapid HPLC-DAD method was developed for simultaneous estimation of lupeol, stigmasterol, and b-sitosterol from C. carandas fruits. Isocratic elution at a flow rate of 1.0 mL/min was employed on C18 Cosmosil column with PDA detection at 210 nm using acetonitrile and ethanol. Different parts of C. carandas and its fruits collected from different geographical region were screened for lupeol, stigmasterol, and b-sitosterol contents. Results: Proposed method for simultaneous estimation of lupeol, stigmasterol and b-sitosterol was found highly specific, reliable, and sensitive which allowed good recovery of all analytes. It was validated as per ICH guidelines and proved to have good linearity, precision, repeatability, and stability. It was efficiently applied to screen different samples of C. carandas providing a mean to assess the quality and authenticity. Conclusion: The HPLC method could be an effective technique for routine analysis of lupeol, stigmasterol, and b-sitosterol simultaneously and monitoring the quality of fruit and its available products in cosmetic and pharmaceutical companies. Key words: Carissa carandas, simultaneous estimation, triterpenoids, validation

An HPLC-DAD method for rapid and high resolution analysis of concentrated BTEX and styrene aqueous samples

A rapid HPLC-DAD method for analysis of concentrated BTEX and styrene (BTEXS) aqueous mixtures is reported. Good resolutions of close to or greater than 1.5 were obtained for high equimolar BTEXS concentrations of up to 2.0 mM. At 5.5 min per sample analysis, this method is also one of the fastest HPLC methods to date, providing high throughput analysis and lowering the analysis price.

Quantitative analysis of isoflavones in 11 Trifolium spec. from Turkish flora and in some commercially available Trifolium products

The genus Trifolium is represented by 96 species in Turkish Flora [1]. Among these species Trifolium pratense L. is marketed for use in alleviating menopausal symptoms. T. pratense contains the isoflavones, daidzein, formononetin, biochanin A, and genistein. It has been shown that the isoflavonoids lower the cholesterol level, play a role in prevention and medication of some cancer types and help to reduce menopausal symptoms [2–5]. The purpose of this study was to quantify four isoflavones in the 11 Trifolium spec. collected from nature and in some herbal products to determine which sample contains the highest isoflavone amount. A rapid HPLC-DAD method used for determination of isoflavonoids [6]. The method was validated according to the guidelines. T. canescens and T. triocephalum were found as the samples contain higher isoflavonoid amount than T. pratense [Fig.1].

Simultaneous determination of nine lignans using pressurized liquid extraction and HPLC-DAD in the fruits of Schisandra chinensis

The pressurized liquid extraction and HPLC-DAD was developed for extraction and determination of bioactive lignans in Schisandra chinensis. The efficient PLE conditions employed methanol as extraction solvent, 125 °C of extraction temperature, 5 min of static extraction time and only one cycle. A rapid HPLC-DAD method was described for simultaneous determination of nine lignans, including schisandrol A, gomisin J, schisandrol B, tigloylgomisin H, angeloylgomisin H, schisandrin A, γ-schisandrin, gomisin N and schisandrin C. The extraction efficiency of PLE was observed to be comparable with reflux and sonication. In addition, the contents of nine lignans in S. chinensis from different regions were analysed by PLE and HPLC-DAD method.

A rapid HPLC-DAD method for the analysis of fluoxetine and norfluoxetine in plasma from overdose patients

There is a need for fast, simple and reliable analytical methods for the analysis of fluoxetine and norfluoxetine in patients who voluntarily or involuntarily have taken an overdose of the drug. A new liquid chromatographic method with diode array detection is presented herein for the determination of fluoxetine and its main active metabolite in human plasma for toxicological purposes. A mobile phase composed of acetonitrile and aqueous tetramethylammonium perchlorate allows to obtain the complete separation of the analytes on a C18 reversed phase column. The fast and accurate sample pre-treatment step is carried out by means of solid-phase extraction using hydrophilic–lipophilic balance cartridges and loading 100 μL of plasma only. This procedure gives satisfactory extraction yield values, as well as good plasma sample purification from matrix interference. Linearity was obtained in the 150–3000 ng/mL range for both analytes. Selectivity with respect to other psychotropic drugs was satisfactory. The method seems to be suitable for the analysis of fluoxetine and its metabolite in human plasma for depressed patients in overdose.

Improved HPLC determination of phenolic compounds in cv. golden delicious apples using a monolithic column.

A rapid HPLC-DAD determination of phenols in apple using an RP monolithic column is reported. Because of the hydrodynamic advantages offered by this kind of column and the use of acidified acetonitrile as eluent, assays of apple extracts can be performed in <21 min. Assays of pulp and peel extracts were carried out without the need for time-consuming sample pretreatment except filtration. Several flavanols, hydroxycinnamic acids, dihydrochalcones, and six quercetin glycosides were identified and quantified. A seventh quercetin derivative, two chalcone-related compounds, and three hydroxycinnamic derivatives were also found. Peels proved to be richer in phenols than pulps, the former being composed mainly of (-)-epicatechin, procyanidin B2, chlorogenic acid, phloridzin, hyperin, and avicularin. In pulps, where the chlorogenic acid was the principal phenolic compound, quercetin glycosides were found in very low amounts.